Secondary literature sources for C1

The following references were automatically generated.

Shindo M et al.
Toward the identification of selective modulators of protein kinase C (PKC) isozymes: establishment of a binding assay for PKC isozymes using synthetic C1 peptide receptors and identification of the critical residues involved in the phorbol ester binding.
Bioorg Med Chem. 2001; 9: 2073-81
Display abstract
Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. We previously synthesized C1 peptides (of approximately 50 residues) corresponding to all PKC isozymes and measured their PDBu binding affinity. While many of these peptide receptors exhibited PDBu affinities comparable to the respective complete isozyme, some of the C1A peptides could not be used because they undergo temperature dependent inactivation. This problem was however eliminated by 4 degrees C incubation or elongation of the 50-mer C1 peptides at both N- and C-termini to increase their folding efficiency and stability. These findings enabled us to determine the K(d)'s of PDBu for all PKC C1 peptides (except for theta-C1A) and establish the value of these peptides as readily available, stable, and easily handled surrogates of the individual isozymes. The resultant C1 peptide receptor library can be used to screen for new ligands with PKC isozyme and importantly C1 domain selectivity. Most of the C1 peptide receptors showed strong PDBu binding affinities with K(d)'s in the nanomolar range (0.45-7.4 nM). Two peptides (delta-C1A and theta-C1A) bound PDBu over 100-fold less tightly. To identify the residues that contribute to this affinity difference, several mutants of delta-C1A and theta-C1A were synthesized. Both the G9K mutant of delta-C1A and the P9K mutant of theta-C1A showed K(d)'s of 2-3 nM. This approach provides a useful procedure to determine the role of each C1 domain of the PKC isozymes by point mutation.

Roaten JB, Kazanietz MG, Sweatman TW, Lothstein L, Israel M, Parrill AL
Molecular models of N-benzyladriamycin-14-valerate (AD 198) in complex with the phorbol ester-binding C1b domain of protein kinase C-delta.
J Med Chem. 2001; 44: 1028-34
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N-Benzyladriamycin-14-valerate (AD 198) is a semisynthetic anthracycline with experimental antitumor activity superior to that of doxorubicin (DOX). AD 198, unlike DOX, only weakly binds DNA, is a poor inhibitor of topoisomerase II, and circumvents anthracycline-resistance mechanisms, suggesting a unique mechanism of action for this novel analogue. The phorbol ester receptors, protein kinase C (PKC) and beta2-chimaerin, were recently identified as selective targets for AD 198 in vitro. In vitro, AD 198 competes with [3H]PDBu for binding to a peptide containing the isolated C1b domain of PKC-delta (deltaC1b domain). In the present study molecular modeling is used to investigate the interaction of AD 198 with the deltaC1b domain. Three models are identified wherein AD 198 binds into the groove formed between amino acid residues 6-13 and 21-27 of the deltaC1b domain in a manner similar to that reported for phorbol-13-acetate and other ligands of the C1 domain. Two of the identified models are consistent with previous experimental data demonstrating the importance of the 14-valerate side chain of AD 198 in binding to the C1 domain as well as current data demonstrating that translocation of PKC-alpha to the membrane requires the 14-valerate substituent. In this regard, the carbonyl of the 14-valerate participates in hydrogen bonding to the deltaC1b while the acyl chain is positioned for stabilization of the membrane-bound protein-ligand complex in a manner analogous to the acyl chains of the phorbol esters. These studies provide a structural basis for the interaction of AD 198 with the deltaC1b domain and a starting point for the rational design of potential new drugs targeting PKC and other proteins with C1 domains.

Slater SJ et al.
Low- and high-affinity phorbol ester and diglyceride interactions with protein kinase C: 1-O-alkyl-2-acyl-sn-glycerol enhances phorbol ester- and diacylglycerol-induced activity but alone does not induce activity.
Biochemistry. 2001; 40: 6085-92
Display abstract
Phorbol ester-induced conventional protein kinase C (PKCalpha, -betaIota/IotaIota, and -gamma) isozyme activities are potentiated by 1,2-diacyl-sn-glycerol. This has been attributed to a "cooperative" interaction of the two activators with two discrete sites termed the low- and high-affinity phorbol ester binding sites, respectively [Slater, S. J., Milano, S. K., Stagliano, B. A., Gergich, K. J., Ho, C., Mazurek, A., Taddeo, F. J., Kelly, M. B., Yeager, M. D., and Stubbs, C. D. (1999) Biochemistry 38, 3804-3815]. Here, we report that the 1-O-alkyl ether diglyceride, 1-O-hexadecyl-2-acetyl-sn-glycerol (HAG), like its 1,2-diacyl counterpart, 1-oleoyl-2-acetyl-sn-glycerol (OAG), also potentiated PKCalpha, -betaI/II, and -gamma activities induced by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA). Similar to OAG, HAG was found to bind to the low-affinity phorbol ester binding site and to enhance high-affinity phorbol ester binding, and to decrease the level of Ca(2+) required for phorbol ester-induced activity, while being without effect on the Ca(2+) dependence of membrane association. Thus, similar to OAG, HAG may also potentiate phorbol ester-induced activity by interacting with the low-affinity phorbol ester binding site, leading to a reduced level of Ca(2+) required for the activating conformational change. However, HAG was found not to behave like a 1,2-diacyl-sn-glycerol in that alone it did not induce PKC activity, and also in that it enhanced OAG-induced activity. The results reveal HAG to be a member of a new class of "nonactivating" compounds that modulate PKC activity by interacting with the low-affinity phorbol ester binding site.

Wang QJ, Fang TW, Nacro K, Marquez VE, Wang S, Blumberg PM
Role of hydrophobic residues in the C1b domain of protein kinase C delta on ligand and phospholipid interactions.
J Biol Chem. 2001; 276: 19580-7
Display abstract
The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity. Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction. [(3)H]Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity. Lipid dependence was determined as a function of lipid concentration and composition. The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity. As expected, Leu-20 and Leu-24 strongly influenced binding. Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect. Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu. In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity. More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.

Schmitz HP, Jockel J, Block C, Heinisch JJ
Domain shuffling as a tool for investigation of protein function: substitution of the cysteine-rich region of Raf kinase and PKC eta for that of yeast Pkc1p.
J Mol Biol. 2001; 311: 1-7
Display abstract
With the completion of the sequences of entire genomes, the need for functional characterisation of proteins and their domains is becoming acute. Conserved regions within proteins often share overlapping functions but despite this conservation may fulfil quite different tasks in different species. In this work, we investigated the cysteine-rich motif (C1 domain) of yeast protein kinase C (Pkc1p) as a model to establish a test system for domain function. C1 domains activate kinases through binding of either diacylglycerol and/or phosphatidylserine, as in many members of the protein kinase C (PKC) family, or by binding small GTPases, as in Raf kinase. In contrast to other members of the protein kinase C superfamily, Pkc1p of Saccharomyces cerevisiae is activated via binding of the small G-protein Rho1p to its C1 domain. We developed a system for domain shuffling to establish the function of C1 domains from human Raf kinase and rat PKC eta in yeast. Only the C1 domain from Raf kinase enabled the chimeric enzyme to bind Rho1p when substituted for the native yeast domain. Accordingly, a chimeric Pkc1p carrying the C1 from Raf kinase, but not that from PKC eta, was able to partially complement the phenotypes of a yeast pkc1 deletion mutant. We interpret these data as further evidence that interaction with a small GTPase is the main regulatory function of the C1 domain in yeast. Copyright 2001 Academic Press.

Pepio AM, Sossin WS
Membrane translocation of novel protein kinase Cs is regulated by phosphorylation of the C2 domain.
J Biol Chem. 2001; 276: 3846-55
Display abstract
Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.

Rey O, Rozengurt E
Protein Kinase D Interacts with Golgi via Its Cysteine-Rich Domain.
Biochem Biophys Res Commun. 2001; 287: 21-6
Display abstract
Protein kinase D (PKD)/protein kinase C&mgr; is a serine/threonine protein kinase that has been localized in the cytosol and in several intracellular compartments including Golgi, mitochondria and plasma membrane. Using real time imaging of fluorescent protein (GFP)-tagged PKD, we have found that the accumulation of PKD in the Golgi compartment, following a temperature shift from 37 to 20 degrees C, was mediated by the cysteine-rich domain (CRD) of PKD. The CRD of PKD also mediates its interaction with the plasma membrane, further supporting the conclusion that the CRD of PKD may act as a subcellular localization signal. Copyright 2001 Academic Press.

Nowicka D
Protein kinase C in the barrel cortex.
Acta Neurobiol Exp (Warsz). 2000; 60: 509-509

Hoyos B et al.
The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors.
J Exp Med. 2000; 192: 835-45
Display abstract
Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH(2)-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms alpha, delta, zeta, and mu, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKC alpha by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.

Acs P, Paragh G
[Protein kinase C as a potential target for drug development in cancer research]
Orv Hetil. 2000; 141: 1767-71
Display abstract
The central role of protein kinase C (PKC) in cellular signaling makes it an attractive and promising target for drug development. With increasing insight into the regulation by ligands, by phosphorylation, and by macromolecular interactions involved in localization, we will be able to predict the consequences of modifications in second messenger levels resulting from changes in oncogenes, growth factors, or the exposures to xenobiotics. The multiplicity of functionally distinct isoforms of PKC and the depth of modification of their properties in a cell-context specific fashion provide abundant opportunities for generating therapeutic specificity. The emerging elucidation of the complexities of regulation of PKC folding and localization presents the opportunity to pursue novel strategies for intervention, complementing the efforts directed at the design of selective catalytic site inhibitors. Although PKC initially attracted the attention of cancer researchers as a potential therapeutic target, its general role in signal transduction affords a much broader field of opportunity.

Irie K et al.
Synthesis and tumor-promoting activities of 12-Epi-phorbol-12,13-dibutyrate.
Biosci Biotechnol Biochem. 2000; 64: 2429-36
Display abstract
12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O2-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the beta-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.

Jaken S, Parker PJ
Protein kinase C binding partners.
Bioessays. 2000; 22: 245-54
Display abstract
Members of the protein kinase C family respond to second messengers and are involved in controlling a broad array of cellular functions. The overlapping specificity and promiscuity of these proteins has promoted the view that specific binding proteins constrain individual family members to create the appropriate specificity of action. It is speculated that such protein kinase C-regulator protein interactions affect substrate availability as well as exposure to allosteric activator(s) and that consequent interactions specify cellular location and impose integration with other signaling systems. These predicted features have been realized in the identification of many protein kinase C interacting proteins and examples of these are discussed.

Wender PA, Lippa B, Park CM, Irie K, Nakahara A, Ohigashi H
Selective binding of bryostatin analogues to the cysteine rich domains of protein kinase C isozymes.
Bioorg Med Chem Lett. 1999; 9: 1687-90
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Designed bryostatin analogues are assayed for binding affinity to individual cysteine rich domains of several protein kinase C (PKC) isozymes. These analogues exhibit significant selectivity for the PKCdelta-C1B peptide in terms of absolute affinity and the PKCdelta-C1A peptide in terms of relative affinity when compared to phorbol-12,13-dibutyrate.

Fukuda H, Irie K, Nakahara A, Ohigashi H, Wender PA
Solid-phase synthesis, mass spectrometric analysis of the zinc-folding, and phorbol ester-binding studies of the 116-mer peptide containing the tandem cysteine-rich C1 domains of protein kinase C gamma.
Bioorg Med Chem. 1999; 7: 1213-21
Display abstract
Tumor-promoting phorbol esters activate protein kinase C (PKC) isozymes by binding to the zinc-finger like cysteine-rich domains in the N-terminal regulatory region. Our recent studies have revealed that only PKCgamma has two high affinity phorbol ester-binding domains, providing a structural blueprint for the rational design of PKCgamma-selective modulators for the treatment of neuropathic pain. To extend this approach, the 116-mer peptide containing the double cysteine-rich motifs of PKCgamma (gamma-C1A-C1B) has been synthesized for the first time using an Fmoc-solid phase strategy with a stepwise chain elongation. This peptide was purified by the reversed phase HPLC to give satisfactory mass data (MALDI-TOF-MS and ESI-TOF-MS). The peptide was successfully folded by zinc treatment and the folded peptide was analyzed intact under neutral conditions by ESI-TOF-MS. The multiple charge mass envelopes shifted to those of the lower mass charge state by addition of 4 molar equiv. ZnCl2, suggesting that gamma-C1A-C1B preserves some higher order structure by the zinc folding. Moreover, the mass spectrum of the zinc-folded peptide in the presence of EDTA clearly showed that gamma-C1A-C1B coordinates exactly four atoms of zinc. This zinc stoichiometry is identical to that of native PKCgamma. Scatchard analysis of the zinc-folded peptide revealed two binding sites of distinctly different affinities (Kd=6.0 +/- 1.5 and 47.0 +/- 6.6 nM) comparable to those reported by Quest and Bell for the GST fusion protein of gamma-C1A-C1B prepared by DNA recombination. These results indicate that gamma-C1A-C1B serves as an effective surrogate for native PKCgamma for the study of the structural characteristics of the binding recognition event and the design, discovery, and development of new PKCgamma-selective modulators.

Ma D, Wang G, Wang S, Kozikowski AP, Lewin NE, Blumberg PM
Synthesis and protein kinase C binding activity of benzolactam-V7.
Bioorg Med Chem Lett. 1999; 9: 1371-4
Display abstract
Benzolactam-V7 (3a), a simplified analogues of (-)-indolactam-V with twist-form conformation, was synthesized and evaluated as a new protein kinase C modulator. Both 3a and its-7-substituted analogue 3c showed weak binding activity to displace PDBU binding from recombinant PKCalpha.

Hori T, Takai Y, Takahashi T
Presynaptic mechanism for phorbol ester-induced synaptic potentiation.
J Neurosci. 1999; 19: 7262-7
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Phorbol ester facilitates transmitter release at a variety of synapses, and the phorbol ester-induced synaptic potentiation (PESP) is a model for presynaptic facilitation. To address the mechanism underlying PESP, we have made paired whole-cell recordings from the giant presynaptic terminal, the calyx of Held, and its postsynaptic target in the medial nucleus of the trapezoid body in rat brainstem slices. Phorbol ester potentiated EPSCs without affecting either presynaptic calcium currents or potassium currents. Protein kinase C inhibitors applied from outside or injected directly into the presynaptic terminal attenuated the PESP. Furthermore, presynaptic loading of a synthetic peptide with the sequence of the N-terminal domain of Doc2alpha interacting with Munc13-1 (Mid peptide) significantly attenuated PESP, whereas mutated Mid peptide had no effect. We conclude that the target of the presynaptic facilitatory effect of phorbol ester resides downstream of calcium influx and may involve both protein kinase C and Doc2alpha - Munc13-1 interaction.

Takasaki A, Hayashi N, Matsubara M, Yamauchi E, Taniguchi H
Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction.
J Biol Chem. 1999; 274: 11848-53
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Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.

Kowara R, Gryckiewicz E, Matecki A, Pawelczyk T
The ultraviolet studies on protein-lipid interaction of a protein kinase C-gamma phorbol-binding domain.
Acta Biochim Pol. 1999; 46: 405-17
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Family of protein kinase C (PKC) isozymes play a key role in transducing a vast number of signals into the cells. The members of classical PKC family are activated by binding of various lipid ligands to one of the several cysteine-rich domains of the enzyme. Second cysteine-rich (Cys2) domain of PKC-gamma was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) using the cDNA sequence from rat brain. The Cys2 protein after cleavage from GST was purified to homogeneity using glutathione-agarose and Mono-S cation exchanger column. In order to investigate the interaction of lipids and calcium with Cys2 protein we used UW spectroscopy. The UV spectrum of Cys2 protein exhibited a maximum at 205 nm. Exposition of Cys2 protein to phosphatidylserine (PS) vesicles resulted in significant decrease in the absorbance in the 210 nm region. Changes in UW spectrum of Cys2 protein induced by phorbol 12,13-dibutyrate (PDB) were smaller than those induced by PS, and addition of PDB with PS had no effect on the PS induced changes in UV spectrum of Cys2. Neither phosphatidylcholine (PC) nor phosphatidylethanolamine (PE) affected UV spectrum of Cys2 but in the presence of phosphatidylinositol 4,5 bisphosphate (PIP2) or phosphatidyliinositol 4-phosphate (PIP) vesicles some changes were observed. Calcium ions alone or in the presence of PS had no effect on the UV spectrum of Cys2 protein. These data indicate that PS comparing to PDB, interacts with a larger area of Cys2 protein, and that the binding sites for these two molecules are at least overlapping. The site of PIP and PIP2 interaction with PKC-gamma is distinct from that of phorbol ester binding site.

Lorenzo PS et al.
Differential roles of the tandem C1 domains of protein kinase C delta in the biphasic down-regulation induced by bryostatin 1.
Cancer Res. 1999; 59: 6137-44
Display abstract
Bryostatin 1 (Bryo), currently in clinical trials, has been shown to induce a biphasic concentration-response curve for down-regulating protein kinase C (PKC) delta, with protection of the enzyme from down-regulation at high Bryo doses. In our ongoing studies to identify the basis for this unique behavior of PKCdelta, we examined the participation of the two ligand binding sites (C1a and C1b) in the regulatory domain of the enzyme. Three mutants of PKCdelta prepared by introducing a point mutation in either C1a or Clb or both C1a and Clb were overexpressed in NIH 3T3 cells. All of the constructs retained a biphasic response to down-regulation assessed after 24-h treatment with Bryo. However, the roles of the individual C1 domains were different for the two phases of the response. For down-regulation, both the C1a and the C1b mutants displayed equivalent 3-4-fold reductions in their affinities for the ligand. For protection from down-regulation, a reduced protection was observed for the C1a mutant, which showed a broader biphasic curve compared with those for wild-type PKCdelta and the Clb mutant. Like wild-type PKCdelta, all of the mutants showed the same subcellular partitioning of the protected enzyme to the particulate fraction of the cells, arguing against changes in sensitivity to Bryo due to differences in localization. Likewise, relatively similar patterns of localization were observed using green fluorescent protein-PKCdelta constructs. We conclude that the C1 domains of PKCdelta do not have equivalent roles in inducing protection against Bryo-induced down-regulation. The C1a domain plays a critical role in conferring the degree of protection at high concentrations of Bryo. Elucidation of the differential effect of Bryo on PKCdelta may suggest strategies for the design of novel ligands with Bryo-like activities.

Maler L
Distribution of protein kinase C in the brain of Apteronotus leptorhynchus as revealed by phorbol ester binding.
J Comp Neurol. 1999; 408: 161-9
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An antibody to the mammalian protein kinase C alpha (PKCalpha) subunit and brain dissection was used for immunoblot analysis of this protein in various brain regions of Apteronotus leptorhynchus. Western blots revealed that the antibody labeled a band of the expected molecular mass (approximately 80 kDa) for this enzyme in mammalian cortex and electric fish brain, suggesting that this protein is also found in gymnotiform brain. The 80-kDa band was enriched in fish forebrain and cerebellum compared with hypothalamus and brainstem areas. [3H]Phorbol 12,13-dibutyrate ([3H]PDBu) binding was used as a marker for the distribution of protein kinase C (PKC). [3H]PDBu binding was nearly completely displaced by excess cold PDBu; specific [3H]PDBu binding sites were heterogenously distributed with high densities in some gray matter regions and negligible densities in fiber tracts. A very high density of [3H]PDBu binding sites were found in the dorsal forebrain with far lower densities in most ventral forebrain nuclei. Low binding densities were observed in preoptic and hypothalamic areas with the exception of the nucleus diffusus and nucleus tuberis anterior. The thalamus and midbrain also had only low levels of binding. The cerebellar molecular layer had dense binding, in contrast to the granule cell layer where binding was negligible. In the electrosensory lateral line lobe (ELL), there was moderate binding in the dorsal molecular layer, which contains cerebellar parallel fibers; the other layers of the ELL had far lower binding densities.

