Secondary literature sources for DAGKc

The following references were automatically generated.

Zhou Y, Lau FW, Nauli S, Yang D, Bowie JU
Inactivation mechanism of the membrane protein diacylglycerol kinase in detergent solution.
Protein Sci. 2001; 10: 378-83
Display abstract
We have examined the irreversible inactivation mechanism of the membrane protein diacylglycerol kinase in the detergents n-octyl-beta-D-glucopyranoside (OG) at 55 degrees C and n-decyl-maltopyranoside (DM) at 80 degrees C. Under no inactivation conditions did we find any direct evidence for the chemical modifications that are commonly found in soluble proteins. Moreover, protein inactivated at 55 degrees C in OG could be reactivated by an unfolding and refolding protocol, suggesting that the protein is inactivated by a stable conformational change, not a covalent modification. We also found that the inactivation rate decreased with both increasing protein concentration and increasing thermodynamic stability, consistent with an inactivation pathway involving transient dissociation and/or unfolding of the protein. Our results suggest that the primary cause of diacylglycerol kinase inactivation is not low solubility, but poor intrinsic stability in the detergent environment.

Ohanian J, Ohanian V
Lipid second messenger regulation: the role of diacylglycerol kinases and their relevance to hypertension.
J Hum Hypertens. 2001; 15: 93-8
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Extracellular stimuli elicit cellular responses through generation of intracellular second messengers. The lipid second messenger diacylglycerol is produced following activation of the phosphoinositide signalling system. Diacylglycerol is the physiological activator of protein kinase C but also interacts indirectly with other signalling molecules such as small G proteins. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid so terminating signalling through diacylglycerol. However, phosphatidic acid itself has a lipid second messenger role, with targets distinct from those of its precursor diacylglycerol. Therefore, diacylglycerol kinases occupy a central position in signal transduction and regulation of their activity is crucial to cellular function. A family of nine mammalian diacylglycerol kinases have been identified. Their structural diversity and complex pattern of tissue expression suggests that they function in distinct cellular processes. In addition to the plasma membrane, diacylglycerol kinases are found at the nucleus and cytoskeleton and translocation between subcellular compartments occurs with agonist stimulation. In small arteries diacylglycerol kinase activity is increased by adrenergic stimulation implying a role in vascular smooth muscle responses. Due to their role as key regulators of protein kinase C activity diacylglycerol kinases may play a role in the cardiovascular changes that occur in hypertension and as such could represent novel therapeutic targets.

Rodriguez de Turco EB et al.
Diacylglycerol kinase epsilon regulates seizure susceptibility and long-term potentiation through arachidonoyl- inositol lipid signaling.
Proc Natl Acad Sci U S A. 2001; 98: 4740-5
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Arachidonoyldiacylglycerol (20:4-DAG) is a second messenger derived from phosphatidylinositol 4,5-bisphosphate and generated by stimulation of glutamate metabotropic receptors linked to G proteins and activation of phospholipase C. 20:4-DAG signaling is terminated by its phosphorylation to phosphatidic acid, catalyzed by diacylglycerol kinase (DGK). We have cloned the murine DGKepsilon gene that showed, when expressed in COS-7 cells, selectivity for 20:4-DAG. The significance of DGKepsilon in synaptic function was investigated in mice with targeted disruption of the DGKepsilon. DGKepsilon(-/-) mice showed a higher resistance to electroconvulsive shock with shorter tonic seizures and faster recovery than DGKepsilon(+/+) mice. The phosphatidylinositol 4,5-bisphosphate-signaling pathway in cerebral cortex was greatly affected, leading to lower accumulation of 20:4-DAG and free 20:4. Also, long-term potentiation was attenuated in perforant path-dentate granular cell synapses. We propose that DGKepsilon contributes to modulate neuronal signaling pathways linked to synaptic activity, neuronal plasticity, and epileptogenesis.

Takeda M et al.
Gene expression and in situ localization of diacylglycerol kinase isozymes in normal and infarcted rat hearts: effects of captopril treatment.
Circ Res. 2001; 89: 265-72
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Diacylglycerol (DG) kinase (DGK) terminates signaling from DG, which serves as an activator of protein kinase C (PKC), by converting DG to phosphatidic acid. DGK is thus regarded as an attenuator of the PKC activity. In rats, five DGK isozymes have been cloned, but little is known about their role in the heart. In this study, the spatiotemporal expression of DGK isozymes was investigated in rat hearts under a normal condition and after myocardial infarction (MI) by in situ hybridization histochemistry and immunohistochemistry. In normal left ventricular myocardium, DGKalpha, DGKepsilon, and DGKzeta mRNAs were expressed evenly throughout the myocardium, although the DGKalpha expression was very low. In infarcted hearts, the expression of DGKzeta was enhanced in the peripheral zone of the necrotic area and at the border zone 3 and 7 days after MI, and to a lesser extent in the middle layer of the granulation tissue 21 days after MI. The enhanced DGKzeta expression in the infarcted and border areas could be attributed to granulocytes and macrophages. In contrast, the expression of DGKepsilon in the infarcted and border areas was lower than that in the viable left ventricle (LV) throughout the postoperation period. Furthermore, DGKepsilon expression in the viable myocardium 21 days after MI decreased significantly compared with left ventricular myocardium in the sham-operated rats and was completely restored by treatment with captopril. Our results demonstrate that three DGK isozymes are expressed in the heart and that each isozyme might have different functional characteristics in the healing and LV remodeling after MI.

Wood NT
Production of a novel calmodulin-binding DGK by alternative splicing.
Trends Plant Sci. 2001; 6: 50-50

Topham MK, Prescott SM
Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.
J Cell Biol. 2001; 152: 1135-43
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Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

Bregoli L, Baldassare JJ, Raben DM
Nuclear diacylglycerol kinase-theta is activated in response to alpha-thrombin.
J Biol Chem. 2001; 276: 23288-95
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Currently, there is substantial evidence that nuclear lipid metabolism plays a critical role in a number of signal transduction cascades. Previous work from our laboratory showed that stimulation of quiescent fibroblasts with alpha-thrombin leads to the production of two lipid second messengers in the nucleus: an increase in nuclear diacylglycerol mass and an activation of phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid. Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid, making it an attractive candidate for a signal transduction component. There is substantial evidence that this activity is indeed regulated in a number of signaling cascades (reviewed by van Blitterswijk, W. J., and Houssa, B. (1999) Chem. Phys. Lipids 98, 95-108). In this report, we show that the addition of alpha-thrombin to quiescent IIC9 fibroblasts results in an increase in nuclear DGK activity. The examination of nuclei isolated from quiescent IIC9 cells indicates that DGK-theta and DGK-delta are both present. We took advantage of the previous observations that phosphatidylserine inhibits DGK-delta (reviewed by Sakane, F., Imai, S., Kai, M., Wada, I., and Kanoh, H. (1996) J. Biol. Chem. 271, 8394-8401), and constitutively active RhoA inhibits DGK-theta (reviewed by Houssa, B., de Widt, J., Kranenburg, O., Moolenaar, W. H., and van Blitterswijk, W. J. (1999) J. Biol. Chem. 274, 6820-6822) to identify the activity induced by alpha-thrombin. Constitutively active RhoA inhibited the nuclear stimulated activity, whereas phosphatidylserine did not have an inhibitory effect. In addition, a monoclonal anti-DGK-theta antibody inhibited the alpha-thrombin-stimulated nuclear activity in vitro. These results demonstrate that DGK-theta is the isoform responsive to alpha-thrombin stimulation. Western blot and immunofluorescence microscopy analyses showed that alpha-thrombin induced the translocation of DGK-theta to the nucleus, implicating that this translocation is at least partly responsible for the increased nuclear activity. Taken together, these data are the first to demonstrate an agonist-induced activity of nuclear DGK-theta activity and a nuclear localization of DGK-delta.

Pilot JD, East JM, Lee AG
Effects of Bilayer Thickness on the Activity of Diacylglycerol Kinase of Escherichia coli.
Biochemistry. 2001; 40: 8188-95
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We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate. When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine [di(C18:1)PC] and a lower activity in bilayers with shorter or longer fatty acyl chains. Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine [di(C14:1)PC] followed from an increase in the K(m) value for DHG and ATP, with no effect on v(max). In contrast, in the long chain lipid dierucoylphosphatidylcholine [di(C24:1)PC], the low activity followed from a decrease in v(max) with no effect on K(m). In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.

Lykidis A, Wang J, Karim MA, Jackowski S
Overexpression of a mammalian ethanolamine-specific kinase accelerates the CDP-ethanolamine pathway.
J Biol Chem. 2001; 276: 2174-9
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Ethanolamine kinase (EKI) is the first committed step in phosphatidylethanolamine (PtdEtn) biosynthesis via the CDP-ethanolamine pathway. We identify a human cDNA encoding an ethanolamine-specific kinase EKI1 and the structure of the EKI1 gene located on chromosome 12. EKI1 overexpression in COS-7 cells results in a 170-fold increase in ethanolamine kinase-specific activity and accelerates the rate of [3H]ethanolamine incorporation into PtdEtn as a function of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to glycerophosphoethanolamine. EKI1 has negligible choline kinase activity in vitro and does not influence phosphatidylcholine biosynthesis. Acceleration of the CDP-ethanolamine pathway also does not change the rate of PtdEtn formation via the decarboxylation of phosphatidylserine. The data demonstrate the existence of separate ethanolamine and choline kinases in mammals and show that ethanolamine kinase can be a rate-controlling step in PtdEtn biosynthesis.

Hogan A et al.
Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions.
J Biol Chem. 2001; 276: 26526-33
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Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.

Thirugnanam S, Topham MK, Epand RM
Physiological implications of the contrasting modulation of the activities of the epsilon- and zeta-isoforms of diacylglycerol kinase.
Biochemistry. 2001; 40: 10607-13
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We have shown that the requirement of the epsilon-isoform of diacylglycerol kinase for diacylglycerols containing arachidonic acid is specific for this substrate and cannot be replaced by the presence of an arachidonoyl group in other places in the membrane; rather, it has to be present on the substrate itself. In addition, we demonstrate that the increased activity shown toward 1-stearoyl-2-arachidonoylglycerol by the epsilon-isoform of diacylglycerol kinase is not a consequence of altered membrane physical properties but is rather a specific interaction with the arachidonoyl group. We have also compared the modulation of the activity of the epsilon-isoform of diacylglycerol kinase with that of the zeta-isoform with regard to some of the intermediates involved in phosphatidylinositol cycling. One of the products of the hydrolysis of phosphatidylinositol diphosphate is diacylglycerol enriched in arachidonic acid. The activity of the epsilon-isoform is known to be specific for this form of diacylglycerol. We show that in contrast, the activity of the zeta-isoform is lower against 1-stearoyl-2-arachidonoylglycerol compared with dioleoylglycerol. We demonstrate that addition of phosphatidylserine, as well as other anionic phospholipids including L-alpha-phosphatidylinositol 4,5-bisphosphate, strongly inhibits the epsilon-isoform, but these anionic lipids increase the activity of the zeta-isoform. Addition of Ca(2+), which is released from internal stores as a consequence of phosphatidylinositol cycling, promotes the activity of the epsilon-isoform of this enzyme but has little effect on the zeta-isoform. The contrasting conditions required for maximal activity of these two isoforms of diacylglycerol kinase, as well as their different substrate specificity, suggest that they have different physiological roles in signal transduction.

Frolov MV, Benevolenskaya EV, Birchler JA
Molecular analysis of a novel Drosophila diacylglycerol kinase, DGKepsilon.
Biochim Biophys Acta. 2001; 1538: 339-52
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Diacylglycerol kinase plays a central role in the metabolism of diacylglycerol by converting diacylglycerol into phosphatidic acid thus initiating resynthesis of phosphatidylinositols. Diacylglycerol is a known second messenger reversibly activating protein kinase C. In addition, diacylglycerol is a potential precursor for polyunsaturated fatty acids. We describe the identification and molecular analysis of a novel type III Drosophila diacylglycerol kinase isoform, DGKepsilon. Drosophila DGKepsilon is mapped to the cytological position 49C1-3. DGKepsilon mRNA is 1.9 kb in length and is broadly distributed throughout development in different cells, primordia and organs, including testes. In embryogenesis, the transcripts are enriched in the cells, which are in S-phase or undergoing endoreplication. Comparison of the Drosophila DGKepsilon with the human homologue revealed that the first zinc finger-like motif is specific for the type III isoform. Although the testis-specific diacylglycerol kinase activity is dependent upon the dose of DGKepsilon gene, the deletion of DGKepsilon does not modulate the total cellular diacylglycerol level. In spite of a proposed key role of diacylglycerol kinase in termination of the diacylglycerol signal, overexpression of a DGKepsilon transgene in flies under the control of a yeast upstream activating sequence promoter does not disrupt normal development in Drosophila.

