Secondary literature sources for PTPc

The following references were automatically generated.

Gil-Henn H, Volohonsky G, Elson A
Regulation of protein-tyrosine phosphatases alpha and epsilon by calpain-mediated proteolytic cleavage.
J Biol Chem. 2001; 276: 31772-9
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The precise subcellular localization of non-receptor tyrosine phosphatases is a major factor in regulating their physiological functions. We have previously shown that cellular processing of protein-tyrosine phosphatase epsilon (PTP epsilon) generates a physiologically distinct, cytoplasmic form of this protein, p65 PTP epsilon. Here we describe a novel protein form of the related receptor-type tyrosine phosphatase alpha (RPTP alpha), p66 PTP alpha, which is detected in nearly all cell types where RPTP alpha is expressed. Both p66 PTP alpha and p65 PTP epsilon are produced by calpain-mediated proteolytic cleavage in vivo. Cleavage is inhibited in living cells by a variety of calpain inhibitors, can be induced in primary cortical neurons treated with calcium chloride, and is observed in lysates of brain or of cultured cells following addition of purified calpain. Cleavage occurs within the intracellular juxtamembrane domain of RPTP alpha, releasing the phosphatase catalytic domains from their membranal anchors and translocating them to the cytoplasm. Translocation reduces the ability of PTPalpha to act on membrane-associated substrates, as it loses its ability to dephosphorylate Src at its C-terminal regulatory site, and its ability to dephosphorylate the Kv2.1 voltage-gated potassium channel is severely impaired. In all, the data indicate that control of phosphatase function via post-translational processing occurs also among receptor-type phosphatases, and demonstrate the molecular complexity of regulating these parameters within the PTP alpha/PTP epsilon phosphatase subfamily.

Czyryca PG, Hengge AC
The mechanism of the phosphoryl transfer catalyzed by Yersinia protein-tyrosine phosphatase: a computational and isotope effect study.
Biochim Biophys Acta. 2001; 1547: 245-53
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In order to evaluate various mechanistic proposals that have been made regarding the mechanism of the first step of the reaction catalyzed by protein-tyrosine phosphatases, new experimental data have been obtained, and some existing data have been carefully reevaluated. New kinetic isotope effect data for the uncatalyzed hydrolysis of p-nitrophenyl phosphate allow a better evaluation of previously reported data from enzymatic reactions with this substrate. The interpretation, and misinterpretation, of pH rate studies is considered. The pathway of this reaction has been modeled computationally and is found to be generally consistent with experimental studies, except for the extent of proton transfer to the leaving group.

Zhao R, Qi Y, Chen J, Zhao ZJ
FYVE-DSP2, a FYVE domain-containing dual specificity protein phosphatase that dephosphorylates phosphotidylinositol 3-phosphate.
Exp Cell Res. 2001; 265: 329-38
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We have recently isolated FYVE-DSP1, a FYVE domain-containing dual specificity protein phosphatase (R. Zhao, Y. Qi, and Z. J. Zhao, Biochem. Biophys. Res. Commun. 270, 222--229 (2000)). Here, we report a novel isozyme that we designated FYVE-DSP2. FYVE-2 contains a single FYVE domain at the C-terminus, and it shares approximately 47% overall sequence identity with FYBE-DSP1. Genomic sequence analyses revealed that the FYVE-DSP1 and FYVE-DSP2 genes share similar intron/exon organization. They are localizedon human chromosome 22q12 and chromosome 17, respectively. Like FYVE-DSP1, recombinant FYVE-DSP2 dephosphorylated low-molecular-weight phosphatase substrate para-nitrophenylphosphate, and its activity was inhibited by sodium vanadate. More importantly, our study also revealed that both FYVE-DSP1 and FYVE-DSP2 efficiently and specifically dephosphorylated phosphotidylinositol 3-phosphate. Subcellular fractionation demonstrated partition of FYVE-DSP1 and FYVE-DSP2 in membrane fractions, and immunofluorescent cell staining showed perinuclear localization of the enzymes. FYVE-DSP2 is expressed in many human tissues with an alternatively spliced isoform expressed in the kidney. Together with two homologous hypothetical proteins found in Caenorhabditis elegans and Drosophila, FYVE-DSP1 and FYVE-DSP2 form a subfamilyof phosphatases that may have an importantrole in cellular processes. Copyright 2001 Academic Press.

Stebbins CE, Galan JE
Structural mimicry in bacterial virulence.
Nature. 2001; 412: 701-5
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An important mechanism underlying the strategies used by microbial pathogens to manipulate cellular functions is that of functional mimicry of host activities. In some cases, mimicry is achieved through virulence factors that are direct homologues of host proteins. In others, convergent evolution has produced new effectors that, although having no obvious amino-acid sequence similarity to host factors, are revealed by structural studies to display mimicry at the molecular level.

Ganesh S, Agarwala KL, Amano K, Suzuki T, Delgado-Escueta AV, Yamakawa K
Regional and developmental expression of Epm2a gene and its evolutionary conservation.
Biochem Biophys Res Commun. 2001; 283: 1046-53
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Lafora's disease, an autosomal recessive progressive myoclonus epilepsy, is caused by mutations in the EPM2A gene encoding a dual-specificity phosphatase (DSP) named laforin. Here, we analyzed the developmental and regional expression of murine Epm2a and discussed its evolutionary conservation. A phylogenetic analysis indicated that laforin is evolutionarily distant from other DSPs. Southern zoo blot analysis suggested that conservation of Epm2a gene is limited to mammals. Laforin orthologs (human, mouse, and rat) display more than 94% similarity. All missense mutations known in Lafora disease patients affect conserved residues, suggesting that they may be essential for laforin's function. Epm2a is expressed widely in various organs but not homogeneously in brain. The levels of Epm2a transcripts in mice brains increase postnatally, attaining its highest level in adults. The most intense signal was detected in the cerebellum, hippocampus, cerebral cortex, and the olfactory bulb. Our results suggest that Epm2a is functionally conserved in mammals and is involved in growth and maturation of neural networks. Copyright 2001 Academic Press.

Rhee J, Lilien J, Balsamo J
Essential tyrosine residues for interaction of the non-receptor protein-tyrosine phosphatase PTP1B with N-cadherin.
J Biol Chem. 2001; 276: 6640-4
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Expression of a dominant-negative, catalytically inactive form of the nonreceptor protein-tyrosine phosphatase PTP1B in L-cells constitutively expressing N-cadherin results in loss of N-cadherin-mediated cell-cell adhesion. PTP1B interacts directly with the cytoplasmic domain of N-cadherin, and this association is regulated by phosphorylation of tyrosine residues in PTP1B. The following three tyrosine residues in PTP1B are potential substrates for tyrosine kinases: Tyr-66, Tyr-152, and Tyr-153. To determine the tyrosine residue(s) that are crucial for the cadherin-PTP1B interaction we used site-directed mutagenesis to create catalytically inactive PTP1B constructs bearing additional single, double, or triple mutations in which tyrosine was substituted by phenylalanine. Mutation Y152F eliminates binding to N-cadherin in vitro, whereas mutations Y66F and Y153F do not. Overexpression of the catalytically inactive PTP1B with the Y152F mutation in L-cells constitutively expressing N-cadherin has no effect on N-cadherin-mediated adhesion, and immunoprecipitation reveals that the mutant Y152F PTP1B does not associate with N-cadherin in situ. Furthermore, among cells overexpressing the Y152F mutant endogenous PTP1B associates with N-cadherin and is tyrosine-phosphorylated.

Sorby M, Sandstrom J, Ostman A
An extracellular ligand increases the specific activity of the receptor-like protein tyrosine phosphatase DEP-1.
Oncogene. 2001; 20: 5219-24
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Cellular growth, differentiation and migration is regulated by protein tyrosine phosphorylation. Receptor-like protein tyrosine phosphatases are thus likely to be key regulators of vital cellular processes. The regulation of these enzymes is in general poorly understood. Ligands have been identified only for a small subset of the receptor-like protein tyrosine phosphatases and in no case has upregulation of the specific activity by extracellular ligands been demonstrated. Prompted by earlier findings of ligands for receptor-like protein tyrosine phosphatases in extracellular matrix we investigated if Matrigel, a preparation of extracellular matrix proteins, contained modulators of the specific activity of the receptor-like protein tyrosine phosphatase DEP-1. Matrigel stimulation of cells increased the specific activity of immunoprecipitated DEP-1. Also, incubation of immunoprecipitated DEP-1 with Matrigel led to an increase in DEP-1 activity, which was blocked by soluble DEP-1 extracellular domain. Finally, immunoprecipitated DeltaECD-DEP-1, a mutant form of DEP-1 lacking most of the extracellular domain, failed to respond to Matrigel stimulation. These experiments identify Matrigel as a source of DEP-1 agonist(s) and provide the first evidence for upregulation of the specific activity of receptor-like protein tyrosine phosphatases by extracellular ligands.

Stoker AW
Receptor tyrosine phosphatases in axon growth and guidance.
Curr Opin Neurobiol. 2001; 11: 95-102
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Receptor-like protein tyrosine phosphatases (RPTPs) continue to emerge as important signalling molecules in axons and their growth cones. Recent findings show that Drosophila RPTPs play key roles in guiding retinal axons and in preventing midline crossing of longitudinal axons. Vertebrate RPTPs are now implicated in controlling axon outgrowth, and preliminary evidence suggests that they too may influence axon guidance.

Tomemori T et al.
Isolation and characterization of murine orthologue of PTP-BK.
Biochem Biophys Res Commun. 2000; 276: 974-81
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We isolated and characterized a murine orthologue of PTP-BK, a receptor-type protein tyrosine phosphatase expressed in brain and kidney. The deduced amino acid sequences showed 89, 95, and 92% identities with human, rabbit, and chicken homologues of PTP-BK. Mouse PTP-BK encodes a polypeptide of 1,226 amino acids with calculated molecular weight of 138,598 Da. The mature form of PTP-BK constitutes of 3 domain structures including extracellular, transmembrane, and a single intracellular PTPase domain. Western blot analysis indicated that anti-PTP-BK antibody specifically immunoreacted to 2 distinct molecules of 200 kDa and 220 kDa in COS cells, possibly due to differential glycosylation. The recombinant PTP-BK showed the phosphatase activity specific for the phosphotyrosine, but not for the phosphoserine residue of phosphopeptides in vitro. Radiation hybrid panel assigned mouse PTP-BK gene to 5.02cR distal to from the marker D6Mit339 on chromosome 6. Mouse PTP-BK was classified in PTPRO family in novel nomenclature. We discuss here the diversity and physiological functions of PTPRO family of PTP.

Avraham H, Avraham S, Taniguchi Y
Receptor protein tyrosine phosphatases in hematopoietic cells.
J Hematother Stem Cell Res. 2000; 9: 425-32
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PTPs and PTKs control the level of tyrosine phosphorylation of cellular proteins. Although many substrates for PTKs have been identified, the specific targets of individual PTP family members, along with the consequences of protein dephosphorylation for cellular physiology, remain largely unknown. Fine regulation of tyrosine phosphorylation events is required for the proper progression of hematopoiesis. In this review, we have summarized the characterization of tyrosine phosphatases in hematopoietic cells and delineated their potential role in the process of hematopoiesis and the development of hematopoietic disorders.

Herrlich P, Bohmer FD
Redox regulation of signal transduction in mammalian cells.
Biochem Pharmacol. 2000; 59: 35-41
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This mini-review addresses the mechanism of ultraviolet-light-induced activation of receptor tyrosine kinases. The experimental approach into this mechanism revealed the existence of redox regulation of signal transduction in mammalian cells. It is postulated that, in addition to responsiveness to oxidative attacks from outside, redox regulation of specific redox-sensitive proteins likely represents an important physiological mechanism.