Irie K et al.
Synthesis and phorbol ester-binding studies of the individual cysteine-rich motifs of protein kinase D.
Bioorg Med Chem Lett. 1999; 9: 2487-90
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To investigate the phorbol ester-binding properties of the individual cysteine-rich motifs of protein kinase D (PKD), the 52-mer peptides containing each cysteine-rich motif of PKD (PKD-C1A, PKD-C1B) have been synthesized. The [3H]phorbol-12,13-dibutyrate (PDBu) binding to PKD-C1A was affected drastically by incubation temperature while that to PKD-C1B was not. Scatchard analysis of [3H]PDBu binding to both PKD C1 peptides gave dissociation constants of 2.5 +/- 0.4 and 2.7 +/- 0.8 nM for PKD-C1A and PKD-C1B, respectively, indicating that the two cysteine-rich motifs of PKD are functionally equivalent like those of PKCgamma.

Yuryev A, Wennogle LP
The RAF family: an expanding network of post-translational controls and protein-protein interactions.
Cell Res. 1998; 8: 81-98
Display abstract
Protein kinase RAF is strategically located in the "Ras-MAP-kinase signal transduction pathway", a principle system which transmits signals from growth factor receptors to the nucleus, resulting in cell proliferation. Growth factor responses are mediated in part by activation of Ras, which in turn activates RAF to phosphorylate MEK, its downstream substrate. MEK activates MAP-kinase to influence nuclear events. It is clear, however, that a network of signals other than those carried by Ras plays a role in RAF regulation. These orthogonal influences are mediated by: serine/threonine kinases, tyrosine kinases, and protein-protein interactions. As a further complication to the RAF network, three isoforms of RAF have been established which have divergent N-terminal regulatory domains. Whereas these divergent regulatory domains implicate isoform-specific functions, no clear evidence or hypothesis for distinct functions for individual isoforms has been presented. Recently, "isoform-specific protein interactions" have been identified among numerous proteins interacting with RAF. These studies may serve to delineate independent functions for RAF isoforms.

Iglesias T, Matthews S, Rozengurt E
Dissimilar phorbol ester binding properties of the individual cysteine-rich motifs of protein kinase D.
FEBS Lett. 1998; 437: 19-23
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Protein kinase D (PKD) is a serine/threonine kinase that binds phorbol esters in a phospholipid-dependent manner via a tandemly repeated cysteine-rich, zinc finger-like motif (the cysteine-rich domain, CRD). Here, we examined whether the individual cysteine-rich motifs of the CRD of PKD (referred to as cysl and cys2) are functionally equivalent in mediating phorbol ester binding both in vivo and in vitro. Our results demonstrate that the cysl and cys2 motifs of the CRD of PKD are functionally dissimilar, with the cys2 motif responsible for the majority of [3H]phorbol 12,13-dibutyrate (PDB) binding, both in vivo and in vitro.

Anton-Tay F, Ramirez G, Martinez I, Benitez-King G
In vitro stimulation of protein kinase C by melatonin.
Neurochem Res. 1998; 23: 601-6
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It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 microM) and protein kinase C inhibitors (100 microM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.

Jacoby JJ, Schmitz HP, Heinisch JJ
Mutants affected in the putative diacylglycerol binding site of yeast protein kinase C.
FEBS Lett. 1997; 417: 219-22
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In an attempt to study the functional similarities between protein kinase C from the yeast Saccharomyces cerevisiae and its human homologues we have started in vitro mutagenesis to alter specific domains. Here we report on the exchange of four cysteine residues by serines in yeast Pkc1p that have been shown to be essential for diacylglycerol (DAG) binding and activation by this compound in humans. The mutant yeast protein leads to sensitivity to caffeine and low concentrations of SDS when expressed in a pkc1 deletion strain. However, sensitivity to staurosporine was not affected. Our data indicate that the conserved DAG binding domain serves an important function in yeast Pkc1p.

Cai H et al.
Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.
Mol Cell Biol. 1997; 17: 732-41
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The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.

de Vries DJ, Rao KS, Willis RH
Application of a radioreceptor assay to the screening and characterisation of compounds from marine organisms with activity at the phorbol ester binding site of protein kinase C.
Toxicon. 1997; 35: 347-54
Display abstract
Protein kinase C is a ubiquitous enzyme with a key role in cellular function, making it an attractive drug target. Utilising a competitive binding assay for the phorbol ester binding site of the enzyme in a rat brain membrane preparation, screening was undertaken on 686 marine macroorganisms representing a broad range of taxa and environments from throughout Australasia. Of these extracts from 28 organisms significantly inhibited [3H]phorbol dibutyrate binding, while two samples enhanced binding. Sponges and echinoderms were particularly well represented in the active specimens. A combination of taxonomic and elution information for individual leads provided a rationale for dereplication and prioritisation. Utilising assay-guided purification, the identity of active compounds from the sponge Agelas axifera was examined in detail. The previously described compounds, the agelasines, were identified. The screening and characterisation methods described provide a method for readily identifying novel probes for protein kinase C.

Naumann U, Eisenmann-Tappe I, Rapp UR
The role of Raf kinases in development and growth of tumors.
Recent Results Cancer Res. 1997; 143: 237-44

Morrison DK, Cutler RE
The complexity of Raf-1 regulation.
Curr Opin Cell Biol. 1997; 9: 174-9
Display abstract
The activation of the serine/threonine kinase Raf-1 is proving to be an intricate multistep process. Recent advances in elucidating how Raf-1 becomes activated in response to signaling events have emphasized the role of phosphorylation and protein interactions in Raf-1 regulation. The picture clearly emerging is that Raf-1 activity can be regulated by multiple mechanisms.

Hagemann C, Kalmes A, Wixler V, Wixler L, Schuster T, Rapp UR
The regulatory subunit of protein kinase CK2 is a specific A-Raf activator.
FEBS Lett. 1997; 403: 200-2
Display abstract
Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two-hybrid screen of a mouse-embryo cDNA library we detected an interaction between A-Raf and CK2beta subunit. This binding was specific, as no interaction between CK2beta and B-Raf or c-Raf-1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A-Raf kinase domain. A-Raf kinase activity was enhanced 10-fold upon coexpression with CK2beta in Sf9 cells. The alpha subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf-isoform-specific activation and a regulatory role for CK2beta independent of the CK2alpha subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.

Hunn M, Quest AF
Cysteine-rich regions of protein kinase Cdelta are functionally non-equivalent. Differences between cysteine-rich regions of non-calcium-dependent protein kinase Cdelta and calcium-dependent protein kinase Cgamma.
FEBS Lett. 1997; 400: 226-32
Display abstract
Regulatory domain elements of the non-calcium-dependent protein kinase Cdelta (nPKCdelta), including either or both of the cysteine-rich regions Cys1(delta) and Cys2(delta), were expressed as fusion proteins with glutathione-S-transferase and characterized using liposomal or mixed micellar phorbol ester binding assays. Fusion proteins containing Cys2(delta) bound phorbol-12,13-dibutyrate (PDBu) efficiently in the assay employing phosphatidylserine (PS) vesicles, while no significant binding was seen for proteins containing only Cys1(delta). Likewise, in mixed micellar assays, fusion proteins with Cys2(delta) bound PDBu with high affinity (Kd: 14-37 nM) and to significant stoichiometric levels (0.23-0.66 mol/mol), but no binding could be detected for proteins with Cys1(delta) only. The PS dependence of PDBu binding to Cys2(delta) was highly cooperative with Hill numbers lying in the range of 2.5-5.2. These results demonstrate the presence of striking functional differences between the cysteine-rich regions of nPKCdelta and the calcium-dependent isoform, cPKCgamma, where both cysteine-rich regions represent functional PDBu binding elements.

Henry SP, Monteith D, Bennett F, Levin AA
Toxicological and pharmacokinetic properties of chemically modified antisense oligonucleotide inhibitors of PKC-alpha and C-raf kinase.
Anticancer Drug Des. 1997; 12: 409-20

Rozengurt E, Sinnett-Smith J, Zugaza JL
Protein kinase D: a novel target for diacylglycerol and phorbol esters.
Biochem Soc Trans. 1997; 25: 565-71

Alexandrov I, Shlyakhova L, Vartanian A, Zajac-Kaye M, Alexandrova N
c-Raf kinase binds to N-terminal domain of c-Myc.
FEBS Lett. 1997; 414: 465-70
Display abstract
We have demonstrated that the 50 N-terminal amino acids of c-Myc bind a kinase activity, which phosphorylates Myc in vitro predominantly on Thr8. We also have shown that c-Raf, a widely known Ser/Thr kinase, involved in the Ras signaling pathway, binds to the same portion of c-Myc in vitro. In addition we were able to precipitate native c-Myc/Raf complex from various cell lysates. Physical interaction of Myc and Raf may potentially be a part of their well-known functional cooperation.

Xing H, Kornfeld K, Muslin AJ
The protein kinase KSR interacts with 14-3-3 protein and Raf.
Curr Biol. 1997; 7: 294-300
Display abstract
BACKGROUND: KSR (kinase suppressor of Ras) is a recently identified putative protein kinase that positively mediates the Ras signaling pathway in the invertebrates Caenorhabditis elegans and Drosophila melanogaster. The function of vertebrate KSR is not well characterized biochemically or biologically. RESULTS: We examined the physiological role of KSR in vertebrate signal transduction using Xenopus laevis oocytes. Overexpression of KSR, in combination with overexpression of the intracellular dimeric protein 14-3-3, induced Xenopus oocyte meiotic maturation and cdc2 kinase activation; the effect of KSR and 14-3-3 on oocyte maturation was blocked by co-expression of dominant-negative Raf-1. We noted that KSR contains multiple potential binding sites for 14-3-3, and we used the yeast two-hybrid system and co-immunoprecipitation experiments to show that KSR can bind to 14-3-3. Furthermore, we demonstrated that KSR can form a complex with Raf kinase both in vitro and in cultured cells. Cell fractionation studies revealed that KSR formed a complex with 14-3-3 in both the membrane and cytoplasmic fractions of cell lysates; however, KSR only formed a complex with Raf-1 in the membrane fraction. CONCLUSIONS: Our finding suggest that KSR, 14-3-3 and Raf form an oligomeric signaling complex and that KSR positively regulates the Ras signaling pathway in vertebrate organisms.

Ghosh S, Bell RM
Regulation of Raf-1 kinase by interaction with the lipid second messenger, phosphatidic acid.
Biochem Soc Trans. 1997; 25: 561-5

Ueno N, Oishi I, Sugiyama S, Nishida Y, Minami Y, Yamamura H
Identification of a novel Drosophila protein kinase highly homologous to protein kinase N (PKN).
Biochem Biophys Res Commun. 1997; 232: 126-9
Display abstract
We identified a novel Drosophila gene, Dpkn (Drosophila protein kinase related to PKN), encoding a putative protein serine/threonine kinase. Although the cDNA obtained was incomplete at its 5'-terminal region, the deduced amino acid sequence of its kinase domain exhibits a high degree of similarity to protein kinase N (PKN), which has a kinase domain related to protein kinase C (PKC) and leucine zipper-like sequences in the amino terminal region. Expression of Dpkn was observed throughout Drosophila development, although its expression level decreased at later stages of embryogenesis. The expression of Dpkn is first detected in the newly formed mesodermal cell layer and is then restricted to the developing somatic musculature, indicating a possible role of Dpkn in the development of somatic muscles in Drosophila.

McCarthy SA, Aziz N, McMahon M
Identification of immediate-early gene targets of the Raf-1 serine/threonine protein kinase using an estradiol-dependent fusion protein, delta Raf-1:ER.
Methods Mol Biol. 1997; 85: 137-51

Gorman C, Delves CJ, Skelly JV, Lowe PN
Interaction of Ras with Raf (residues 136-189) and related cysteine-rich protein fragments.
Biochem Soc Trans. 1997; 25: 511-511

Emerson SD et al.
Structure of the Ras-binding domain of c-Raf-1 as determined by NMR spectroscopy and identification of the region that interacts with Ras.
Drug Des Discov. 1996; 13: 83-93
Display abstract
The structure of the Ras-binding domain of human c-Raf-1 (residues 55 to 132) as determined in solution by NMR spectroscopy is presented. It consists of a five-stranded beta-sheet, a twelve residue alpha-helix, and an additional one-turn helix. The fold belongs to a known family whose members include ubiquitin and protein G. The surface of Raf55-132 that interacts with Ras has been identified by resonance perturbation mapping. The binding site is a spatially contiguous patch comprised of the two-N-terminal beta-strands, the loop between them, and the C-terminal end of the alpha-helix. A model of the Raf-Ras complex is presented, which was derived by analogy to the complex between protein G and a Fab fragment of IgG. In the model, edge beta-strands of each protein align in an antiparallel orientation, forming a unified beta-sheet, and side chains from both proteins are able to participate in ionic and hydrophobic interactions at the interface.

Radziwill G, Steinhusen U, Aitken A, Moelling K
Inhibition of Raf/MAPK signaling in Xenopus oocyte extracts by Raf-1-specific peptides.
Biochem Biophys Res Commun. 1996; 227: 20-6
Display abstract
Raf-1 is an upstream element of the mitogen-activated protein kinase (MAPK) pathway which leads to cell proliferation and differentiation. In this study Raf-1 derived peptides comprising the conserved amino acid residues Arg89 and Ser259, involved in binding of activated Ras and 14-3-3 proteins, respectively, were shown to interfere with MAPK activation in extracts from immature Xenopus oocytes. Lipids prepared from oocyte extracts can stimulate MAPK in a Ras- and protein kinase C-independent manner. This lipid-induced MAPK activation is blocked by a Raf-1 derived peptide comprising Ser259.

Ghosh S, Strum JC, Sciorra VA, Daniel L, Bell RM
Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.
J Biol Chem. 1996; 271: 8472-80
Display abstract
Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

Janosch P et al.
Characterization of IkappaB kinases. IkappaB-alpha is not phosphorylated by Raf-1 or protein kinase C isozymes, but is a casein kinase II substrate.
J Biol Chem. 1996; 271: 13868-74
Display abstract
The NF-kappaB transcription factor is activated by a wide variety of stimuli, including phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate. In its inactive state, NF-kappaB is sequestered in the cytoplasm tethered to an inhibitor protein, IkappaB. Activation comprises the rapid phosphorylation of IkappaB-alpha at N-terminal sites, which presumably marks IkappaB-alpha for proteolytic degradation and leads to release of NF-kappaB into the nucleus. In addition, IkappaB-alpha is constitutively phosphorylated at the C terminus, which may be a prerequisite for proper IkappaB function. Protein kinase C (PKC) is activated by 12-O-tetradecanoylphorbol-13-acetate and has been previously reported to phosphorylate IkappaB-alpha in vitro. As PKC has turned out to constitute a multigene family encoding isozymes with different biological functions, we have reinvestigated IkappaB-alpha phosphorylation by PKC using recombinant PKC isozymes expressed in insect cells. While crude PKC preparations were efficient IkappaB-alpha kinases, highly purified PKC isozymes completely failed to phosphorylate IkappaB-alpha. Biochemical separation of porcine spleen yielded at least two fractions with IkappaB-alpha kinase activity, both of which were devoid of detectable PKC isozymes. One peak contained both Raf-1 and casein kinase II (CKII). Purified Raf-1 does not phosphorylate IkappaB-alpha directly, but associates with CKII, which efficiently phosphorylates the C terminus of IkappaB-alpha. Two-dimensional phosphopeptide mapping and high pressure liquid chromatography-mass spectroscopy analysis showed that all IkappaB-alpha kinases induced phosphorylation at the same prominent sites in the C terminus. Our results clearly indicate that PKC isozymes alpha, beta, gamma, delta, epsilon, eta, and zeta as well as Raf-1 are not IkappaB-alpha kinases. They furthermore demonstrate that IkappaB-alpha is targeted by several kinases, one of which appears to be CKII.

Lee J et al.
Conformationally constrained analogues of diacylglycerol. 12. Ultrapotent protein kinase C ligands based on a chiral 4,4-disubstituted heptono-1,4-lactone template.
J Med Chem. 1996; 39: 36-45
Display abstract
Conformationally constrained analogues of diacylglycerol (DAG) built on a 5(-)[(acyloxy)methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template (1, Chart 1) were shown previously to bind tightly to protein kinase C alpha (PK-C alpha) in a stereospecific manner. These compounds, however, racemized readily through rapid acyl migration and lost biological potency. In order to circumvent this problem, the "reversed ester" analogues were designed as a new set of PK-C ligands. This reversal of the ester function produced some new DAG mimetics that are embedded in a C-4 doubly-branched heptono-1,4-lactone template. The reversed ester analogues were impervious to racemization, and their chemically distinct branches facilitated the enantiospecific syntheses of all targets. Compound 2, the simplest reversed ester analogue of 1 (Chart 1), exhibited a 3.5-fold reduction in binding affinity toward PK-C alpha which we attributed to the loss of a stabilizing gauche interaction that caused the ester branch in 2 to be more disordered than in the normal ester 1. However, conversion of the propanoyl branch of 2 into a propenoyl branch restored binding affinity (3 versus 5). As expected, the compounds bound to the enzyme with strict enantioselectivity (3 and 5 versus 4 and 6). Functionalization of the propenoyl-branched compounds as alpha-alkylidene lactones, in a manner which proved successful with the 5(-)[(acyloxy)methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template (9 and 10), produced stable compounds with equivalent ultrapotent binding affinities for PK-C alpha (7 and 8). The additional incorporation of the propenoyl-branched carbonyl into a gamma-lactone ring was performed (11-14) not only to derive a possible additional entropic advantage but also to confirm the spatial disposition of this carbonyl function in the ligand-enzyme complex. Although no additional entropic advantage was derived, the high binding affinities displayed by compounds 11 and 12 helped to establish the correct orientation of the equivalent carbonyl group in PK-C-bound DAG. As expected, these DAG analogues activated PK-C alpha. The most potent agonist, compound 8, stimulated phosphorylation of the alpha-pseudosubstrate peptide, and in primary mouse keratinocytes it caused inhibition of binding of epidermal growth factor with an ED50 of approximately 1 microM. In contrast to the phorbol esters, compound 8 did not induce acute edema or hyperplasia in skin of CD-1 mice, and its pattern of downregulation with several PK-C isozymes was different from that of phorbol 12-myristate 13-acetate (PMA).

Lee J et al.
Conformationally constrained analogues of diacylglycerol. 11. Ultrapotent protein kinase C ligands based on a chiral 5-disubstituted tetrahydro-2-furanone template.
J Med Chem. 1996; 39: 29-35
Display abstract
Conformationally constrained analogues of diacylglycerol (DAG) built on a racemic 5(-)[(acyloxy)-methyl]-5-(hydroxymethyl)tetrahydro-2-furanone template were shown previously to have excellent binding affinities for protein kinase C (PK-C). Since the interaction of PK-C with DAG is stereospecific, it was anticipated that PK-C would bind tightly to only one enantiomeric form of the compounds constructed with this new lactone template. Separation of enantiomers by chiral HPLC was discarded due to the ease with which acyl migration occurs in these class of compounds, and a total chiral synthesis was undertaken. Prior to chemical synthesis, the selection of the "correct" enantiomeric template was predicted by a molecular conformational analysis that compared the two enantiomers of DAG in their presumed "active" conformation with the two enantiomeric lactone templates. This presumed "active" conformation for DAG was derived from a previously developed pharmacophore model that uses the molecule of a potent phorbol diester as the ideal rigid template. The results from this analysis indicated that the "correct" lactone template corresponded to the inactive (R)-isomer of DAG. This analysis also predicted that the lactone template corresponding to the active (S)-DAG enantiomer would not fit adequately into the pharmacophore. The chiral syntheses of target compounds 2, 4, and 6, constructed on the selected, and presumably "correct" lactone template, were achieved from a common bicyclic intermediate (5R,8R,9R)-8,9-O-isopropylidene-2-keto-1,7-dioxaspiro[4.4]nonane (10) that was synthesized from commercially available 1,2:3,5-di-O-isopropylidene-alpha-D-threo-apiofuranose (7) by a very effective spirolactonization approach. On the basis of their ability to inhibit the binding of [3H-20]phorbol 12,13-dibutyrate (PDBU) to PK-C alpha, the enantiomeric ligands 2, 4, and 6 were twice as potent as the corresponding racemates. These results confirm that binding of these lactones is stereospecific and consistent with a binding mechanism similar to that of DAG.