Mansson E, Jonsson K, Albertioni F
Identification and characterization of human deoxyguanosine kinase cDNA fragments.
Cancer Lett. 2001; 170: 147-52
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Mitochondria require deoxyribonucleoside triphosphates for the synthesis of their DNA and one of the enzymes responsible for the initial phosphorylation of purine deoxyribonucleoside is deoxyguanosine kinase (dGK; EC 2.7.1.113). Recent studies have suggested that dGK in addition to deoxycytidine kinase phosphorylates several anti-cancer agents, such as 9-beta-D-arabinofuranosylguanine (Ara-G), cladribine (CdA), and fludarabine. There appear to coexist several mRNA fragments of dGK. In the present study we found 10 fragments, the longest fragment had 834bp, and represented the entire open reading frame of dGK (780bp). The nine additional fragments detected ranged from 807 to 269bp. All the fragments were found to contain the specific mitochondria translocation signal sequence. Expression of these fragments in Escherichia coli demonstrated that only the full-length dGK resulted in a protein that could phosphorylate CdA and Ara-G. Given the difficulty to measure the full-length dGK, these data are of value for studying the mRNA gene expression of dGK in cell lines and in leukemic cells from patients.

Hinderlich S, Berger M, Schwarzkopf M, Effertz K, Reutter W
Molecular cloning and characterization of murine and human N-acetylglucosamine kinase.
Eur J Biochem. 2000; 267: 3301-8
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N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases. It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase. In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme. An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa. The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase. The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity. The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags. An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa. Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level. Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present. Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis. The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development. mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines. In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size.

Snedden WA, Blumwald E
Alternative splicing of a novel diacylglycerol kinase in tomato leads to a calmodulin-binding isoform.
Plant J. 2000; 24: 317-26
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Calmodulin is a regulatory protein activated during Ca2+ signalling. We have isolated a cDNA, designated LeCBDGK (Lycopersicon esculentum calmodulin-binding diacylglycerol kinase) encoding a novel calmodulin-binding protein with sequence similarity to diacylglycerol kinases from animals. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid. We delineated the calmodulin-binding domain to approximately 25 residues near the C-terminus of LeCBDGK. We have also isolated a second diacylglycerol kinase cDNA, designated LeDGK1, identical to LeCBDGK, except that it lacks the calmodulin-binding domain. Both recombinant LeCBDGK and LeDGK1 were catalytically active in vitro. Anti-DGK antiserum detected two immunoreactive proteins associated with microsomal and plasma membrane fractions from cell suspensions. The higher molecular weight immunoreactive protein was also present in soluble extracts and bound to calmodulin-agarose in the presence of calcium, demonstrating that native LeCBDGK is a calmodulin-binding protein. In the presence of calcium, LeCBDGK associated with membrane cell fractions in vitro, but calmodulin antagonists disrupted this association, suggesting a possible role of calcium in the recruitment of LeCBDGK from soluble to membrane cell fractions. Native LeCBDGK and calmodulin co-immunoprecipitated from tomato soluble cell extracts, suggesting their interaction in vivo. The same gene encodes both LeCBDGK and LeDGK1 and the calmodulin-binding domain of LeCBDGK is encoded by a separate exon. Thus, alternative transcript splicing leads to calmodulin-binding and non-binding forms of diacylglycerol kinases in tomato. Possible roles of LeCBDGK and LeDGK1 in calcium and lipid signalling are discussed.

Parekh DB, Ziegler W, Parker PJ
Multiple pathways control protein kinase C phosphorylation.
EMBO J. 2000; 19: 496-503

Balasubramanyam M, Mohan V
Signal transduction during cardiac hypertrophy: new insights.
Indian Heart J. 2000; 52: 226-32

van Blitterswijk WJ, Houssa B
Properties and functions of diacylglycerol kinases.
Cell Signal. 2000; 12: 595-605
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Diacylglycerol kinases (DGKs) phosphorylate the second-messenger diacylglycerol (DAG) to phosphatidic acid (PA). The family of DGKs is well conserved among most species. Nine mammalian isotypes have been identified, and are classified into five subgroups based on their primary structure. DGKs contain a conserved catalytic domain and an array of other conserved motifs that are likely to play a role in lipid-protein and protein-protein interactions in various signalling pathways dependent on DAG and/or PA production. DGK is therefore believed to be activated at the (plasma) membrane where DAG is generated. Some isotypes are found associated with and/or regulated by small GTPases of the Rho family, presumably acting in cytoskeletal rearrangements. Others are (also) found in the nucleus, in association with other regulatory enzymes of the phosphoinositide cycle, and have an effect on cell cycle progression. Most DGK isotypes show high expression in the brain, often in distinct brain regions, suggesting that each individual isotype has a unique function.

Shirai Y, Segawa S, Kuriyama M, Goto K, Sakai N, Saito N
Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C.
J Biol Chem. 2000; 275: 24760-6
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We examined the translocation of diacylglycerol kinase (DGK) alpha and gamma fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKalpha and gamma were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKgamma, but not DGKalpha, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKgamma was inhibited by the mutation of C1A but not C1B domain of DGKgamma and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKgamma from the cytoplasm to the plasma membrane, whereas DGKalpha showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKgamma and alpha to the plasma membrane. The timing of the ATP-induced translocation of DGKgamma roughly coincided with that of PKCgamma re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCgamma was obviously hastened by co-expression with DGKgamma and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.

Jiang Y, Qian W, Hawes JW, Walsh JP
A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha.
J Biol Chem. 2000; 275: 34092-9
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Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.

Kitada T, Asakawa S, Minoshima S, Mizuno Y, Shimizu N
Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene.
Mamm Genome. 2000; 11: 417-21
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We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3'-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals.

Nava VE et al.
Functional characterization of human sphingosine kinase-1.
FEBS Lett. 2000; 473: 81-4
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Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N, N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.

Endo H, Persson P, Watanabe T
Molecular cloning of the crustacean DD4 cDNA encoding a Ca(2+)-binding protein.
Biochem Biophys Res Commun. 2000; 276: 286-91
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A cDNA, named DD4, was identified in the prawn Penaeus japonicus in a search for genes that were expressed during calcification of the crustacean exoskeleton. DD4 transcripts were detected in the epidermal cells underlying the exoskeleton specifically during the postmolt stage, when the calcification takes place. In the DD4 cDNA an open reading frame of 542 amino acids was found. The deduced protein was acidic and proline-rich, and exhibited similarity to the Drosophila Ca(2+)-binding protein calphotin in the amino acid sequence and composition. The DD4 cDNA was expressed in Escherichia coli to characterize Ca(2+)-binding of the encoded protein, and Ca(2+) was found to bind to a central segment of 186 amino acids. The DD4 protein is suggested to play a role in the calcification of the crustacean exoskeleton.

Park KW et al.
Molecular cloning and characterization of a protein tyrosine phosphatase enriched in testis, a putative murine homologue of human PTPMEG.
Gene. 2000; 257: 45-55
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Protein tyrosine phosphorylation is regulated by protein tyrosine kinase and protein tyrosine phosphatase activities. These two counteracting proteins are implicated in cell growth and transformation. Using polymerase chain reaction with degenerate primers, we have identified a novel mouse protein tyrosine phosphatase (PTP). This cDNA contains a single open reading frame of the predicted 926 amino acids. Those predicted amino acids showed significant identity with human megakaryocyte protein-tyrosine phosphatase by 91% in nucleotide sequences and 94% in amino acid sequences. We have identified that expression of this PTP is highly enriched in the testis in mouse and human and has been termed here as a 'testis-enriched phosphatase' (TEP). Northern analysis detected two mRNA species of 3.7 and 3.2kb for this PTP in mouse testis and the expression of TEP is regulated during development. The recombinant phosphatase domain possesses protein tyrosine phosphatase activity when expressed in Escherichia coli. Immunohistochemical analysis of the cellular localization of TEP on mouse testis sections showed that this PTP is specifically expressed in spermatocytes and spermatids within seminiferous tubules, suggesting an important role in spermatogenesis.

Melendez AJ, Carlos-Dias E, Gosink M, Allen JM, Takacs L
Human sphingosine kinase: molecular cloning, functional characterization and tissue distribution.
Gene. 2000; 251: 19-26
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Sphingosine-1-phosphate (SPP), the product of sphingosine kinase, is an important signaling molecule with intra- and extracellular functions. The cDNA for the mouse sphingosine kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human sphingosine kinase (huSPHK1). Sequence analysis comparison revealed that this kinase is evolutionarily very conserved, having a high degree of homology with the murine enzyme, and presenting several conserved regions with bacteria, yeast, plant, and mammalian proteins. Expressed huSPHK1 cDNA specifically phosphorylates D-erythro-sphingosine and, to a lesser extent, D, L-erythro-dihydrosphingosine, and not at all the 'threo' isoforms of dihydrosphingosine; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-phosphate (PIP); or phosphatidylinositol-4, 5-bisphosphate (PIP(2)). huSPHK1 shows typical Michaelis-Menten kinetics (V(max)=56microM and K(m)=5microM). The kinase is inhibited by D,L-threo-dihydrosphingosine (K(i)=3microM), and by N, N-dimethyl-sphingosine (K(i)=5microM). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respectively. It is also expressed in brain and heart. In addition, database searches with the stSG2854 sequence indicate that huSPHK1 is also expressed in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

Petrakis TG, Ktistaki E, Wang L, Eriksson S, Talianidis I
Cloning and characterization of mouse deoxyguanosine kinase. Evidence for a cytoplasmic isoform.
J Biol Chem. 1999; 274: 24726-30
Display abstract
Deoxyguanosine kinase (dGK) is a nuclear gene product that catalyzes the phosphorylation of purine deoxyribonucleosides and their analogues. The human enzyme is located predominantly in the mitochondria, as shown by biochemical fractionation studies and in situ localization of the overexpressed recombinant protein. Here we describe the cloning of mouse dGK cDNA and the identification of a novel amino-terminally truncated isoform that corresponds to about 14% of the total dGK mRNA population in mouse spleen. In situ fluorescence assays suggest that the new isoform cannot translocate into the mitochondria and thus may represent a cytoplasmic enzyme. Expression of mouse dGK mRNA was highly tissue-specific and differed from the tissue distribution observed in humans. Recombinant mouse dGK showed similar specific activity and substrate specificity as compared with the human enzyme. The broad specificity, restricted tissue distribution, and location of mouse dGK in multiple cellular compartments raise new considerations with respect to the role of the individual deoxynucleoside kinases in nucleotide metabolism.

Pettitt TR, Wakelam MJ
Diacylglycerol kinase epsilon, but not zeta, selectively removes polyunsaturated diacylglycerol, inducing altered protein kinase C distribution in vivo.
J Biol Chem. 1999; 274: 36181-6
Display abstract
Porcine aortic endothelial cells have previously been shown to contain particularly high basal levels of polyunsaturated diacylglycerol (DAG) together with a very high degree of membrane-associated protein kinase C (PKC), which is largely insensitive to further activation (Pettitt, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., and Wakelam, M. J. O. (1997) J. Biol. Chem. 272, 17354-17359). To investigate the possibility that the high polyunsaturated DAG levels were constitutively activating PKC, we transfected porcine aortic endothelial cells with two different forms of human diacylglycerol kinase, epsilon and zeta. In vitro, the former is specific for polyunsaturated structures, whereas the latter shows no apparent selectivity. Overexpression of DAGKepsilon specifically reduced the level of polyunsaturated DAG in the transfected cells while having little effect on the more saturated structures. It also caused the redistribution of PKCalpha and epsilon from the membrane to the cytosol. Overexpression of DAGKzeta caused a general reduction in DAG levels but had little effect on PKC distribution. These results for the first time show that DAGKepsilon specifically phosphorylates polyunsaturated DAG in vivo and that in so doing it regulates PKC localization and activity. This provides support for the proposal that it is the polyunsaturated DAGs that function as messengers and convincing evidence for DAGKepsilon being a physiological terminator of DAG second messenger signaling.

Topham MK, Prescott SM
Mammalian diacylglycerol kinases, a family of lipid kinases with signaling functions.
J Biol Chem. 1999; 274: 11447-50

Meton I, Caseras A, Mediavilla D, Fernandez F, Baanante IV
Molecular cloning of a cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from liver of Sparus aurata: nutritional regulation of enzyme expression.
Biochim Biophys Acta. 1999; 1444: 153-65
Display abstract
A cDNA clone encoding full-length 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) was isolated and sequenced from a Sparus aurata liver cDNA library. The 2527 bp nucleotide sequence of the cDNA contains a 73 bp 5'-untranslated region (5'-UTR), an open reading frame that encodes a 469 amino acid protein and 1041 bp at the 3'-UTR. The deduced amino acid sequence is the first inferred 6PF-2-K/Fru-2, 6-P2ase in fish. The kinase and bisphosphatase domains, where the residues described as crucial for the mechanism of reaction of the bifunctional enzyme are located, present a high degree of homology with other liver isoenzymes. However, within the first 30 amino acids at the N-terminal regulatory domain of the fish enzyme a low homology is found. Nutritional regulation of the 6-phosphofructo-2-kinase activity, together with immunodetectable protein and mRNA levels of 6PF-2-K/Fru-2,6-P2ase, was observed after starvation and refeeding. In contrast to results previously described for rat liver, the decrease in immunodetectable protein and kinase activity caused by starvation was associated in the teleostean fish to a decrease in mRNA levels.