Tsuruta H, Aizono Y
Cloning of phosphatase I gene from a psychrophile, Shewanella sp., and some properties of the recombinant enzyme.
J Biochem (Tokyo). 2000; 127: 143-9
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Psychrophilic phosphatase I from Shewanella sp. is a cold enzyme that was found as a novel protein-tyrosine-phosphatase (PTPase, EC 3. 1.3.48) with a histidine as its catalytic residue [Tsuruta and Aizono (1999) J. Biochem. 125, 690-695]. Here, we determined the nucleotide sequence of a DNA fragment (2,004 bp) containing the phosphatase I gene by cloning with polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence, of the enzyme contained a conserved region of protein-serine/threonine-phosphatase (PPase). The 38.5 kDa-recombinant protein expressed in Escherichia coli was purified to homogeneity by glutathione-Sepharose 4B column chromatography, treatment with endoproteinase and Mono-Q column chromatography. The recombinant enzyme had a specific activity of 49.4 units and, like native psychrophilic phosphatase I, exhibited high catalytic activity at low temperature and PTPase activity.

Wang H et al.
Isolation of a novel protein tyrosine phosphatase inhibitor, 2-methyl-fervenulone, and its precursors from Streptomyces.
J Nat Prod. 2000; 63: 1641-6
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High-throughput screening identified an extract from Streptomyces sp. IM 2096 with inhibitory activity toward several protein tyrosine phosphatases (PTPs). Four 1,2,4-triazine compounds 2096A-D (1-4) were isolated from this extract and their structures elucidated by interpretation of spectroscopic data and confirmed by degradation and synthesis. The novel glycocyamidine derivatives 1 and 2 are diastereomers and may interconvert. Both are inactive in the PTP inhibition assay. Compounds 1 and 2 are unstable and partially decompose to 3 and glycocyamidine (5) at room temperature. Compound 3, known as MSD-92 or 2-methyl-fervenulone, is a broad-specificity PTP inhibitor with comparable potency to vanadate. The imidazo[4, 5-e]-1,2,4-triazine (4), inactive in the PTP-inhibition assay, may be a degradation product of 3.

Justement LB
Signal transduction via the B-cell antigen receptor: the role of protein tyrosine kinases and protein tyrosine phosphatases.
Curr Top Microbiol Immunol. 2000; 245: 1-51

Augustine KA et al.
Protein tyrosine phosphatase (PC12, Br7,S1) family: expression characterization in the adult human and mouse.
Anat Rec. 2000; 258: 221-34
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Protein tyrosine phosphatases (PTPs) play important roles in modulating signals transduced by tyrosine kinases. Certain phosphatases have been implicated as having important roles in embryonic development as well as in adult physiology. Although both kinases and phosphatases are equally important in regulating signal transduction, phosphatases as a group have not been well characterized. Thus, characterization of sequence, expression, and biological function for additional phosphatases is informative. PTPBr7/PC12 and PTPSl are mouse receptor PTPs sharing similar amino acid sequences. Northern blot analysis demonstrated expression of these genes in adult rodent brain and revealed previously uncharacterized transcripts in the brain and other tissues. Our results demonstrate that PTPBr7/PC12 and PTPSl are members of a larger family of PTPs. We have identified two novel family members as well as several novel transcriptional splice variants from both human and mouse colon cDNA libraries. Expression analysis demonstrated that the various mRNA transcripts are differentially expressed, with the highest levels found in the brain, intestinal tract, uterus, and placenta. In situ hybridization analysis of mouse brain and intestinal tissues established that each isoform has a unique expression pattern in specific cell populations as well as in tissue regions. Furthermore, these restricted patterns suggest that the encoded family of phosphatases may play roles in modulating signal transduction pathways important for specific cell types and biological processes.

Johnson TR, Biggs JR, Winbourn SE, Kraft AS
Regulation of dual-specificity phosphatases M3/6 and hVH5 by phorbol esters. Analysis of a delta-like domain.
J Biol Chem. 2000; 275: 31755-62
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Treatment of leukemic cells with phorbol 12-myristate 13-acetate (PMA) induces a short-lived phosphorylation and activation of stress-activated protein kinase (SAPK) and cellular differentiation. To investigate whether the rapid deactivation of SAPK results from dephosphorylation by dual-specificity phosphatases (DSPs), we studied regulation of the DSP hVH5 and its murine orthologue M3/6 in K562 human leukemia cells. PMA treatment rapidly induced hVH5 transcripts in these cells, and induced expression of M3/6 completely inhibited PMA-stimulated phosphorylation of SAPK, suggesting a feedback loop to control SAPK activity. Using both stable cell lines and transient transfection we demonstrate that activation of SAPK rapidly stimulated phosphorylation of M3/6. This phosphorylation did not regulate the half-life of total cellular M3/6. hVH5 and M3/6 shares with all sequenced mammalian DSPs an amino acid motif, XILPXLXL, located approximately 80 amino acids from the active site. The hVH5-M3/6 sequence, RILPHLYL, shares significant homology with the SAPK binding site of the c-Jun protein, called the delta domain. This motif was found to be important for DSP function, because deletion of RILPHLYL inhibits SAPK-mediated phosphorylation of M3/6, and deletion of this sequence or mutation of the LYL portion blocks the ability of this phosphatase to dephosphorylate SAPK.

Ibarra-Sanchez MJ, Simoncic PD, Nestel FR, Duplay P, Lapp WS, Tremblay ML
The T-cell protein tyrosine phosphatase.
Semin Immunol. 2000; 12: 379-86
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In recent years, the T-cell protein tyrosine phosphatase (TC-PTP) has become an important member of the protein-tyrosine phosphatase (PTP) family in two aspects. Firstly, TC-PTP has been reported to act on downstream signalling events initiated by the epidermal growth receptor, suggesting that it may act as an important modulator of receptor tyrosine kinases and mitogenic signalling. Secondly, the finding of immune deficiency and lethality observed in TC-PTP null mice emphasizes the importance of this small PTP in the hematopoietic system. In this review, we provide a summary of the recent literature published on the TC-PTP and its various orthologs. Although much remains to be uncovered, some recent findings on the function of this small PTP suggest that it plays a critical role in regulating mammalian cell signalling.

Dadke S, Kusari J, Chernoff J
Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region.
J Biol Chem. 2000; 275: 23642-7
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Protein-tyrosine phosphatases (PTPs) play a major role in regulating insulin signaling. Among the PTPs that regulate this signaling pathway, PTP1B plays an especially prominent role. PTP1B inhibits insulin signaling and has previously been shown to bind to the activated insulin receptor (IR), but neither the mechanism nor the physiological importance of such binding have been established. Here, we show that a previously undefined region in the N-terminal, catalytic half of PTP1B contributes to IR binding. Point mutations within this region of PTP1B disrupt IR binding but do not affect the catalytic activity of this phosphatase. This binding-defective mutant of PTP1B does not efficiently dephosphorylate the IR in cells, nor does it effectively inhibit IR signaling. These results suggest that PTP1B targets the IR through a novel binding element and that binding is required for the physiological effects of PTP1B on IR signal transduction.

Pasquali C, Vilbois F, Curchod ML, Hooft van Huijsduijnen R, Arigoni F
Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting.
Electrophoresis. 2000; 21: 3357-68
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Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.

Hoff RH, Hengge AC, Wu L, Keng YF, Zhang ZY
Effects on general acid catalysis from mutations of the invariant tryptophan and arginine residues in the protein tyrosine phosphatase from Yersinia.
Biochemistry. 2000; 39: 46-54
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General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

Avraham H, Park SY, Schinkmann K, Avraham S
RAFTK/Pyk2-mediated cellular signalling.
Cell Signal. 2000; 12: 123-33
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Intracellular signal transduction following extracellular ligation by a wide variety of surface molecules involves the activation and tyrosine phosphorylation of protein tyrosine kinases (PTKs). Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and tyrosine kinases, is a critical regulatory mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion PTK family consists of the focal adhesion kinase (FAK) and the RAFTK/Pyk2 kinase (also known as CAK-beta and CADTK). RAFTK/Pyk2 can be activated by a variety of extracellular signals that elevate intracellular calcium concentration, and by stress signals. RAFTK/Pyk2 is expressed mainly in the central nervous system and in cells derived from hematopoietic lineages, while FAK is widely expressed in various tissues and links transmembrane integrin receptors to intracellular pathways. This review describes the role of RAFTK/Pyk2 in various signalling cascades and details the differential signalling by FAK and RAFTK/Pyk2.

Zhan XL, Guan KL
A specific protein-protein interaction accounts for the in vivo substrate selectivity of Ptp3 towards the Fus3 MAP kinase.
Genes Dev. 1999; 13: 2811-27
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The mitogen-activated protein kinases (MAPKs) play critical roles in many signal transduction processes. Several MAPKs have been found in Saccharomyces cerevisiae, including Fus3 in the mating pathway and Hog1 in the osmotic-stress response pathway. Cells lacking Fus3 or Hog1 activity are deficient in mating or adaptation to osmotic shock, respectively. However, constitutive activation of either Fus3 or Hog1 is lethal. Therefore, yeast cells have to tightly regulate both the activation and inactivation of Fus3 and Hog1 MAPKs, which are controlled mainly by phosphorylation and dephosphorylation. Previous studies have shown that Fus3 activity is negatively regulated by protein tyrosine phosphatase Ptp3. In contrast, the Hog1 MAPK is mainly dephosphorylated by Ptp2 even though the two phosphatases share a high degree of sequence similarity. To understand the mechanisms of MAPK regulation, we examined the molecular basis underlying the in vivo substrate specificity between phosphatases and MAPKs. We observed that the amino-terminal noncatalytic domain of Ptp3 directly interacts with Fus3 via CH2 (Cdc25 homology) domain conserved among yeast PTPases and mammalian MAP kinase phosphatases and is responsible for the in vivo substrate selectivity of the phosphatase. Interaction between Ptp3 and Fus3 is required for dephosphorylation and inactivation of Fus3 under physiological conditions. Mutations in either Ptp3 or Fus3 that abolish this interaction cause a dysregulation of the Fus3 MAPK. Our data demonstrate that the specificity of MAP kinase inactivation in vivo by phosphatases is determined by specific protein-protein interactions outside of the phosphatase catalytic domain.

den Hertog J, Blanchetot C, Buist A, Overvoorde J, van der Sar A, Tertoolen LG
Receptor protein-tyrosine phosphatase signalling in development.
Int J Dev Biol. 1999; 43: 723-33
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Receptor Protein-Tyrosine Phosphatases (RPTPs) belong to the superfamily of protein-tyrosine phosphatases and have the intrinsic ability to transduce signals across the cell membrane. We are beginning to understand the role of RPTPs in development of invertebrates, due to elegant genetic studies. In contrast, relatively little is known about the role of RPTPs in vertebrate development. Signalling by RPTPs has predominantly been studied in mammalian cell systems, which has led to important insights into potential ligands, into regulation of RPTP activity and into potential RPTP substrates. Here, we will introduce the RPTPs, and discuss the function of the LAR-subfamily of RPTPs. In addition, we focus on the function and signalling of the haematopoietic RPTP, CD45. Finally, we will discuss the structure and function of RPTPalpha, the RPTP that is the subject of our studies.

Vincent C, Doublet P, Grangeasse C, Vaganay E, Cozzone AJ, Duclos B
Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb.
J Bacteriol. 1999; 181: 3472-7
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Two proteins of Escherichia coli, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate p-nitrophenyl phosphate into p-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that E. coli cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.