Sharma R et al.
Conformationally constrained analogues of diacylglycerol. 10. Ultrapotent protein kinase C ligands based on a racemic 5-disubstituted tetrahydro-2-furanone template.
J Med Chem. 1996; 39: 19-28
Display abstract
5,5-Bis(hydroxymethyl)tetrahydro-2-furanone and its isomer 4,4-bis(hydroxymethyl)tetrahydro-2-furanone were investigated as possible templates for the construction of conformationally constrained analogues of the biologically important second messenger, diacylglycerol (DAG). The former lactone contains embedded within its structure an exact glycerol moiety, while in the latter the ring oxygen has been transposed to the other side of the carbonyl group. All target compounds were synthesized as racemates from 1,3-dihydroxy-2-propanone. The 5,5-bis(hydroxymethyl)tetrahydro-2-furanone proved to be the better template for the construction of DAG surrogates that were demonstrated to have high binding affinities for the biological target, protein kinase C (PK-C). The simplest target compounds derived from this template (3e and 3f) have one of the hydroxyl moieties functionalized either as a myristate or as an oleate ester. The simplest target compound (9c) derived from the ineffective 4,4-bis-(hydroxymethyl)tetrahydro-2-furanone template was investigated only with a myristoyl acyl chain. Reducing the long acyl chain to an acetyl moiety and attaching a compensating lipophilic chain to the lactone ring as an alpha-alkylidene moiety produced compounds 10e and 10f (Z-isomers) and 11e and 11f (E-isomers), which were constructed on the more effective 5,5-bis(hydroxymethyl)tetrahydro-2-furanone template. Targets 14c (Z-isomer) and 15c (E-isomer) were derived, in turn, from 4,4-bis(hydroxymethyl)tetrahydro-2-furanone. The affinities of these ligands for PK-C were assessed in terms of their ability to displace bound [3H-20]phorbol 12,13-dibutyrate (PDBU) from the single isozyme PK-C alpha. The biological data support the hypothesis that the increase in binding affinity for PK-C shown by some of these constrained DAG mimetics appears to be entropic in nature. Two of the designed ligands (10e and 10f) showed the highest affinities (34 and 24 nM, respectively) reported so far for a DAG analogue. Assuming that the interaction between these racemic compounds and PK-C is stereospecific, the potency of the active enantiomer is anticipated to double.

Irie K
[Structural studies of phorbol ester-binding domain of protein kinase C by photoaffinity labeling]
Seikagaku. 1996; 68: 129-32

Marais R, Marshall CJ
Control of the ERK MAP kinase cascade by Ras and Raf.
Cancer Surv. 1996; 27: 101-25
Display abstract
Activation of ERKs plays a central part in the control of cell proliferation. In this chapter, we have concentrated on the identification and regulation of the kinases that activate Mek. We have chosen to focus on this area because of the many puzzles associated with it. Key issues for the future are the need to derive methods to determine what contribution each individual activator makes to both the magnitude and duration of Mek activation and understanding the mechanisms of regulation. Furthermore, we have considered the roles of Raf and the other kinases solely as Mek activators; they may well have other substrates including cdc25A (Galaktionov et al, 1995), I kappa B (Li and Sedivy, 1993) and TP53 (Jamal and Ziff, 1995).

Diaz-Meco MT et al.
The product of par-4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C.
Cell. 1996; 86: 777-86
Display abstract
The atypical PKCs are involved in a number of important cellular functions, including cell proliferation. We report here that the product of the par-4 gene specifically interacts with the regulatory domains of zeta PKC and lambda/LPKC, which dramatically inhibits their enzymatic activity. This is particularly challenging, because expression of par-4 has been shown to correlate with growth inhibition and apoptosis. Results are shown here demonstrating that the expression of par-4 in NIH-3T3 cells induces morphological changes typical of apoptosis, which are abrogated by cotransfection of either wild-type zeta PKC or lambda/LPKC, but not by their respective kinase-inactive mutants. These findings support a role for the atypical PKC subspecies in the control of cell growth and survival.

Dieterich S, Herget T, Link G, Bottinger H, Pfizenmaier K, Johannes FJ
In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu.
FEBS Lett. 1996; 381: 183-7
Display abstract
To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu displays high affinity phorbol ester binding (Kd=7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKC mu kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKC mu too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKC mu.

Wang S, Kazanietz MG, Blumberg PM, Marquez VE, Milne GW
Molecular modeling and site-directed mutagenesis studies of a phorbol ester-binding site in protein kinase C.
J Med Chem. 1996; 39: 2541-53
Display abstract
The protein kinase C (PKC) binding site used by PKC activators such as phorbol esters and diacylglycerols (DAGs) has been characterized by means of molecular modeling and site-directed mutagenesis studies. Based upon a NMR-determined solution structure of the second cysteinerich domain of PKC alpha, molecular modeling was used to study the structures of the complexes formed between the PKC receptor and a number of PKC ligands, phorbol esters, and DAGs. Site-directed mutagenesis studies identified a number of residues important to the binding of phorbol esters to PKC. Analysis of the molecular modeling and mutagenesis results allows the development of a binding model for PKC ligands for which the precise binding nature is defined. The calculated hydrogen bond energies between the protein and various ligands in this binding model are consistent with their measured binding affinities. The binding site for phorbol esters and DAGs is located in a highly conserved, hydrophobic loop region formed by residues 6-12 and 20-27. For the binding elements in phorbol esters, the oxygen at C20 contributes most to the overall binding energy, and that at C3 plays a significant role. The oxygen atom at C12 is not directly involved in the interaction between phorbol esters and PKC. Our results also suggest that the oxygens at C9 and C13 are involved in PKC binding, while the oxygen at C4 is of minimal significance. These results are consistent with known structure-activity relationships in the phorbol ester family of compounds. Comparisons with the X-ray structure showed that although the X-ray data support the results for oxygens at C3, C12, and C20 of phorbol esters, they suggest different roles for oxygens at C4, C9, and C13. Several factors which may contribute to these discrepancies are discussed.

Sugita K, Sawada D, Sodeoka M, Sasai H, Shibasaki M
Novel phorbol analogs which bind to protein kinase C (PKC) without activation.
Chem Pharm Bull (Tokyo). 1996; 44: 463-5
Display abstract
13-Deacetoxy-11-demethyl-phorbol derivatives with acyl groups of various lengths (from hexanoyl to tetradecanoyl) at the C-12 position were synthesized in an optically active form. Although considerable binding affinities to PKC were observed by analogs 3-7, no activation of PKC was seen even at 10 microM.

Pap EH, Ketelaars M, Borst JW, van Hoek A, Visser AJ
Reorientational properties of fluorescent analogues of the protein kinase C cofactors diacylglycerol and phorbol ester.
Biophys Chem. 1996; 58: 255-66
Display abstract
The reorientational properties of the fluorescently labelled protein kinase C (PKC) cofactors diacylglycerol (DG) and phorbol ester (PMA) in vesicles and mixed micelles have been investigated using time-resolved polarised fluorescence. The sn-2 acyl chain of DG was replaced by diphenylhexatriene- (DPH) propionic acid, while a dansyl labelled analogue of phorbol ester was used. The extent of ordering of DPH-DG in vesicles turned out to be slightly different from that of the control choline lipid DPH-PC. Addition of PKC to vesicles containing 30 mole% brain PS considerably slowed down the DPH-DG anisotropy decay. This was not observed when DPH-DG was replaced by DPH-PC. Analysis of the fluorescence anisotropy decays of these DPH-lipids in micelles polyoxyethylene-9-laurylether mixed with 10 mole% of the essential phosphatidylserine allowed estimation of their lateral diffusion, orientation distribution and reorientational dynamics within the micelles. Addition of PKC resulted in a significantly slower decay of the fluorescence anisotropy of both DPH-DG and DPH-PC even in the absence of calcium, indicating a calcium independent complexation of PKC with the PS containing micelles. Addition of calcium resulted in a further reduction of the decay of anisotropy of DPH-DG but not of DPH-PC indicating that the Ca2+ dependent immobilisation is cofactor-specific. Similar specific interactions with PKC resulted in a slower decay of dansylated PMA when calcium and PS were present.

Szallasi Z, Bogi K, Gohari S, Biro T, Acs P, Blumberg PM
Non-equivalent roles for the first and second zinc fingers of protein kinase Cdelta. Effect of their mutation on phorbol ester-induced translocation in NIH 3T3 cells.
J Biol Chem. 1996; 271: 18299-301
Display abstract
Classical and novel protein kinase C (PKC) isozymes contain two, so-called cysteine-rich zinc finger domains that represent the binding sites for phorbol esters and the diacylglycerols. X-ray crystallographic, mutational, and modeling studies are providing detailed understanding of the interactions between the phorbol esters and individual PKC zinc fingers. In the present study, we explore the roles of the individual zinc fingers in the context of the intact enzyme. Our approach was to mutate either the first, the second, or both zinc fingers of PKCdelta, to express the mutated enzyme in NIH 3T3 cells, and to monitor the effect of the mutations on the dose-response curve for translocation induced by phorbol 12-myristate 13-acetate. The introduced mutations change into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation causes a 125-fold decrease in phorbol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the dose-response curve for translocation; mutation in the second zinc finger caused a 21-fold shift, whereas mutation in both zinc fingers caused a 138-fold shift. We conclude that the zinc fingers in the intact PKC are not equivalent and that the second zinc finger plays the predominant role in translocation of protein kinase Cdelta in response to phorbol 12-myristate 13-acetate. Our findings have important implications for the understanding and design of PKC inhibitors targeted to the zinc finger domains.

Lee J, Lewin NE, Acs P, Blumberg PM, Marquez VE
Conformationally constrained analogues of diacylglycerol. 13. Protein kinase C ligands based on templates derived from 2,3-dideoxy-L-erythro(threo)-hexono-1,4-lactone and 2-deoxyapiolactone.
J Med Chem. 1996; 39: 4912-9
Display abstract
In the present investigation, the last two possible modes of generating conformationally semirigid diacylglycerol (DAG) analogues embedded into five-membered ring lactones as templates III and IV are investigated. The first two templates studied in previous investigations corresponded to 2-deoxyribonolactone (template I) and 4,4-disubstituted gamma-butyrolactone (template II), with the latter producing potent protein kinase C (PK-C) ligands with low nanomolar binding affinities. The templates reported in this work correspond to 2,3-dideoxy-L-erythro- or -threo-hexono-1,4-lactone (template III) and 2-deoxyapiolactone (template IV). Compounds constructed with the dideoxy-L-erythro- or -threo-hexono-1,4-lactone template were synthesized stereospecifically from tri-O-acetyl-L-glucal and L-galactono-1,4-lactone, respectively. Compounds constructed with the 2-deoxyapiolactone template were synthesized stereoselectively from di-O-isopropylidene-alpha-D-apiose. Inhibition of the binding of [3H]phorbol-12,13-dibutyrate to PK-C alpha showed that only the threo-isomer, 5-O-tetradecanoyl-2,3-dideoxy-L-threo-hexono-1,4-lactone (2) was a good PK-C ligand (Ki = 1 microM). The rest of the ligands had poorer affinities with Ki values between 10 and 28 microM. With these results, the order of importance of five-membered ring lactones as competent templates for the construction of semirigid DAG surrogates with effective PK-C binding affinity can be established as II >> I approximately III > IV.

Feng Y, Narita M, Makimura M, Hoskins B, Ho IK
Effects of repeated administration of a kappa-opioid agonist on phorbol ester binding to membrane-bound protein kinase C in rat brain.
Biol Pharm Bull. 1996; 19: 303-4
Display abstract
The dissociation constant (KD) of [3H]phorbol 12,13-dibutyrate (PDBu) binding to protein kinase C (PKC) in membranes of rat cortex and midbrain was significantly decreased with no change in the receptor density (Bmax) following 7-d treatment with a kappa-opioid agonist, U-50,488 (20 mg/kg/d, i.p.). Neither the Bmax nor KD values in pons/medulla were altered by repeated U-50,488 treatment. These results suggest that repeated administration of a kappa-opioid agonist increases the affinity for PDBu binding to the membrane-bound PKC in rat cortex and midbrain, but not in pons/medulla.

Moruzzi MS, Marverti G, Piccinini G, Frassineti C, Monti MG
The effect of spermine on calcium requirement for protein kinase C association with phospholipid vesicles.
Int J Biochem Cell Biol. 1995; 27: 783-8
Display abstract
We have previously reported that polyamines interfere with protein kinase C-membrane interactions. With the aim of clarifying the influence of the relationship between calcium and polyamines on this process we have investigated the effect of spermine on the formation of active protein kinase C-membrane complexes as a function of Ca++ concentrations. Protein kinase C, purified from rat brain, was allowed to interact with phospholipid vesicles of defined composition. The active complex protein kinase C-liposomes was determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The results show that, at Ca++ levels below 0.1 microM, spermine inhibits the formation of complexes between protein kinase C and membranes. At higher Ca++ concentrations, spermine does not prevent the association process but does influence the ratio between the enzyme molecules irreversibly inserted into the membrane and those reversibly associated with it. We have also demonstrated that spermine, by reducing the density of acidic component of liposomes, influences the calcium requirement for protein kinase C-membrane binding. This study indicates that spermine may regulate the activation of protein kinase C and affects the calcium requirement for the association of this enzyme with the phospholipid bilayer. The results suggest a possible role for polyamines in signal transduction when protein kinase C is involved.

Kazanietz MG, Wang S, Milne GW, Lewin NE, Liu HL, Blumberg PM
Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis.
J Biol Chem. 1995; 270: 21852-9
Display abstract
Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. A 50-amino-acid peptide comprising the second cysteine-rich region of PKC delta, expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound [3H]phorbol 12,13-dibutyrate (PDBu) with high affinity (Kd = 0.8 nM). Using the cDNA of that cysteine-rich region as a template, a series of 37 point mutations was generated by site-directed mutagenesis, and the mutated proteins were analyzed quantitatively for binding of [3H]PDBu and, as appropriate, for binding of the ultrapotent analog [3H]bryostatin 1. Mutants displayed one of three patterns of behavior: phorbol ester binding was completely abolished, binding affinity was reduced, or binding was not significantly modified. As expected, five of the six cysteines as well as the two histidines involved in Zn2+ coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21 and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.

Kazanietz MG, Lewin NE, Bruns JD, Blumberg PM
Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand-binding interactions, lipid cofactor requirements, and inhibitor sensitivity.
J Biol Chem. 1995; 270: 10777-83
Display abstract
The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [3H]Phorbol 12,13-dibutyrate [3H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (Kd = 1.3 +/- 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some "specific" antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.

Zhang G, Kazanietz MG, Blumberg PM, Hurley JH
Crystal structure of the cys2 activator-binding domain of protein kinase C delta in complex with phorbol ester.
Cell. 1995; 81: 917-24
Display abstract
Protein kinase Cs (PKCs) are a ubiquitous family of regulatory enzymes that associate with membranes and are activated by diacylglycerol or tumor-promoting agonists such as phorbol esters. The structure of the second activator-binding domain of PKC delta has been determined in complex with phorbol 13-acetate, which binds in a groove between two pulled-apart beta strands at the tip of the domain. The C3, C4, and C20 phorbol oxygens form hydrogen bonds with main-chain groups whose orientation is controlled by a set of highly conserved residues. Phorbol binding caps the groove and forms a contiguous hydrophobic surface covering one-third of the domain, explaining how the activator promotes insertion of PKC into membranes.

Aitken A, Jones D, Soneji Y, Howell S
14-3-3 proteins: biological function and domain structure.
Biochem Soc Trans. 1995; 23: 605-11

Datta K et al.
AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.
Mol Cell Biol. 1995; 15: 2304-10
Display abstract
The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

Quest AF, Bardes ES, Xie WQ, Willott E, Borchardt RA, Bell RM
Expression of protein kinase C gamma regulatory domain elements containing cysteine-rich zinc-coordinating regions as glutathione S-transferase fusion proteins.
Methods Enzymol. 1995; 252: 153-67

Fukuda K, Ueno M, Saitoh M, Nishioka S, Tanaka S
Activation of protein kinase C and the involvement of prostaglandin E2 in the inhibition of osteosarcoma-derived cell alkaline phosphatase activity.
J Lab Clin Med. 1995; 126: 269-74
Display abstract
We present evidence for the presence of specific, high-affinity binding sites for tritiated phorbol 12,13-dibutyrate on osteosarcoma-derived (HT-3) cells. Activation of protein kinase C by a phorbol ester resulted in an inhibition of alkaline phosphatase activity and the accumulation of prostaglandin E2. Indomethacin blocked prostaglandin E2 production and enhanced alkaline phosphatase activity. These data suggest that prostaglandin E2 is enhanced by activation of protein kinase C, and in turn, alkaline phosphatase activity is reduced.

Rozengurt E, Sinnett-Smith J, Van Lint J, Valverde AM
Protein kinase D (PKD): a novel target for diacylglycerol and phorbol esters.
Mutat Res. 1995; 333: 153-60
Display abstract
A novel serine/threonine protein kinase regulated by phorbol esters and diacylglycerol (named PKD) has been identified. PKD contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed NH2-terminal domain of PKD exhibited high affinity phorbol ester binding activity (Kd = 35 nM). Expression of PKD cDNA in COS cells conferred increased phorbol ester binding to intact cells. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide-2 in serine but did not catalyse significant phosphorylation of a variety of other substrates utilised by PKCs and other major second messenger regulated kinases. PKD expressed in COS cells showed syntide-2 kinase activity that was stimulated by phorbol esters in the presence of phospholipids. We propose that PKD may be a novel component in the transduction of diacylglycerol and phorbol ester signals.

Wender PA, Irie K, Miller BL
Identification, activity, and structural studies of peptides incorporating the phorbol ester-binding domain of protein kinase C.
Proc Natl Acad Sci U S A. 1995; 92: 239-43
Display abstract
The family of homologous enzymes known as protein kinase C (PKC) has been the object of intense interest because of its crucial role in cellular signal transduction. Although considerable information about the activation of PKC has been gained through structure-activity, molecular modeling, and synthetic studies of both natural and designed activators, information about the structure of PKC itself has been limited by its large size and requirement for phospholipid cofactors. Additionally, difficulties in the purification of truncated mutants of PKC have thus far prevented their analysis by nuclear magnetic resonance (NMR) or x-ray crystallographic methods. We describe the identification, synthesis, ligand-binding analysis, cofactor requirements, and preliminary NMR evaluation of two subdomains (peptides B and C) of the regulatory domain of PKC-gamma. Peptides B and C bind [3H]phorbol 12,13-dibutyrate with good affinity (Kd = 6.4 microM and 414 nM, respectively) in the presence of phosphatidylserine. In comparison, the binding affinity of [3H]phorbol 12,13-dibutyrate for PKC was found to be 2.6 nM. Like PKC itself, these peptides also recognize other PKC activators, including dioctanoylglycerol and teleocidin B-4, and exhibit an ability to differentiate phorbol ester from its C-4 epimer. NMR studies of PKC subdomains are also described, indicating that both peptides B and C are well behaved in solution and do not exhibit any concentration-dependent changes. Finally, these studies reveal that peptide B becomes conformationally ordered only in the presence of phospholipid, suggesting that the regulatory domain of PKC itself might be organized for activation only when associated with the lipid bilayer, where its activator (diacylglycerol) is encountered.