Lucka L, Krause M, Danker K, Reutter W, Horstkorte R
Primary structure and expression analysis of human UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, the bifunctional enzyme in neuraminic acid biosynthesis.
FEBS Lett. 1999; 454: 341-4
Display abstract
N-Acetylneuraminic acid is a main constituent of glycoproteins and gangliosides. In many membrane-bound receptors it is the target for external stimuli. The key enzyme for its biosynthesis is the bifunctional enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, catalysing the first two steps of the biosynthesis in the cytosol. The rat enzyme was previously isolated and characterised. In this report we present the corresponding human cDNA sequence, compare it with the primary structure of the rodent enzyme, and report the analysis of its expression in different human tissues and cell lines.

Goto K, Kondo H
[Diacylglycerol kinase: molecular diversity and gene expression in central nervous system]
Tanpakushitsu Kakusan Koso. 1999; 44: 976-82

Lee WK et al.
Molecular cloning and expression analysis of a mouse phospholipase C-delta1.
Biochem Biophys Res Commun. 1999; 261: 393-9
Display abstract
We describe here the molecular cloning and expression analysis of mouse PLC-delta1 (mPLC-delta1), a key enzyme in cell signal transduction. A mouse brain cDNA library was screened in order to isolate the mPLC-delta1 cDNA. The mPLC-delta1 cDNA was 2660 bp in length. The predicted open reading frame encodes a protein of 756 amino acids with an estimated molecular mass of 85 kDa. The deduced amino acid sequence exhibits 96.9% and 92.7% identity with the sequence of rat and human PLC-delta1, respectively. The mPLC-delta1 mRNA was highly expressed in brain, heart, lung, and testis. We found that transcripts of mPLC-delta1 are present in almost all regions of mouse brain examined, implying that the enzyme may play a role in some fundamental cellular process in brain. In male reproductive tract, mPLC-delta1 mRNA was widely expressed in the epididymis as well as in the testis. In situ hybridization studies indicate that distribution of mPLC-delta1 mRNA in mouse testis is discrete and unique. The expression of mPLC-delta1 mRNA was defined in the periphery of each seminiferous tubule, especially in spermatogonia, which might imply that mPLC-delta1 plays a role in proliferation of spermatogonia. To the best our knowledge, this is the first report to demonstrate the high expression of mPLC-delta1 mRNA in spermatogonia of testis. Taken together, these results suggest that mPLC-delta1 may carry out fundamental roles in almost all of mouse tissues, especially in brain and specific roles in testis.

Parker PJ
Inhibition of protein kinase C--do we, can we, and should we?
Pharmacol Ther. 1999; 82: 263-7
Display abstract
The second messenger responsive protein kinase C (PKC) family has promised much as therapeutic targets. Indeed, there has been excellent progress in the development of highly selective inhibitors for this class of kinases. The key to the further development and use of inhibitors selective within the family depends upon our understanding of the involvement of PKC in human disease. Recent work in model systems ex vivo has modified the simplistic view of PKC control and broadened the spectrum of signalling pathway involvement. The consequence is that there is a need to reassess the role of PKC that to date has coloured our view of PKC and disease. These developments, their implications, and the future assessment of PKC as a target are discussed.

Saito A, Ozaki K, Fujiwara T, Nakamura Y, Tanigami A
Isolation and mapping of a human lung-specific gene, TSA1902, encoding a novel chitinase family member.
Gene. 1999; 239: 325-31
Display abstract
Using differential display technique, we have isolated a novel human gene expressed specifically in the lung. Two forms of the gene, designated TSA1902, were transcribed by alternate mRNA splicing. The transcribed mRNAs, termed TSA1902-L and TSA1902-S, putatively encode proteins of 368 and 315 amino acids, respectively, which show high similarity to human chitotriosidase protein. The N-terminal region of TSA1902-L protein contains the conserved active site residues (DXXDXDXE) of the catalytic center of various chitinases which are essential for chitinase activity. The deduced protein sequence of TSA1902-S, however, does not possess this active site, with the N-terminal 54 amino acids present in TSA1902-L protein having been deleted. Both proteins lacked the secretory sequence of N-termini and, judging from the hydropathy profile, may be soluble proteins in the cytoplasm. Chromosomal mapping by radiation hybrid analysis localized this gene to the chromosome 1p13.1-p21.3.

van Blitterswijk WJ, Houssa B
Diacylglycerol kinases in signal transduction.
Chem Phys Lipids. 1999; 98: 95-108
Display abstract
Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. A family of nine mammalian isotypes have been identified. Their primary structure shows a diverse array of conserved domains, such as a catalytic domain, zinc fingers, pleckstrin homology domains and EF-hand structures, known to interact with other proteins, lipids or Ca2+, in signal transduction processes. DGK is believed to act in the phosphoinositide cycle in which DAG is enriched with arachidonoyl moieties, but the majority of DGK isotypes do not show specificity for this DAG species in vitro. This could imply that DGKs may also have other functions in the cell. DGK activity is not only found in membranes, but also in the nucleus and at the cytoskeleton. Agonist-induced translocations of DGK to or from these subcellular sites are known to occur. Some isotypes are contained in signaling complexes in specific association with members of the Rho family of small GTP binding proteins, suggesting that they are involved in Rho-mediated processes such as cytoskeletal reorganization.

Goto K, Kondo H
Diacylglycerol kinase in the central nervous system--molecular heterogeneity and gene expression.
Chem Phys Lipids. 1999; 98: 109-17
Display abstract
Diacylglycerol (DAG) is one of the important second messengers, which serves as an activator of protein kinase C (PKC). DAG kinase (DGK) phosphorylates DAG to generate phosphatidic acid, thus DGK is considered to be a regulator of PKC activity through attenuation of DAG. Recent studies have revealed molecular structures of several DGK isozymes from mammalian species, and showed that most of the isozymes are expressed in the brain in various amounts. We have cloned four DGK isozyme cDNAs from rat brain library (DGK alpha, -beta, -gamma, and -zeta) (previously also designated DGK-I, -II, -III, and -IV, respectively) and examined their mRNA expressions in rat brain by in situ hybridization histochemistry. Interestingly, it is revealed that the mRNA for each isozyme is expressed in a distinct pattern in the brain; DGK alpha is expressed in oligodendrocytes, glial cells that form myelin; DGK beta in neurons of the caudate-putamen; DGK gamma predominantly in the cerebellar Purkinje cells; and DGK zeta in the cerebellar and cerebral cortices. Molecular diversity and distinct expression patterns of DGK isozymes suggest a physiological importance for the enzyme in brain function. Furthermore, functional implications of these DGK isozymes are briefly discussed.

Tamari M, Daigo Y, Ishikawa S, Nakamura Y
Genomic structure of a novel human gene (XYLB) on chromosome 3p22-->p21.3 encoding a xylulokinase-like protein.
Cytogenet Cell Genet. 1998; 82: 101-4
Display abstract
We isolated a novel human cDNA of 1963 nucleotides that included an open reading frame encoding a protein of 528 amino acids. Homology analysis indicated that the predicted gene product, XYLB, bore 22% identity to the xylulokinase of Haemophilus influenzae that plays an important role in energy metabolism. The gene consists of 18 exons and spans about 28 kb of genomic DNA on chromosome 3p21.3.

Mohapatra B, Verma S, Shankar S, Suri A
Molecular cloning of human testis mRNA specifically expressed in haploid germ cells, having structural homology with the A-kinase anchoring proteins.
Biochem Biophys Res Commun. 1998; 244: 540-5
Display abstract
We have cloned a human testis specific gene (hi), which encodes for a protein having regional homologies to the domain of A-kinase anchoring proteins. Open reading frame encodes a protein of 860 amino acids. The predicted protein has a molecular weight 95.8 kD and contains 32 cysteine residues and 34 potential phosphorylation sites. The deduced protein analysis revealed an eukaryotic secretory signal with a potential cleavage site. Northern blot analysis detected a single transcript of approximately 3.0 kb in testis and not in any other somatic tissues. Expression of hi transcript was observed only in round spermatids indicating post meiotic haploid gene expression.

Zaucha JA, Westphal RS, Wadzinski BE
protein phosphatase 2A regulatory subunits. cDNA cloning and analysis of mRNA expression.
Methods Mol Biol. 1998; 93: 279-91

Yanagisawa K et al.
cDNA cloning, expression, and characterization of the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase enzyme.
Biosci Biotechnol Biochem. 1998; 62: 1037-40
Display abstract
A cDNA encoding the human bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase was cloned and sequenced. The enzyme contains an APS kinase domain in its N-terminal portion and an ATP sulfurylase domain in its C-terminal portion. Recombinant full-length enzyme and its constituent APS kinase and ATP sulfurylase domains were individually expressed, purified, and shown to have their respective enzymatic activities.

Ding L, McIntyre TM, Zimmerman GA, Prescott SM
The cloning and developmental regulation of murine diacylglycerol kinase zeta.
FEBS Lett. 1998; 429: 109-14
Display abstract
Diacylglycerol kinases (DGKs) regulate the key signaling intermediates diacylglycerol (DAG) and phosphatidic acid (PA). We isolated cDNA clones of mouse diacylglycerol kinase zeta (mDGKzeta) and found that it shares 88% identity at the nucleic acid level and 95.5% identity at the amino acid level with human DGKzeta (hDGKzeta). Murine DGKzeta protein rose gradually during embryonic development, and was abundant in newborn and adult brains. By RNA whole-mount in situ hybridization, mDGKzeta was shown to be expressed in spinal ganglia and limb buds at low level in E11.5 embryos and at higher level in E12.5 embryos. In E13.5 embryos, DGKzeta mRNA was highly expressed in vibrissa follicles, in spinal ganglia, and in the interdigital regions of the developing limbs. Northern blotting showed that DGKzeta expression was limited to specific anatomical regions of the brain. Thus, the expression of DGKzeta is regulated temporally and spatially during mammalian development and correlates with the development of sensory neurons and regions undergoing apoptosis.

Mochly-Rosen D, Kauvar LM
Modulating protein kinase C signal transduction.
Adv Pharmacol. 1998; 44: 91-145

Kohama T, Olivera A, Edsall L, Nagiec MM, Dickson R, Spiegel S
Molecular cloning and functional characterization of murine sphingosine kinase.
J Biol Chem. 1998; 273: 23722-8
Display abstract
Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

Miranda-Vizuete A, Gustafsson JA, Spyrou G
Molecular cloning and expression of a cDNA encoding a human thioredoxin-like protein.
Biochem Biophys Res Commun. 1998; 243: 284-8
Display abstract
This report describes the cloning of a human cDNA that encodes a new protein (Txl, Thioredoxin-like) that belongs to the expanding family of thioredoxins based on sequence comparison of the deduced amino acid sequence. This cDNA, with a total length of 1,278 bp, consists of 205 bp of 5'-untranslated sequence (including an in frame stop codon), an open reading frame of 870 bp and a 203 bp fragment of 3'-untranslated sequence. The coding sequence predicts a protein of 289 amino acids with two distinct domains: an N-terminal domain of 105 residues homologous to the rest of mammalian thioredoxins containing the conserved active site (CGPC) and a C-terminal domain of 184 residues with no homology with any other protein in the database. Northern blot analysis indicates that the txl probe hybridizes to a 1.3 Kb mRNA and is ubiquitously expressed in human tissues with the highest expression in stomach, testis and bone marrow.

Ding L, Traer E, McIntyre TM, Zimmerman GA, Prescott SM
The cloning and characterization of a novel human diacylglycerol kinase, DGKiota.
J Biol Chem. 1998; 273: 32746-52
Display abstract
Diacylglycerol (DAG) plays a central role in both the synthesis of complex lipids and in intracellular signaling; diacylglycerol kinase (DGK) catalyzes the phosphorylation of DAG, which yields phosphatidic acid. A family of DGKs has been identified in multicellular organisms over the past few years, but the physiological function(s) of this diversity is not clear. One clue has come from the Drosophila DGK2, rdgA, since mutations in this gene cause retinal degeneration. We isolated a novel DGK, which we designated DGKiota, from human retina and brain libraries. DGKiota contains two cysteine-rich repeats, a region similar to the phosphorylation site domain of myristoylated alanine-rich C kinase substrate, a conserved catalytic domain, and four ankyrin repeats at its C terminus. By primary structure, it is most similar to human DGKzeta and Drosophila rdgA. An >12-kilobase mRNA for DGKiota was detected only in brain and retina among the tissues examined. In cells transfected with the DGKiota cDNA, we detected an approximately 130-kDa protein by immunoassay, and activity assays demonstrated that it encodes a functional DAG kinase. The protein was found to be in both the cytoplasm and nucleus with the localization controlled by PKC isoforms alpha and gamma. The gene encoding DGKiota was localized to human chromosome 7q32.3-33, which is known to be a locus for an inherited form of retinitis pigmentosa. These results have defined a novel isoform of DAG kinase, which may have important cellular functions in the retina and brain.