Umemori H, Hayashi T, Inoue T, Nakanishi S, Mikoshiba K, Yamamoto T
Involvement of protein tyrosine phosphatases in activation of the trimeric G protein Gq/11.
Oncogene. 1999; 18: 7399-402
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A variety of receptors coupled to the heterotrimeric guanine nucleotide-binding protein Gq/11 stimulate intracellular Ca2+ release through inositol (1,4,5)-trisphosphate (IP3) formation. We previously reported that tyrosine phosphorylation of the alpha subunit of the Gq/11 protein by protein tyrosine kinases (PTKs) regulates the activation of Gq/11 protein. Here we show that protein tyrosine phosphatases (PTPs) are also essential for Gq/11 protein activation. We find that Gq/11 protein-coupled receptor-mediated formation of IP3 is blocked by PTP inhibitors as well as PTK inhibitors. These inhibitors act prior to Gq/11 protein activation. Tyrosine phosphorylation of the alpha subunit of Gq/11 appears to inhibit its interaction with receptors. Thus, PTP is required for controlling the level of tyrosine phosphorylation of the alpha subunit of Gq/11 to promote its interaction with receptors. Therefore, we conclude that PTKs and PTPs co-operate to proceed activation cycle of the Gq/11 protein through tyrosine phosphorylation and de-phosphorylation of the alpha subunit of Gq/11.

Li C, Friedman JM
Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction.
Proc Natl Acad Sci U S A. 1999; 96: 9677-82
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Leptin exerts its weight-reducing effects by binding to its receptor and activating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an approach was developed in which bacterially expressed phosphorylated fragments of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of the cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosines of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin treatment. Affinity chromatography using a tyrosine-phosphorylated fragment spanning Tyr 985 of Ob-Rb was used to identify proteins that bind to this site. The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was isolated from bovine and mouse hypothalamus by using this method. After cotransfection of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2 into 293T cells, leptin results in direct binding of SHP-2 to the phosphorylated Tyr 985. The bound SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y-->F 985 receptor mutant is cotransfected. In the absence of SHP-2 phosphorylation, the level of JAK2 phosphorylation was increased. Tyrosyl phosphorylation of the leptin receptor and signal transducer and activater of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation of SHP-2 by the leptin receptor results in a decreased phosphorylation of JAK2 and may act to attenuate leptin signal transduction. The method used in this report can in principle be used to isolate additional components of the leptin, or other, signal transduction pathway.

Deshpande T, Takagi T, Hao L, Buratowski S, Charbonneau H
Human PIR1 of the protein-tyrosine phosphatase superfamily has RNA 5'-triphosphatase and diphosphatase activities.
J Biol Chem. 1999; 274: 16590-4
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A human cDNA was isolated encoding a protein with significant sequence similarity (41% identity) to the BVP RNA 5'-phosphatase from the Autographa californica nuclear polyhedrosis virus. This protein is a member of the protein-tyrosine phosphatase (PTP) superfamily and is identical to PIR1, shown by Yuan et al. (Yuan, Y., Da-Ming, L., and Sun, H. (1998) J. Biol. Chem. 272, 20347-20353) to be a nuclear protein that can associate with RNA or ribonucleoprotein complexes. We demonstrate that PIR1 removes two phosphates from the 5'-triphosphate end of RNA, but not from mononucleotide triphosphates. The specific activity of PIR1 with RNA is several orders of magnitude greater than that with the best protein substrates examined, suggesting that RNA is its physiological substrate. A 120-amino acid segment C-terminal to the PTP domain is not required for RNA phosphatase activity. We propose that PIR1 and its closest homologs, which include the metazoan mRNA capping enzymes, constitute a subgroup of the PTP family that use RNA as a substrate.

Keng YF, Wu L, Zhang ZY
Probing the function of the conserved tryptophan in the flexible loop of the Yersinia protein-tyrosine phosphatase.
Eur J Biochem. 1999; 259: 809-14
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The involvement of the strictly conserved Trp354 residue in the catalysis of the Yersinia protein tyrosine phosphatase (PTPase) has been investigated by site-directed mutagenesis and kinetic studies. Crystallographic structural data have revealed that Trp354 interacts with the active site Arg409 and is located at one of the hinge positions of the flexible surface loop (WpD loop) which also harbors the general acid/base (Asp356) essential for catalysis [Schubert, H. L., Fauman, E. B., Stuckey, J. A., Dixon, J. E. & Saper, M. A. (1995) Protein Sci. 4, 1904-1913]. Two mutants were constructed and expressed that contained the Trp354-->Phe and Trp354-->Ala substitutions. The K(m) of the W354F and W354A mutants were not significantly different from that of the wild-type. However, a major decrease in the affinity for oxyanions was observed for the mutants, which is consistent with Trp354 playing a role in aligning Arg409 for oxyanion binding. In addition replacement of Trp354 with Phe or Ala caused a decrease in kcat of 200-fold and 480-fold, respectively, and impaired the ability of the mutant enzymes to stabilize the negative charge in the leaving group at the transition state. In fact, the W354F and W354A mutants exhibited catalytic efficiency and leaving group dependency similar to those observed for the general acid-deficient PTPase D356N. These results indicate that Trp354 is an important residue that keeps the WpD loop in a catalytically competent conformation and positions the general acid/base Asp356 in the correct orientation for proton transfer.

Hermel JM, Dirkx R Jr, Solimena M
Post-translational modifications of ICA512, a receptor tyrosine phosphatase-like protein of secretory granules.
Eur J Neurosci. 1999; 11: 2609-20
Display abstract
The autoantigen of type I diabetes ICA512 is a receptor tyrosine phosphatase-like protein enriched in the secretory granule membranes of neurons and peptide secreting endocrine cells. While the function of ICA512 remains unknown, it is thought to link regulated neuropeptide and peptide hormone secretion with signal transduction pathways involving tyrosine phosphorylation/dephosphorylation. To characterize further its biochemical properties, we conducted studies in the bovine pituitary, an abundant source of native ICA512, as well as in fibroblasts transfected with various human ICA512 cDNA constructs. Based on these studies we have established that the signal peptide of ICA512 encompasses residues 1-34 and that the ectodomain of ICA512 undergoes multiple post-translation modifications, including N-glycosylation. Newly synthesized ICA512 appears first as a pro-protein of 110 kDa that is then converted by post-translational modifications into a 130-kDa species. Cleavage of pro-ICA512 at a consensus for furin-like convertases generates a 60-66-kDa ICA512 transmembrane fragment (amino acids 449-979). Such processing ICA512 is not restricted to neuroendocrine cells, as it can also occur in transfected fibroblasts. Finally, the predicted N-terminal fragment of ICA512 resulting from this cleavage (amino acids 35-448) or parts thereof are present in the neurosecretosomes of posterior pituitary, raising the possibility that they may be secreted upon exocytosis of secretory granules.

Gaya A et al.
CD148, a new membrane tyrosine phosphatase involved in leukocyte function.
Leuk Lymphoma. 1999; 35: 237-43
Display abstract
Protein tyrosine phosphatases play an essential role in the control of leucocyte cell growth an differentiation. Recently a new receptor type membrane tyrosine phosphatase named CD148 has been identified. This molecule is present on the membrane of all the hematopoietic lineages as well as on several other cell types, mainly epithelial cells and its expression increases after cell activation. This molecule is able to act as a transducing molecule. Moreover, CD148 is able to modulate the signal transduction through the TCR/CD3 complex, in a manner similar to CD45. It has also been suggested that CD148 could be involved in mechanisms of differentiation and inhibition of cell growth. In addition, CD148 seems to be associated with a serine/threonine kinase in certain epithelial cell lines and leucocytes. Here, we review recent data on the expression and function of CD148 in both human, mouse and rat.

Thomas ML
The regulation of antigen-receptor signaling by protein tyrosine phosphatases: a hole in the story.
Curr Opin Immunol. 1999; 11: 270-6
Display abstract
Antigen-receptor signaling requires Src-family kinases to initiate tyrosine phosphorylation. CD45 dephosphorylates the inhibitory site in Src-family kinases before antigen-receptor engagement and thus serves to 'prime' the kinases. It has been unclear why CD45 does not also dephosphorylate 'activated' kinases or motifs within the cytoplasmic domains of antigen-receptors and thus prevent signal transduction. Recent reports raise the possibility that CD45 is excluded from engaged antigen-receptors by mechanisms that may include the formation of lipid microdomains.

Roknabadi SM, Bose SK, Taneja V
A histidine thiol 100 kDa, tetrameric acid phosphatase from lentil, Lens esculenta, seeds with the characteristics of protein tyrosine phosphatases.
Biochim Biophys Acta. 1999; 1433: 272-80
Display abstract
A non-specific acid phosphatase (APase) hydrolysing L-tyrosine-O-phosphate and 3'-AMP was purified to electrophoretic homogeneity from mature lentil seeds with apparent native molecular mass of 100 kDa and subunit molecular mass of 24 kDa. These activities appear to reside on the same protein which shows a single band in native and SDS-PAGE. The pH optimum is 5.5, while the K(m) (mM) and V(max) (micromoles/min/mg protein) for p-nitrophenyl phosphate (pNPP) are 0.7 and 9.2 and for L-tyrosine-O-phosphate 1.4 and 10.1, respectively, at 30 degrees C and for 3'-AMP, 2 and 4.4 at 37 degrees C. The protein also hydrolyses other phosphomonoesters to a lesser extent. L-Tyrosine-O-phosphate, 3'-AMP and pNPP hydrolysis is potently inhibited by micromolar orthovanadate and also to nearly the same extent by sodium fluoride, potassium tartrate and metal ions. Histidine and cysteine are likely to be involved in the catalysis. Thermal inactivation studies indicate that the active site conformations for pNPP and 3'-AMP hydrolytic activities are different. The enzyme shows the characteristics of the animal protein tyrosine phosphatase.

Zhang YL et al.
Impaired transition state complementarity in the hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases.
Biochemistry. 1999; 38: 12111-23
Display abstract
The hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases (PTPases) was studied with the aim of providing a mechanistic framework for the reactions of this important class of substrate analogues. O-arylphosphorothioates are hydrolyzed 2 to 3 orders of magnitude slower than O-aryl phosphates by PTPases. This is in contrast to the solution reaction where phosphorothioates display 10-60-fold higher reactivity than the corresponding oxygen analogues. Kinetic analyses suggest that PTPases utilize the same active site and similar kinetic and chemical mechanisms for the hydrolysis of O-arylphosphorothioates and O-aryl phosphates. Thio substitution has no effect on the affinity of substrate or product for the PTPases. Bronsted analyses suggest that like the PTPase-catalyzed phosphoryl transfer reaction the transition state for the PTPase-catalyzed thiophosphoryl transfer is highly dissociative, similar to that of the corresponding solution reaction. The side chain of the active-site Arg residue forms a bidentate hydrogen bond with two of the terminal phosphate oxygens in the ground state and two of the equatorial oxygens in a transition state analog complex with vanadate [Denu et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2493-2498; Zhang, M. et al. (1997) Biochemistry 36, 15-23; Pannifer et al. (1998) J. Biol. Chem. 273, 10454-10462]. Replacement of the active-site Arg409 in the Yersinia PTPase by a Lys reduces the thio effect by 54-fold, consistent with direct interaction and demonstrating strong energetic coupling between Arg409 and the phosphoryl oxygens in the transition state. These results suggest that the large thio effect observed in the PTPase reaction is the result of inability to achieve precise transition state complementarity in the enzyme active site with the larger sulfur substitution.