Dekker LV, Palmer RH, Parker PJ
The protein kinase C and protein kinase C related gene families.
Curr Opin Struct Biol. 1995; 5: 396-402
Display abstract
Protein kinase C is an important target enzyme for lipid second messengers. Recent developments have focused on the tertiary structure analysis of domains present in protein kinase C and in combination with functional approaches such as mutagenesis and domain expression have generated a detailed understanding of the modular mechanism by which lipids cause activation. This provides a reference for the study of the protein kinase C-related kinases, a recently identified new class of kinases that are highly related to protein kinase C in their catalytic characteristics but have distinct regulatory features.

Emerson SD et al.
Solution structure of the Ras-binding domain of c-Raf-1 and identification of its Ras interaction surface.
Biochemistry. 1995; 34: 6911-8
Display abstract
The structure of the Ras-binding domain of human c-Raf-1 (residues 55-132) has been determined in solution by nuclear magnetic resonance (NMR) spectroscopy. Following complete assignment of the backbone and side-chain 1H, 15N, and 13C resonances, the structure was calculated using the program CHARMM. Over 1300 NOE-derived constraints were applied, resulting in a detailed structure. The fold of Raf55-132 consists of a five-stranded beta-sheet, a 12-residue alpha-helix, and an additional one-turn helix. It is similar to those of ubiquitin and the IgG-binding domain of protein G, although the three proteins share very little sequence identity. The surface of Raf55-132 that interacts with Ras has been identified by monitoring perturbation of line widths and chemical shifts of 15N-labeled Raf55-132 resonances during titration with unlabeled Ras-GMPPNP. The Ras-binding site is contained within a spatially contiguous patch comprised of the N-terminal beta-hairpin and the C-terminal end of the alpha-helix.

Ahmed S, Kozma R, Hall C, Lim L
GTPase-activating protein activity of n(alpha 1)-Chimaerin and effect of lipids.
Methods Enzymol. 1995; 256: 114-25

Kazanietz MG, Barchi JJ Jr, Omichinski JG, Blumberg PM
Low affinity binding of phorbol esters to protein kinase C and its recombinant cysteine-rich region in the absence of phospholipids.
J Biol Chem. 1995; 270: 14679-84
Display abstract
Binding of phorbol esters to protein kinase C (PKC) has been regarded as dependent on phospholipids, with phosphatidylserine being the most effective for reconstituting binding. By using a purified single cysteine-rich region from PKC delta expressed in Escherichia coli we were able to demonstrate that specific binding of [3H]phorbol 12,13-dibutyrate to the receptor still takes place in the absence of the phospholipid cofactor. However, [3H]phorbol 12,13-dibutyrate bound to the cysteine-rich region with 80-fold lower affinity in the absence than in the presence of 100 micrograms/ml phosphatidylserine. Similar results were observed with the intact recombinant PKC delta isolated from insect cells. When different phorbol derivatives were examined, distinct structure-activity relations for the cysteine-rich region were found in the presence and absence of phospholipid. Our results have potential implications for PKC translocation, for inhibitor design, and for PKC structural determination.

Janssen-Bienhold U, Buschmann-Gebhardt B, Weiler R
Phorbol ester binding sites in the fish retina: correlation with stimulation of endogenous phosphorylation and protein kinase C activation.
J Neurochem. 1995; 65: 744-53
Display abstract
The injection of phorbol esters into the eyes of dark-adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs). It is therefore very likely that protein kinase C (PKC) mediates light-induced synaptic plasticity. In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritated phorbol 12,13-dibutyrate ([3H]PDBu). The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [3H]PDBu binding is time dependent, specific, saturable, and reversible. Binding sites in HCs displayed a dissociation constant of 11.5 nM and a total number of 2.8 pmol/mg of protein. Autoradiography revealed that [3H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata. Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation. In HCs, the treatment with PDBu in particular affected the amount of 32P incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation. These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation.

Marechal E, Miege C, Block MA, Douce R, Joyard J
The catalytic site of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes. Biochemical analysis of the structure and of the metal content.
J Biol Chem. 1995; 270: 5714-22
Display abstract
We have analyzed the structure of the active site of monogalactosyldiacylglycerol (MGDG) synthase from spinach chloroplast envelope. Since purification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far impossible, we used indirect strategies. Analyses of the inhibition of MGDG synthase by UDP and of its inactivation by citraconic anhydride first indicated that the enzyme contained two functionally independent and topologically distinct binding sites for each substrate. Whereas MGDG synthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to UDP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme. The UDP-Gal-binding site contains lysine residues, as demonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far. Radiolabeling of MGDG synthase by sulfur labeling reagent, a 35S-labeled lysine-blocking reagent, confirmed that MGDG synthase was a polypeptide with a low molecular mass (around 20 kDa). The 1,2-diacylglycerol-binding site contains reduced cysteines and at least one metal. The divalent cation(s) associated to apo-MGDG synthase was not unambiguously identified, but the results suggest that it could be zinc. Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinase C and 1,2-diacylglycerol kinase, which are characterized by the presence of a ubiquitous Cys6His2 domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.

Ichikawa S, Hatanaka H, Takeuchi Y, Ohno S, Inagaki F
Solution structure of cysteine-rich domain of protein kinase C alpha.
J Biochem (Tokyo). 1995; 117: 566-74
Display abstract
The three-dimensional structure of the second cysteine-rich domain of protein kinase C alpha (residues 95-159) was determined in aqueous solution by two-dimensional proton nuclear magnetic resonance and simulated annealing based calculations. On the basis of 687 distance constraints derived from assigned nuclear Overhauser effect (NOE) connectivities, a total of 10 converged structures were obtained from 40 runs of calculations. The atomic root-mean-square (RMS) difference about the mean coordinate positions (excluding residues 1-7, 16-17, 30-34, and 55-65) is 0.55 A for backbone atoms (N, C alpha, C') and 1.07 A for all non-hydrogen atoms. The molecular scaffold is maintained by triple-stranded and double-stranded twisted beta-sheets packed against an alpha-helix and two independent zincs are coordinated by His8, Cys38, Cys41, Cys57 and Cys21, Cys24, His46, Cys49, respectively. It should be noted that the metal ligands from the two sites are interleaved and this is thought to be a new structural motif of a zinc finger domain. Based on the resultant structure, we propose an interaction site of the cysteine-rich domain of protein kinase C with diacylglycerols and phorbol esters.

Diaz-Meco MT et al.
Evidence for the in vitro and in vivo interaction of Ras with protein kinase C zeta.
J Biol Chem. 1994; 269: 31706-10
Display abstract
The zeta isoform of protein kinase C (zeta PKC) has been shown to be involved in the maturation of Xenopus oocytes and mitogenic signaling in fibroblasts. zeta PKC also regulates the important transcription factor nuclear factor kappa B, most probably by phosphorylation of the inhibitory molecule I kappa B. The mechanisms that control zeta PKC activity are still poorly characterized. This kinase is not activated by diacylglycerol but is potently stimulated in vitro by the products of phosphatidylinositol 3-kinase (PI 3-kinase), which suggests that zeta PKC is at least one of the critical targets of PI 3-kinase-triggered signals, and strengthens its role in cell proliferation. PI 3-kinase has been shown, like Raf, to be a direct effector of Ras. zeta PKC is a required step for Ras mitogenic signaling. Therefore, it is possible that zeta PKC directly interacts with Ras during mitogenic activation. We demonstrate here that Ras interacts in vitro with the regulatory domain of zeta PKC as well as that the association of zeta PKC with Ras in vivo is triggered by platelet-derived growth factor. It is also shown here that the expression of a dominant negative mutant of Ras (Asn-17) severely impairs the activation of zeta PKC in mouse fibroblasts.

Wang S, Milne GW, Nicklaus MC, Marquez VE, Lee J, Blumberg PM
Protein kinase C. Modeling of the binding site and prediction of binding constants.
J Med Chem. 1994; 37: 1326-38
Display abstract
A detailed examination of the mode of binding of phorbol esters to protein kinase C led to a model of the phorbol binding site in the enzyme. The efficacy with which various synthetic diacylglycerol analogs and ribonolactones are able to bind to this site was determined by means of semiempirical quantum mechanical calculations using PM3, and an estimate of the binding energy was made in each case. Sixteen synthetic analogs of 1,2-diacylglycerol and two natural products were studied, and their calculated energies of binding to this model were correlated with the measured Ki values. The binding energies calculated for this receptor model, together with solubility and entropy considerations, allow prediction through regressive fit of free energies of binding which correlate very well with the measured binding constants.

Palmer RH, Ridden J, Parker PJ
Identification of multiple, novel, protein kinase C-related gene products.
FEBS Lett. 1994; 356: 5-8
Display abstract
A PCR approach has been employed to screen two cDNA libraries for PKC(-related) sequences. In each case multiple cDNAs were identified, including known PKC isotypes, a previously unknown PKC-eta related sequence and three members of what appears to be a protein kinase C related kinase (PRK 1-3) family. The origin and relationships of these predicted proteins is discussed.

Franke TF, Tartof KD, Tsichlis PN
The SH2-like Akt homology (AH) domain of c-akt is present in multiple copies in the genome of vertebrate and invertebrate eucaryotes. Cloning and characterization of the Drosophila melanogaster c-akt homolog Dakt1.
Oncogene. 1994; 9: 141-8
Display abstract
The Akt proto-oncogene encodes a serine-threonine protein kinase whose carboxyterminal catalytic domain is closely related to the catalytic domains of all the known members of the protein kinase C (PKC) family. Akt, however, differs from PKC in its N-terminal region which contains a domain related distantly to the SH2 domain of cytoplasmic tyrosine kinases and other signalling proteins, which we have named Akt homology (AH) domain. Low stringency hybridization of a c-akt AH probe to a panel of genomic DNAs from vertebrate and invertebrate eucaryotes detected multiple DNA bands (perhaps multiple genes) in all tested species. Drosophila DNA contains at least three hybridizing DNA bands. One of them was cloned, and found by sequence analysis, to define an Akt related gene (Dakt1). Comparison between the coding regions of c-akt and Dakt1 revealed 64.6% identity at the nucleotide level and 76.5% similarity at the amino acid level. The highest degree of homology was detected in the AH domain (68.3% similarity at the amino acid level) and the catalytic domain (83.3% similarity). Additional sequence comparisons revealed that the amino acid similarity between the catalytic domains of Dkt1 and the three known members of the Drosophila protein kinase C (PKC) family, Dpkc1, Dpkc2 and Dpkc3, is 68%, 63.6% and 67.1%, respectively. Dakt1 was mapped to Drosophila chromosome 3R 89BC. Its expression is subject to developmental regulation with the highest levels detected within the fourth hour of embryonic development. These results confirm that the AH domain of Akt defines new protein families in both vertebrate and invertebrate eucaryotes. The high degree of homology between the catalytic domains of Dkt1 and the three known members of the Drosophila PKC family suggests an evolutionarily conserved functional relationship between the members of the two families.

Areces LB, Kazanietz MG, Blumberg PM
Close similarity of baculovirus-expressed n-chimaerin and protein kinase C alpha as phorbol ester receptors.
J Biol Chem. 1994; 269: 19553-8
Display abstract
n-Chimaerin is a recently described phorbol ester receptor that shares homology in its N-terminal region with the cysteine-rich zinc finger domain of protein kinase C. We have expressed n-chimaerin in insect cells using the baculovirus system and have used the isolated, recombinant n-chimaerin to characterize phorbol ester binding and structure-activity relations, lipid requirements, and inhibitor sensitivity. We find that n-chimaerin expressed in the baculovirus system bound [3H]phorbol 12,13-dibutyrate with high affinity (0.17 +/- 0.01 nM). Although having only a single cysteine-rich zinc finger region compared to two for protein kinase C, n-chimaerin thus closely resembled protein kinase C alpha. n-Chimaerin was likewise virtually indistinguishable from protein kinase C alpha in phorbol ester structure-activity relations, in phospholipid requirements, and in inhibition of binding by sphingosine and calphostin C, protein kinase C inhibitors acting on the regulatory domain. We conclude that a number of typical approaches used to implicate protein kinase C in biological function in cells do not discriminate between the n-chimaerin and protein kinase C classes of phorbol ester receptors.

Konishi H, Kuroda S, Kikkawa U
The pleckstrin homology domain of RAC protein kinase associates with the regulatory domain of protein kinase C zeta.
Biochem Biophys Res Commun. 1994; 205: 1770-5
Display abstract
Binding proteins to the pleckstrin homology domain of RAC protein kinase were screened by using glutathione S-transferase fusion protein system. Proteins in CHO cell extract of approximate molecular mass of 76 kD and 200 kD bound specifically to the pleckstrin homology domain of RAC protein kinase in vitro. The 76 kD protein was identified as protein kinase C zeta by immunoblot analysis. Studies of the association between the pleckstrin homology domain-truncated mutants and protein kinase C zeta indicated that the amino-terminal portion of the pleckstrin homology domain is essential for the binding and the whole structure of the domain is important for the efficient binding to protein kinase C zeta. The pleckstrin homology domain of RAC protein kinase was shown to recognize the regulatory domain of protein kinase C zeta. The protein-protein interaction between RAC protein kinase and protein kinase C through the pleckstrin homology domain might be important for the regulation of these protein kinases.

Quest AF, Bell RM
The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations.
J Biol Chem. 1994; 269: 20000-12
Display abstract
The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function.

Zhu J, Hansen H, Su L, Shieh HL, Riedel H
Ligand regulation of bovine protein kinase C alpha response via either cysteine-rich repeat of conserved region C1.
J Biochem (Tokyo). 1994; 115: 1000-9
Display abstract
Based on the finding by others that the conserved region C1 of conventional protein kinase C isoforms carries two independent, cysteine-rich phorbol ester binding sites, we have mapped the structural elements of the C1 region for their role in the phorbol ester- and phospholipid regulation of PKC alpha responses. We have prepared two amino terminal truncation mutants of bovine PKC alpha, ND91 lacking the first Cys-repeat of C1, and ND153 lacking both Cys-repeats of C1, as well as two internal deletion mutants, D162-245 lacking most of C2, and D109-263 lacking most of C2 and the second Cys-repeat of C1. The mutants were expressed in the yeast Saccharomyces cerevisiae which allows the rapid biochemical and physiological characterization of mammalian PKC isoforms. We found that all mutants displayed an elevated basal level of enzymatic activity in vitro but retained the basic catalytic PKC characteristics: regulation by Ca2+ and (except for ND153) by phospholipid or phorbol ester. In vivo we observed proportional physiological responses, the stimulation of Ca2+ uptake, and an increase in the cell doubling time for all mutants upon phorbol ester stimulation (constitutive for ND153) similar to the response of normal PKC alpha. Our findings indicate that after partial PKC activation by deletion mutagenesis, the presence of either Cys-repeat in C1 still allows phospholipid- and phorbol ester regulation of protein kinase C alpha responses.

Valverde AM, Sinnett-Smith J, Van Lint J, Rozengurt E
Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain.
Proc Natl Acad Sci U S A. 1994; 91: 8572-6
Display abstract
A serine/threonine protein kinase that binds phorbol esters and diacylglycerol (named protein kinase D, PKD) has been identified. PKD contains membrane localization signals and a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC). A bacterially expressed N-terminal domain of PKD exhibited high-affinity phorbol ester binding activity (Kd = 35 nM). The diacylglycerol analog 1-oleoyl-2-acetylglycerol inhibited phorbol ester binding in a dose-dependent manner. The catalytic domain of PKD contains all characteristic sequence motifs of serine protein kinases but shows only a low degree of sequence similarity to PKCs. The highest identity is with the catalytic domain of myosin light-chain kinase from Dictyostelium (41%). The bacterially expressed catalytic domain of PKD efficiently phosphorylated the exogenous peptide substrate syntide 2 in serine but did not catalyze significant phosphorylation of a variety of other substrates used by PKCs and other major second messenger regulated kinases. PKD may be an unusual component in the transduction of diacylglycerol and phorbol ester signals.

Kazanietz MG et al.
Zinc finger domains and phorbol ester pharmacophore. Analysis of binding to mutated form of protein kinase C zeta and the vav and c-raf proto-oncogene products.
J Biol Chem. 1994; 269: 11590-4
Display abstract
The phorbol ester binding domain consists of a cysteine-rich region with a postulated consensus sequence for binding that includes 15 amino acids (Ahmed, S., Kozma, R., Lee, J., Monfries, C., Harden, N., and Lim, L. (1991) Biochem. J. 280, 233-241). In PKC zeta, the only PKC isoform lacking phorbol ester binding, this region differs in a single residue from the consensus (proline in position 11 of the motif). Restoration of this proline by site-directed mutagenesis of PKC zeta does not restore binding of either [3H]phorbol 12,13-dibutyrate or of the ultrapotent ligand [3H]bryostatin 1, suggesting that even a low affinity ligand interaction is absent. In addition, the vav and c-raf proto-oncogene products, proteins that possess cysteine-rich regions with high homology to PKC isozymes and other phorbol ester receptors, are unable to bind any of these ligands. Instead, all of these cysteine-rich regions bind zinc. Our results suggest that other amino acids besides those postulated for the consensus must be necessary for ligand binding and argue against direct modulation of PKC zeta, Vav, and c-Raf by phorbol esters.

Scheffler JE et al.
Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP.
J Biol Chem. 1994; 269: 22340-6
Display abstract
Four overlapping peptide fragments of human c-Raf-1 (residues 55-132, 55-117, 77-132, and 77-117) were expressed in Escherichia coli as carboxyl-terminal extensions of maltose binding protein (MBP). The MBP-Raf fusions were purified by affinity chromatography on amylose resin and tested for binding to Ras.GTP indirectly by measuring their ability to inhibit the stimulation of Ras GTPase activity by GTPase activating protein (GAP120) in vitro. MBP-Raf(55-132) was a potent inhibitor in this assay (50% inhibition at 100 nM concentration), but the other fusion proteins had no measurable effect. The fusion partners were cleaved with Factor Xa protease and separated by gel filtration. The 8960-dalton Raf(55-132) fragment retained full activity as a competitive inhibitor of GAP120. It also blocked Ras-stimulated germinal vesicle breakdown in frog oocytes. Raf(55-132) was further characterized by circular dichroism and nuclear magnetic resonance spectroscopy. The results indicate that this fragment of c-Raf-1 adopts a highly structured, monomeric conformation in solution.

Johannes FJ, Prestle J, Eis S, Oberhagemann P, Pfizenmaier K
PKCu is a novel, atypical member of the protein kinase C family.
J Biol Chem. 1994; 269: 6140-8
Display abstract
We have isolated the full-length cDNA of a novel human serine/threonine protein kinase gene. The deduced protein sequence shows strong homology to conserved domains of members of the protein kinase C (PKC) subfamily. Homologies reside in the duplex zinc-finger-like cysteine-rich motif and in the protein kinase domain. The lack of the C2 domain of the Ca(2+)-dependent PKCs and the presence of a unique NH2-terminal sequence with a potential signal peptide and a transmembrane domain suggest that PKC mu is a novel member of the subgroup of atypical PKCs. An open reading frame coding for 912 amino acids directs an in vitro translation product with an apparent M(r) of 115,000. In vitro phorbol ester binding studies and kinase assays with lysates of cells overexpressing PKC mu showed phorbol ester-independent kinase activity, autophosphorylation, and, in normal rat kidney (NRK) cells, predominant phosphorylation of a 30-kDa protein at serine residues. Southern analysis revealed that PKC mu is a single copy gene located on human chromosome 21. There is constitutive low level expression of the human PKC mu gene in normal tissues with a single transcript of 3.8 kilobases and elevated expression levels in selected tumor cell lines. These data suggest a role of PKC mu in signal transduction pathways related to growth control.