Sofer L, Kampa D, Burnside J
Molecular cloning of a chicken JAK homolog from activated T cells.
Gene. 1998; 215: 29-36
Display abstract
A cDNA encoding a JAK-related protein was isolated from a chicken Tcell library by screening with a PCR-generated probe that corresponds to a conserved region in the kinase domain. Sequence analysis reveals an ORF of 3318nt, encoding a protein with a calculated molecular weight of 123000. Chicken JAK (cJAK) contains a double catalytic domain that is characteristic of the JAK family of tyrosine kinases. Compared with mammalian JAKs, the kinase domain shows 70% sequence identity with the corresponding region of the mammalian JAKs. Overall, cJAK shows approximately 59% amino acid identity with mammalian JAK3s, and 52% amino acid identity with mammalian JAK2s. cJAK is expressed predominantly in thymus and spleen, with lower levels in kidney, thyroid and liver. cJAK is also expressed at low levels in unstimulated splenic Tcells, whereas mRNA levels are increased after activation of the Tcells with Con A. The sequence analysis and pattern of expression suggests that this is an avian homolog of JAK3.

Aoyama C, Nakashima K, Matsui M, Ishidate K
Complementary DNA sequence for a 42 kDa rat kidney choline/ethanolamine kinase.
Biochim Biophys Acta. 1998; 1390: 1-7
Display abstract
By means of peptide sequence information, several cDNA clones encoding a 42 kDa choline/ethanolamine kinase were isolated from a rat kidney cDNA library. Eight clones were sequenced with all of them resulting in identical overlapping nucleotide sequences. Four of them possessed entire open reading frame which could encode 394 amino acids with a calculated molecular size of 45 100. The predicted amino acid sequence contained all of the internal peptide fragment sequences derived from the purified 42 kDa enzyme. When the open reading frame was introduced into pGEX-2T vector and transfected into E. coli cells, a significant choline/ethanolamine kinase activity did appear in the cell lysate. A homology comparison with the previously reported choline kinase cDNAs (CKR1 and CKR2) from rat liver showed 66%-68% in entire nucleotide sequences and 57%-59% in amino acid sequences, indicating that the cloned cDNA here must be a novel CK/EK gene product. (c) 1998 Elsevier Science B. V.

Aoyama C, Nakashima K, Ishidate K
Molecular cloning of mouse choline kinase and choline/ethanolamine kinase: their sequence comparison to the respective rat homologs.
Biochim Biophys Acta. 1998; 1393: 179-85
Display abstract
Complementary DNAs homologous to a rat 42-kDa choline/ethanolamine kinase [C. Aoyama et al., Biochim. Biophys. Acta 1390 (1998) 1-7] and to a 50-kDa choline kinase [T. Uchida and S. Yamashita, J. Biol. Chem. 267 (1992) 10156-10162] were isolated from a 17-day post coitum mouse embryo cDNA library and their sequences were compared with the two murine species, respectively. The nucleotide sequence homology (within the coding sequence) between mouse and rat 50-kDa choline kinases (96.0%) was considerably higher than that between their 42-kDa choline/ethanolamine kinases (92.4%). Northern blot and RT-PCR studies on several rat tissues demonstrated that both isozymes are expressed ubiquitously with the highest level in testis.

Huang AM, Chang TJ, Cho WL, Chou CK
From mosquito to man: identification of a novel protein kinase, HsHPK, which is highly expressed in human hepatoma tissues.
J Biomed Sci. 1998; 5: 135-40
Display abstract
Protein kinases play an important role in the signaling pathway of growth factors in most of the higher organisms. During the study of protein kinase profiles of mosquitoes using RT-PCR and degenerate primers for consensus catalytic domain motifs to amplify protein kinase genes, we have noticed that a novel mosquito kinase, AaPK-38, shares a stretch of amino acids identical to the corresponding domain in Tousled gene of Arabidopsis thaliana that is required for leaf and flower development. A 2.1-kb cDNA encoding human HsHPK gene, which is a homolog of AaPK-38, was isolated from human testis cDNA library. This cDNA contains an open reading frame of 563 amino acids, with a complete kinase domain in its carboxyl terminus. The expressed Flag-tagged HsHPK was shown to have kinase activity based on in vitro autophosphorylation. Northern blot analysis revealed that human HsHPK mRNA is most abundant in testes, much less in heart and skeletal muscle and almost undetectable in liver and lung. Finally, we found that the expression of HsHPK in 4 out of 6 human hepatoma tissues is much higher than that in the adjacent normal counterpart. This result suggests HsHPK may play a role in the development of human hepatoma.

Divecha N
Signal transduction. Marked for nuclear export?
Nature. 1998; 394: 619-20

Ishihara H et al.
Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family.
J Biol Chem. 1998; 273: 8741-8
Display abstract
Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.

Johansson M, Karlsson A
Cloning of the cDNA and chromosome localization of the gene for human thymidine kinase 2.
J Biol Chem. 1997; 272: 8454-8
Display abstract
Human thymidine kinase 2 (TK2) is a deoxyribonucleoside kinase that phosphorylates thymidine, deoxycytidine, and deoxyuridine. The enzyme also phosphorylates anti-viral and anti-cancer nucleoside analogs. We have identified an expressed sequence tag cDNA that encoded a 27.5-kDa protein approximately 30% similar to the human deoxycytidine kinase and deoxyguanosine kinase. The protein was expressed in Escherichia coli and shown to have similar substrate specificity as reported for purified native human TK2. The recombinant TK2 was shown to phosphorylate the anti-cancer nucleoside analog 2',2'-difluorodeoxycytidine. Northern blot analysis showed two mRNA species at 2.4 and 4.0 kilobases predominantly expressed in liver, pancreas, muscle, and brain. We identified a sequence-tagged site designed from the 3' region of the TK2 cDNA. The sequence-tagged site has been mapped to 81-84 centimorgans from the top linkage group of chromosome 16, which corresponds to the 16q22 region. Our data show that deoxycytidine kinase, deoxyguanosine kinase, and TK2 belong to a family of closely related enzymes. At the time of this report all four of the known human deoxyribonucleoside kinases have been cloned. This provides the opportunity to characterize their individual contribution to therapeutic and toxic effects of nucleoside analogs.

Houssa B et al.
Cloning of a novel human diacylglycerol kinase (DGKtheta) containing three cysteine-rich domains, a proline-rich region, and a pleckstrin homology domain with an overlapping Ras-associating domain.
J Biol Chem. 1997; 272: 10422-8
Display abstract
Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794-800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGKtheta, of 941 amino acids with an apparent molecular mass of 110 kDa. DGKtheta contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGKtheta contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGK-specific. DGKtheta furthermore contains various domains for protein-protein interaction, such as a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGKtheta is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGKtheta mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGKtheta activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.

Sakane F
[Diverse structures of diacylglycerol kinase gene family]
Seikagaku. 1997; 69: 176-81

Honke K, Tsuda M, Hirahara Y, Ishii A, Makita A, Wada Y
Molecular cloning and expression of cDNA encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase.
J Biol Chem. 1997; 272: 4864-8
Display abstract
We have isolated a cDNA clone encoding human 3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8- to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.

Lu Y, Durkin JP
Protein kinase C in IL-2 signal transduction.
Immunol Res. 1997; 16: 355-74
Display abstract
Interleukin-2 (IL-2), secreted principally by activated helper T-cells, plays a pivotal role in the generation and regulation of the immune response. The various biologic functions of IL-2 have been the focus of intensive study over the years and have been well worked out. By contrast, an understanding of the intracellular signals coupled to the IL-2 receptor and responsible for mediating IL-2 effects in T-cells is far less developed, and the role that protein kinase C (PKC) may play in the various cellular responses to IL-2 receptor activation is unclear. In this article we will discuss IL-2, its receptors, and IL-2 signal transduction in relation to the physiological roles PKC activation may play in IL-2-mediated activation of T-cells and other hematopoietic cells.

Stirdivant SM et al.
Cloning and mutagenesis of the p110 alpha subunit of human phosphoinositide 3'-hydroxykinase.
Bioorg Med Chem. 1997; 5: 65-74
Display abstract
Activation of phosphoinositide 3'-hydroxykinase (P13K) is required for mitogenic signal transduction by several growth factors and oncogenes. P13K is a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit. In the current study, we report the cloning and characterization of the p110 alpha catalytic subunit of human P13K. This clone is highly homologous (> 99% amino acid identity) to bovine brain p110 alpha, but contains 10 amino acid differences from the human p110 alpha sequence previously reported. Comparison of this sequence with known Ser/Thr kinases and p110 homologs highlighted several conserved residues within the putative kinase domain. Mutational analysis of these residues (Asp915, (Asp933 + Phe934)) yielded P13K mutants with virtually complete loss of phosphoinositide phosphorylating activity. Expression of the wild-type p110 alpha protein in CHO cells is sufficient to activate the serum response element derived from the promoter of c-fos, an immediate early gene product. In contrast, the catalytically impaired p110 alpha mutants as well as the p85 alpha subunit of P13K were inactive in the fos assay. These studies suggest that the mitogenic signal transduction pathway mediated by P13K is dependent upon the enzymatic activity of the p110 alpha subunit of P13K.

Nobe K, Ohata H, Momose K
Receptor-mediated diacylglycerol kinase translocation dependent on both transient increase in the intracellular calcium concentration and modification by protein kinase C.
Biochem Pharmacol. 1997; 53: 1683-94
Display abstract
Diacylglycerol kinase (DG kinase) is activated by various stimuli in many types of cells. We reported earlier that carbachol (CCh) induced DG kinase translocation from the cytosolic fraction to the membrane fraction in guinea pig taenia coli (Biochem. Pharmacol., 50: 591-599, 1995). In this study, the regulation mechanisms of DG kinase translocation are reported, based on the following findings: 1) CCh sustained an increase in DG kinase in the membrane fraction and a decrease in the cytosolic fraction; 2) blocking calcium influx by removing extracellular calcium did not affect the CCh-induced sustained DG kinase translocation; 3) exposing purified protein kinase C (PKC) to DG kinase increased DG kinase affinity to octylglycoside micelles only with the enzyme extracted from the cytosolic fraction; and 4) CCh-induced DG kinase translocation was reversed by removing CCh, and the serine/threonine phosphatase inhibitor, okadaic acid, blocked the reversal of the translocation. These results suggest that CCh-induced DG kinase translocation is promoted by both a transient increase in intracellular calcium, which may be released from the intracellular store, and by DG kinase phosphorylation by PKC.

Balla T, Downing GJ, Jaffe H, Kim S, Zolyomi A, Catt KJ
Isolation and molecular cloning of wortmannin-sensitive bovine type III phosphatidylinositol 4-kinases.
J Biol Chem. 1997; 272: 18358-66
Display abstract
Agonist-sensitive phosphoinositide pools are maintained by recently-identified wortmannin (WT)-sensitive phosphatidylinositol (PI) 4-kinase(s) (Nakanishi, S., Catt, K. J., and Balla, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5317-5321). Two loosely membrane-associated WT-sensitive type III PI 4-kinases were isolated from bovine adrenal cortex as [3H]WT-labeled 110- and 210-kDa proteins. Based on peptide sequences from the smaller enzyme, a 3. 9-kilobase pair (kb) cDNA with an open reading frame encoding a 90-kDa protein was isolated from a bovine brain cDNA library. Expression of this cDNA in COS-7 cells yielded a 110-kDa protein with WT-sensitive PI 4-kinase activity. Northern blot analysis of a human mRNA panel showed a single approximately 3.8-kb transcript. Peptide sequences obtained from the 210-kDa enzyme corresponded to those of a recently described rat 230-kDa PI 4-kinase. A 6.5-kb cDNA containing an open reading frame of 6129 nucleotides that encoded a 230-kDa protein, was isolated from brain cDNA. Northern blot analysis of human mRNA revealed a major 7.5-kb transcript. The molecular cloning of these novel WT-sensitive type III PI 4-kinases will allow detailed analysis of their signaling and other regulatory functions in mammalian cells.