Cohen S, Dadi H, Shaoul E, Sharfe N, Roifman CM
Cloning and characterization of a lymphoid-specific, inducible human protein tyrosine phosphatase, Lyp.
Blood. 1999; 93: 2013-24
Display abstract
Protein tyrosine phosphatases act in conjunction with protein kinases to regulate the tyrosine phosphorylation events that control cell activation and differentiation. We have isolated a previously undescribed human phosphatase, Lyp, that encodes an intracellular 105-kD protein containing a single tyrosine phosphatase catalytic domain. The noncatalytic domain contains four proline-rich potential SH3 domain binding sites and an NXXY motif that, if phosphorylated, may be recognized by phosphotyrosine binding (PTB) domains. Comparison of the Lyp amino acid sequence with other known proteins shows 70% identity with the murine phosphatase PEP. The human Lyp gene was localized to chromosome 1p13 by fluorescence in situ hybridization analysis. We also identified an alternative spliced form of Lyp RNA, Lyp2. This isoform encodes a smaller 85-kD protein with an alternative C-terminus. The lyp phosphatases are predominantly expressed in lymphoid tissues and cells, with Lyp1 being highly expressed in thymocytes and both mature B and T cells. Increased Lyp1 expression can be induced by activation of resting peripheral T lymphocytes with phytohemagglutinin or anti-CD3. Lyp1 was found to be constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. Overexpression of lyp1 reduces Cbl tyrosine phosphorylation, suggesting that it may be a substrate of the phosphatase. Thus, Lyp may play a role in regulating the function of Cbl and its associated protein kinases.

Blery M, Vivier E
How to extinguish lymphocyte activation, immunotyrosine-based inhibition motif (ITIM)-bearing molecules a solution?
Clin Chem Lab Med. 1999; 37: 187-91
Display abstract
ITIM-bearing molecules represent a novel family of inhibitory receptors expressed widely through the hematopoietic compartment. These molecules share certain features such as the presence in their intracytoplasmic domain of the so-called motif ITIM (ImmunoTyrosine-based Inhibition Motif). These molecules are able to recruit phosphatases on their phosphorylated ITIM and thus mediate a localized inhibition of the transduction pathways. The molecular basis of this inhibitory pathway is discussed below.

Hunter T
The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease.
Philos Trans R Soc Lond B Biol Sci. 1998; 353: 583-605
Display abstract
The reversible phosphorylation of tyrosines in proteins plays a key role in regulating many different processes in eukaryotic organisms, such as growth control, cell cycle control, differentiation cell shape and movement, gene transcription, synaptic transmission, and insulin action. Phosphorylation of proteins is brought about by enzymes called protein-tyrosine kinases that add phosphate to specific tyrosines in target proteins; phosphate is removed from phosphorylated tyrosines by enzymes called protein-tyrosine phosphatases. Phosphorylated tyrosines are recognized by specialized binding domains on other proteins, and such interactions are used to initiate intracellular signaling pathways. Currently, more than 95 protein-tyrosine kinases and more than 55 protein-tyrosine phosphatase genes are known in Homo sapiens. Aberrant tyrosine phosphorylation is a hallmark of many types of cancer and other human diseases. Drugs are being developed that antagonize the responsible protein-tyrosine kinases and phosphatases in order to combat these diseases.

Wang Q, Huang Z, Ramachandran C, Dinaut AN, Taylor SD
Naphthalenebis[alpha,alpha-difluoromethylenephosphonates] as potent inhibitors of protein tyrosine phosphatases.
Bioorg Med Chem Lett. 1998; 8: 345-50
Display abstract
A series of naphthalenebis(difluoromethylenephosphonates) were prepared and compared to their monosubstituted counterparts as inhibitors of the protein phosphatases, PTP1B, CD45 and PP2A. In general, the bissubstituted compounds were better inhibitors than the mono derivatives and some of these are among the most potent, nonpeptidyl inhibitors of protein tyrosine phosphatases, PTP1B and CD45, reported to date.

Cui X, De Vivo I, Slany R, Miyamoto A, Firestein R, Cleary ML
Association of SET domain and myotubularin-related proteins modulates growth control.
Nat Genet. 1998; 18: 331-7
Display abstract
Several proteins that contribute to epigenetic mechanisms of gene regulation contain a characteristic motif of unknown function called the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain. We have demonstrated that SET domains mediate highly conserved interactions with a specific family of proteins that display similarity with dual-specificity phosphatases (dsPTPases). These include myotubularin, the gene of which is mutated in a subset of patients with X-linked myotubular myopathy, and Sbf1, a newly isolated homologue of myotubularin. In contrast with myotubularin, Sbf1 lacks a functional catalytic domain which dephosphorylates phospho-tyrosine and serine-containing peptides in vitro. Competitive interference of endogenous SET domain-dsPTPase interactions by forced expression of Sbf1 induced oncogenic transformation of NIH 3T3 fibroblasts and impaired the in vitro differentiation of C2 myoblast cells. We conclude that myotubularin-type phosphatases link SET-domain containing components of the epigenetic regulatory machinery with signalling pathways involved in growth and differentiation.

Zhang YL, Keng YF, Zhao Y, Wu L, Zhang ZY
Suramin is an active site-directed, reversible, and tight-binding inhibitor of protein-tyrosine phosphatases.
J Biol Chem. 1998; 273: 12281-7
Display abstract
The effect of suramin, a well known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers, on protein-tyrosine phosphatases (PTPases) has been examined. Suramin is a reversible and competitive PTPase inhibitor with Kis values in the low microM range, whereas the Kis for the dual specificity phosphatase VHR is at least 10-fold higher. Although suramin can also inhibit the activity of the potato acid phosphatase at a slightly higher concentration, it is 2-3 orders of magnitude less effective against the protein Ser/Thr phosphatase 1alpha and the bovine intestinal alkaline phosphatase. Suramin binds to the active site of PTPases with a binding stoichiometry of 1:1. Furthermore, when suramin is bound to the active site of PTPases, its fluorescence is enhanced approximately by 10-fold. This property has allowed the determination of the binding affinity of suramin for PTPases and several catalytically impaired mutant PTPases by fluorescence titration techniques. Thus, the active site Cys to Ser mutants bind suramin with similar affinity as the wild type, while the active site Arg to Ala mutant exhibits a 20-fold reduced affinity toward suramin. Interestingly, the general acid deficient Asp to Ala mutant PTPases display an enhanced affinity toward suramin, which is in accord with their use as improved "substrate-trapping" agents. That suramin is a high affinity PTPase inhibitor is consistent with the observation that suramin treatment of cancer cell lines leads to an increase in tyrosine phosphorylation of several cellular proteins. Given the pleiotropic effects of suramin on many enzyme systems and growth factor-receptor interactions, the exact in vivo actions of suramin require further detailed structure-activity investigation of suramin and its structural analogs.

Takada T, Matozaki T, Kasuga M
[The signal transduction of receptor tyrosine kinase]
Nippon Rinsho. 1998; 56: 1756-62
Display abstract
Polypeptides such as growth factors, differentiation factors, and hormones are crucial components of the regulatory system that coordinates development of multicellular organisms. Many of these factors mediate their pleiotropic actions by binding to and activating cell surface receptors with an intrinsic protein tyrosine kinase activity. The receptor activation due to ligands binding are translated across the membrane barrier into activation of intracellular domain functions. All receptor tyrosine kinase are composed of three major domains; an extracellular domain connected via a single membrane-spanning domain to a cytoplasmic domain. The extracellular domain is responsible for ligand binding and transmission of the biological signal to the cytoplasmic domain, whose role is to transmit the biological signal to intracellular target proteins. The cytoplasmic domain contains, in addition to the catalytic protein tyrosine kinase, distinct regulatory sequences with tyrosine, serine, and threonine phosphorylation sites. It appears that ligand-induced activation of the kinase domain and its signaling potential are mediated by receptor oligomerization. Ligand binding and the subsequent conformational alteration of the extracellular domain induce receptor oligomerization, which stabilizes interaction between adjacent cytoplasmic domains and leads to activation of kinase function and autophosphorylation of themselves. These receptor and substrate phosphorylation create binding sites for SH2 containing signaling molecule, such as Grb2, Shc, PI3 kinase and SHP-2. Binding of SH2 domains to tyrosine-phosphorylated regions of receptors or adaptor proteins, and a number of protein, such as SH3 containing protein, cytosol protein tyrosine kinase, protein tyrosine phosphatase and serine/threonine kinase, mediate intracellular signaling cascade and play critical roles in activated receptor protein tyrosine kinase to downstream signaling pathways.

Drake PG, Posner BI
Insulin receptor-associated protein tyrosine phosphatase(s): role in insulin action.
Mol Cell Biochem. 1998; 182: 79-89
Display abstract
Protein tyrosine phosphatases (PTPs) play a critical role in regulating insulin action in part through dephosphorylation of the active (autophosphorylated) form of the insulin receptor (IRK) and attenuation of its tyrosine kinase activity. Following insulin binding the activated IRK is rapidly internalized into the endosomal apparatus, a major site at which the IRK is dephosphorylated in vivo. Studies in rat liver suggest a complex regulatory process whereby PTPs may act, via selective IRK tyrosine dephosphorylation, to modulate IRK activity in both a positive and negative manner. Use of peroxovanadium (pV) compounds, shown to be powerful PTP inhibitors, has been critical in delineating a close relationship between the IRK and its associated PTP(s) in vivo. Indeed the in vivo administration of pV compounds effected activation of IRK in parallel with an inhibition of IRK-associated PTP activity. This process was accompanied by a lowering of blood glucose levels in both normal and diabetic rats thus implicating the IRK-associated PTP(s) as a suitable target for defining a novel class of insulin mimetic agents. Identification of the physiologically relevant IRK-associated PTP(s) should facilitate the development of drugs suitable for managing diabetes mellitus.

Goldstein BJ, Ahmad F, Ding W, Li PM, Zhang WR
Regulation of the insulin signalling pathway by cellular protein-tyrosine phosphatases.
Mol Cell Biochem. 1998; 182: 91-9
Display abstract
Protein-tyrosine phosphatases (PTPases) have been implicated in the physiological regulation of the insulin signalling pathway. In cellular and molecular studies, the transmembrane, receptor-type PTPase LAR and the intracellular, non-receptor enzyme PTP1B have been shown to have a direct impact on insulin action in intact cell models. Since insulin signalling can be enhanced by reducing the abundance or activity of specific PTPases, pharmaceutical agents directed at blocking the interaction between individual PTPases and the insulin receptor may have potential clinical relevance to the treatment of insulin-resistant states such as obesity and Type II diabetes mellitus.

Byon JC, Kusari AB, Kusari J
Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction.
Mol Cell Biochem. 1998; 182: 101-8
Display abstract
Insulin signaling involves a dynamic cascade of protein tyrosine phosphorylation and dephosphorylation. Most of our understanding of this process comes from studies focusing on tyrosine kinases, which are signal activators. Our knowledge of the role of protein-tyrosine phosphatases (PTPases), signal attenuators, in regulating insulin signal transduction remains rather limited. Protein-tyrosine phosphatase 1B (PTP-1B), the prototypical PTPase, is ubiquitously and abundantly expressed. Work from several laboratories, including our own, has implicated PTP-1B as a negative regulator of insulin action and as a potentially important mediator in the pathogenesis of insulin-resistance and non-insulin dependent diabetes mellitus (NIDDM).