Carroll MP, May WS
Protein kinase C-mediated serine phosphorylation directly activates Raf-1 in murine hematopoietic cells.
J Biol Chem. 1994; 269: 1249-56
Display abstract
We have previously found that Raf-1, which is activated by hematopoietic growth factors in association with phosphorylation, is required for hematopoietic cell proliferation. Recently, 12-O-tetradecanoylphorbol 13-acetate has been found to mediate Raf-1 phosphorylation, suggesting that protein kinase C (PKC) may be involved in the Raf-1 activation mechanism(s). Since PKC can be activated by hematopoietic growth factors, it was investigated as a potential "Raf-1 kinase-kinase." Results demonstrate that bryostatin 1, a pharmacologic activator of PKC, induces activation of Raf-1 in FDC-P1 cells. PKC inhibitors H7 and staurosporine block both bryostatin 1- and interleukin-3-mediated Raf-1 phosphorylation and FDC-P1 cell proliferation. Additionally, an antisense c-raf oligodeoxyribonucleotide specifically inhibits bryostatin 1-mediated proliferation, indicating a necessary role for Raf-1 in PKC signaling. Purified PKC can phosphorylate Raf-1 serine residues to high stoichiometry in vitro. Comparative phosphopeptide maps localize two PKC phosphorylation sites to Raf-1 phosphopeptides isolated from hematopoietic growth factor- or bryostatin 1-stimulated cells. The sites of PKC-mediated Raf-1 phosphorylation are deduced to be Ser497 and Ser619. Furthermore, PKC-mediated serine phosphorylation is sufficient to activate the enzymatic function of Raf-1 in vitro. These findings demonstrate that activated PKC can promote hematopoietic cell growth by regulating the enzymatic activity of Raf-1 through direct serine phosphorylation.

Quest AF, Bardes ES, Bell RM
A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein.
J Biol Chem. 1994; 269: 2953-60
Display abstract
Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity. The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography. This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM). The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D. J., and Bell, R. M. (1991) J. Biol. Chem. 266, 18330-18338). The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer. Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding. The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine. Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8. These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8. Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion. Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC.

Quest AF, Bardes ES, Bell RM
A phorbol ester binding domain of protein kinase C gamma. Deletion analysis of the Cys2 domain defines a minimal 43-amino acid peptide.
J Biol Chem. 1994; 269: 2961-70
Display abstract
Cysteine-rich regions of protein kinase C (PKC) are critical for the lipid-dependent regulation of activity and are implicated in the coordination of zinc. A glutathione S-transferase fusion protein containing the second cysteine-rich region, Cys2, of PKC gamma with bound zinc with a stoichiometry of 1.8 +/- 0.1 mol of zinc/mol of protein. Deletion analysis within this cysteine-rich region defined amino acids essential for zinc coordination. An NH2-terminal histidine (His102) and a COOH-terminal cysteine (Cys151) were both critical for the coordination of distinct zinc atoms. Both represent the ultimate residues of a 50-amino acid consensus motif with six conserved cysteines and two conserved histidines present in the cysteine-rich regions of all PKC isoforms. Removal of histidine His102 abolished phorbol ester binding, while deletion of cysteine Cys151 did not. Deletion of valine (Val147) greatly diminished phorbol ester binding, which was completely lost only when valine (Val144) was also deleted. No significant further reduction in zinc stoichiometry below one resulted even when three COOH-terminal conserved cysteines (Cys151, Cys143, and Cys135) and a conserved histidine (His140) were deleted. These results are consistent with a model in which two zinc atoms are tetracoordinated per cysteine-rich region in two independent coordination spheres that are not functionally equivalent. These analyses determine a minimal peptide (residues 102-144) of 43 amino acids capable of [3H]PDBu binding.

Mukai H, Ono Y
A novel protein kinase with leucine zipper-like sequences: its catalytic domain is highly homologous to that of protein kinase C.
Biochem Biophys Res Commun. 1994; 199: 897-904
Display abstract
A novel protein kinase, designated PKN, was identified by molecular cloning from a human hippocampus cDNA library. PKN consists of 942 amino acids with a calculated molecular mass of 103,925 daltons. PKN has leucine zipper-like sequences in its amino terminal region and contains a catalytic domain that shows strong similarity to that of protein kinase C family. Northern blot analysis indicates PKN is expressed ubiquitously in human tissues. Antisera against PKN identified a 120K dalton protein on SDS polyacrylamide gel electrophoresis when PKN was expressed in the insect cells or COS7 cells. Recombinant PKN revealed an intrinsic protein kinase activity associated with a 120K protein. This activity was abolished by mutation of the lysine residue in the potential ATP binding site.

Riedel H, Parissenti AM, Hansen H, Su L, Shieh HL
Stimulation of calcium uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha.
J Biol Chem. 1993; 268: 3456-62
Display abstract
Ca2+ plays essential roles as a second messenger often in synergism with the calcium- and phospholipid-dependent phorbol ester receptor, protein kinase C (PKC), which stimulates Ca2+ influx in various cell types in a potential positive feedback mechanism. To address the compatibility of these mechanisms between lower eukaryotes and mammals, we have stably expressed bovine PKC alpha in the yeast Saccharomyces cerevisiae. We find that phorbol ester binding sites are created which stimulate a specific calcium- and phospholipid-dependent catalytic activity in vitro. Phorbol ester activation in vivo stimulates PKC down-regulation, uptake of extracellular Ca2+, Ca2+ dependence of cell viability, and changes in cell morphology. This may represent activation of a putative PKC-mediated signaling pathway utilized by functional yeast homologs of mammalian PKC isoforms. These are suggested by some protein data; however, their genes have not yet been characterized (Simon, A. J., Milner, Y., Saville, S. P., Dvir, A., Mochly-Rosen, D., and Orr, E. (1991) Proc. R. Soc. Lond. B 243, 165-171). Our findings indicate that bovine PKC alpha is functional in yeast and stimulates calcium uptake in a manner similar to some of its responses in mammalian cells, which suggests compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.

Hanson-Painton O, Morgenstern K, Cooper DR, Moore B, Botchlet T, Grammas P
Protein kinase C in rat cerebral microvessels.
Mol Chem Neuropathol. 1993; 20: 245-61
Display abstract
Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.

Kano-Sueoka T, Nicks ME
Abnormal function of protein kinase C in cells having phosphatidylethanolamine-deficient and phosphatidylcholine-excess membranes.
Cell Growth Differ. 1993; 4: 533-7
Display abstract
When rat mammary carcinoma 64-24 cells are grown in the absence of ethanolamine, their membrane phospholipid composition changes significantly, becoming phosphatidylethanolamine-deficient and phosphatidylcholine-excess due to a reduced de novo rate of phosphatidylethanolamine synthesis, and growth stops. We have assumed that this membrane phospholipid environment is not suitable for membrane-associated functions. We have previously demonstrated that functions normally stimulated by tumor-promoting phorbol ester, phorbol 12,13-dibutyrate, are not stimulated in ethanolamine-deprived cells, suggesting that function of protein kinase C may be abnormal under the altered membrane environment. In the present study, the behavior of protein kinase C in 64-24 cells grown in the presence and absence of ethanolamine (having normal and phosphatidylethanolamine-deficient/phosphatidylcholine-excess phospholipid) was compared by enzyme assay as well as Western blotting. The results show that the nature of association of protein kinase C to the membrane, which is induced by phorbol ester, is abnormal when cells have the altered membrane phospholipid, and thus argue that membrane phospholipid environment is important in the function of protein kinase C.

Ohmori Y et al.
Protein kinase C imaging using carbon-11-labeled phorbol esters: 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate as the potential ligand for positron emission tomography.
J Nucl Med. 1993; 34: 431-9
Display abstract
Protein kinase C plays a crucial role in signal transduction for a variety of biologically active substances which activates cellular functions and their proliferation. The actions are closely related to both normal and abnormal functions in the nervous system. Tumor-promoting phorbol esters can substitute for diacylglycerols which are important ligands that bind to protein kinase C. Three typical phorbol esters, phorbol 13-[1-11C]butyrate, phorbol 12,13-[1-11C]dibutyrate and 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate, were synthesized by using [11C]ethylketene with a high specific activity (186GBq/mumol). Their in vivo autoradiograms demonstrated a heterogenous distribution in rat brain. 12-deoxyphorbol 13-isobutyrate-20-[1-11C]butyrate was particularly suited for in vivo use due to its nontumor-promoting activity and its ready permeability to the blood-brain barrier. High optical density was observed in the cortex, amygdala and hippocampus. The in vivo binding properties of this compound to protein kinase C were confirmed by in vivo displacement studies with unlabeled 12-deoxyphorbol 13-isobutyrate-20-butyrate and unlabeled phorbol 12,13-dibutyrate. This suggests that 12-deoxyphorbol 13-isobutyrate-20-[1-11C] butyrate has a specific binding affinity for protein kinase C.

Chiou SH, Raynor RL, Zheng B, Chambers TC, Kuo JF
Cobra venom cardiotoxin (cytotoxin) isoforms and neurotoxin: comparative potency of protein kinase C inhibition and cancer cell cytotoxicity and modes of enzyme inhibition.
Biochemistry. 1993; 32: 2062-7
Display abstract
Effects of cobra cardiotoxin (cytotoxin) and its isoforms, and neurotoxin, on protein kinase C (PKC) and cancer cells were investigated. A positive correlation existed between hydrophobicities and activities of the toxins to inhibit PKC activity (assayed using phosphatidylserine vesicle, arachidonate monomer, and Triton/phosphatidylserine mixed micelle systems), phorbol ester binding to PKC, proliferation of several cancer cell lines, and phorbol ester-induced HL60 cell differentiation. Their relative hydrophobicities and activities, in a decreasing order, were cardiotoxin-1 approximately cardiotoxin-3 > cardiotoxin (a mixture of isoforms) > cardiotoxin-4 >> neurotoxin (inactive). Under the mixed micelle assay system (containing 0.3% Triton X-100, 8 mol % phosphatidylserine, 2 mol % diolein, and 200 microM CaCl2), cardiotoxin inhibited PKC competitively with respect to phosphatidylserine (apparent Ki of about 0.06 mol % or 2.5 microM), and in a mixed-type manner with respect to both diolein (apparent Ki of about 0.04 mol % or 1.7 microM) and Ca2+ (apparent Ki of about 2.9 microM). On the basis of findings that IC50 (approximately 0.3 microM) of cardiotoxin for inhibition of HL60 cell proliferation and differentiation was lower than its IC50 (9 microM) for PKC inhibition in vitro in the phosphatidylserine vesicle system and that PKC inhibition was the only known molecular mechanism of cardiotoxin, it was suggested that cardiotoxin might be highly membrane interacting and that the observed cellular effects of cardiotoxin might be mediated, in part, via PKC inhibition.

Kolch W et al.
Protein kinase C alpha activates RAF-1 by direct phosphorylation.
Nature. 1993; 364: 249-52
Display abstract
The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

Barbee JL, Deutscher SL, Loomis CR, Burns DJ
The cDNA sequence encoding human protein kinase C-zeta.
Gene. 1993; 132: 305-6
Display abstract
A 1779-bp complementary DNA (cDNA) that encodes protein kinase C-zeta (PKC-zeta) has been isolated from a human frontal cortex library using traditional plaque-screening methods and PCR screening. The deduced 592-amino-acid sequence of the human PKC-zeta clone has a 95-96% identity to those deduced from the previously described rat and mouse PKC-zeta clones.

Filipuzzi I, Fabbro D, Imber R
Unphosphorylated alpha-PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro.
J Cell Biochem. 1993; 52: 78-83
Display abstract
Expression of the alpha-isoform of protein kinase C (alpha-PKC) in E. coli yielded the unphosphorylated 74 kD precursor molecule. This precursor form exhibited phospholipid- and calcium-dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity. In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of alpha-PKC is not required for binding of substrates, cofactors, or activators. These results, therefore, suggest that posttranslational phosphorylation of alpha-PKC is not needed for the formation of a functional enzyme-substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.

Zhang DC, Cao CX, Zhang CJ, Zhou BN
Yuanhuacin A is a selective antagonist of phorbol ester receptor in protein kinase C.
Sci China B. 1993; 36: 803-8
Display abstract
Protein kinase C with a molecular weight of 82 kD has been purified to electrophoresis homogeneous from rat brain through a series of chromatography columns including DE-52, Sepharose G-200 and phenyl-Sepharose. The enzyme possessed autophosphorylation activity. Yuanhuacin A inhibited the 3H-phorbol-12, 13-dibutyrate (3H-PdBu) binding of PKC with an IC50 value of 1.48 +/- 0.28 x 10(-8) mol/L when the concentration of 3H-PdBu was 1.5 x 10(-9) mol/L (Ki = 1.2 x 10(-8) mol/L). Yuanhuacin A inhibited the PdBu-stimulated PKC activity in the catalysis of the phosphorylation of Histone III-S with an IC50 of 2.82 +/- 0.37 x 10(-9) mol/L (PdBu = 10(-6) mol/L), while it had no effect on the basal and Ca(2+)-stimulated PKC activity in the same assay system. This result suggests that Yuanhuacin A is a selective antagonist of the phorbol ester receptor in protein kinase C.

Morrison DK, Heidecker G, Rapp UR, Copeland TD
Identification of the major phosphorylation sites of the Raf-1 kinase.
J Biol Chem. 1993; 268: 17309-16
Display abstract
Treatment of cells with various growth factors and mitogens results in the rapid hyperphosphorylation and activation of the Raf-1 kinase. To determine if phosphorylation events affect Raf-1 activity, we have initiated experiments to identify the phosphorylation sites of Raf-1. In this report, we find that Ser43, Ser259, and Ser621 are the major sites of Raf-1 which are phosphorylated in mammalian cells and in Sf9 insect cells infected with a recombinant baculovirus encoding human Raf-1. Mutant Raf-1 proteins lacking kinase activity are also phosphorylated on these sites in vivo, indicating that these phosphorylation events are not a consequence of autophosphorylation. Furthermore, we find that Thr268 is the predominant Raf-1 residue phosphorylated in in vitro autokinase assays. In addition, we have examined the biochemical activity of baculovirus-expressed Raf-1 proteins containing mutations at these phosphorylation sites. In in vitro protein kinase assays Ser259 mutant proteins were 2-fold more active than wild-type Raf-1 and Ser621 mutant proteins were inactive as kinases. Analysis of the residues surrounding Ser259 and Ser621 indicates that RSXSXP may be a consensus sequence for the kinase responsible for phosphorylation of Raf-1 at these sites. Interestingly, these RSXSXP sequences are completely conserved throughout evolution in all Raf family members.

Blackshear PJ
The MARCKS family of cellular protein kinase C substrates.
J Biol Chem. 1993; 268: 1501-4

Osada S, Mizuno K, Saido TC, Suzuki K, Kuroki T, Ohno S
A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.
Mol Cell Biol. 1992; 12: 3930-8
Display abstract
A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.

Raynor RL, Kim YS, Zheng B, Vogler WR, Kuo JF
Membrane interactions of mastoparan analogues related to their differential effects on protein kinase C, Na, K-ATPase and HL60 cells.
FEBS Lett. 1992; 307: 275-9
Display abstract
Membrane interactions of tetradecapeptide toxin mastoparan (MP) and analogues (MP-3, MP-X and polistes MP), as indicated by inhibition of various enzymatic and cellular activities, were investigated. MP-3 was found to be the least active in inhibiting protein kinase C (PKC; activated by phosphatidylserine vesicles, synaptosomal membranes or phorbol ester), synaptosomal membrane Na,K-ATPase and proliferation and viability of leukemia HL60 cells. MP-3, however, was as active as others in inhibiting PKC activated by arachidonate monomers and phorbol ester binding. The unique properties of MP-3, the [des-Ile1-Asn2]-analogue of MP, might be related to its low functional amphiphilicity compared to others and useful in further delineating biological activities associated with or regulated by membranes.

Quest AF, Bloomenthal J, Bardes ES, Bell RM
The regulatory domain of protein kinase C coordinates four atoms of zinc.
J Biol Chem. 1992; 267: 10193-7
Display abstract
Protein kinase C (PKC) was found to be a zinc metallo-enzyme. Atomic absorption measurements on the intact enzyme indicated that four zinc atoms (4.2 +/- 0.5) were bound per PKC alpha molecule. Similar stoichiometric ratios were determined for PKC beta II and PKC gamma, other PKC isoforms individually expressed in the baculovirus-insect cell expression system, as well as for purified rat brain PKC. By trypsin treatment of PKC alpha, a 32-kDa lipid binding regulatory and a 50-kDa catalytic domain were generated that were subsequently completely separated by gel filtration in the presence of Triton X-100/phosphatidylserine mixed micelles. Zinc was present at levels significantly above background in fractions that contained the 32-kDa fragment and displayed phorbol ester binding activity. Lipid association and phorbol ester binding did not lead to displacement of zinc from the protein. The stoichiometry determined for this fragment (4.7 +/- 0.9) suggested that zinc was bound exclusively within the lipid binding regulatory domain of intact PKC. Furthermore, this stoichiometry is consistent with zinc being coordinated between 6 cysteine residues in a structural motif related to the Zn(II)2Cys6 binuclear cluster identified in the GAL4 transcriptional factor (Pan, T., and Coleman, J.E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077-2081).

Lee J, Ahmed S, Kozma R, Teo M, Monfries C, Lim L
The N-terminal region of n-Chimaerin allows lipid modulation of the C-terminal p21rac-GTPase activating domain.
Biochem Soc Trans. 1992; 20: 310-310

Mizukawa K, Otsuka N, Ogawa N, Haba K, Mori A
Changes of phorbol ester binding sites in rat brain following intracerebroventricular administration of thyrotropin-releasing hormone (TRH): an in vitro macroautoradiographic investigation.
Neurochem Res. 1992; 17: 361-5
Display abstract
We examined the influence of the intracerebroventricular (icv) administration of thyrotropin-releasing hormone (TRH) on protein kinase C (PKC) activities in various rat forebrain regions in order to cast light on the mechanism of extra-pituitary non-endocrine physiological actions of TRH in the central nervous system. An in vitro macroautoradiographic method, with [3H]phorbol 12, 13-dibutyrate (PDBu) as the radioactive ligand, was used to investigate quantitative alterations of PKC activities. The optical densities for PDBu binding sites in the striatum and hippocampal formation were significantly increased after the icv administration of TRH, while those in the frontal cortex and septum were unchanged. These findings suggest that TRH may exert some of its non-endocrine functions through striatal and hippocampal neurons which used PKC in their second messenger systems.

James G, Olson E
Deletion of the regulatory domain of protein kinase C alpha exposes regions in the hinge and catalytic domains that mediate nuclear targeting.
J Cell Biol. 1992; 116: 863-74
Display abstract
Members of the protein kinase C (PKC) family are characterized by an NH2-terminal regulatory domain containing binding sites for calcium, phosphatidylserine, and diacylglycerol (or tumor-promoting phorbol esters), a small central hinge region and a COOH-terminal catalytic domain. We have constructed fusion proteins in which the regulatory domain of PKC alpha was removed and replaced by a 19-amino acid leader sequence containing a myristoylation consensus or by the same sequence in which the amino-terminal glycine was changed to alanine to prevent myristoylation. The goal was to generate constitutively active mutants of PKC that were either membrane bound, due to their myristoylation, or cytoplasmic. Western blotting of fractions from COS cells transfected with plasmids encoding wild-type and mutant proteins revealed that PKC alpha resided entirely in a Triton X-100 soluble (TS) fraction, whereas both the myristoylated and nonmyristoylated mutants were associated primarily with the nuclear envelope fraction. A similar mutant that lacked the 19 amino acid leader sequence was also found almost entirely in the nuclear envelope, as was a truncation mutant containing only the regulatory domain, hinge region, and a small portion of the catalytic domain. However, an additional truncation mutant consisting of only the regulatory domain plus the first one-third of the hinge region was almost entirely in the TS fraction. A nonmyristoylated fusion protein containing only the catalytic domain was also found in the nuclear envelope. Immunostaining of cells transfected with these constructs revealed that both the myristoylated and nonmyristoylated mutants were localized in nuclei, whereas wild-type PKC alpha was primarily cytoplasmic and perinuclear. Phorbol dibutyrate treatment of PKC alpha-transfected cells resulted in increased perinuclear and nuclear staining. The results are consistent with a model in which activation of PKC, by phorbol esters or by deletion of the regulatory domain, exposes regions in the hinge and catalytic domains that interact with a PKC "receptor" present in the nuclear envelope, and may explain the ability of wild-type PKC to be translocated to the nucleus under certain conditions.