Kobayashi K, Shimada A, Yamano Y, Umemura T
Molecular cloning of a canine metallothionein cDNA.
J Vet Med Sci. 1997; 59: 819-23
Display abstract
A canine metallothionein cDNA obtained from the liver of a cadmium-treated beagle was cloned and sequenced. Asn at position 4 conserved among all mammalian metallothionein-1 and metallothionein-2 is replaced by Asp in the canine metallothionein cDNA clone. Because the acidic amino acid doesn't exist at either position 10 or 11 in the deduced amino acid sequence, it is supposed that this cDNA is derived from canine metallothionein-1 mRNA. Northern blot analysis using the cDNA as a probe revealed the induction of the canine metallothionein mRNA expression in the liver and kidney of a cadmium-treated beagle. Thus, the canine metallothionein cDNA obtained in the present study should provide an useful tool for the molecular investigation of metallothionein in dog.

Meyers R, Cantley LC
Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase.
J Biol Chem. 1997; 272: 4384-90
Display abstract
Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P, the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel, ubiquitously expressed PtdIns 4-kinase (PI4Kbeta). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kbeta are characteristic of a type III enzyme. Interestingly, both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi, S., Catt, J. K., and Balla, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5317-5321).

Kohyama-Koganeya A, Watanabe M, Hotta Y
Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in rat retina.
FEBS Lett. 1997; 409: 258-64
Display abstract
We have cloned and characterized a new diacylglycerol kinase (DGK) isozyme which is expressed in the retina and the brain of rat. The cDNA contains an open reading frame of 567 amino acid residues with a predicted protein of 64 kDa and shows very high homology to human DGK epsilon. The new DGK isozyme contains two distinctive zinc-finger structures and a putative catalytic domain. This DGK expressed predominantly in the inner and outer nuclear layers of retina. This expression pattern is different from those of the previously cloned DGKs including the human DGK epsilon, suggesting that this DGK isozyme has potential importance in visual functions as was the case in Drosophila retinal cells.

Ding L, Bunting M, Topham MK, McIntyre TM, Zimmerman GA, Prescott SM
Alternative splicing of the human diacylglycerol kinase zeta gene in muscle.
Proc Natl Acad Sci U S A. 1997; 94: 5519-24
Display abstract
Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from the 3.5-kb clone originally identified in endothelial cells. One transcript, which was 3.4 kb long, was shown to be nonfunctional; it had a 77-bp deletion that included the translation initiation site. The other was 4.1 kb long with a unique 5' sequence of 853 bp. We also isolated a genomic clone of DGKzeta and determined its organization and location; it contains 32 exons, spans approximately 50 kb of genomic sequence, and maps to chromosome 11p11.2. The protein encoded by the 4.1-kb transcript contains two cysteine-rich regions, a catalytic domain, and ankyrin repeats like the endothelial form of DGKzeta, as well as a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with the 4.1-kb clone, we detected a 130-kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. We conclude that alternative splicing generates tissue-specific variants of DGKzeta that share some properties but may have unique ones as well.

Koivu T, Laitinen S, Riento K, Olkkonen VM
Sequence of a human cDNA encoding Cab45, a Ca2+-binding protein with six EF-hand motifs.
DNA Seq. 1997; 7: 217-20
Display abstract
We report the sequence of a human cDNA encoding a deduced 362 amino acid protein with six EF-hand calcium-binding motifs. The protein is a likely human counterpart of the Cab45 protein recently identified in the 3T3-L1 mouse adipocyte cell line [Scherer et al. (1996), J. Cell Biol. 133, 257-268], displaying 87% aa and 83% nt identity with this sequence. The mRNA for human Cab45 is detected ubiquitously in tissue Northern blots.

Ho LK, Liu D, Rozycka M, Brown RA, Fry MJ
Identification of four novel human phosphoinositide 3-kinases defines a multi-isoform subfamily.
Biochem Biophys Res Commun. 1997; 235: 130-7
Display abstract
Phosphoinositide (PI) 3-kinases have critical roles in diverse cellular signalling processes and in protein trafficking. This suggests that like other intracellular signalling molecules, e.g., phospholipase C and protein kinase C, there might be a large family of PI 3-kinase isoforms with the individual members having discrete signalling roles. Reverse transcription-polymerase chain reaction methods, using degenerate oligonucleotide primers against the lipid kinase consensus region, revealed eight sequences from human cDNA containing a high degree of identity to the family of PI 3-kinases. The sequences obtained included the previously described p110 alpha, p110 beta, and p110 gamma isoforms and HsVps34. Additionally, we have identified four novel sequences which are related to PI 3-kinases. Three of the novel sequences would appear to form a distinct sub-family of PI 3-kinases. We report the expression of these novel PI 3-kinases in human tissues and in cells derived from normal breast.

Wilson MP, Majerus PW
Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme.
J Biol Chem. 1996; 271: 11904-10
Display abstract
Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified.

Gehrmann T et al.
Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing.
Biochim Biophys Acta. 1996; 1311: 53-63
Display abstract
Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.

Ishihara H et al.
Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase.
J Biol Chem. 1996; 271: 23611-4
Display abstract
Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic beta-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic beta-cell line MIN6. Two novel cDNAs, designated PI4P5K-Ialpha and PI4P5K-Ibeta, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles.

Molz L, Chen YW, Hirano M, Williams LT
Cpk is a novel class of Drosophila PtdIns 3-kinase containing a C2 domain.
J Biol Chem. 1996; 271: 13892-9
Display abstract
We report the identification of a novel class of phosphatidylinositol (PtdIns) 3-kinases whose members contain C-terminal C2 domains. We have isolated Drosophila and murine genes (termed cpk and cpk-m respectively) by polymerase chain reaction amplification of cDNA libraries with degenerate primers corresponding to conserved regions of PtdIns kinases. The amino acid sequences of Cpk and Cpk-m are most similar to that of p110, a family of PtdIns 3-kinases that mediates the responses of cells to mitogenic stimuli. The Cpk and Cpk-m sequences are similar to a large, central region of p110, but differ from p110 at their N and C termini. The N termini of the Cpk proteins do not contain any recognizable protein motif, while the C termini contain "C2 domains," a feature unique among PtdIns kinases. Cpk has an intrinsic PtdIns kinase activity and can phosphorylate PtdIns and PtdIns-4-P, but not PtdIns(4,5)P2, at the D3 position of the inositol ring. Cpk is the first PtdIns 3-kinase identified with this particular substrate specificity. We have identified two potential Cpk-binding proteins, p90 and p190, and have determined that both Cpk and p190 may be tyrosine phosphorylated. This finding suggests that Cpk function may be regulated by tyrosine kinases.

Kanoh H, Kai M, Wada I
Molecular properties of enzymes involved in diacylglycerol and phosphatidate metabolism.
J Lipid Mediat Cell Signal. 1996; 14: 245-50
Display abstract
Diacylglycerol and phosphatidate serve as key lipid intermediates of glycerolipid biosynthesis and also as the second messengers liberated in signal-stimulated cells. The molecular properties of enzymes involved in the metabolic processing of the two lipids are largely unknown. We have revealed by cDNA cloning the presence of a novel enzyme family of diacylglycerol kinase (DGK) having unique structural characteristics. Furthermore, we recently identified the plasma membrane-bound phosphatidate phosphatase (PAP) as a 35 kDa glycoprotein. Detailed studies on the two enzymes would contribute to understanding the molecular mechanisms underlying the control of both glycerolipid biosynthesis and signal transduction.

Bunting M, Tang W, Zimmerman GA, McIntyre TM, Prescott SM
Molecular cloning and characterization of a novel human diacylglycerol kinase zeta.
J Biol Chem. 1996; 271: 10230-6
Display abstract
Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Whatley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGKzeta displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.

Goto K, Kondo H
Heterogeneity of diacylglycerol kinase in terms of molecular structure, biochemical characteristics and gene expression localization in the brain.
J Lipid Mediat Cell Signal. 1996; 14: 251-7
Display abstract
Three different cDNA clones for diacylglycerol (DG) kinase were isolated from a rat brain cDNA library and designated DGK-I, DGK-II and DGK-III. These three encode distinct polypeptides with 58% identity to each other and contain EF-hand motifs, cysteine-rich zinc finger-like sequences and putative ATP-binding sites. A high kinase activity is shown in COS cells transfected with either one of the three cDNAs without substrate specificity among DG species, and the kinase activity is Ca-dependent. The activity for DGK-I is recovered dominantly in the soluble fraction of the cell, that for DGK-II in the particulate fraction; and that for DGK-III equally in both of the fractions. The difference in their expression localization is most noticeable: DGK-I is expressed in oligodendrocytes of the brain as well as T-lymphocytes in the thymus and spleen; DGK-II is expressed in neurons of the caudate-putamen, accumbens nucleus and olfactory tubercle; and DGK-III in the cerebellar Purkinje cells and granule cells. The functional significance of the discovery of three DG kinase isozymes is briefly discussed.

Razdan K, Kroll MH
Molecular cloning of a novel platelet protein showing homology to the angiotensin II receptor C-terminal domain.
J Biol Chem. 1996; 271: 2221-4
Display abstract
Oligoscreening of a cDNA library obtained from 4 beta-phorbol 12-myristate 13-acetate-stimulated human erythroleukemia (HEL) cells resulted in the isolation of a novel clone coding for a protein with a calculated molecular mass of 8110 Da. This protein of 71 amino acids shows significant homology to the carboxyl-terminal regulatory domain of angiotensin II type 1 receptors. The homology encompasses four regions of amino acid residues thought to serve as consensus sequences for phosphorylation by serine/threonine kinases such as protein kinase C, which are key mediators of intracellular signaling. Reverse transcription-polymerase chain reaction identified the transcript in human platelets, human megakaryocytic DAMI cells, and HEL cells. High stringency Northern blotting revealed a tissue-specific distribution of three transcript species, with predominant expression in skeletal muscle and pancreas. Rabbit anti-peptide antiserum was used to immunoblot protein lysates from washed resting platelets and from 4 beta-phorbol 12-myristate 13-acetate-stimulated DAMI and HEL cells. These immunoblots revealed the presence of an intense approximately 8-kDa protein band in platelets and HEL cells and a faint band of identical size in DAMI cells.

Virbasius JV, Guilherme A, Czech MP
Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain.
J Biol Chem. 1996; 271: 13304-7
Display abstract
Phosphatidylinositol (PI) 3-kinases catalyze the formation of 3?-phosphoinositides, which appear to promote cellular responses to growth factors and such membrane trafficking events as insulin-stimulated translocation of intracellular glucose transporters. We report here the cloning of a novel PI 3-kinase, p170, from cDNA of insulin-sensitive mouse 3T3-L1 adipocytes. Mouse p170 utilizes PI and to a limited extent PI 4-P as substrates, in contrast to the PI-specific yeast VPS34 homolog PtdIns 3-kinase and the p110 PI 3-kinases, which phosphorylate PI, PI 4-P, and PI 4,5-P2. Mouse p170 is also distinct from PtdIns 3-kinase or the p110 PI 3-kinases in exhibiting a 10-fold lower sensitivity to wortmannin. Unique structural elements of p170 include C-terminal sequences strikingly similar to the phosphoinositide-binding C2 domain of protein kinase C isoforms, synaptotagmins, and other proteins. These features of mouse p170 are shared with a recently cloned Drosophila PI 3-kinase, DmPI3K_68D. Together, these proteins define a new class of PI 3-kinase likely influenced by cellular regulators distinct from those acting upon p110- or VPS34-like PI 3-kinases.

Mertsalov IB, Gundelfinger E, Tsetlin VI
[Cloning cDNA for human neurogranin]
Bioorg Khim. 1996; 22: 366-9
Display abstract
By using the human fetal brain library, a 1.2-kb cDNA coding for human neurogranin, a neurospecific endogenous substrate of protein kinase C, was cloned. The results of the Northern blot analysis of the human brain cortex mRNA show that the human neurogranin mRNA exists as a single transcript. Its gene thereby differs from the rat neurogranin gene (RC3), which was shown to produce two mRNAs of different lengths. cDNAs of the human and rat neurogranins show a 70% homology, whereas the protein sequences only differ in three amino acid residues out of the total of 78. The 3'-untranslated region of the human neurogranin cDNA lacks the purine-rich stretch characteristic of the rat neurogranin cDNA.