Yano H, Sato T
[FAP-1: a protein tyrosine phosphatase that is involved in Fas-mediated apoptosis]
Tanpakushitsu Kakusan Koso. 1998; 43: 1193-9

Hinoda Y, Idogawa M, Imai K
[Involvement of protein tyrosine phosphatases in cancer development]
Tanpakushitsu Kakusan Koso. 1998; 43: 1186-92

Kuroyanagi H, Tagawa M, Shirasawa T
[Structure, expression and functions of receptor-type protein tyrosine phosphatase RPTP-BK in central nervous system]
Tanpakushitsu Kakusan Koso. 1998; 43: 1162-8

Seimiya H, Tsuruo T
[Involvement of PTP-U2 in terminal differentiation and apoptosis of leukemia cells]
Tanpakushitsu Kakusan Koso. 1998; 43: 1153-61

Watanabe T, Kume T, Mukouyama Y
[The roles of protein tyrosine phosphatases during hematopoietic progenitor cell development and differentiation]
Tanpakushitsu Kakusan Koso. 1998; 43: 1144-52

Mizuno K
[Role of tyrosine phosphatases in neuronal differentiation]
Tanpakushitsu Kakusan Koso. 1998; 43: 1136-43

Bucciantini M, Stefani M, Taddei N, Chiti F, Rigacci S, Ramponi G
Sequence-specific recognition of peptide substrates by the low Mr phosphotyrosine protein phosphatase isoforms.
FEBS Lett. 1998; 422: 213-7
Display abstract
A number of phosphotyrosine-containing peptides derived from the PDGF receptor phosphorylation sites have been synthesised. The peptides were assayed as substrates of the two isoforms (IF1 and IF2) of the low Mr PTPase. The calculated k(cat), Km, and k(cat)/Km values indicate that only one peptide is best hydrolysed by IF2 (but not IF1), whose catalytic efficiency averages those previously reported for most PTPases (except the Yersinia enzyme). This peptide is the only one containing a couple of no bulky hydrophobic residues at the phosphotyrosine N-side. The determination of the same catalytic parameters in the presence of analogues of the best hydrolysed peptide in which one or both hydrophobic residues were replaced by Asp or Lys residues confirmed the importance of the hydrophobic cluster at the phosphotyrosine N-side for optimal enzymatic hydrolysis. These findings are discussed in the light of the known IF2 X-ray structure.

Black DS, Montagna LG, Zitsmann S, Bliska JB
Identification of an amino-terminal substrate-binding domain in the Yersinia tyrosine phosphatase that is required for efficient recognition of focal adhesion targets.
Mol Microbiol. 1998; 29: 1263-74
Display abstract
YopH is a protein tyrosine phosphatase (PTP) that is delivered into host mammalian cells via a type III secretion pathway in pathogenic Yersinia species. Although YopH is a highly active PTP, it preferentially targets a subset of tyrosine-phosphorylated proteins in host cells, including p130Cas. Previous in vitro studies have indicated that the carboxy-terminal PTP domain contributes specificity to the interaction of YopH with substrates. However, it is not known if the PTP domain is sufficient for substrate recognition by YopH. Here, we have identified paxillin as an additional substrate of YopH in HeLa cells. In addition, we have identified a domain in the amino-terminal region of YopH that binds to both p130Cas and paxillin and is required for the efficient recognition of substrates by the wild-type enzyme. This 'substrate-binding' domain exhibits a ligand specificity that is similar to that of the Crk Src homology 2 (SH2) domain, and it binds substrates directly in a phosphotyrosine-dependent manner. The substrate-binding domain of YopH may represent a novel type of protein-protein interaction module, as it lacks significant sequence similarity with any known SH2 or phosphotyrosine-binding (PTB) domain.

Liu F, Sells MA, Chernoff J
Protein tyrosine phosphatase 1B negatively regulates integrin signaling.
Curr Biol. 1998; 8: 173-6
Display abstract
Protein tyrosine phosphatase (PTP) 1B has long been known to regulate cell proliferation negatively, but the mechanism by which this inhibition occurs is poorly defined. We have shown previously that PTP1B binds to, and dephosphorylates, p130(Cas) (Crk-associated substrate) [1], a protein that is thought to play a role in integrin signaling [2,3]. In this report, we present evidence that PTP1B interferes specifically with cell-adhesion-stimulated, but not growth-factor-stimulated, signaling pathways. In rat fibroblasts that overexpress PTP1B, the activation of mitogen-activated protein (MAP) kinase by growth factors was not affected, but activation by cell adhesion was markedly impaired. The inhibition of adhesion-dependent MAP kinase activation by PTP1B required an intact proline-rich region in the carboxyl terminus of PTP1B, a region we have shown to mediate binding to the Src-homology 3 (SH3) domain of p130Cas [1]. Overexpression of wild-type PTP1B, but not of a proline-to-alanine mutant form (PA-PTP1B) that is unable to bind or dephosphorylate p130Cas, interfered with cell spreading, cytoskeletal architecture, and the formation of focal adhesion complexes. Cells overexpressing wild-type PTP1B also displayed markedly reduced migration in response to a fibronectin gradient, whereas cells expressing the PA-PTP1B mutant migrated normally. These data indicate that PTP1B exerts its inhibitory effects via proline-dependent interactions with one or more critical components of the adhesion-dependent signaling apparatus, and suggest that one of these components may be p130Cas.

Bell SD, Denu JM, Dixon JE, Ellington AD
RNA molecules that bind to and inhibit the active site of a tyrosine phosphatase.
J Biol Chem. 1998; 273: 14309-14
Display abstract
Protein tyrosine phosphatases (PTPases) are essential proteins in many cellular processes. In vitro selection was used to evolve high affinity RNA aptamers to the Yersinia PTPase from two random pools varying in length. Selected aptamers from the two different pools share a 21-residue conserved sequence. They bind to their target with dissociation constants of 18 and 28 nM and inhibit the enzyme with IC50 values of 10 and 35 nM, but do not bind a related PTPase. Modification of the PTPase's active site cysteine with the alkylating agent iodoacetate results in a loss of binding affinity. These experiments suggest that the selected aptamers act by binding at or near the active site and might therefore be useful in defining the interactions between PTPases and their targets.

Denu JM, Tanner KG
Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation.
Biochemistry. 1998; 37: 5633-42
Display abstract
Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphotyrosine from specific signal-transducing proteins. Although regulatory mechanisms for protein kinases have been described, no general mechanism for controlling PTPs has been demonstrated. Numerous reports have shown that cellular redox status plays an important role in tyrosine phosphorylation-dependent signal transduction pathways. This study explores the proposal that PTPs may be regulated by reversible reduction/oxidation involving cellular oxidants such as hydrogen peroxide (H2O2). Recent reports indicated that H2O2 is transiently generated during growth factor stimulation and that H2O2 production is concomitant with relevant tyrosine phosphorylation. By use of recombinant enzymes, the effects of H2O2 on three PTPs [PTP1, LAR (leukocyte antigen-related), and VHR (vaccinia H1-related)] and three distinct serine/threonine protein phosphatases (PPs: PP2Calpha, calcineurin, and lambda phosphatase) were determined. Hydrogen peroxide had no apparent effect on PP activity. In contrast, PTPs were rapidly inactivated (kinact = 10-20 M-1 s-1) with low micromolar concentrations of H2O2 but not with large alkyl hydroperoxides. PTP inactivation was fully reversible with glutathione and other thiols. Because of the slower rate of reduction, modification occurred even in the presence of physiological thiol concentrations. By utilization of a variety of biochemical techniques including chemical modification, pH kinetic studies, and mutagenesis, the catalytic cysteine thiolate of PTPs was determined to be the selective target of oxidation by H2O2. By use of the electrophilic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl), it was shown that a cysteine sulfenic acid intermediate (Cys-SOH) is formed after attack of the catalytic thiolate on H2O2. A chemical mechanism for reversible inactivation involving a cysteine sulfenic acid intermediate is proposed.

Hunter T
The role of tyrosine phosphorylation in cell growth and disease.
Harvey Lect. 1998; 94: 81-119

Fitzgerald LR, Walton KM, Dixon JE, Largent BL
PTP NE-6: a brain-enriched receptor-type protein tyrosine phosphatase with a divergent catalytic domain.
J Neurochem. 1997; 68: 1820-9
Display abstract
A receptor-type protein tyrosine phosphatase, PTP NE-6, was identified from rat olfactory epithelial cDNA and cloned from a rat brain cDNA library. PTP NE-6 mRNA is abundant in brain and expressed at lower levels in olfactory tissue and adrenal gland. In situ hybridization demonstrates that PTP NE-6 mRNA is expressed throughout the brain, with highest levels in the medial habenula and at intermediate levels in layer IV of cortex, medial geniculate nucleus, inferior colliculus, hypothalamus, and thalamus. The predicted amino acid sequence demonstrates that PTP NE-6 contains a single catalytic domain that diverges from the consensus protein tyrosine phosphatase catalytic domain by expressing an aspartate instead of the conserved alanine residue in the catalytic site. Recombinantly expressed PTP NE-6 does not exhibit detectable phosphatase activity. Upon mutation of the aspartate to the consensus alanine, phosphatase activity toward p-nitrophenyl phosphate is observable with a k(cat) value of 3.7 s(-1) and a Km of 980 microM. These data demonstrate that the inactivity of native PTP NE-6 toward p-nitrophenyl phosphate is due to the divergent aspartate in the catalytic site and not to variant amino acids within the phosphatase domain.

Desai CJ, Krueger NX, Saito H, Zinn K
Competition and cooperation among receptor tyrosine phosphatases control motoneuron growth cone guidance in Drosophila.
Development. 1997; 124: 1941-52
Display abstract
The neural receptor tyrosine phosphatases DPTP69D, DPTP99A and DLAR are involved in motor axon guidance in the Drosophila embryo. Here we analyze the requirements for these three phosphatases in growth cone guidance decisions along the ISN and SNb motor pathways. Any one of the three suffices for the progression of ISN pioneer growth cones beyond their first intermediate target in the dorsal muscle field. DLAR or DPTP69D can facilitate outgrowth beyond a second intermediate target, and DLAR is uniquely required for formation of a normal terminal arbor. A different pattern of partial redundancy among the three phosphatases is observed for the SNb pathway. Any one of the three suffices to allow SNb axons to leave the common ISN pathway at the exit junction. When DLAR is not expressed, however, SNb axons sometimes bypass their ventrolateral muscle targets after leaving the common pathway, instead growing out as a separate bundle adjacent to the ISN. This abnormal guidance decision can be completely suppressed by also removing DPTP99A, suggesting that DLAR turns off or counteracts a DPTP99A signal that favors the bypass axon trajectory. Our results show that the relationships among the tyrosine phosphatases are complex and dependent on cellular context. At growth cone choice points along one nerve, two phosphatases cooperate, while along another nerve these same phosphatases can act in opposition to one another.

Lohse DL, Denu JM, Santoro N, Dixon JE
Roles of aspartic acid-181 and serine-222 in intermediate formation and hydrolysis of the mammalian protein-tyrosine-phosphatase PTP1.
Biochemistry. 1997; 36: 4568-75
Display abstract
Protein tyrosine phosphatases (PTPases) share a number of conserved amino acid residues, including the active site sequence HCXXGXXRS(T), which are strongly implicated in catalysis. The roles of two conserved active site residues, Asp-181 and Ser-222, were investigated using a combination of site-directed mutagenesis and kinetic analysis in the mammalian PTPase PTP1. The pH profiles for k(cat)/K(m) and k(cat) of the wild-type enzyme indicate that two ionizable groups, of pK(a) values 5.1 and 5.44, must be deprotonated and one group with a pK(a) value of 4.93 must be protonated for maximal activity. The group of pK(a) value 5.1 is the second ionization of the substrate phosphate moiety. Selective thiolate anion inactivation indicates the residue with pK(a) value of 5.44 is C215. The pH-dependent profiles of the D181N mutant during establish the residue with pK(a) value of 4.93 to be Asp-181 and suggest that it functions as a general acid phosphoryl transfer to the enzyme. Rapid reaction kinetics of wild-type and D181N mutant enzymes indicate that the formation of the phospho-enzyme intermediate is rate-limiting at pH 7.0 and 30 degrees C. Enzymes containing the S222A mutation exhibited rapid reaction burst kinetics, strongly suggesting that phospho-enzyme intermediate hydrolysis is fully rate-limiting. The role of the active site S222 is to accelerate the rate of phospho-enzyme intermediate hydrolysis. The kinetic analysis of a third mutant, containing both the D181N and S222A mutations, suggests that D181 also serves as a general base in the breakdown of the phospho-enzyme intermediate.