McGivern J, Scholfield CN
Phorbol ester and lignocaine or pentobarbitone interactions at presynaptic axons.
Neuroreport. 1992; 3: 139-42
Display abstract
The interaction between anaesthetics and protein kinase C activation was studied in unclamped field currents from unmyelinated axons which give rise to en passant synapses. Electrical responses could be resolved into Na, K and Ca components revealed by electrode polarisation pretreatment with blockers of K-conductances. In the presence of phorbol dibutyrate, there was an increase in the potency of lignocaine, pentobarbitone and tetrodotoxin: for the Na current, the potency increased by 2.67 +/- 0.64, 2.35 and 2.47 fold respectively. The potentiation does not appear to be any indirect result of changed membrane potential. It is suggested that protein kinase C phosphorylation of membrane channel proteins increases the effectiveness of these substances.

Burns DJ, Bell RM
Protein kinase C contains two phorbol ester binding domains.
J Biol Chem. 1991; 266: 18330-8
Display abstract
A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.

Gopalakrishna R, Anderson WB
Reversible oxidative activation and inactivation of protein kinase C by the mitogen/tumor promoter periodate.
Arch Biochem Biophys. 1991; 285: 382-7
Display abstract
The oxidant mitogen/tumor promoter, periodate, was used to selectively modify either the regulatory domain or the catalytic domain of protein kinase C (PKC) to induce oxidative activation or inactivation of PKC, respectively. Periodate, at micromolar concentrations, modified the regulatory domain of PKC as determined by the loss of ability to stimulate kinase activity by Ca2+/phospholipid, and also by the loss of phorbol ester binding. This modification resulted in an increase in Ca2+/phospholipid-independent kinase activity (oxidative activation). However, at higher concentrations (greater than 100 microM) periodate also modified the catalytic domain, resulting in complete inactivation of PKC. The oxidative modification induced by low periodate concentrations (less than 0.5 mM) was completely reversed by a brief treatment with 2 mM dithiothreitol. In this aspect, the modification induced by periodate was different from that of the previously reported irreversible modification of PKC induced by H2O2. However, the inactivation of PKC induced by periodate at concentrations greater than 1 mM was not reversed by dithiothreitol. Among the phospholipids and ligands of the regulatory domain tested, only phosphatidylserine protected the regulatory domain from oxidative modification. In the presence of phosphatidylserine, the catalytic site was selectively modified by periodate, resulting in formation of a form of PKC that exhibited phorbol ester binding but not kinase activity. Both reversible and irreversible oxidative activation and inactivation of PKC also were observed in intact cells treated with periodate. Taken together these results suggest that periodate, by virtue of having a tetrahedral structure, binds to the phosphate-binding regions present within the phosphatidylserine-binding site of the regulatory domain and the ATP-binding site of the catalytic domain, and modifies the vicinal thiols present within these sites. This results in the formation of intramolecular disulfide bridge(s) within the regulatory domain or catalytic domain leading to either reversible activation or inactivation of PKC, respectively. Thus, oxidant mitogen/tumor promoters such as periodate may be able to bypass normal transmembrane signalling systems to directly activate pathways involved in cellular regulation.

Maruyama IN, Brenner S
A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans.
Proc Natl Acad Sci U S A. 1991; 88: 5729-33
Display abstract
Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode Caenorhabditis elegans. Molecular cloning of the gene and sequencing of the cDNA revealed that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium; diacylglycerol inhibited the binding in a competitive manner. These findings confirm that the unc-13 gene product has binding sites similar to those of protein kinase C and may be a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system.

Sullivan JP, Connor JR, Tiffany C, Shearer BG, Burch RM
NPC 15437 interacts with the C1 domain of protein kinase C. An analysis using mutant PKC constructs.
FEBS Lett. 1991; 285: 120-3
Display abstract
We recently demonstrated that 2,6,diamino-N-[( 1-(oxotridecyl)-2-piperidinyl]methyl)-hexanamide (NPC 15437) is a selective inhibitor of PKC interacting at the regulatory domain of the enzyme. To further investigate the interaction of NPC 15437 with PKC we expressed a series of cDNAs encoding mutant PKC molecules in COS7 cells. NPC 15437 had no effect on the protein kinase activity of mutants lacking the N-terminal region of the C1 domain. Further, NPC 15437 was a competitive inhibitor of the activation of PKC alpha by phorbol ester and attenuated the binding of phorbol ester to the enzyme in intact cells. The present study demonstrates that mutant enzyme constructs can be used to localize the site of interaction of NPC 15437 with PKC to residues 12-42, which encodes the pseudosubstrate binding domain and part of the first cysteine-rich repeat sequence.

Gatti A, Watsuji T, Hanley M
Apparent calcium-independent phorbol ester binding to protein kinase C.
Biochem Soc Trans. 1991; 19: 150-150

Hubbard SR, Bishop WR, Kirschmeier P, George SJ, Cramer SP, Hendrickson WA
Identification and characterization of zinc binding sites in protein kinase C.
Science. 1991; 254: 1776-9
Display abstract
Metal ion coordination in the regulatory domain of protein kinase C (PKC) is suggested by the conservation of six cysteines and two histidines in two homologous regions found therein. By monitoring x-ray fluorescence from a purified sample of rat PKC beta I overexpressed in insect cells, direct evidence has been obtained that PKC beta I tightly binds four zinc ions (Zn2+) per molecule. Extended x-ray absorption fine structure (EXAFS) data are best fit by an average Zn2+ coordination of one nitrogen and three sulfur atoms. Of the plausible Zn2+ coordination models, only those featuring nonbridged Zn2+ sites accommodate the EXAFS data and all of the conserved potential ligands.

Zalewski PD, Forbes IJ, Giannakis C, Betts WH
Regulation of protein kinase C by Zn(2+)-dependent interaction with actin.
Biochem Int. 1991; 24: 1103-10
Display abstract
Zn2+ influences diverse cellular processes by poorly understood mechanisms. Some of these effects may be mediated by the protein kinase C (PKC) family of enzymes, since an influx of Zn2+ greatly increases their binding of regulatory ligand phorbol ester and induces their translocation from cytosol to the cytoskeleton. Using a model with purified components, we now show that Zn2+ acts by forming a phospholipid-dependent complex of PKC with filamentous actin, which results in expression of new binding sites for phorbol ester and phosphorylation of actin. These results provide a basis for the observed localization of PKC at actin-membrane junctions, in-vivo.

Finkenzeller G, Marme D, Hug H
Sequence of human protein kinase C alpha.
Nucleic Acids Res. 1990; 18: 2183-2183

Ohno S
[Identification and functional analysis of novel proteins deduced from the cDNA sequences: protein kinase C related proteins]
Tanpakushitsu Kakusan Koso. 1990; 35: 2450-6

Cazaubon S, Webster C, Camoin L, Strosberg AD, Parker P
Effector-dependent conformational changes in protein kinase C gamma through epitope mapping with inhibitory monoclonal antibodies.
Eur J Biochem. 1990; 194: 799-804
Display abstract
Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase C gamma (PKC gamma); 15G4, 5A2 and 36G9, were shown to display distinct properties with respect to PKC gamma kinase activity [Cazaubon, S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Eur. J. Biochem. 182, 401-406]. The mAb 5A2 and 36G9, which act as potent inhibitors of the cofactor-dependent kinase activity, can no longer bind PKC gamma in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 15G4 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb- and cofactor-binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKC gamma. For this purpose, several deletions were introduced at the 5' end of the PKC gamma cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKC gamma proteins shows that the amino acid residues essential to the binding of 5A2 and 36G9 are directly adjacent to the second cysteine-rich motif: these are contained in the sequences at positions 151-163 and 164-197, respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine-rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 36G9 are distinct from the cofactor-binding sites. This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 36G9 immunoreactivity of the native protein. In this conformational state, PKC gamma conserves its ability to interact with the non-inhibitory mAb 15G4. By using synthetic peptides, the 15G4 epitope was localized to the sequence 297-310 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C-terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC. In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates.

Ahmed S et al.
Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor.
Biochem J. 1990; 272: 767-73
Display abstract
A human brain-specific cDNA encoding n-chimaerin, a protein of predicted molecular mass 34 kDa, has sequence identity with two different proteins: protein kinase C (PKC) at the N-terminus and BCR protein [product of the breakpoint-cluster-region (BCR) gene, involved in Philadelphia chromosome translocation] at the C-terminus [Hall, Monfries, Smith, Lim, Kozma, Ahmed, Vannaisungham, Leung & Lim (1990) J. Mol. Biol. 211, 11-16]. The sequence identity of n-chimaerin with PKC includes the cysteine-rich motif CX2CX13CX2CX7CX7C, and amino acids upstream of the first cysteine residue, but not the kinase domain. This region of PKC has been implicated in the binding of diacylglycerol and phorbol esters in a phospholipid-dependent fashion. Part of this cysteine-rich motif (CX2CX13CX2C) has the potential of forming a 'Zn-finger' structure. Phorbol esters cause a variety of physiological changes and are among the most potent tumour promoters that have been described. PKC is the only known protein target for these compounds. We now report that n-chimaerin cDNA encodes a novel phospholipid-dependent phorbol ester receptor, with the cysteine-rich region being responsible for this activity. This finding has wide implications for previous studies equating phorbol ester binding with the presence of PKC in the brain.

Osada S et al.
A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin.
J Biol Chem. 1990; 265: 22434-40
Display abstract
Protein kinase C (PKC)-related cDNA clones isolated from mouse epidermis cDNA library encoded a 78-kDa protein, nPKC eta. nPKC eta contains a characteristic cysteine-rich repeat sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are conserved among PKC family members. However, nPKC eta lacks a putative Ca2+ binding region (C2 region) that is seen in conventional PKCs (alpha, beta I, beta II, gamma), but not in novel PKCs (nPKC delta, -epsilon, -zeta). nPKC eta shows the highest sequence similarity to nPKC epsilon (59.4% identity). The similarity extends to the NH2-terminal sequence (E region) which corresponds to one of the divergent regions (D1 region). Northern blot analysis showed that the mRNA for nPKC eta is highly expressed in the lung and skin but, in contrast to other members of the PKC family, only slightly expressed in the brain. nPKC eta expressed in COS cells shows phorbol ester binding activity with a similar affinity to nPKC epsilon. Antiserum raised against a COOH-terminal peptide of nPKC eta identified an 82-kDa protein in mouse lung extract as well as in an extract from COS cells transfected with the nPKC eta-cDNA expression plasmid. Autophosphorylation of nPKC eta immunoprecipitated with the specific antiserum was observed, indicating that nPKC eta is a protein kinase. These results clearly demonstrate the existence and the possible importance of nPKC eta as a member of the phorbol ester receptor/protein kinase, PKC, family.

Huang KP, Huang FL
Differential sensitivity of protein kinase C isozymes to phospholipid-induced inactivation.
J Biol Chem. 1990; 265: 738-44
Display abstract
Interactions of types I, II, and III protein kinase C (PKC) with phospholipids were investigated by following the changes in protein kinase activity and phorbol ester binding. The acidic phospholipids such as phosphatidylserine (PS), phosphatidic acid, phosphatidyl-glycerol, and cardiolipin, which are activators of PKC in the assay of protein phosphorylation, could differentially inactivate PKC I, II, and III during preincubation in the absence of divalent cation. The phospholipid-induced inactivation of PKC was concentration and time dependent and only affected the kinase activity without influencing phorbol ester binding. PKC I was the most susceptible to the phospholipid-induced inactivation, and PKC III was the least. The IC50 values of PS for PKC I, II, and III were 5, 45, and greater than 120 microM, respectively. Addition of divalent cation such as Ca2+ or Mg2+ suppressed the phospholipid-induced inactivation of PKC. In the absence of divalent cation, PKC I, II, and III all formed complexes with PS vesicles, although to a slightly different degree, as analyzed by molecule sieve chromatography. [3H]Phorbol 12,13-dibutyrate binding for PKC I, II, and III was recovered after chromatography; however, the kinase activities of all these enzymes were greatly reduced. In the presence of Ca2+, all three PKCs formed complexes with PS vesicles, and both the kinase and phorbol ester-binding activities of PKC II and III were recovered following chromatography. Under the same conditions, the phorbol ester-binding activity of PKC I was also recovered, but the kinase activity was not. The phospholipid-induced inactivation of PKC apparently results from a direct interaction of phospholipid with the catalytic domain of PKC; this interaction can be suppressed by divalent cations. In the presence of divalent cations, PS interacted preferentially with the regulatory domain of PKC and resulted in the activation of the kinase.

Walters JD, Johnson JD
Terbium as a luminescent probe of metal-binding sites in protein kinase C.
J Biol Chem. 1990; 265: 4223-6
Display abstract
In the present report, we demonstrate that Tb3+ binds to protein kinase C and serves as a luminescent reporter of certain cationic metal-binding sites. Tb3+ titration of 50 nM protein kinase C results in a 20-fold enhancement of Tb3+ luminescence which is half-maximal at 12 microM Tb3+. A Kd of approximately 145 nM was determined for Tb3+ binding to the enzyme. The excitation spectrum of bound Tb3+ exhibits a peak at 280 nm characteristic of energy transfer from protein tryptophan or tyrosine residues. The luminescence of this complex can be markedly decreased by other metals, including Pb2+ (IC50 = 25 microM), La3+ (IC50 = 50 microM), Hg2+ (IC50 = 300 microM), Ca2+ (IC50 = 6 mM), and Zn2+ (IC50 greater than 10 mM), and chelation of Tb3+ by 2 mM EGTA. Tb3+ binding to protein kinase C is correlated with its inhibition of protein kinase activity (IC50 = 8 microM), r = 0.99) and phorbol ester binding (IC50 = 15 microM, r = 0.98). Tb3+ inhibition of protein kinase C activity cannot be overcome by excess Ca2+, but can be partially overcome with excess phosphatidylserine or by chelation of Tb3+ with EGTA. Tb3+ noncompetitively inhibits phorbol ester binding by decreasing the maximal extent of binding without significantly altering binding affinity. The results suggest that the Tb3(+)-binding site is at or allosterically related to the enzyme's phosphatidylserine-binding site, but is distinct from the phorbol ester-binding domain and the Ca2(+)-binding site that regulates enzyme activity.

el Touny S, Khan W, Hannun Y
Regulation of platelet protein kinase C by oleic acid. Kinetic analysis of allosteric regulation and effects on autophosphorylation, phorbol ester binding, and susceptibility to inhibition.
J Biol Chem. 1990; 265: 16437-43
Display abstract
The regulation of protein kinase C by oleic acid was studied, and parameters that characterize the activation of protein kinase C by oleic acid and distinguish its effects from those of diacylglycerol (DAG) and phosphatidylserine (PS) were delineated. Activation of protein kinase C by sodium oleate required the presence of calcium and showed mild cooperative behavior (Hill number of 1.25) suggesting that Ca(oleate)2 is the active species. Kinetic analysis of the interaction of sodium oleate with substrates indicated that sodium oleate acted to increase the activity of the enzyme without modulating the KM for either MgATP or histone substrates. In this respect, sodium oleate action resembled that of DAG but not PS. However, multiple parameters distinguished the effects of sodium oleate from those of DAG. Unlike DAG, sodium oleate was unable to inhibit phorbol dibutyrate binding to protein kinase C. Sodium oleate also failed to interact with micelle-bound protein kinase C and preferentially activated "soluble" protein kinase C. The addition of histone caused protein/lipid aggregation in the presence of DAG but not in the presence of oleate. Activation of protein kinase C by sodium oleate or by PS/DAG demonstrated differential susceptibility to the action of inhibitors. Sphingosine and NaCl were more potent in inhibiting activation of protein kinase C by PS/DAG than by sodium oleate. Sodium oleate also expressed PS-like activity in that calcium and oleate acted as cofactors in activation of protein kinase C by DAG. Similar to PS, the ability of oleate to act in synergy with DAG resulted from "competitive" activation with a decrease in KM(app) of protein kinase C for DAG. Finally, sodium oleate was unable to induce autophosphorylation of protein kinase C. These studies demonstrate that oleate activates protein kinase C by a mechanism that is distinct from PS/DAG but partially overlaps the kinetic effects of both PS and DAG. The significance of these studies is discussed in relation to mechanisms of protein kinase C activation and to the possible physiological relevance of activation of protein kinase C by fatty acids.

Zalewski PD, Forbes IJ, Giannakis C, Cowled PA, Betts WH
Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton.
FEBS Lett. 1990; 273: 131-4
Display abstract
Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of [3H]phorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of alpha-, beta- and gamma-isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2-50 microM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten-fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by 1,10-phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.

Erusalimsky JD, Morris C, Perks K, Brown R, Brooks S, Rozengurt E
Internal amino acid sequence analysis of the 80 kDa protein kinase C substrate from rat brain: relationship to the 87 kDa substrate from bovine brain.
FEBS Lett. 1989; 255: 149-53
Display abstract
We have obtained sequence data from five proteolytic peptides totalling 102 amino acid residues of the 80 kDa protein kinase C substrate purified from rat brain. The amino acid sequences of these five peptides were compared with that deduced from a cDNA encoding the 87 kDa protein kinase C substrate from bovine brain. The overall amino acid sequence identity within the regions covered by our peptides is 54%. Two peptides aligned at the C- and N-termini of the bovine protein kinase C substrate with a very high degree of homology (more than 80% identity). Two other peptides exhibited 62% and 46% identity with two regions located in the C-terminal half of the bovine protein. The fifth peptide which contains the sequence PEQPEQPEQ did not reveal any similarity with the bovine protein. Based on the homologies of our experimentally determined sequences, which represent about 30% of the deduced sequence of the bovine protein, we suggest that although these protein kinase C substrates are not identical, they may belong to a family of related proteins.

Kaibuchi K, Fukumoto Y, Oku N, Takai Y, Arai K, Muramatsu M
Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C.
J Biol Chem. 1989; 264: 13489-96
Display abstract
We have constructed the expression plasmids harboring protein kinase C (PKC) mutant cDNAs with a series of deletions in the PKC coding region. These plasmids were transfected into COS7 cells to characterize the PKC mutants. Immunoblot analysis using the anti-PKC antibody identified proteins with the Mr values expected from the PKC mutant cDNAs in the extracts from COS7 cells. The wild-type PKC, when expressed in COS7 cells, conferred increased phorbol ester binding activity on intact cells; but the PKC mutants with the deletion around the C1 region did not show this activity. The wild-type PKC showed protein kinase activity dependent on phospholipid, Ca2+, and phorbol ester, whereas these PKC mutants exhibited protein kinase activity independent of the activators in a cell-free system. A PKC mutant cDNA with the deletion in the C2 region gave increased phorbol ester binding activity. Protein kinase activity of this mutant was much less dependent on Ca2+ compared with the wild-type PKC. A PKC mutant cDNA with the deletion in the C3 region conferred increased phorbol ester binding activity, but neither activator-dependent nor -independent protein kinase activity. These results indicate that elimination of the C1 region of PKC gives rise to constitutively active PKC independent of phospholipid, Ca2+, and phorbol ester and that the C1-C3 regions play distinct roles in the regulatory and catalytic function of PKC. In another series of experiments, transfection of some PKC mutant cDNAs with the deletions around the C1 region into Chinese hamster ovary and Jurkat cells activated the activator protein-1-binding element or the c-fos gene enhancer linked to the chloramphenicol acetyltransferase reporter gene in the absence of phorbol ester. Microinjection of these constructs into Xenopus oocytes induced initiation of germinal vesicle breakdown, indicating that they stimulated the PKC pathway in vivo. Thus, the phorbol ester-independent PKC mutant cDNAs could be a powerful tool to investigate the transmembrane signaling pathway mediated by PKC.