Nakagawa T, Goto K, Kondo H
Cloning, expression, and localization of 230-kDa phosphatidylinositol 4-kinase.
J Biol Chem. 1996; 271: 12088-94
Display abstract
A phosphatidylinositol (PI) 4-kinase cDNA was cloned from a rat brain cDNA library. This cDNA encoded a protein of 2041 amino acids with a calculated molecular weight of 231,317. The deduced amino acid sequence shared the identity of 52.3 and 34.4% in the presumed catalytic domain with two yeast PI 4-kinases, STT4 and PIK1, respectively, and showed 31.7% identity to p110alpha subunit of rat PI 3-kinase in the same domain. In addition, a 3' half coding region of the present cDNA was 89.6% identical to and its deduced amino acid sequence was 98.2% identical to the sequence for P14Kalpha, a recently reported human PI 4-kinase of type II, suggesting that P14Kalpha is an alternative form of the present PI 4-kinase molecule. The present cDNA contained sequences encoding the ankyrin repeat domain, lipid kinase unique domain, pleckstrin homology domain, presumed lipid kinase/protein kinase homology domain, proline-rich region, and SH3 domain. By examining PI kinase activity in transfected COS-7 cells using the epitope tag immunoprecipitation as well as the conventional way, the product phosphatidylinositol phosphate was identified as phosphatidylinositol 4-phosphate but not phosphatidylinositol 3-phosphate. This PI 4-kinase activity was markedly enhanced in the presence of Triton X-100 but relatively insensitive to inhibition by adenosine. By epitope tag immunohistochemistry, the immunoreactivity for this PI 4-kinase molecule was largely localized in close association with the membranes of the Golgi vesicles and vacuoles. By in situ hybridization analysis, the expression of mRNA for this PI 4-kinase was evident throughout the gray matter of entire brain with higher expression intensity in fetal brain. These data imply that this novel PI 4-kinase is involved in some processes essential to neuronal differentiation and maturation including the synaptogenesis and synaptic plasticity.

Katagiri T, Mizoguchi T, Shinozaki K
Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana.
Plant Mol Biol. 1996; 30: 647-53
Display abstract
Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 CDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.

Hodgkin MN, Pettitt TR, Martin A, Wakelam MJ
Regulation of 'signalling diacylglycerol' in cells: the importance of diacylglycerol kinase.
Biochem Soc Trans. 1996; 24: 991-4

Itano N, Kimata K
Molecular cloning of human hyaluronan synthase.
Biochem Biophys Res Commun. 1996; 222: 816-20
Display abstract
We report here the cDNA sequence of human hyaluronan synthase. The cDNA clone was isolated from a human fetal brain cDNA library by screening with a DNA probe generated from the coding sequence of murine hyaluronan synthase. It contains a 2107 bp cDNA insert which is composed of a 1629 bp open reading frame and a short segment of the 3'-untranslated region. Comparative analysis of the predicted coding region with the murine hyaluronan synthase sequence indicates 84.4% and 96.0% identity in nucleotide sequence and amino acid sequences, respectively. Northern analysis indicated that the gene was ubiquitously expressed in various human tissues.

Klauck TM, Xu X, Mousseau B, Jaken S
Cloning and characterization of a glucocorticoid-induced diacylglycerol kinase.
J Biol Chem. 1996; 271: 19781-8
Display abstract
Diacylglycerol kinase (DGK) plays a key role in cellular processes by regulating the intracellular concentration of the second messenger diacylglycerol. We screened a hamster DDT1 smooth muscle cell library and isolated a unique, glucocorticoid-inducible cDNA with substantial homology to known DGKs. DGK activity was increased in lysates of insect cells infected with recombinant baculovirus containing this cDNA. Antibodies raised against expressed sequences recognized a glucocorticoid-inducible 130-140-kDa protein on immunoblots of DDT1 cell lysates. Thus, this sequence appears to be a new member of the DGK family that we refer to as DGKeta. Homology to other DGKs was apparent in domains that are thought to be important for DGK function including the cysteine-rich motifs and potential catalytic domains. DGKeta shares substantial homology with DGKdelta including the N-terminal pleckstrin homology domain. The tissue distribution of DGKeta message (determined by ribonuclease protection assays) and protein (determined by immunoblots) was broader than reported for other DGKs, indicating that DGKeta may play a more general role in regulating cellular DG levels than other DGKs. Heterogeneity among DGK family members indicates that individual DGKs may have unique functions.

Loijens JC, Anderson RA
Type I phosphatidylinositol-4-phosphate 5-kinases are distinct members of this novel lipid kinase family.
J Biol Chem. 1996; 271: 32937-43
Display abstract
Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) synthesize phosphatidylinositol-4,5-bisphosphate, a key precursor in phosphoinositide signaling that also regulates some proteins and cellular processes directly. Two distinct PIP5Ks have been characterized in erythrocytes, the 68-kDa type I (PIP5KI) and 53-kDa type II (PIP5KII) isoforms. Using peptide sequences from the erythroid 68-kDa PIP5KI, we have isolated cDNAs encoding PIP5KIalpha from human brain. Partial cDNAs obtained for a second isoform, PIP5KIbeta, established that the human STM7 gene encoded a previously unrecognized PIP5KI. However, the peptide sequences demonstrated that erythroid PIP5KI corresponded to PIP5KIalpha. Recombinant, bacterially expressed PIP5KIalpha possessed PIP5K activity and was immunoreactive with erythroid PIP5KI antibodies. By Northern analysis, PIP5KIalpha and PIP5KIbeta had wide tissue distributions, but their expression levels differed greatly. PIP5KIs had homology to the kinase domains of PIP5KIIalpha, yeast Mss4p and Fab1p, and a new Caenorhabditis elegans Fab1-like protein identified in the data base. These new isoforms have refined the sequence requirements for PIP5K activity and, potentially, regulation of these enzymes. Furthermore, the limited homology between PIP5KIs and PIP5KIIalpha, which was almost exclusively within the kinase domain core, provided a molecular basis for distinction between type I and II PIP5Ks.

Goto K, Kondo H
A 104-kDa diacylglycerol kinase containing ankyrin-like repeats localizes in the cell nucleus.
Proc Natl Acad Sci U S A. 1996; 93: 11196-201
Display abstract
The cDNA corresponding to a fourth species of diacylglycerol (DG) kinase (EC 2.7.1.107) was isolated from cDNA libraries of rat retina and brain. This cDNA encoded a 929-aa, 104-kDa polypeptide termed DGK-IV. DGK-IV was different from previously identified mammalian DG kinase species, DGK-I, DGK-II, and DGK-III, in that it contained no EF-hand motifs but did contain four ankyrin-like repeats at the carboxyl terminus. These structural features of DGK-IV closely resemble the recently cloned, eye-specific DG kinase of Drosophila that is encoded by the retinal degeneration A (rdgA) gene. However, DGK-IV was expressed primarily in the thymus and brain with relatively low expression in the eye and intestine. Furthermore, the primary structure of the DGK-IV included a nuclear targeting motif, and immunocytochemical analysis revealed DGK-IV to localize in the nucleus of COS-7 cells transfected with the epitope-tagged cDNA, suggesting an involvement of DGK-IV in intranuclear processes.

Johansson M, Karlsson A
Cloning and expression of human deoxyguanosine kinase cDNA.
Proc Natl Acad Sci U S A. 1996; 93: 7258-62
Display abstract
A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.

Wang L, Hellman U, Eriksson S
Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA.
FEBS Lett. 1996; 390: 39-43
Display abstract
Mammalian mitochondrial deoxyguanosine kinase (dGK) is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix. Using a RT-PCR-generated probe, based on amino acid sequence information from proteolytic fragments of purified bovine dGK, we have cloned a cDNA from a human brain cDNA library that encodes a 30 kDa protein. The deduced amino acid sequence of this protein included the sequence of all six peptides isolated and sequenced from purified dGK. Expression and purification of recombinant protein from induced Escherichia coli extracts revealed that it catalyses efficient phosphorylation of dGuo, arabinosyl guanine, dAdo, 2-chloro-2'-deoxyadenosine and dIno similar to purified dGK. Northern blot analysis demonstrated one dominant positive mRNA of 1.35 kb and it was found in several tissues at similar levels. The coding sequence of dGK showed 46% identity to the coding sequence of cytosolic deoxycytidine kinase, and conserved sequence motifs among the known deoxynucleoside kinase were identified.

Machida T, Higashi K, Ogawara H
Cochlioquinone A, an inhibitor of diacylglycerol kinase.
J Antibiot (Tokyo). 1995; 48: 1076-80
Display abstract
The effects of cochlioquinone A, isolated from Drechslera sacchari, were studied in vitro and in vivo. This compound specifically inhibited diacylglycerol kinase activity with Ki = 3.1 microM. The kinetics revealed that cochlioquinone A inhibited diacylglycerol kinase in competition with ATP, and non-competitively with diacylglycerol. The compound inhibited neither protein kinase C, epidermal growth factor receptor-associated protein tyrosine kinase, nor phospholipase C. Cochlioquinone A reduced the concentration of phosphatidic acid in T cell lymphoma with a half maximal concentration of 3 microM, and simultaneously augmented the phosphorylation of 80 kDa protein, a known substrate of protein kinase C. The degree of the phosphorylation of 80 kDa protein in the presence of cochlioquinone A was similar to that in the presence of phorbol myristate acetate (0.1 microgram/ml). These results demonstrate that cochlioquinone A is a specific inhibitor of diacylglycerol kinase, which regulates the activity of protein kinase C.

Goto K
[Heterogenity of diacylglycerol Kinase--Structure, biochemical characteristics and gene expression in the brain]
Kaibogaku Zasshi. 1995; 70: 509-11

Burbelo PD, Hall A
14-3-3 proteins. Hot numbers in signal transduction.
Curr Biol. 1995; 5: 95-6
Display abstract
14-3-3 proteins have been known about for a long while but their functions have been poorly understood; recent results indicate that they play a role in both signal transduction and the cell cycle.

Ma GT, Hong YS, Ives DH
Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26.
J Biol Chem. 1995; 270: 6595-601
Display abstract
Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.

Redman C, Lefevre J, MacDonald ML
Inhibition of diacylglycerol kinase by the antitumor agent calphostin C. Evidence for similarity between the active site of diacylglycerol kinase and the regulatory site of protein kinase C.
Biochem Pharmacol. 1995; 50: 235-41
Display abstract
Calphostin C is an anti-tumor agent that binds to the regulatory domain of protein kinase C and inhibits the binding of phorbol dibutyrate. Recent studies suggest that there may be structural similarities between protein kinase C (PKC) and diacylglycerol kinase (DGK). Both enzymes bind diacylglycerol and phosphatidylserine, and sequencing of the 80 kDa diacylglycerol kinase shows that it contains zinc finger-like sequences, similar to those occurring in PKC. Similarities in some enzymatic properties of PKC and DGK led us to examine whether regulatory-site inhibitors of PKC also might inhibit DGK. For these studies, the membrane-bound DGK was partially purified from porcine testis membranes. Calphostin C inhibited DGK with an IC50 in the micromolar range. The inhibition of DGK by calphostin C was competitive with respect to diacylglycerol and was not affected by the presence or absence of phosphatidylserine. Other inhibitors of protein kinase C were without effect, with the exception of Adriamycin, which inhibited at millimolar concentrations. Staurosporine, which binds to the catalytic domain of protein kinase C, did not inhibit DGK. The results suggest that there are functional similarities between the substrate binding site of DGK and the regulatory site of protein kinase C.

Goto K, Funayama M, Kondo H
Cloning and expression of a cytoskeleton-associated diacylglycerol kinase that is dominantly expressed in cerebellum.
Proc Natl Acad Sci U S A. 1994; 91: 13042-6
Display abstract
A third species of diacylglycerol kinase (EC 2.7.1.107) cDNA was cloned from a rat brain cDNA library. The isolated cDNA encoded a 788-amino acid, 88-kDa polypeptide. This isozyme shared 58% identity with the previously isolated rat 80-kDa and 90-kDa diacylglycerol kinases. EF hand motifs, cysteine-rich zinc finger-like sequences, and putative ATP-binding site were all conserved among these isozymes. The 88-kDa diacylglycerol kinase was expressed specifically in brain and localized predominantly in cerebellar Purkinje cells. This isozyme was associated equally with particulate and supernatant fractions in cDNA-transfected COS-7 cells and dominantly with the particulate fraction in the brain. After Triton X-100 extraction, this isozyme remained in the detergent-insoluble cytoskeletal fraction of the brain and transfected COS-7 cells.

Watanabe F, Sakai A, Furuya E, Uyeda K
Molecular cloning and tissue specific expression of fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase of rat brain.
Biochem Biophys Res Commun. 1994; 198: 335-40
Display abstract
A cDNA clone (3591 base pairs) encoding an isozyme of Fructose 6-phosphate, 2-kinase:Fructose 2,6-bisphosphatase was isolated from a rat brain cDNA library. The 5' sequence of this clone (1241 base pairs) was identical to that of the heart type isozyme cDNA (except for 7 base pair mismatches), but the 3' nucleotide sequence (2024 base pairs) was completely different. Its deduced amino acid sequence showed that the enzyme lacked a regulatory domain which contained phosphorylation sites for protein kinase A and C. The results of Northern blotting and polymerase chain reaction demonstrated that the mRNA, 7.4 kilobases long, was expressed also in heart, testis, liver, and skeletal muscle.