Unkeless JC, Jin J
Inhibitory receptors, ITIM sequences and phosphatases.
Curr Opin Immunol. 1997; 9: 338-43
Display abstract
A diverse group of inhibitory receptors, including FcgammaRII, killer cell inhibitory receptors, and B22, shares an immunoreceptor tyrosine-based inhibition motif (ITIM). Recent studies have shown that this motif, when phosphorylated on tyrosine, forms a docking site for the Src homology 2 recognition domains of the protein tyrosine phosphatase SHP-1 and the inositol 5-phosphatase SHIP. A similar motif in cytotoxic T-lymphocyte antigen-4 recruits the related tyrosine phosphatase SHP-2. These three enzymes act to inhibit signaling cascades resulting from ligation of the BCR, TCR, FcgammaRIII, and FcepsilonRI, although the relative importance of the tyrosine phosphatases and the inositol phosphatase differs depending on the cell type.

Black DS, Bliska JB
Identification of p130Cas as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions.
EMBO J. 1997; 16: 2730-44
Display abstract
A number of pathogenic bacteria utilize type III secretion pathways to translocate virulence proteins into host eukaryotic cells. We identified a host target of YopH, a protein tyrosine phosphatase that is translocated into mammalian cells by Yersiniae. A catalytically inactive 'substrate-trapping' mutant, YopHC403S, was used as a probe to determine where YopH substrates localize in eukaryotic cells. Immunofluorescence microscopy demonstrated that YopHC403S localized to focal adhesions in human epithelial cells infected with Y. pseudotuberculosis. YopHC403S stabilized focal adhesions, as shown by its dominant-negative effect on focal adhesion disassembly mediated by YopE, a translocated protein which disrupts actin stress fibers. Conversely, YopH destabilized focal adhesions, even in the absence of YopE, as shown by loss of phosphotyrosine staining. Immunoprecipitation revealed that YopHC403S was trapped in a complex with a hyperphosphorylated 125-135 kDa protein, identified by immunoblotting as the focal adhesion protein p130Cas. YopHC403S bound directly to p130Cas in a phosphotyrosine-dependent manner in vitro. Translocation of YopH into cells plated on fibronectin resulted in rapid and selective dephosphorylation of p130Cas. These results demonstrate that YopH targets focal adhesions in host cells and that p130Cas, a docking protein for multiple SH2 domains, is a direct substrate of this enzyme in vivo.

Nanney LB, Davidson MK, Gates RE, Kano M, King LE Jr
Altered distribution and expression of protein tyrosine phosphatases in normal human skin as compared to squamous cell carcinomas.
J Cutan Pathol. 1997; 24: 521-32
Display abstract
Amounts and subcellular localizations of 4 protein tyrosine phosphatases (PTPs) were compared in cultured normal human keratinocytes, an immortalized keratinocyte cell line, and 2 squamous cell carcinoma (SCC) lines. Cellular localizations for PTPs were determined in biopsies of normal human skin and SCCs. Compared to normal keratinocytes, SCC cell lines had higher levels of PTP-1B and T-cell PTP and comparable levels of PTP-1C or PTP-1D. The subcellular localization of each PTP was similar in the 3 types of keratinocytes with PTP-1B localizing to the endoplasmic reticulum, T-cell PTP exclusively found in the nucleus, PTP-1C localized to the plasma membrane, cytosol and nucleus, and PTP-1D present in both cytosol and nucleus. Compared to normal skin, immunoreactive PTP-1B was markedly increased in the invasive margins of SCCs while T-cell PTP was generally increased in tumors. PTP-1C immunostaining varied between cells with no obvious difference between normal and neoplastic tissues. The intensity and distribution of immunoreactive PTP-1D varied greatly between cells within tumors. These differences in amounts and in cellular and subcellular localization of these PTPs, especially those differences in invasive margins of SCCs, may reflect the diverse roles these PTPs play in the proliferation and invasive potential of neoplastic keratinocytes.

Lim KL, Lai DS, Kalousek MB, Wang Y, Pallen CJ
Kinetic analysis of two closely related receptor-like protein-tyrosine-phosphatases, PTP alpha and PTP epsilon.
Eur J Biochem. 1997; 245: 693-700
Display abstract
Among transmembrane protein-tyrosine-phosphatases, the membrane distal catalytic domain (D2) of protein-tyrosine-phosphatase alpha (PTP alpha) is unusual in having low but detectable activity in the absence of the membrane proximal catalytic domain (D1). To investigate the catalytic properties of PTP alpha D2 in association with D1, kinetic parameters of activity were established for PTP alpha D1D2 proteins containing an inactivating point mutation in D1 and/or D2. In this context, D2 activity was unchanged by the presence (N-terminal or C-terminal) or absence of inactive D1, and the presence or absence of inactive D2 affected the velocity but not the Km of D1 catalysis. While D1 appears to be the major catalytic contributor to PTP alpha activity, D2 possesses a significantly higher substrate-specific activity relative to wild-type D1D2 than the D2 domains of other protein-tyrosine-phosphatases. Also, PTP alpha D2 is an active phosphatase with comparable or better efficiency, on the basis of k(cat)/Km criteria, to some of the dual specificity phosphatases. Kinetic parameters of a closely related receptor-like protein-tyrosine-phosphatase, PTP epsilon, were determined. PTP epsilon D1 is the major, if not the only, catalytic moiety of PTP epsilon, and has much higher turnover numbers than D1 of PTP alpha. The PTP epsilon D2 activity is insignificant compared to that of PTP epsilon-D1D2, with lower turnover numbers than PTP alpha D2. Thus, the intrinsic activity of PTP alpha D2 is high compared to other D2 domains and, more outstandingly, its activity relative to D1 appears unique. These are also apparent upon in vitro assay of full-length PTP alpha catalytic mutants expressed in mammalian cells. Together. these results suggest potential catalytic and regulatory roles for PTP alpha D2, and that PTP alpha may be an optimal model transmembrane protein-tyrosine-phosphatase for investigating the former within the cell.

Zhang ZY, Wu L
The single sulfur to oxygen substitution in the active site nucleophile of the Yersinia protein-tyrosine phosphatase leads to substantial structural and functional perturbations.
Biochemistry. 1997; 36: 1362-9
Display abstract
Protein-tyrosine phosphatases (PTPases) feature an essential nucleophilic thiol group which attacks the phosphorus atom in a substrate. A single S to O atom substitution in the nucleophile (via Cys to Ser mutation) renders PTPases catalytically inactive. We suggest that the lack of activity in the Cys to Ser mutant may be caused by structural and/or conformational perturbations in the active site. Yersinia PTPase contains a single tryptophan residue, Trp354, which is invariant among all PTPases and is located in the vicinity of the active site nucleophile Cys403. Thus, Trp354 serves as an intrinsic probe of the PTPase active site conformation. We show that although C403S displays a nearly identical circular dichroism spectrum to that of the wild type enzyme, its ultraviolet spectrum in the region attributed to Trp is significantly different from that of the wild-type enzyme. In addition, the intrinsic fluorescence intensity of C403S is enhanced 3-fold and exhibits different ionic strength dependency from that of the wild-type enzyme. Trp354 also has different accessibilities to quenchers in the wild-type and the C403S mutant PTPases. Furthermore, unfolding experiments demonstrate that the structure of C403S is significantly less stable than the wild-type PTPase and displays a different sensitivity to urea and guanidine hydrochloride. Finally, binding of tungstate enhances the fluorescence of the wild-type Yersinia PTPase with a Kd of 55 microM, whereas binding of tungstate quenches the fluorescence of the C403S mutant with a Kd of 690 microM. Collectively, these results indicate that the single sulfur to oxygen change in the active site nucleophile leads to substantial structural/conformational and functional alterations in the Yersinia PTPase.

Hengge AC, Zhao Y, Wu L, Zhang ZY
Examination of the transition state of the low-molecular mass small tyrosine phosphatase 1. Comparisons with other protein phosphatases.
Biochemistry. 1997; 36: 7928-36
Display abstract
The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 +/- 0.0005 for 18(V/K)bridge, 1.0007 +/- 0.0001 for 15(V/K), and 1.0018 +/- 0.0003 for 18(V/K)nonbridge. The values for 18(V/K)nonbridge and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)bridge of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)nonbridge of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

Skorey K et al.
How does alendronate inhibit protein-tyrosine phosphatases?
J Biol Chem. 1997; 272: 22472-80
Display abstract
Alendronate (4-amino-1-hydroxybutylidene 1,1-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The inhibition of protein-tyrosine phosphatases (PTPs) by alendronate suggests that PTPs may be molecular targets. As a clear understanding of the inhibition mechanism is lacking, our aim was to analyze the mechanism to provide further insight into its therapeutic effect. We show here that the inhibition of PTPs by alendronate in the presence of calcium followed first-order kinetic behavior, and kinetic parameters for the process were determined. Evidence is presented that the inhibition by alendronate/calcium is active site-directed. However, this process was very sensitive to assay constituents such as EDTA and dithiothreitol. Furthermore, the inhibition of PTPs by alendronate/calcium was eliminated by the addition of catalase. These observations suggest that a combination of alendronate, metal ions, and hydrogen peroxide is responsible for the inhibition of PTPs. The individual effects of alendronate, calcium, or hydrogen peroxide on the inactivation of CD45 were determined. Electrospray ionization mass spectrometry demonstrated that the mass of PTP1B increased by 34 +/- 2 units after the enzyme was inactivated with alendronate/calcium, due to the oxidization of the catalytic cysteine to sulfinic acid (Cys-SO2H). The inhibited PTP1B could be partially reactivated by treatment with reducing agents such as hydroxylamine (NH2OH) and N,N'-dimethyl-N, N'-bis(mercaptoacetyl)hydrazine, indicating the presence of other oxidized forms such as sulfenic acid (Cys-SOH). This further confirms that the inhibition is the result of oxidation of the catalytic cysteine. The relevance of this oxidative inhibition mechanism in a biological system is discussed.

Garton AJ, Burnham MR, Bouton AH, Tonks NK
Association of PTP-PEST with the SH3 domain of p130cas; a novel mechanism of protein tyrosine phosphatase substrate recognition.
Oncogene. 1997; 15: 877-85
Display abstract
The protein tyrosine phosphatase PTP-PEST displays remarkable substrate specificity, in vitro and in vivo for p130cas a signalling intermediate implicated in mitogenic signalling, cell-adhesion induced signalling, and in transformation by a variety of oncogenes. We have identified a high affinity interaction between the SH3 domain of p130cas and a proline-rich sequence (P335PPKPPR) within the C-terminal segment of PTP-PEST. Mutation of proline 337 within this sequence to alanine significantly impairs the ability of PTP-PEST to recognise tyrosine phosphorylated p130cas as a substrate, without qualitatively affecting the selectivity of the interaction. Thus the highly specific nature of the interaction between PTP-PEST and p130cas appears to result from a combination of two distinct substrate recognition mechanisms; the catalytic domain of PTP-PEST contributes specificity to the interaction with p130cas, whereas the SH3 domain-mediated association of p130cas and PTP-PEST dramatically increases the efficiency of the interaction. Furthermore, our results indicate that one important function of the p130cas SH3 domain is to associate with PTP-PEST and thereby facilitate the dephosphorylation of p130cas, resulting in the termination of tyrosine phosphorylation-dependent signalling events downstream of p130cas.

Bone H, Dechert U, Jirik F, Schrader JW, Welham MJ
SHP1 and SHP2 protein-tyrosine phosphatases associate with betac after interleukin-3-induced receptor tyrosine phosphorylation. Identification of potential binding sites and substrates.
J Biol Chem. 1997; 272: 14470-6
Display abstract
The cytoplasmic tyrosine phosphatases, SHP1 and SHP2, are implicated in the control of cellular proliferation and survival. Here we demonstrate that both SHP1 and SHP2 associate with the betac subunit of the human interleukin-3 (IL-3) receptor following IL-3 stimulation and that the src homology region 2 (SH2) domains of these phosphatases mediate this interaction. Sequential immunoprecipitation analyses suggest this interaction is direct. Competition studies, using phosphotyrosine-containing peptides based on sequences surrounding key tyrosine residues within betac, suggest that phosphorylation of tyrosine 612 is the key event mediating the association of betac with SHP1 and SHP2. However, inhibition of SHP2 binding to betac, did not prevent tyrosine phosphorylation of SHP2. Interestingly, this same phosphopeptide served as a substrate for the tyrosine phosphatase activity of both SHP1 and SHP2. Binding of these protein-tyrosine phosphatases to the IL-3 receptor may regulate IL-3 signal transduction pathways, both through their catalytic activity and through the recruitment of other molecules to the receptor complex.