Bazzi MD, Nelsestuen GL
Properties of the protein kinase C-phorbol ester interaction.
Biochemistry. 1989; 28: 3577-85
Display abstract
The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. The initial calcium-dependent and reversible form of membrane-associated PKC was capable of binding PDBu. Both PKC and PDBu were released from this complex by calcium chelation. Sustained interaction with phospholipid vesicles resulted in a PKC-membrane complex that could not be dissociated by calcium chelation and appeared to result from insertion of PKC into the hydrocarbon portion of the phospholipid bilayer. Membrane insertion was observed at calcium concentrations of 2-500 microM and with membrane compositions of 10-50% acidic phospholipid. However, the extent of insertion was dependent on the binding conditions and was promoted by high phospholipid to PKC ratios, high calcium, the presence of phorbol esters, high membrane charge, and long incubations.(ABSTRACT TRUNCATED AT 250 WORDS)

Lee MH, Bell RM
Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles.
J Biol Chem. 1989; 264: 14797-805
Display abstract
The specificity of the phospholipid cofactor requirement of rat brain protein kinase C was investigated using Triton X-100 mixed micellar methods. Sixteen analogues of phosphatidylserine were prepared and tested for their ability to support protein kinase C activity, [3H]phorbol 12,13-dibutyrate binding, and protein kinase C binding to mixed micelles. Phosphatidylserinol, -L-serine methyl ester, -N-acetyl-L-serine, -2-hydroxyacetate, -3-hydroxypropionate, and -4-hydroxybutyrate did not activate protein kinase C in mixed micelles containing 2 mol % of sn-1,2-dioleoylglycerol. This indicates that both the carboxyl and amino moieties are important for activation. Phosphatidyl-D-serine and -L-homoserine were incapable of supporting full activation; this demonstrates stereospecificity and the importance of the distance between the phosphate and carboxyl and amino moieties. Since 1,2-rac-phosphatidyl-L-serine and 1,3-phosphatidyl-L-serine fully supported protein kinase C activity, the stereochemistry within the glycerol backbone at the interface was not necessary for maximal activation. Neither lysophosphatidyl-L-serine nor 1-oleoyl-2-acetyl-sn-glycero-3-phospho-L-serine supported protein kinase C activity implying that the interfacial conformation is critical to the activation process. The phospholipid dependencies of [3H]phorbol 12,13-dibutyrate binding and of protein kinase C binding to mixed micelles containing sn-1,2-dioleoylglycerol did not mirror those for activation. The data demonstrate that protein kinase C possesses a high degree of specificity with respect to phospholipid activation and implicate several functional groups within the phospho-L-serine polar head group in binding and activation.

Ohno S
[Studies on the molecular multiplicity, structure, and function of the protein kinase C family]
Seikagaku. 1989; 61: 569-87

Battaini F, Del Vesco R, Govoni S, Trabucchi M
Chronic alcohol intake modifies phorbol ester binding in selected rat brain areas.
Alcohol. 1989; 6: 169-72
Display abstract
3H-Phorbol 12,13 dibutyrate binding to rat brain was modified by chronic ethanol treatment. Among the areas examined hippocampus and cortex showed a decrease in Bmax values of 32 and 24% respectively. No significant effect was observed in hypothalamus and cerebellum. In vitro ethanol did not modify the binding in all the areas except at molar concentrations. In hippocampus and cortex the direct measurement of protein kinase C activity indicated that the decrease in phorbol ester binding was accompanied with a concomitant decrease in kinase activity. The results indicate that chronic ethanol treatment leads to an inhibition of brain protein kinase C function.

Baker ME
Similarity between phospholipase C and the regulatory domain of protein kinase C.
Mol Cell Endocrinol. 1989; 61: 129-31
Display abstract
We report that a 100 residue segment in the carboxy-terminus half of phosphatidylinositol-specific phospholipase C is similar to a segment in the amino-terminus half of protein kinase C. The computer-based comparison score is 9.5 standard deviations higher than that of 2500 comparisons of randomized sequences of these segments (P = 10(-21), suggesting that the two segments have similar biological properties. Phospholipase C has a segment that is homologous to part of the non-catalytic domain of src kinase and other tyrosine protein kinases. The similarity of phospholipase C with protein kinase C, a serine/threonine kinase suggests that novel exon shuffling occurred among serine/threonine and tyrosine kinases and phospholipase C.

Schaap D, Parker PJ, Bristol A, Kriz R, Knopf J
Unique substrate specificity and regulatory properties of PKC-epsilon: a rationale for diversity.
FEBS Lett. 1989; 243: 351-7
Display abstract
PKC-epsilon was isolated from a murine brain cDNA library. The clone, lambda 61PKC-epsilon, encoded a polypeptide of 737 amino acids that is homologous to other PKCs. Northern analysis showed that the 7 kb mRNA for this cDNA is widely expressed. The protein when expressed in COS-1 cells displayed phorbol ester-binding activity. However in order to detect the kinase activity of PKC-epsilon, it was necessary to employ a synthetic peptide substrate based upon the pseudosubstrate site. Subsequent analysis demonstrated that PKC-epsilon, while showing certain properties characteristic of the PKC family, has a quite distinct substrate specificity and is independent of Ca2+.

Hannun YA, Foglesong RJ, Bell RM
The adriamycin-iron(III) complex is a potent inhibitor of protein kinase C.
J Biol Chem. 1989; 264: 9960-6
Display abstract
Using Triton X-100/lipid mixed micellar methods, we observed that the adriamycin-iron(III) complex was a potent inhibitor of protein kinase C while uncomplexed adriamycin itself was a poor inhibitor in the absence of heavy metal contaminants. The 3:1 adriamycin-iron complex was more potent than 2:1, 1:1, and 1:0 complexes. Inhibition of protein kinase C was reversible, and 50% inhibition occurred at 13 microM (adriamycin)3Fe3+. Both the catalytic and the regulatory domain of protein kinase C were affected by adriamycin-iron(III). Adriamycin-iron(III) was a competitive inhibitor of the catalytic domain of protein kinase C with respect to MgATP but not with respect to magnesium (IC50 350 microM). The predominant interaction of adriamycin-iron(III) with native protein kinase C was as a competitive inhibitor with respect to diacylglycerol. Inhibition was not competitive with respect to phosphatidylserine, calcium, magnesium, MgATP, or histone. Interaction with the regulatory domain was demonstrated by the ability of adriamycin-iron(III) to inhibit phorbol dibutyrate binding. Other adriamycin transitional metal complexes showed little inhibition of protein kinase C activity. Acetylation of the amine on the daunosamine moeity of adriamycin did not preclude the formation of a ferric complex but resulted in total loss of inhibitory activity. These results suggest that the presence of free amines in a highly structured adriamycin-iron complex is necessary for inhibition. The implications of inhibition of protein kinase C by adriamycin-iron(III) are discussed.

Csermely P, Szamel M, Resch K, Somogyi J
Zinc increases the affinity of phorbol ester receptor in T lymphocytes.
Biochem Biophys Res Commun. 1988; 154: 578-83
Display abstract
In the primary structure of the major phorbol ester receptor, protein kinase C the presence of putative metal (zinc) binding sites has been suggested. We have demonstrated earlier that zinc activates protein kinase C and contributes to its binding to plasma membranes in T lymphocytes. Here we report that zinc increases the phorbol ester binding affinity of cytosolic protein kinase C. The effect of zinc on the membrane-bound enzyme is much less pronounced. Our results raise the possibility that cytosolic protein kinase C is a mixture of isoenzymes with different sensitivity towards zinc ions.

Chida K, Kato N, Yamada S, Kuroki T
Protein kinase C activities and bindings of a phorbol ester tumor promoter in 41 cell lines.
Biochem Biophys Res Commun. 1988; 157: 1-8
Display abstract
The activities of protein kinase C (PKC) and the bindings to phorbol-12,13-dibutyrate (PDBu) of 41 cell lines were measured. The activities of PKC varied from 0.2 to 37 mU/10(6) cells in different cell lines, and in general were high in normal or untransformed cells and low in malignant, or transformed cells. The PDBu binding also varied considerably in different cell lines, and was again higher in normal or untransformed cells. In some cell lines, the binding was much higher at 4 degrees C than at 37 degrees C, suggesting rapid down-regulation of the binding. A correlation between PKC activity and PDBu binding was found only within certain cell types, i.e., epithelial cell lines derived from human tumors.

Bonser RW, Thompson NT, Hodson HF, Beams RM, Garland LG
Evidence that a second stereochemical centre in diacylglycerols defines interaction at the recognition site on protein kinase C.
FEBS Lett. 1988; 234: 341-4
Display abstract
The interaction of novel diacylglycerol analogues at the recognition site on protein kinase C has been evaluated using a modified [3H]phorbol dibutyrate binding assay and an established kinase activation assay. Studies with the 3-methyl analogues of 1,2-dihexanoyl-sn-glycerol have revealed a preferred stereochemical configuration at the C-3 position. Other chemical modifications have extended existing structure/activity relationships by showing that carbamates and sulphonyl esters cannot substitute for carboxylate esters and that cyclic acyl groups are active. Thus, most, if not all of the functionalities in the diacylglycerol molecule are required for interaction at the receptor on protein kinase C. Stereochemical specificity is required at C2 and C3.

Talor Z, Mejicano GC
Phorbol-ester is bound specifically to renal luminal membranes and stimulates the Na-H antiporter.
Res Commun Chem Pathol Pharmacol. 1988; 61: 335-43
Display abstract
We characterized binding of [3H]-phorbol-12,13-dibutyrate (an activator of protein kinase C) to highly purified rabbit cortical renal luminal membranes and measured its effect on the kinetics of the Na-H antiporter. There was 95% specific binding to luminal membranes and this binding was time, temperature and pH dependent with optimal binding at 4 degrees C and pH 7.4. Scatchard analysis of the binding revealed Kd of 0.8 microM and Bmax of 19.4 pmoles/mg protein. Phorbol-12,13-dibutyrate stimulated the Vmax of the Na-H antiporter by 16.5% +/- 4.7 at 10(-6) M and by 19.9% +/- 5.4 at 10(-5) M. The protein kinase C inhibitor H7(1) prevented the observed stimulation. Thus, there is a correlation between phorbol ester binding and phorbol ester stimulation of the Na-H antiporter. These data demonstrate that protein kinase C plays a role in the stimulation of the Na-H antiporter in renal luminal membranes.

Nakabayashi H, Huang KP
Monoclonal antibodies against type II rat brain protein kinase C.
J Biol Chem. 1988; 263: 298-304
Display abstract
Monoclonal antibodies (8/1, 10/10, and 25/3) against rat brain type II protein kinase C were used for the immunochemical characterization of this kinase. These antibodies immunoprecipitated the type II protein kinase C in a dose-dependent manner but did neither to the type I nor III isozyme. Immunoblot analysis of the tryptic fragments from protein kinase C revealed that all three antibodies recognized the 27-38-kDa fragments, the phospholipid/phorbol ester-binding domain, but not the 45-48-kDa fragments, the kinase catalytic domain. The immune complexes of the kinase and the antibodies retained 70-80% of the kinase activity which was dependent on Ca2+ and phosphatidylserine and further activated by diacylglycerol or tumor-promoting phorbol ester. With antibody 8/1, the kinetic parameters with respect to Km for ATP and histone and K alpha for phosphatidylserine and phorbol 12,13-dibutyrate were not significantly influenced. However, the antibody causes variable effects on the K alpha for Ca2+ under different assay conditions. When determined in the presence of phosphatidylserine, the K alpha for Ca2+ was reduced by an order of magnitude (37 +/- 8 to 2.0 +/- 1.8 microM); in the presence of phosphatidylserine and phorbol 12,13-dibutyrate, the K alpha for Ca2+ was not significantly altered; and in the presence of phosphatidylserine and dioleoylglycerol, the kinase became an apparently Ca2+-independent enzyme. The effects of antibody 8/1 on the kinetic parameters of the enzyme for phorbol ester binding were different from those for kinase activity. This antibody causes a 20-30% reduction in phorbol ester binding and a 2-fold increase (1.9 +/- 0.2 to 3.9 +/- 0.3 micrograms/ml) in the concentration of phosphatidylserine required for half-maximal binding, but is without significant influence on those parameters for Ca2+ and phorbol 12,13-dibutyrate. The differential effects of antibody 8/1 on kinase activity and phorbol ester binding with respect to the kinetic parameter of phosphatidylserine suggest that the roles of this phospholipid in supporting phorbol ester binding and kinase activation are different. In the presence of the antibody, the autophosphorylations of the phospholipid/phorbol ester-binding domain and the kinase domain were reduced; the reduction was more pronounced for the former than for the latter. These results suggest that the epitope for antibody 8/1 is localized within the phospholipid/phorbol ester-binding domain at the region adjacent to the kinase domain so that the autophosphorylations of both domains are affected.

Ono Y, Fujii T, Igarashi K, Kikkawa U, Ogita K, Nishizuka Y
Nucleotide sequences of cDNAs for alpha and gamma subspecies of rat brain protein kinase C.
Nucleic Acids Res. 1988; 16: 5199-200

Hidaka H, Onoda K
[Heterogeneity of protein kinase C]
Nippon Rinsho. 1988; 46: 2751-61

Koenen M, Sippel AE, Trachmann C, Bister K
Primary structure of the chicken c-mil protein:identification of domains shared with or absent from the retroviral v-mil protein.
Oncogene. 1988; 2: 179-85
Display abstract
The complete primary structure of the protein product of the proto-oncogene c-mil was deduced from the nucleotide sequence of chicken c-mil cDNA clones. The c-mil protein contains 647 amino acid residues and has a calculated molecular weight of 73,132. Based on sequence comparisons with proteins of known or presumed biochemical function, two domains were recognized on the c-mil protein. In the carboxyl-terminal half of the protein, a 250-amino acid segment displays significant homology to the protein kinase domains of the src oncogene protein or of protein kinase C. In the amino-terminal half, a cysteine-rich segment (Cys-X2-Cys-X9-Cys-X2-Cys-X7-Cys-X7-Cys) of the c-mil protein shares significant homology with two similar repetitive domains of protein kinase C. Of the two structural and presumably functional domains of the c-mil protein, only the kinase domain is contained within the carboxyl-terminal 379-amino acid polypeptide encoded by the transduced v-mil allele of avian oncogenic retrovirus MH2. Hence, truncation of the 5' coding region in the course of the transduction and the resulting lack of the authentic amino-terminal domain in the protein product of the transduced allele may be a critical event in changing mil function from physiologic to oncogenic.

Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y
The structure, expression, and properties of additional members of the protein kinase C family.
J Biol Chem. 1988; 263: 6927-32
Display abstract
In rat brain three members of the protein kinase C family encoded by cDNAs termed delta, epsilon, and zeta were newly identified by molecular cloning and sequence analysis. The new members have a common structure that is closely related to but clearly distinct from the four members of the family previously isolated having alpha-, beta I-, beta II-, and gamma-sequences, although the zeta-cDNA available at present does not appear to contain a complete reading frame for protein kinase C. The delta-, epsilon-, and zeta-cDNAs all encode a characteristic cysteine-rich sequence and protein kinase domain sequence, both of which are highly homologous among the protein kinase C family. However, the new members lack one of the conserved regions that is present in alpha-, beta I-, beta II-, and gamma-sequences. An additional cDNA clone termed epsilon' was isolated, which is identical with epsilon-cDNA except for a short sequence at the 5'-terminal end region. The two members having delta- and epsilon-sequences were expressed in COS 7 cells, and partially purified and characterized. The enzymes having delta- and epsilon-sequences depend on phospholipid and diacylglycerol for the enzymatic activity, but their properties slightly differ from the previously known members of protein kinase C. Northern blot analysis suggests that the new members of protein kinase C exist in the brain and some other tissues.

Louis JC, Magal E, Yavin E
Protein kinase C alterations in the fetal rat brain after global ischemia.
J Biol Chem. 1988; 263: 19282-5
Display abstract
Marked changes in the intracellular localization of brain protein kinase C are evident after global ischemia generated by the restriction of the placental blood flow in the near-term rat embryo. A rapid (5 min) ischemia-dependent translocation of the enzyme from the cytosol to the particulate membrane fraction, which is completely reversible upon reperfusion, is observed. After 30 min of ischemia, substantial losses in protein kinase C activity and content as measured by [3H]phorbol dibutyrate binding are apparent. This is accompanied by a marked increase of a Ca2+-phosphatidylserine-independent kinase activity, already evident after 5 min of ischemia. By 15 or 30 min the total activity of the latter enzyme is equally distributed between the particulate and the cytosol fractions and is more than 3-fold higher in ischemic in comparison to naive animals. Activation and possible deregulation of protein kinase C are proposed to represent an initial step in the pathophysiology of brain ischemia.

Caramelo C, Tsai P, Okada K, Schrier RW
Protein kinase C activity in compensatory kidney growth.
Biochem Biophys Res Commun. 1988; 152: 315-21
Display abstract
Very little information exists about the intracellular mechanisms mediating remnant kidney hypertrophy after reduction of renal mass. The present experiments demonstrate that the activity of a key enzyme in cellular proliferation, protein kinase C, is enhanced in the kidney during post-uninephrectomy hypertrophy. As additional evidence of "in vivo" enzyme activation, the enzyme activity migrates from the cytosolic to the particulate fraction of the cells and the number of sites of phorbol dibutyrate binding is enhanced in kidney membranes 24 hr after contralateral nephrectomy. The stimulation of the enzyme is higher than the increase in kidney weight, although both phenomena are linearly related.

Nakadate T, Jeng AY, Blumberg PM
Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action.
Biochem Pharmacol. 1988; 37: 1541-5
Display abstract
The effects of inhibitors of protein kinase C on the activities of the intact enzyme, the proteolytically-generated catalytic domain, and [3H]phorbol 12,13-dibutyrate (PDBu) binding were compared in an effort to evaluate this approach for clarifying mechanisms of inhibitor action. Staurosporine, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], and quercetin inhibited the catalytic fragment with similar potencies as for the intact enzyme while having little or no effect on binding, consistent with reports that they are competitive with ATP. Adriamycin, trifluoperazine, and tamoxifen, suggested to disrupt hydrophobic interactions between the regulatory domain of protein kinase C and phospholipid, were all most effective on the intact enzyme. They appear to possess a mixed mechanism, however, inhibiting activity of the catalytic domain with approximately 3-fold lower potencies. Gossypol inhibited intact enzyme, catalytic fragment, and PDBu binding with similar potencies. In light of multiple apparent sites of action for such protein kinase C inhibitors, comparison of their activities on the individual functional domains of the kinase may provide a useful complement to studies with the intact enzyme.

Huang KP, Huang FL, Nakabayashi H, Yoshida Y
Biochemical characterization of rat brain protein kinase C isozymes.
J Biol Chem. 1988; 263: 14839-45
Display abstract
Biochemical characteristics of three rat brain protein kinase C isozymes, types I, II, and III, were compared with respect to their protein kinase and phorbol ester-binding activities. All three isozymes appeared to be alike in their phorbol ester-binding activities as evidenced by their similar Kd for phorbol 12,13-dibutyrate and requirements for Ca2+ and phospholipids. However, differences with respect to the effector-mediated stimulation of protein kinase activity were detectable among these isozymes. The type I enzyme could be stimulated by cardiolipin to a greater extent than those of the type II and III enzymes. In the presence of cardiolipin, the concentrations of dioleoylglycerol or phorbol 12,13-dibutyrate required for half-maximal activation (A1/2) of the type I enzyme were nearly an order of magnitude lower than those for the type II and III enzymes. In the presence of phosphatidylserine, differences in the A1/2 of dioleoylglycerol and phorbol 12,13-dibutyrate for the three isozymes of protein kinase C were less significant than those measured in the presence of cardiolipin. Nevertheless, the A1/2 of these two activators for the type I enzyme were lower than those for the type II and III enzymes. At high levels of phosphatidylserine (greater than 15 mol %), binding of phorbol 12,13-dibutyrate to the type I enzyme evoked a corresponding stimulation of the kinase activity, whereas binding of this phorbol ester to the type II and III enzymes produced a lesser degree of kinase stimulation. For all three isozymes, the concentrations of phosphatidylserine required for half-maximum [3H]phorbol 12,13-dibutyrate binding were almost an order of magnitude less than those for kinase stimulation. Consequently, neither isozyme exhibited a significant kinase activity at lower levels of phosphatidylserine (less than 5 mol %) and phorbol 12,13-dibutyrate (50 nM), a condition sufficient to promote near maximal phorbol ester binding. In addition to their different responses to the various activators, the three protein kinase C isozymes also have different Km values for protein substrates. The type I enzyme appeared to have lower Km values for histone IIIS, myelin basic protein, poly(lysine, serine) (3:1) polymer, and protamine than those for the type II and III enzymes. These results documented that the three protein kinase C isozymes were distinguishable in their biochemical properties. In particular, the type I enzyme, which is a brain-specific isozyme, is distinct from the type II and III enzymes, both have a widespread distribution among different tissues.