Nachiappan V, Rajasekharan R
Enzymatic synthesis of [32P]acyl-sn-glycerol 3-phosphate using diacylglycerol kinase.
Anal Biochem. 1994; 222: 283-5

Kai M, Sakane F, Imai S, Wada I, Kanoh H
Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in human retina with a truncated and inactive enzyme expression in most other human cells.
J Biol Chem. 1994; 269: 18492-8
Display abstract
In order to clone novel diacylglycerol kinase (DGK) isozymes, we first obtained a DGK-related cDNA fragment by polymerase chain reaction using the human hepatoma cell line HepG2 mRNA and degenerated primers. The amplified fragment was subsequently used as a probe for screening the cDNA library from HepG2 cells. We obtained a cDNA clone coding for a novel DGK isozyme (designated DGK gamma) comprised of 791 amino acid residues. The amino acid sequence of DGK gamma was 52 and 62% identical to those of previously sequenced porcine 80-kDa and rat 90-kDa enzymes, respectively. DGK gamma, although initially cloned from the HepG2 cDNA libraries, was unexpectedly expressed in the human retina abundantly and to a much lesser extent in the brain. Other human tissues, including the liver and HepG2 cells, contained extremely low levels of DGK gamma mRNA. Furthermore, HepG2 cells and most of the human tissues except for the retina and brain expressed a truncated DGK gamma with an internal deletion of 25 amino acid residues (Ile451-Gly475). When transfected into COS-7 cells, the nontruncated cDNA gave phosphatidylserine-dependent DGK activity with no apparent specificity with regard to the acyl compositions of diacylglycerol. In contrast the truncated cDNA failed to give DGK activity in spite of the expression of its mRNA and enzyme protein in COS cells, thus demonstrating that the truncated DGK gamma is catalytically inactive. The sequence comparison of the three cloned DGKs revealed the presence of four highly conserved regions including the two sets each of EF-hand and zinc finger structures. Although the implication of the catalytically inactive form of DGK gamma remains unknown, this work further demonstrates the occurrence of multiple animal DGK isozymes with a conserved basic structure but with markedly different expression patterns depending on the cell types.

Hodgkin M, Gardner S, Wakelam M
The identification of several stearoyl-arachidonyl selective diacylglycerol kinases in the particulate fraction of porcine testes.
Biochem Soc Trans. 1993; 21: 490-490

Toth M, Gimes G
Phosphocholine transferase is more efficient than diacylglycerol kinase as possible attenuator of diacylglycerol signals in primordial human placenta.
Acta Physiol Hung. 1993; 81: 341-54
Display abstract
Diacylglycerols (DAG) are assumed to play intracellular signal roles in rapidly growing tissues like those present in the primordial human placenta. The aim of the present study was to gain information on the capacity of DAG-kinase and CDP-choline: DAG phosphocholine transferase enzymes to eliminate DAG and behave as possible signal attenuators in these tissues. Previous and present results has provided evidence that Triton X-100 (0.05%, v/v), when incubated with placenta mince, (I) causes the accumulation of DAG by inhibiting DAG-acyltransferase and by a suggested acceleration of phosphatidylcholine (PC) decomposition, (II) inhibits the synthesis of PC and (III) increases the formation of phosphatidic acid (PA) without decreasing the conversion of PA into acylglycerols. Inhibition by the synthetic DAG: dioctanoylglycerol (DOCG) and by the DAG-analog: dioctanoylethyleneglycol (DOEG), of the Triton-induced (3H)PA-accumulation from (3H)glucose and of the increased labeling of PA with (32P)phosphate indicated some DAG-attenuating roles of DAG-kinase, but this mechanism appeared not to be very efficient. The low rate of conversion of DOCG into (32P)PADOCG species in incubations with (32P)phosphate confirmed this view. Compared to the rate of formation of PADOCG, DOCG was eliminated in the form of PCDOCG more than one order of magnitude faster. In addition, DOCG stimulated, whereas DOEG inhibited the formation of endogenous PC. These findings suggest that in the primordial human placenta transformation of DAG into PC by phosphocholine transferase and an attending stimulation of PC synthesis represent a more effective attenuator mechanism than the conversion of DAG into PA by DAG-kinase.

Masai I, Okazaki A, Hosoya T, Hotta Y
Drosophila retinal degeneration A gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc-finger motifs and ankyrin repeats.
Proc Natl Acad Sci U S A. 1993; 90: 11157-61
Display abstract
The Drosophila visual mutant, carrying the retinal degeneration A gene (rdgA), has photoreceptor cells that degenerate within a week after eclosion. Morphological studies suggested that this mutant harbors abnormalities in membrane turnover of the photoreceptor cells. Biochemically, the rdgA mutant lacks an eye-specific and membrane-associated diacylglycerol kinase (DGK; EC 2.7.1.107) activity in a gene-dosage-dependent manner, suggesting that rdgA gene encodes a DGK. We report the molecular cloning and characterization of a DGK gene, which maps to the rdgA locus. This gene, designated as DGK2, has a single open reading frame that encodes 1454 amino acids. Like porcine DGK, DGK2 has two cysteine-rich zinc-finger motifs as well as a DGK catalytic domain. The DGK2 protein contains four ankyrin-like repeats at the C-terminal region, suggesting that DGK2 is likely anchored to the membrane or cytoskeleton. Northern blot analysis and tissue in situ hybridization to adult sections revealed that DGK2 is expressed exclusively in the adult retina and that the amount of its mRNA is reduced in some of the rdgA mutant alleles. Furthermore, in two rdgA alleles, rdgA1 and rdgA2, nonsense and missense mutations occur within their DGK2 gene, respectively. Thus, we conclude that rdgA encodes an eye-specific DGK, the absence of which leads to rhabdomere degeneration due to defective phospholipid turnover.

Goto K, Kondo H
Molecular cloning and expression of a 90-kDa diacylglycerol kinase that predominantly localizes in neurons.
Proc Natl Acad Sci U S A. 1993; 90: 7598-602
Display abstract
A diacylglycerol kinase cDNA was isolated from a rat brain cDNA library. This cDNA encoded an 801-amino acid protein of 90,287 Da. This 90-kDa diacylglycerol kinase showed 58% identity in deduced amino acid sequence with a previously isolated rat 80-kDa diacylglycerol kinase. EF-hand motifs, cysteine-rich zinc-finger-like sequences, and putative ATP-binding sites were all conserved between the two kinase species. However, mRNA encoding the 90-kDa kinase was confined to restricted neuronal populations such as the caudate-putamen, the accumbens nucleus, and the olfactory tubercle. Further, the 90-kDa kinase was found to exhibit high phosphorylation activity for long-chain diacylglycerols and was mainly associated with the membrane fraction when the cDNA was transfected into COS-7 cells.

Kanoh H, Sakane F, Imai S, Wada I
Diacylglycerol kinase and phosphatidic acid phosphatase--enzymes metabolizing lipid second messengers.
Cell Signal. 1993; 5: 495-503

Yamada K, Sakane F
The different effects of sphingosine on diacylglycerol kinase isozymes in Jurkat cells, a human T-cell line.
Biochim Biophys Acta. 1993; 1169: 211-6
Display abstract
We studied the effect of sphingosine on the activities of soluble and membrane-bound isozymes from Jurkat cells using combinations of different substrates (arachidonoyl- and didecanoyl DGs) and assay methods (octylglucoside mixed micellar and deoxycholate suspension assays). The results suggested the presence of at least four DGK isoforms, which could be distinguished from each other with respect to intracellular localization, specificity to DG molecular species, responsiveness to sphingosine, and reactivity to anti-80 kDa DGK antibody. We confirmed the presence of arachidonoyl DG-specific DGK in membranes, though this isozyme was not activated by sphingosine. We detected in the cytosol at least two species of sphingosine-activatable and non-activatable DGK isoforms, the major species being the 80 kDa DGK. We postulate that both or either of the two soluble DGKs may be the target of the sphingosine action.

Ohanian J, Heagerty AM
Diacylglycerol kinase activity in intact small arteries.
Biochem Soc Trans. 1993; 21: 349-349

Chen Q, Klemm N, Jeng I
Characterization of two cytosolic diacylglycerol kinase forms.
J Neurochem. 1993; 60: 1212-9
Display abstract
Two forms of rat brain cytosolic diacylglycerol kinase (EC2.7.1.107) were separated by heparin-agarose column chromatography. These forms, designated DGK-I and DGK-II, were not interconvertible as determined by rechromatography. DGK-I and DGK-II had respective molecular masses of 88 and 180 kDa, as measured by Sepharose 6B chromatography. Both forms preferred diacylglycerol over monoacylglycerol and were insensitive to R59022. DGK-II, but not DGK-I, was activated by an activator substance prepared from chicken egg yolk. DGK-II was activated by a rat brain cytosolic activator and was exclusively sensitive to 5'-AMP-mediated inactivation. Further studies revealed that these two forms had the following distinct characteristics: (a) substrate specificity, (b) inhibition by heparin, (c) sensitivity to lysine-containing polyamino acids, and (d) responses to different phospholipids. In general, DGK-II was more responsive to various inhibitors and activators, making it a prime candidate for a regulatable enzyme.

Yamada K, Sakane F, Imai S, Takemura H
Sphingosine activates cellular diacylglycerol kinase in intact Jurkat cells, a human T-cell line.
Biochim Biophys Acta. 1993; 1169: 217-24
Display abstract
Sphingosine is known to regulate a variety of cellular functions through protein kinase C-dependent or independent pathways. In an attempt to investigate differential functions of diacylglycerol kinase (DGK) isozymes, we tested the effect of sphingosine on DGK operating in intact Jurkat cells, a human T-cell line. We found that phosphatidic acid (PA) synthesized from endogenous diacylglycerol (DG) and exogenously added short-chain DGs like dioctanoylglycerol were markedly enhanced by approx. 20 microM sphingosine. Further studies such as the use of protein kinase C down-regulated cells, mass measurements of cellular DGs, analysis of molecular species of PA and the effect of exogenous DG on the conversion of endogenous DG to PA suggested that sphingosine directly activated cellular DGK having a broad specificity toward DG molecular species.

Goto K, Watanabe M, Kondo H, Yuasa H, Sakane F, Kanoh H
Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain.
Brain Res Mol Brain Res. 1992; 16: 75-87
Display abstract
A 3.1 kbp cDNA clone encoding diacylglycerol (DG) kinase of 80 kDa (80K-DG kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence was 82% homologous to previously identified porcine 80K-DG kinase and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites similar to the porcine counterpart. By in situ hybridization histochemistry of rat brain at postnatal week 3, the expression signals for 80K-DG kinase mRNA appeared predominantly on somata of discrete cells in the white matter, and the expression pattern was similar to that of the myelin-specific proteins. In immunohistochemistry using the antibody against bacterially expressed DG kinase-fusion protein, numerous fibrous or dot-like structures exhibiting the immunoreactivity were concentrated in the white matter and they were arranged to radiate in the cerebral cortex and the cerebellar granular layer in a pattern almost identical to that of oligodendrocytes. No neuronal cells exhibited the immunoreactivity. The present finding thus strongly suggests that 80K-DG kinase is expressed specifically in the oligodendrocytes, but not neurons, and may be involved in the myelin formation and metabolism. In addition, the intense hybridization signals and the immunoreactivity for this protein were detected in the entire medulla of the thymus and the periarterial lymphatic area of the splenic white pulp both of which represent T-cell-dependent areas.

Fu T, Sugimoto Y, Okano Y, Kanoh H, Nozawa Y
Abolishment of bradykinin-induced calcium oscillations in ras-transformed fibroblasts by the expression of 80 kDa diacylglycerol kinase.
FEBS Lett. 1992; 307: 301-4
Display abstract
Our previous study showed bradykinin-induced periodic Ca2+ changes (Ca2+ oscillations) in v-Ki-ras-transformed NIH/3T3 (DT) cells in which protein kinase C (PKC) activity is partially down-regulated by a sustained high level of 1,2-diacylglycerol (DAG) [FEBS Lett. (1991) 281, 263-266]. In the present study, DAG kinase with 80 kDa mass (80K DGK) has been successfully transfected in DT cells, which exhibited enhanced cellular DAG kinase activities, decreased cellular DAG contents, and increased PKC activities compared to the control vector-transfected cells. Furthermore, these DGK-transfectants showed strong inhibition in bradykinin-induced Ca2+ oscillations. The results suggest that the sustained DAG increase down-regulates the PKC activity, thereby leading to the induction of Ca2+ oscillations in DT cells.

Kanoh H, Sakane F, Yamada K
Diacylglycerol kinase isozymes from brain and lymphoid tissues.
Methods Enzymol. 1992; 209: 162-72

Lundberg GA, Sommarin M
Diacylglycerol kinase in plasma membranes from wheat.
Biochim Biophys Acta. 1992; 1123: 177-83
Display abstract
Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg2+ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl2. The Mg2+ requirement could be substituted only partially by Mn2+ and not at all by Ca2+. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.