Adamson RH
Protein tyrosine phosphorylation modulates microvessel permeability in frog mesentery.
Microcirculation. 1996; 3: 245-7

Hendriks W et al.
Protein-tyrosine phosphatases expressed in mouse epidermal keratinocytes.
J Invest Dermatol. 1996; 106: 972-6
Display abstract
The importance of growth factors acting via receptor-type protein-tyrosine kinases in the continuous renewal of the epidermis from the keratinocyte stem cell population has been well established. Protein-tyrosine phosphatases (PTPases), which dephosphorylate phosphotyrosine-containing proteins, may therefore be expected to play an equally important role in the control of epidermal growth and differentiation. In this study, we have made an inventory of the various PTPases that are expressed during mouse keratinocyte proliferation and maturation. A panel of 13 different PTPases probes was obtained by combining a set of PTPase cDNAs previously cloned from mouse brain and a set of PTPase probes obtained from a normalized keratinocyte PTPase cDNA library. This PTPase cDNA panel, spanning probes for receptor-type as well as cytoplasmic-type family members, was used to monitor RNA expression levels in keratinocyte fractions isolated from murine epidermis and in keratinocyte cell cultures. No overt changes were observed in PTPase mRNA levels in all strata of mouse epidermis, but comparison of cultured cells with freshly isolated keratinocytes revealed several conspicuous differences. In the cultured Balb/MK cell line, absence of PTP delta expression and upregulation of PTP kappa and, to a lesser extent, PTP gamma mRNA ratios were observed compared to the freshly isolated cells. These results provide a basis for further research on the impact of PTPase activity on epidermal growth control.

Gottlin EB et al.
Kinetic analysis of the catalytic domain of human cdc25B.
J Biol Chem. 1996; 271: 27445-9
Display abstract
The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118-5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the kcat/Km for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is approximately 20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s-1) and dephosphorylation (1.5 s-1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

MacKintosh C et al.
Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation.
FEBS Lett. 1996; 397: 235-8
Display abstract
The gamma1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1gamma) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates. In contrast, PP1gamma dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1gamma were inhibited by vanadate with IC50 values (30-100 microM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1gamma lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72. These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.

Galvan B, Christopoulos TK
Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity.
Clin Biochem. 1996; 29: 125-31
Display abstract
OBJECTIVE: To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity. METHODS: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer. RESULTS: The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay. CONCLUSIONS: The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

Gates RE, Miller JL, King LE Jr
Activity and molecular weight of protein tyrosine phosphatases in cell lysates determined by renaturation after gel electrophoresis.
Anal Biochem. 1996; 237: 208-15
Display abstract
A procedure for renaturing and detecting the activity of protein tyrosine phosphatases (PTPases) after sodium dodecyl sulfate (SDS)-gel electrophoresis with greatly improved sensitivity and resolution is described. Epidermal growth factor receptor-kinase, c-src kinase, and focal adhesion kinase were phosphorylated on tyrosine with 32PO4 and incorporated into gels prior to electrophoresis. These proteins are dephosphorylated when cellular proteins are electrophoresed and the separated PTPases are renatured in the gel by removing SDS with extensive washing. With whole cell lysates, at least eight separate bands of decreased radioactivity corresponding to PTPase activity with molecular weights between 110 and 34 kDa are seen in autoradiographs of the dried gels. PTPases detected are similar with different cell types and with the three 32P-labeled protein substrates, although they are different in cytosolic and membrane-associated fractions. A PTPase detected above 200 kDa in wheat germ agglutinin eluates from solubilized cells suggests that some receptor PTPases can be renatured. While microgram levels of recombinant PTP-1C are required for detection, nanogram levels of recombinant PTP-1B are easily detected. Assaying the activity of renatured PTPases after they have been separated by molecular weight in SDS gel electrophoresis provides a simple and rapid means of determining the activity of individual PTPases in cell fractions.

Lee K, Nichols J, Smith A
Identification of a developmentally regulated protein tyrosine phosphatase in embryonic stem cells that is a marker of pluripotential epiblast and early mesoderm.
Mech Dev. 1996; 59: 153-64
Display abstract
A sensitive RT-PCR display technique was used to examine the expression of protein tyrosine phosphatases (PTPs) during the differentiation of mouse embryonic stem (ES) cells. The majority of PTPs are expressed constitutively but one is present only in undifferentiated ES cells. This PTP was cloned and named ES cell phosphatase (ESP). ESP mRNA is detectable in oocytes and throughout early mouse embryo development. At early egg cylinder stages, transcripts are localised in the pluripotential epiblast. As gastrulation commences, however, epiblast expression is lost. Transcripts are present transiently in newly formed embryonic mesoderm. These data suggest that this transmembrane signaling molecule is associated with developmental lability in early embryogenesis.

Zhang ZY, Walsh AB, Wu L, McNamara DJ, Dobrusin EM, Miller WT
Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1.
J Biol Chem. 1996; 271: 5386-92
Display abstract
Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a phosphotyrosine-containing peptide derived from epidermal growth factor autophosphorylation site Tyr992 (EGFR988 998). This peptide photoinactivated PTP1 in a time- and concentration-dependent manner. Three lines of evidence indicate that the interaction between PTP1 and the photoaffinity label was specific: 1) photoinactivation was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr992 in molar excess. 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic constants similar to those of the non-Bpa-containing peptide under identical conditions. 3) After complete photoinactivation, the level of incorporation of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry of photolabeling. Radiolabeled peptide was used to identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and radioactive fragments were purified by high performance liquid chromatography (HPLC) and analyzed by Edman sequencing. In two parallel experiments which were analyzed using different HPLC columns, a site in the alpha2 region of PTP1, most likely Ile23, was labeled by the Tyr992-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of substrate recognition must exist for PTP1 catalysis.

den Hertog J, Overvoorde J, de Laat SW
Expression of receptor protein-tyrosine phosphatase alpha mRNA and protein during mouse embryogenesis.
Mech Dev. 1996; 58: 89-101
Display abstract
Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is a transmembrane member of the family of protein-tyrosine phosphatases (PTPs) that has been implicated in neuronal differentiation in vitro. Here we demonstrate that RPTP alpha is differentially expressed during mouse embryogenesis in a spatio-temporal manner. RPTP alpha expression was detectable in 6 days post coitum (dpc) embryos, but not in 7.5 dpc embryos. From 10.5 dpc onwards a striking RPTP alpha expression pattern was observed with elevated levels in the dorsal root ganglia, cranial ganglia and adrenal gland, suggesting that RPTP alpha levels are specifically enhanced in neural crest derivatives. Marked differences between RPTP alpha mRNA and protein levels indicated that RPTP alpha expression is regulated by transcriptional and (post-) translational mechanisms. The expression pattern of RPTP alpha suggests that RPTP alpha may play a role in neural crest cell differentiation in vivo.

Monteiro HP, Stern A
Redox modulation of tyrosine phosphorylation-dependent signal transduction pathways.
Free Radic Biol Med. 1996; 21: 323-33
Display abstract
The main purpose of this review article is to provide a better understanding of the role of oxidants as modulators/mediators of tyrosine phosphorylation-dependent signal transduction pathways. It is generally accepted that reversible phosphorylation of protein tyrosine residues by polypeptide growth factor receptor protein tyrosine kinases (e.g., epidermal growth factor receptor, platelet derived growth factor receptor, insulin receptor) is a signalling mechanism implicated in cell proliferation, adhesion, differentiation, transformation, and apoptosis. It is controlled by the opposing actions of protein tyrosine kinases and protein tyrosine phosphatases. Nevertheless, increasing amounts of experimental data indicate that intracellular redox state plays a major role in the mechanisms underlying the actions of growth factors. Furthermore, redox active species mediate signalling processes on their own. Thus, in this article we attempted to discuss these points, presenting our published as well as unpublished contribution to the field.

Bult A et al.
STEP61: a member of a family of brain-enriched PTPs is localized to the endoplasmic reticulum.
J Neurosci. 1996; 16: 7821-31
Display abstract
The STEP family of protein tyrosine phosphatases is highly enriched within the CNS. Members of this family are alternatively spliced to produce both transmembrane and cytosolic variants. This manuscript describes the distinctive intracellular distribution and enzymatic activity of the membrane-associated isoform STEP61. Transfection experiments in fibroblasts, as well as subcellular fractionations, sucrose density gradients, immunocytochemical labeling, and electron microscopy in brain tissue, show that STEP61 is an intrinsic membrane protein of striatal neurons and is associated with the endoplasmic reticulum. In addition, structural analysis of the novel N-terminal region of STEP61 reveals several motifs not present in the cytosolic variant STEP46. These include two putative transmembrane domains, two sequences rich in Pro, Glu, Asp, Ser, and Thr (PEST sequences), and two polyproline-rich domains. Like STEP46, STEP61 is enriched in the brain, but the recombinant protein has less enzymatic activity than STEP46. Because STEP46 is contained in its entirety within STEP61 and differs only in the extended N terminus of STEP61, this amino acid sequence is responsible for the association of STEP61 with membrane compartments and may also regulate its enzymatic activity.

Chien CB
PY in the fly receptor-like tyrosine phosphatases in axonal pathfinding.
Neuron. 1996; 16: 1065-8

Wu L, Zhang ZY
Probing the function of Asp128 in the lower molecular weight protein-tyrosine phosphatase-catalyzed reaction. A pre-steady-state and steady-state kinetic investigation.
Biochemistry. 1996; 35: 5426-34
Display abstract
The role of Asp128 in the catalytic mechanism of the low Mr protein-tyrosine phosphatase (PTPase) from the fission yeast Schizosaccharomyces pombe has been investigated by a combination of site-directed mutagenesis and pre-steady-state and steady-state kinetic analysis. The corresponding aspartic acid in the bovine enzyme is located on a loop adjacent to the phosphate-binding loop and forms a hydrogen bond with the oxygen atom of the bound sulfate or phosphate that is structurally homologous to the ester oxygen in substrates [Su et al. (1994) Nature 370, 575-578; Zhang, M., et al. (1994) Biochemistry 33, 11097-11105]. Asp128 has been replaced by a Glu, an Asn, and an Ala. The kcat for the hydrolysis of p-nitrophenyl phosphate (pNPP) decreases by factors of 6.7, 400, and 650 for the mutants D128E, D128N, and D128A, respectively. Compared to the wild type, the binding affinity for phosphate is decreased 2 and 4.3-fold, respectively, for the D128A and D128N mutants, whereas no change in affinity is observed for the D128E mutant. An evaluation of the burst kinetics demonstrates that Asp128 plays a role in both the phosphoenzyme intermediate formation (k2) and breakdown (k3). Thus, substitution at Asp128 by a Glu, an Asn, or an Ala reduces k2 by 17, 7480, and 11900 and reduces k3 by 6.2, 380, and 40. The greater effect on k2 and k3 is consistent with a dissociative transition-state for the low M(r)PTPase-catalyzed reaction. Results from the rapid kinetics, partition experiments, and leaving group dependence experiments suggest that for the wild type and D128E mutant, the rate-limiting step is k3, whereas k2 has become rate-limiting for the D128N mutant. With the exception of pNPP, k2 may also be rate-limiting for D128A. Taken together, these results are consistent with Asp128 or Glu128 acting as a general acid to donate a proton to the phenolate leaving group in the phosphorylation step, and the carboxylate side chain plays a role as a general base to activate a nucleophilic water molecule in the dephosphorylation step. The presence of the general acid ensures productive partitioning toward phosphoenzyme formation. In the absence of the general acid, the nature of the transition-state for the phosphorylation step is sensitive to the pKa of the attacking active site thiol group and changes with the structure of the leaving group.