Zalewski PD, Forbes IJ, Valente L, Apostolou S, Hurst NP
Translocation of protein kinase C to a Triton-insoluble sub-cellular compartment induced by the lipophilic gold compound auranofin.
Biochem Pharmacol. 1988; 37: 1415-7

Chen KH, Peng ZG, Lavu S, Kung HF
Molecular cloning and sequence analysis of two distinct types of Xenopus laevis protein kinase C.
Second Messengers Phosphoproteins. 1988; 12: 251-60
Display abstract
Two distinct types of protein kinase C cDNA clones were isolated from a Xenopus laevis oocyte cDNA library, and the complete nucleotide sequences were determined. The sequences encode a single open reading frame with a domain structure that consists of four constant (designated C1-C4) and five variable (designated V1-V5) regions. Comparison of the two sequences shows good homology at the nucleotide and deduced amino acid level. The differences reside primarily in the variable regions. Each clone encodes 671 and 676 amino acids, respectively, having extensive homology with published sequences of human, rat, and bovine protein kinase C. These results provide evidence that these two distinct types of protein kinase C are members of a multigene family in amphibian and mammalian species.

Ohno S, Akita Y, Konno Y, Imajoh S, Suzuki K
A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family.
Cell. 1988; 53: 731-41
Display abstract
Protein kinase C (PKC)-related cDNA clones encode an 84 kd protein, nPKC. nPKC contains a cysteine-rich repeat sequence homologous to that seen in conventional PKCs (alpha, beta I, beta II, and gamma), which make up a family of 77-78 kd proteins with closely related sequences. nPKC, when expressed in COS cells, confers increased high-affinity phorbol ester receptor activity to intact cells. Antibodies raised against nPKC identified a 90 kd protein in rabbit brain extract as well as in extracts from COS cells transfected with the cDNA construct. nPKC shows protein kinase activity that is regulated by phospholipid, diacylglycerol, and phorbol ester but is independent of Ca2+. The structural and enzymological characteristics of nPKC clearly distinguish it from conventional PKCs, which until now have been the only substances believed to mediate the various effects of diacylglycerol and phorbol esters. These results suggest an additional signaling pathway involving nPKC.

Ellis CA, Aitken A, Takayama K, Qureshi N
Substitution of phosphatidylserine by lipid A in the activation of purified rabbit brain protein kinase C.
FEBS Lett. 1987; 218: 238-42
Display abstract
Three lipid A derivatives (hexaacyl monophosphoryl lipid A, hexaacyl diphosphoryl lipid A, and disaccharide precursor IVA) were shown to activate protein kinase C from rabbit brain. These derivatives substituted for phosphatidylserine in a concentration-dependent manner and did not compete for binding of [3H]phorbol dibutyrate to its receptor site. Instead, phorbol dibutyrate binding was increased on raising the concentration of the derivatives in a similar manner to phosphatidylserine. The phorbol ester 12-0-tetra-decanol 13-acetate augmented the activation of protein kinase C by the lipid A derivatives.

Nakadate T, Blumberg PM
Modulation by palmitoylcarnitine of protein kinase C activation.
Cancer Res. 1987; 47: 6537-42
Display abstract
Palmitoylcarnitine, a reported protein kinase C inhibitor, enhanced the phorbol ester dependency of the enzyme, augmenting protein kinase C activity in the presence of phorbol esters such as phorbol 12,13-dibutyrate while inhibiting the basal activity measured in the presence of calcium plus phosphatidylserine. Weakly potent phorbol esters such as phorbol 12,13-diacetate and 4-O-methylphorbol 12-myristate 13-acetate were full agonists like phorbol 12,13-dibutyrate for activation of protein kinase C in the presence of palmitoylcarnitine. On the other hand, 1,2-diacylglycerols such as 1,2-diolein were only partially stimulatory. Palmitoylcarnitine did not interfere with the association of protein kinase C with phosphatidylserine, suggesting that its action was on protein kinase C activation per se rather than on priming. A long fatty acid ester, quaternary amine, and anionic charge were needed for the palmitoylcarnitine-like action. Phosphatidylcholine, which possesses these features, partially mimicked the action of palmitoylcarnitine. Palmitoylcarnitine thus appears to be a lipophilic modulator of protein kinase C rather than a simple inhibitor. The results raise the possibility that differences in response between phorbol esters and diacylglycerols may reflect differential ability to activate protein kinase C in the appropriate lipid environment rather than the existence of unique targets for one or the other compound.

Moruzzi M, Barbiroli B, Monti MG, Tadolini B, Hakim G, Mezzetti G
Inhibitory action of polyamines on protein kinase C association to membranes.
Biochem J. 1987; 247: 175-80
Display abstract
Physiological activation of protein kinase C requires the interaction of this enzyme with cellular membranes [Nishizuka (1986) Science 233, 305-312]. In the present work a reconstituted system of protein kinase C and human inside-out erythrocyte vesicles was utilized to study the effect in vitro of naturally occurring polyamines on the activation process of protein kinase C. The active membrane-associated complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The association reaction of the enzyme to membrane was rapid, being complete within 1 min at 25 degrees C. The addition of polyamines, particularly spermine, greatly decreased in a dose-dependent manner the amount of protein kinase C bound to membranes (i.e. in the activated form). The effect observed was quite specific, since it was dependent on the chemical structure of the polyamine and it was manifest at micromolar concentrations of the polycation; the order of potency was spermine greater than spermidine greater than putrescine. A characterization of this effect is presented and possible physiological implications are discussed.

Coussens L, Rhee L, Parker PJ, Ullrich A
Alternative splicing increases the diversity of the human protein kinase C family.
DNA. 1987; 6: 389-94
Display abstract
Isolation of two protein kinase C (PKC) cDNA clones containing divergent carboxy-terminal sequences suggested a common genetic origin for these cDNAs. Partial characterization of the hPKC beta chromosomal gene provided direct evidence for the existence of two adjacent carboxy-terminal exons (beta 1 and beta 2) that are alternatively spliced to generate two types of hPKC beta sequences. PKC beta 1 and beta 2 mRNAs are expressed in a selective manner in both human hematopoietic cells and bovine brain tissues.

Ohno S et al.
Tissue-specific expression of three distinct types of rabbit protein kinase C.
Nature. 1987; 325: 161-6
Display abstract
We examined the structure of protein kinase C in an attempt to understand the molecular events connecting protein kinase C activation with the cellular response. Rabbit complementary DNA clones coding for three distinct types of protein kinase C, named alpha, beta and gamma, have been identified and sequenced. The deduced amino acid sequence for alpha, beta and gamma (673, 671 and 672 amino acids, respectively) are closely related. Kinases alpha and beta share an identical N-terminal sequence of 621 amino acid residues and their messenger RNAs arise from a single gene. The C-terminal halves of alpha, beta and gamma are protein kinase domains and are highly homologous to other protein kinases. The mRNAs for alpha, beta and gamma are expressed in various tissues with strikingly different tissue specificities. The one for gamma is found ubiquitously among various tissues, while those for alpha and beta predominate in the brain.

Kubo K, Ohno S, Suzuki K
Primary structures of human protein kinase C beta I and beta II differ only in their C-terminal sequences.
FEBS Lett. 1987; 223: 138-42
Display abstract
Two types of cDNA clones encoding human protein kinase C (PKC) were isolated from a spleen cDNA library using rabbit protein kinase C beta I/beta II cDNA as a hybridization probe. Nucleotide sequence analyses of these cDNA inserts revealed complete primary structures of two distinct types of human protein kinase C beta I and beta II which differ only in their C-terminal 50 or 52 amino acid residues. It was concluded that there exist four distinct types of PKC, PKC alpha, beta I, beta II and gamma, in human as well as rabbit, and that the corresponding sequences are strictly conserved among mammalian species.

Kikkawa U et al.
The common structure and activities of four subspecies of rat brain protein kinase C family.
FEBS Lett. 1987; 223: 212-6
Display abstract
Elucidation of the complete sequences of four cDNA clones (alpha, beta I, beta II, and gamma) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C1-C4) and five variable (V1-V5) regions. Although these sequences are highly homologous and closely related to one another V3-, V4-, and V5-regions of gamma-subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C1, contains a tandem repeat of cysteine-rich sequence (6, total 12 cysteine residues). The third constant region, C3, has an ATP-binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of alpha-, beta I-, beta II-, and gamma-subspecies are roughly 16, 8, 55, and 21%, respectively. gamma-Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.

Jaken S
Phorbol ester receptor and protein kinase C heterogeneity: evidence for PKC type specific biochemical properties.
Prog Clin Biol Res. 1987; 249: 205-13

Kubo K, Ohno S, Suzuki K
Nucleotide sequence of the 3' portion of a human gene for protein kinase C beta I/beta II.
Nucleic Acids Res. 1987; 15: 7179-80

Stabel S, Rodriguez-Pena A, Young S, Rozengurt E, Parker PJ
Quantitation of protein kinase C by immunoblot--expression in different cell lines and response to phorbol esters.
J Cell Physiol. 1987; 130: 111-7
Display abstract
Antisera have been raised against human protein kinase C and also against a synthetic peptide based on the sequence of the bovine brain enzyme (LLNQEE-GEYYNVPIPE). These antibodies react with protein kinase C from a number of species (human, murine, rat, rabbit, bovine), indicating substantial conservation of epitopes. These antisera have been used to quantitate directly protein kinase C by immunoblot analysis. We show here that there is a strict correlation between the levels of immunoreactive polypeptide and extractable calcium- and phospholipid-dependent kinase activity for various cell lines. Treatment of murine, rat, and human cells with phorbol dibutyrate was found to deplete levels of immunoreactive protein kinase C severely. A detailed study of the time course of this depletion in Swiss 3T3 cells shows that it follows precisely the loss of extractable activity. On exposure to 400 nM phorbol 12,13-dibutyrate protein kinase C was essentially undetectable by 40 hours; the half-life of this down-regulation was 6.7 hours. This data thus demonstrate that the loss of immunoreactive protein kinase C and of extractable calcium- and phospholipid-dependent kinase activity precisely parallels the phorbol ester induced down-regulation of binding and responsiveness in Swiss 3T3 cells.

Hoshijima M, Kikuchi A, Tanimoto T, Kaibuchi K, Takai Y
Formation of a phorbol ester-binding fragment from protein kinase C by proteolytic digestion.
Cancer Res. 1986; 46: 3000-4
Display abstract
When washed human platelets were disrupted by sonication in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, both the catalytic and [3H]phorbol-12,13-dibutyrate (PDBu)-binding activities of protein kinase C were recovered in the soluble fraction and were not separable from each other upon several column chromatographies. Platelet protein kinase C required diacylglycerol, Ca2+, and phospholipid for its activation and showed a molecular weight of about 87,000 as estimated by gel filtration analysis. However, when platelets were first incubated with 2 microM Ca2+-ionophore A23187 for 5 min at 37 degrees C in the medium containing 3 mM CaCl2 and then disrupted under the same conditions, the catalytic and [3H]phorbol-12,13-dibutyrate-binding activities were separately recovered in the soluble and particulate fractions, respectively; moreover, the catalytic activity recovered in the soluble fraction became independent of diacylglycerol, Ca2+, and phospholipid, and showed a molecular weight of about 50,000 as estimated by gel filtration analysis. The kinetic properties of this Mr 50,000 enzyme were similar to those of the catalytic fragment of rat brain protein kinase C described previously. In a cell-free system, digestion with trypsin of protein kinase C highly purified from rat brain caused the generation of a fragment which had no catalytic activity but showed full [3H]phorbol-12,13-dibutyrate-binding activity. The molecular weight of this fragment was estimated to be about 35,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that protein kinase C consists of at least two functionally different domains, a hydrophobic phorbol ester- or diacylglycerol-binding and hydrophilic catalytic domains.

Parker PJ et al.
The complete primary structure of protein kinase C--the major phorbol ester receptor.
Science. 1986; 233: 853-9
Display abstract
Protein kinase C, the major phorbol ester receptor, was purified from bovine brain and through the use of oligonucleotide probes based on partial amino acid sequence, complementary DNA clones were derived from bovine brain complementary DNA libraries. Thus, the complete amino acid sequence of bovine protein kinase C was determined, revealing a domain structure. At the amino terminal is a cysteine-rich domain with an internal duplication; a putative calcium-binding domain follows, and there is at the carboxyl terminal a domain that shows substantial homology, but not identity, to sequences of other protein kinase.

Tanaka Y, Miyake R, Kikkawa U, Nishizuka Y
Rapid assay of binding of tumor-promoting phorbol esters to protein kinase C1.
J Biochem (Tokyo). 1986; 99: 257-61
Display abstract
Protein kinase C is generally accepted to be a receptor protein of tumor-promoting phorbol esters. The binding of [3H]phorbol-12,13-dibutyrate to protein kinase C can be assayed by a rapid filtration procedure using a glass-fiber filter that has been treated with a cationic polymer, polyethylenimine. The phorbol ester specifically binds to the protein kinase only in the presence of phosphatidylserine and calcium. Non-specific binding is less than 10%, at most, of the total binding. The binding is linear with respect to the concentration of protein kinase C, is dependent on the concentrations of phorbol ester and phosphatidylserine in a saturative manner, and is inhibited by diacylglycerol (an endogenous activator of the protein kinase).

Nagao M, Ishikawa F, Tahira T, Ochiai M, Sugimura T
Activation of rat and human c-raf(-1) by rearrangement.
Princess Takamatsu Symp. 1986; 17: 75-84
Display abstract
Activated c-raf(-1) gene was found in three transformants obtained by transfecting DNAs from rat hepatocellular carcinoma, metastasis of human colon cancer in mesocolon and normal mucosa from a different colon cancer patient. Rat and human activated c-raf(-1) genes were cloned into cosmid vectors; restriction enzyme mapping revealed both activated c-raf(-1) genes to have rearrangement in the center of the normal form of the gene, and the upstream sequences were replaced by unrelated sequences. Using genomic DNA fragments located immediately downstream of the recombination points, the activations of all these c-raf(-1) were shown to have occurred during the transfection process. The recombination points in both the rat and human clones isolated were located in the intron between exons 7 and 8, and nucleotide sequencing around these recombination points showed there to be an inverted repeat which could be involved in inducing in vitro recombination. Nucleotide sequencing of rat and human c-raf(-1) cDNAs revealed the upstream sequences, recombined to the 3' half of c-raf(-1), to be expressed as fusion mRNAs; the production of fused proteins was predicted from a long open reading frame, which is in-frame with the kinase domain encoded from the 3' half of the c-raf(-1) gene. There is a cysteine clustering region in an N-terminal region of the c-raf(-1) product deduced from the nucleotide sequence, and this cysteine clustering region was found to be highly homologous to that present in an N-terminal region of protein kinase C, although, in the latter cysteine clusters are present in duplicate. From analogy with the activation mechanism of protein kinase C, the N-terminal region of serine/threonine kinase coded by the c-raf(-1) gene is suggested to be a regulatory part of the enzyme activity, and it proposed that the replacement or truncation of this regulatory part could be the mechanism whereby c-raf(-1) is activated.

Huang KP, Huang FL
Immunochemical characterization of rat brain protein kinase C.
J Biol Chem. 1986; 261: 14781-7
Display abstract
Polyclonal antibodies against rat brain protein kinase C (the Ca2+/phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca2+, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca2+/phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of [3H]phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. This degraded form of the phorbol ester-binding protein still requires phospholipid for activity but, unlike the native enzyme, becomes less dependent on Ca2+. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca2+-independent/phospholipid-dependent phorbol ester-binding protein.

Knopf JL et al.
Cloning and expression of multiple protein kinase C cDNAs.
Cell. 1986; 46: 491-502
Display abstract
Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.

Lee MH, Bell RM
The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity.
J Biol Chem. 1986; 261: 14867-70
Display abstract
Trypsinization of rat brain protein kinase C (80 kDa) into 50- and 32-kDa fragments occurred without inhibition of [3H]phorbol dibutyrate ([3H]PDBu) binding activity. The 50-kDa fragment, the catalytic domain (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616), was further degraded by trypsin, whereas the 32-kDa fragment was resistant. Protein kinase activity and the [3H]PDBu binding activity were completely separated upon gel filtration of a solution containing Triton X-100/phosphatidylserine mixed micelles and trypsinized protein kinase C. Pooled fractions of the [3H]PDBu binding activity contained a 32-kDa fragment exclusively. The binding of [3H]PDBu to this fragment was dependent on calcium and phosphatidylserine and was of high affinity (Kd = 2.8 nM) and of essentially identical specificity to that of native protein kinase C. It is concluded that the 32-kDa fragment represents a lipid binding, regulatory domain of protein kinase C.

Ono Y et al.
Cloning of rat brain protein kinase C complementary DNA.
FEBS Lett. 1986; 203: 111-5
Display abstract
Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Ishikawa F, Takaku F, Nagao M, Sugimura T
Cysteine-rich regions conserved in amino-terminal halves of raf gene family products and protein kinase C.
Jpn J Cancer Res. 1986; 77: 1183-7
Display abstract
The predicted amino acid sequences of rat c-raf and A-raf products were subjected to homology search against other known amino acid sequences. We found that characteristic, cysteine-rich regions present in the amino-terminal halves of c-raf and A-raf products had striking homologies with the cysteine-rich regions present in protein kinase C. These cysteine-rich regions had been shown to be deleted in the activated raf products and in the catalytically active protein kinase C produced by proteolysis. These facts suggest the significance of the cysteine-rich regions of raf products as well as those of protein kinase C in the regulation of kinase activities.

Sharkey NA, Blumberg PM
Kinetic evidence that 1,2-diolein inhibits phorbol ester binding to protein kinase C via a competitive mechanism.
Biochem Biophys Res Commun. 1985; 133: 1051-6
Display abstract
Diacylglycerols inhibit binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDBu) to protein kinase C (the phorbol ester receptor). This inhibition could reflect competitive binding by the diglyceride. Alternatively, it might simply represent perturbation of the lipid environment required for binding activity. As predicted for a competitive mechanism, we report here that inhibitory concentrations of the diglyceride 1,2-diolein do not affect the off-rate of [3H]PDBu from its receptor. This behavior contrasts with that of arachidonic acid, which appears to interact via a mixed mechanism.

Miyake R, Tanaka Y, Tsuda T, Kaibuchi K, Kikkawa U, Nishizuka Y
Activation of protein kinase C by non-phorbol tumor promoter, mezerein.
Biochem Biophys Res Commun. 1984; 121: 649-56
Display abstract
Mezerein, classified as a second-stage tumor promoter, has no diacylglycerol-like structure in its molecule, but can activate protein kinase C both in vitro and in vivo. This non-phorbol diterpene competitively inhibits the specific binding of a radioactive tumor-promoting phorbol ester to the enzyme. It is suggestive that tumor-promoting phorbol esters and mezerein cause analogous changes in the membrane to activate protein kinase C, and utilize this protein kinase as a common receptive protein for tumor promotion.