Lemaitre RN, Glomset JA
Arachidonoyl-specific diacylglycerol kinase.
Methods Enzymol. 1992; 209: 173-82

Inoue H, Yoshioka T, Hotta Y
Partial purification and characterization of membrane-associated diacylglycerol kinase of Drosophila heads.
Biochim Biophys Acta. 1992; 1122: 219-24
Display abstract
A membrane-associated diacylglycerol kinase of Drosophila heads was purified to near homogeneity from the KCl extract of Drosophila heads. The purification procedure involved chromatography on Q-Sepharose, ammonium sulfate fractionation, Superose 12, hydroxyapatite and ATP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions after the ATP-agarose column chromatography showed that only a 115 kDa protein correlated well with the enzyme activity. The apparent Km values of partially purified DG kinase were 220 microM for ATP and 540 microM for diolein, respectively. The activity of the DG kinase was inhibited by deoxycholate and was not activated by Ca2+.

Ohanian J, Heagerty AM
The phosphoinositide signaling system and hypertension.
Curr Opin Nephrol Hypertens. 1992; 1: 73-82
Display abstract
The phosphoinositide signaling system is common to many vasoconstrictor agents and as such is influential in the regulation of blood pressure. Recently, there have been major advances in our understanding of these lipids and their metabolism. Characterization of the phospholipase C isozymes and protein kinase C isozymes involved in transmembrane signaling has progressed rapidly. The role of diacylglycerol kinase as a regulator of protein kinase C activity has been established, and phosphatidic acid has been recognized as a cellular messenger. Studies in the spontaneously hypertensive rat have shown abnormalities of phospholipase C that could result in enhanced activity and explain changes in sensitivity reported in rats with this disease. During agonist activation of inositol lipid hydrolysis, levels of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol are elevated in spontaneously hypertensive rats compared with Wistar-Kyoto control rats. These changes are observed early, prior to blood pressure stabilization, and may be downregulated once hypertension is established. In addition, there is evidence for reduced diacylglycerol kinase activity and enhanced protein kinase C activity in the spontaneously hypertensive rat. These data provide evidence for hyperresponsiveness of the phosphoinositide signaling system in the developmental stages of hypertension. However, confirmatory experiments in nongenetic animal models of hypertension and in human tissues are needed to establish that this is not just a phenotypic phenomenon of the spontaneously hypertensive rat.

Sakane F, Yamada K, Kanoh H
[The primary structure of diacylglycerol kinase and regulation of its activity]
Tanpakushitsu Kakusan Koso. 1991; 36: 290-8

Chen Q, Klemm N, Jeng IM
Irreversible inactivation of diacylglycerol kinase-II requires a mediator.
Biochem Int. 1991; 25: 775-81
Display abstract
Cytosolic diacylglycerol kinase was irreversibly inactivated by 5'-AMP since the enzyme remained less active after the removal of 5'-AMP by P-10 gel chromatography. The inactivation was time-dependent, suggesting the involvement of a covalent bond modification. A reconstitution experiment detected a rat brain cytosolic mediator for the effect of 5'-AMP. A protein kinase rich fraction prepared from rat liver was also capable of restoring the sensitivity of diacylglycerol kinase-II to 5'-AMP. We propose that 5'-AMP-activated protein kinase is the mediator which inactivates diacylglycerol kinase-II, possibly by phosphorylation.

Sakane F, Imai S, Yamada K, Kanoh H
The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants.
Biochem Biophys Res Commun. 1991; 181: 1015-21
Display abstract
To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.

Sakane F, Yamada K, Imai S, Kanoh H
Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipid-dependent enzyme and undergoes calcium-dependent translocation.
J Biol Chem. 1991; 266: 7096-100
Display abstract
We attempted to assess the regulatory role of EF-hand motifs recently detected in the primary structure of porcine 80-kDa diacylglycerol kinase (DGK) (Sakane, F., Yamada, K., Kanoh, H., Yokoyama, C., and Tanabe, T. (1990) Nature 344, 345-348). By using 80-kDa DGK purified from porcine thymus cytosol, we found that this isozyme indeed bound 2 mol Ca2+ per mol enzyme with high affinity (apparent dissociation constant, kd = 0.3 microM). The Ca2+ binding was cooperative with a Hill coefficient of 1.4. We next studied the effect of 1 x 10(-5) M Ca2+ on the kinetic properties of DGK employing a beta-octyl glucoside mixed micellar assay system. In the absence of Ca2+, phosphatidylserine, so far used as an enzyme activator in various assay systems, was rather inhibitory, and Ca2+ alone activated enzyme to a limited extent. However, phosphatidylserine plus Ca2+ markedly activated the enzyme, giving approximately 4-fold higher Vmax and 10-fold less Km values for ATP. In contrast, the apparent Km values for diacylglycerol were not significantly affected (approximately 3 mol %). Furthermore, by immunoblotting using anti-80 kDa DGK antibodies we found that the soluble DGK in the homogenate of porcine thymocytes was translocated to membranes in a Ca2(+)-dependent manner. Indeed we noted the presence of a 33-residue amphipathic alpha-helix in the DGK sequence, which may account for the protein-lipid interaction. The results demonstrate that Ca2+ plays a key role in the regulation of DGK action by controlling enzyme interaction with membrane phospholipids.

Kolesnick RN, Hemer MR
Characterization of a ceramide kinase activity from human leukemia (HL-60) cells. Separation from diacylglycerol kinase activity.
J Biol Chem. 1990; 265: 18803-8
Display abstract
This laboratory first provided evidence for a potential signal transduction pathway involving sphingomyelin and its derivatives (Kolesnick, R.N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). Recently, this laboratory demonstrated the existence of the novel sphingolipid ceramide 1-phosphate in human leukemia (HL-60) cells. Ceramide 1-phosphate was synthesized from ceramide derived from sphingomyelin but not glycosphingolipids. This suggested that a specific pathway extended from sphingomyelin to ceramide 1-phosphate. The present studies provide additional support for this notion by demonstrating the existence of a ceramide kinase activity distinct from diacylglycerol (DG) kinase in HL-60 cells. Microsomal membranes contained a kinase activity that phosphorylated ceramide but not 1,2-DG in the presence of physiologic and higher Ca2+ concentrations (60 nM-3 mM). Kinetic analyses demonstrated an apparent Vmax for ceramide and ATP of 70 pmol.min-1.mg protein-1; apparent Km values were 45 and 25 microM, respectively. The pH optimum was within the physiologic range (pH 6-8). Magnesium but not other divalent cations (Mn2+, Ba2+, Cd2+, Zn2+) also stimulated ceramide phosphorylation. Magnesium also induced 1,2-DG phosphorylation. Since DG kinase is a Mg2(+)-stimulable enzyme that may utilize ceramide as substrate, additional studies separated calcium-dependent ceramide kinase from DG kinase activity. 1,2-DGs competitively inhibited magnesium- but not calcium-dependent ceramide phosphorylation. Hence, calcium-dependent ceramide kinase activity neither utilized DG as substrate nor was inhibited by DG. These activities were physically separable. Both activities were solubilized by n-octyl-beta-D-glucopyranoside and stabilized by glycerol. Ceramide kinase activity bound weakly to a DEAE-cellulose anion exchange column and eluted with 4-fold purification as a single peak of activity in the flow-through and 0.05 M NaCl elutions. In contrast, the majority of DG kinase activity bound more tightly and was recovered as a broad peak in the 0.2-0.35 M NaCl elutions. These studies demonstrate the existence of a ceramide kinase activity in HL-60 cells which is functionally and physically separable from DG kinase. These studies provide further support for the notion of a specific pathway from sphingomyelin to ceramide 1-phosphate.

Lemaitre RN, King WC, MacDonald ML, Glomset JA
Distribution of distinct arachidonoyl-specific and non-specific isoenzymes of diacylglycerol kinase in baboon (Papio cynocephalus) tissues.
Biochem J. 1990; 266: 291-9
Display abstract
We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.

Kanoh H, Sakane F, Yamada K
[Diacylglycerol kinase--a novel enzyme having both zinc finger and E-F hand motifs]
Tanpakushitsu Kakusan Koso. 1990; 35: 1558-63

Tang YM, Ashendel CL
Isolation of cloned mouse protein kinase C beta-II cDNA and its sequence.
Nucleic Acids Res. 1990; 18: 5310-5310

Kageyama H, Shimizu M, Tokunaga K, Hiwasa T, Sakiyama S
A partial cDNA for a novel protein which has a typical E-F hand structure.
Biochim Biophys Acta. 1989; 1008: 255-7
Display abstract
We cloned a partial cDNA which includes an open reading frame of 459 bp long from a mouse fibroblast cDNA library. The deduced amino-acid sequence showed a partial homology with several calcium-binding proteins. The clone possibly encodes a novel calcium-binding protein whose domain adopts the E-F hand structure.

Kikkawa U, Ono Y
[Study of the heterogeneity and structures of protein kinase C]
Seikagaku. 1989; 61: 551-68

Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y
Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties.
Proc Natl Acad Sci U S A. 1989; 86: 3099-103
Display abstract
The primary structure of the zeta subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The zeta subspecies of protein kinase C consists of 592 amino acid residues with the calculated molecular mass of 67,740 Da and has regulatory and protein kinase domains in its amino- and carboxyl-terminal halves, respectively. Although all members of the protein kinase C family so far identified have a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence in the regulatory domain, the zeta subspecies contains only one set of this sequence. Northern (RNA)-blot hybridization analysis indicated that two major RNA transcripts of the zeta subspecies with different lengths may be generated by the use of different polyadenylylational signals. The enzyme was expressed in COS-7 cells by transfection with the cDNA construct encoding its whole sequence. It showed an approximate molecular mass of 64,000 Da upon SDS/PAGE. The enzyme activity was significantly dependent on phospholipid but was independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein. The zeta subspecies expressed in COS-7 cells did not appear to show binding activity of phorbol ester. The structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other subspecies of protein kinase C. Perhaps, this subspecies belongs to another entity of the enzyme family.

Yamada K, Sakane F, Kanoh H
Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells.
FEBS Lett. 1989; 244: 402-6
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80 kDa diacylglycerol kinase (DGK) was immunoquantitated in cell homogenates and subcellular fractions. It was extremely abundant in the cytosol of various lymphocytes and comprised, in the highest case, more than 0.2% of the total soluble protein in T cell-enriched pig splenocytes. The lymphocyte membrane contained less than 10% of the total cellular DGK protein. The content of 80 kDa DGK in the human T cell leukemic cell line, Jurkat (360 ng/mg homogenate protein), was similar to those in pig and human peripheral blood lymphocytes. In contrast, the enzyme level was very low in the human promyeloblastic cell line, HL-60 (less than 10 ng/mg homogenate protein), and was undetectable in human polymorphonuclear leukocytes. These findings indicate that the content of 80 kDa DGK is markedly variable depending on the type of cells, even though all these cells are known to accumulate phosphatidate rapidly upon cell stimulation.

Russ E, Kaiser U, Sandermann H Jr
Lipid-dependent membrane enzymes. Purification to homogeneity and further characterization of diacylglycerol kinase from Escherichia coli.
Eur J Biochem. 1988; 171: 335-42
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1. Diacylglycerol kinase apoprotein was purified from membranes of Escherichia coli K12 by a six-step procedure that included HPLC. The proposed assignment of the enzyme to the dgkA gene [Lightner et al. (1983) J. Biol. Chem. 258, 10856-10861] could be supported by molecular mass determination (approximately 14 kDa), N-terminal sequencing (Met-Ala-Asn), cyanogen bromide fragmentation and amino acid analysis. As predicted, proline was absent. 2. The membrane-associated as well as the butan-1-ol-dissolved enzyme survived heating to 100 degrees C. 3. Alkylglycoside detergents were found to constitute an additional class of lipid activators. 4. The enzyme apoprotein in a non-activating substrate/detergent solution was capable of autocatalytic self-activation which was attributed to a novel feedback activation mechanism involving phosphatidic acid (diacylglycerol 3-phosphate).

Jeng IM, Klemm N, Wu CZ
An endogenous regulator of diacylglycerol kinase.
Biochem Biophys Res Commun. 1988; 151: 1088-92
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During the initial steps of the subcellular fractionation of rat brain homogenate, we recovered more than 100% of diacylglycerol kinase activity. The unusually high yields prompted us to examine the possibility that we had removed an endogenous inhibitor from diacylglycerol kinase during those steps. Our study revealed the existence of a potent inhibitor of diacylglycerol kinase in the crude synaptosomal-mitochondrial fraction (P2 pellet). The inhibitory substance was water soluble upon organic solvent extraction. The inhibitory activity of the substance was retained after extensive dialysis, suggesting the macromolecular nature of this compound. This substance may represent an important physiological regulator of diacylglycerol kinase.

Kanoh H, Ohno K
Partial purification and properties of diacylglycerol kinase from rat liver cytosol.
Arch Biochem Biophys. 1981; 209: 266-75

Konig B, Bohnenberger E, Sandermann H Jr
Hexamethylphosphoric triamide as a solubilizing agent. Purification and reactivation of diglyceride kinase.
FEBS Lett. 1981; 129: 301-4