Ng DH, Harder KW, Clark-Lewis I, Jirik F, Johnson P
Non-radioactive method to measure CD45 protein tyrosine phosphatase activity isolated directly from cells.
J Immunol Methods. 1995; 179: 177-85
Display abstract
Preparation of radioactive phosphorylated substrates is laborious, yields a limited amount of substrate with a short half-life and generates a low percentage of phosphorylated product which then has to be separated from non-phosphorylated material. These factors limit the usefulness of radioactive phosphorylated substrates in phosphatase assays and prohibit their use for kinetic analysis, which often requires large amounts of substrate. An alternative method for the kinetic analysis of purified or recombinant soluble phosphatases uses the malachite green reagent which can detect nanomoles of phosphate released from chemically synthesized phosphorylated peptides. In this report we describe a rapid and sensitive non-radioactive method that can be used to measure protein tyrosine phosphatase (PTP) activities of both transmembrane and soluble phosphatases immunoprecipitated directly from cells. This colorimetric microassay is performed in 96 well microtitre plates and can reliably detect 100 pmol of free phosphate released, using a standard microplate reader. The phosphatase activity of CD45, a transmembrane PTP, was determined from as few as 1 x 10(4) lymphoid cells. The development of this colorimetric assay to measure immunoprecipitated CD45 PTP activity isolated from very small numbers of cells has general applicability for other PTPs and will help identify the cellular situations and conditions that result in changes in PTP activity.

Burridge K, Nelson A
An in-gel assay for protein tyrosine phosphatase activity: detection of widespread distribution in cells and tissues.
Anal Biochem. 1995; 232: 56-64
Display abstract
A method is described for the detection of protein tyrosine phosphatase activity in sodium dodecyl sulfate-polyacrylamide gels. A radiolabeled substrate, 32P-labeled poly(glutamic acid-tyrosine) (random copolymer) is incorporated into gels prior to polymerization. Following electrophoresis, the sodium dodecyl sulfate is removed; the proteins are fully denatured by soaking gels in 6 M guanidine hydrochloride and then renatured by incubation in buffers containing 0.04% Tween 40 and high concentrations of reducing agents. Protein tyrosine phosphatase activity is detected in autoradiographs of dried gels as regions from which the 32P has been selectively removed. Electrophoresis of known cytoplasmic protein tyrosine phosphatases indicates activity as the predicted molecular weights. As little as 10 pg of some cytoplasmic phosphatases is detectable. However, transmembrane tyrosine phosphatases, such as CD45, are detected only at very high protein loadings in this assay. Electrophoresis of whole cell lysates indicates multiple bands of tyrosine phosphatase activity, some of which comigrate with known cytoplasmic protein tyrosine phosphatases. The activity is inhibited by sodium orthovanadate or the omission of reducing agents during the renaturation process. The assay has been used to analyze embryonic and adult tissues, as well as whole cell lysates. A similar profile of bands of tyrosine phosphatase activity is seen with many different cells and tissues. However, some that are highly differentiated, such as adult skeletal muscle, erythrocytes, or sperm, reveal either a reduced level of tyrosine phosphatase activity or a simplified profile of bands.

Lorenzen JA, Dadabay CY, Fischer EH
COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus.
J Cell Biol. 1995; 131: 631-43
Display abstract
The noncatalytic domain of the human T cell protein tyrosine phosphatase (TCPTP) is alternatively spliced to generate a 45-kD form, p45TC, and a 48-kD form, p48TC (Champion-Arnaud et al., 1991; Mosinger et al., 1992). This manuscript concerns structural motifs in the noncatalytic segment of the enzyme responsible for targeting the two forms to different subcellular compartments. Endogenous and transiently expressed p48TC associates with the ER, as determined by sucrose gradient fractionation and indirect immunofluorescence, respectively. By contrast, p45TC localizes in the nucleus even though upon cell lysis it is not retained and fractionates with markers for soluble enzymes. Using fusion proteins consisting of beta-galactosidase and COOH-terminal fragments of p48TC, two motifs necessary for ER retention within a 70-residue targeting segment have been identified. These include the terminal 19 hydrophobic residues which comprise a potential membrane-spanning segment and residues 346-358 which encompass a cluster of basic amino acids that may represent another type of ER retention motif. The sequence RKRKR, which immediately precedes the splice junction, functions as a nuclear localization signal for p45TC.

Zhang ZY
Kinetic and mechanistic characterization of a mammalian protein-tyrosine phosphatase, PTP1.
J Biol Chem. 1995; 270: 11199-204
Display abstract
The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat protein-tyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques. Product inhibition and 18O exchange experiments are consistent with the enzymatic reaction proceeding through two chemical steps, i.e. formation and breakdown of a covalent phosphoenzyme intermediate. The variation of kcat/Km with pH indicates that three ionizable groups are involved in enzyme substrate binding and catalysis. The first group must be deprotonated and is attributed to the second ionization of the substrate. The other two groups with pK alpha values of 5.1 and 5.5 correspond to two enzyme active site residues. The kcat-pH profiles for both p-nitrophenyl phosphate and beta-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK alpha values derived from the acidic limb and the basic limb of the profile being 4.4 and 6.8, respectively. This suggests that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate at all pH values. Results from leaving group dependence of kcat at two different pH values support the above conclusion. Furthermore, burst kinetics have been demonstrated with PTP1 using p-nitrophenyl phosphate as a substrate. The rate constants for the formation and the breakdown of the intermediate are 241 and 12 s-1, respectively, at pH 6.0 and 3.5 degrees C. A normal D2O solvent isotope effect (kcatH/kcatD = 1.5) is associated with the breakdown of the phosphoenzyme intermediate, indicating a solvent-derived proton in the transition state. The leaving group dependence of kcat/Km suggests that there is a strong electrophilic interaction between the enzyme and the leaving group oxygen in the transition state of the phosphorylation event. These results are compared with those of the Yersinia PTPase and suggest that the mechanism for PTPase-catalyzed phosphate monoester hydrolysis is conserved from bacterial to mammals.

Martell KJ, Seasholtz AF, Kwak SP, Clemens KK, Dixon JE
hVH-5: a protein tyrosine phosphatase abundant in brain that inactivates mitogen-activated protein kinase.
J Neurochem. 1995; 65: 1823-33
Display abstract
A novel protein tyrosine phosphatase [homologue of vaccinia virus H1 phosphatase gene clone 5 (hVH-5)] was cloned; it shared sequence similarity with a subset of protein tyrosine phosphatases that regulate mitogen-activated protein kinase. The catalytic region of hVH-5 was expressed as a fusion protein and was shown to hydrolyze p-nitrophenylphosphate and inactivate mitogen-activated protein kinase, thus proving that hVH-5 possessed phosphatase activity. A unique proline-rich region distinguished hVH-5 from other closely related protein tyrosine phosphatases. Another feature that distinguished hVH-5 from related phosphatases was that hVH-5 was expressed predominantly in the adult brain, heart, and skeletal muscle. In addition, in situ hybridization histochemistry of mouse embryo revealed high levels of expression and a wide distribution in the central and peripheral nervous system. Some specific areas of abundant hVH-5 expression included the olfactory bulb, retina, layers of the cerebral cortex, and cranial and spinal ganglia. hVH-5 was induced in PC12 cells upon nerve growth factor and insulin treatment in a manner characteristic of an immediate-early gene, suggesting a possible role in the signal transduction cascade.

Pulido R, Krueger NX, Serra-Pages C, Saito H, Streuli M
Molecular characterization of the human transmembrane protein-tyrosine phosphatase delta. Evidence for tissue-specific expression of alternative human transmembrane protein-tyrosine phosphatase delta isoforms.
J Biol Chem. 1995; 270: 6722-8
Display abstract
Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of cell activation, proliferation, and differentiation. A major subfamily of these enzymes is the transmembrane-type PTPases that contain extracellular regions comprised of Ig-like and fibronectin type III (FN-III)-like domains. Characterization of the human transmembrane PTPase delta (HPTP delta) revealed the existence of multiple HPTP delta isoforms that vary in their extracellular regions. The full-length HPTP delta isoform has an extracellular region containing three Ig-like and eight FN-III-like domains connected via a transmembrane peptide to an intracellular region with two PTPase domains, whereas another isoform lacks four of the eight FN-III like domains. Furthermore, other HPTP delta isoforms exist that lack 9 amino acids within the second Ig-like domain and 4 amino acids at the junction of the second and third Ig-like domains or 9 amino acids within the fifth FN-III-like domain. Reverse transcription polymerase chain reaction analysis demonstrated that HPTP delta isoforms lacking these short peptides are expressed in kidney, whereas isoforms containing these peptides are expressed in the brain. Analysis of HPTP delta biosynthesis demonstrated that HPTP delta is expressed as a complex of two noncovalently associated subunits derived from a proprotein and that the HPTP delta ectodomain is shed from the cell surface. Mutational analysis of the HPTP delta proprotein cleavage site revealed the existence of two or three functional and overlapping furin-like endoprotease cleavage sites.

Zhang SH, Eckberg WR, Yang Q, Samatar AA, Tonks NK
Biochemical characterization of a human band 4.1-related protein-tyrosine phosphatase, PTPH1.
J Biol Chem. 1995; 270: 20067-72
Display abstract
PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeleton-associated proteins. Here, we report the purification and biochemical characterization of this enzyme from baculovirus-infected insect cells. The purified protein exhibited an apparent M(r) of 120,000 on SDS gels. The native enzyme dephosphorylated both myelin basic protein (MBP) and reduced, carboxamidomethylated, and maleylated lysozyme (RCML) but was over 5-fold more active on MBP. The Km values for the two substrates were similar (1.45 microM for MBP and 1.6 microM for RCML). Phosphorylation of PTPH1 by protein kinase C in vitro resulted in a decrease in Km but had no effect on Vmax. Removal of the NH2-terminal band 4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated dephosphorylation of RCML but inhibited its activity toward MBP. The dephosphorylation of RCML by full-length PTPH1 was enhanced up to 6-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M NaCl, whereas trypsinized preparations of PTPH1 containing the isolated catalytic domain were unaffected. These results suggest that in addition to a potential role in controlling subcellular localization, the NH2-terminal band 4.1 homology domain of PTPH1 may exert a direct effect on catalytic function.

Sahin M, Dowling JJ, Hockfield S
Seven protein tyrosine phosphatases are differentially expressed in the developing rat brain.
J Comp Neurol. 1995; 351: 617-31
Display abstract
Regulation of protein function through tyrosine phosphorylation is critical in the control of many developmental processes, such as cellular proliferation and differentiation. Growing evidence suggests that tyrosine phosphorylation also regulates key events in neural development. Although a large body of data has demonstrated that protein tyrosine kinases play an important role in neural development, much less is known about their counterparts, protein tyrosine phosphatases (PTPases). Using polymerase chain reaction (PCR) with degenerate primers and a neonatal rat cortex cDNA library, we have identified seven PTPases expressed in the developing rat brain. Four of these are transmembrane PTPases: LAR, LRP, RPTP gamma, and CPTP1. Three are nonreceptor PTPases: PTP-1, P19-PTP, and SHP. Northern hybridization analysis demonstrates that only CPTP1 is preferentially expressed in neural tissues, whereas the others are found abundantly in nonneural tissues as well as in the brain. Within the embryonic and early postnatal brain, the seven PTPases have overlapping, yet unique, distributions. For example, LAR mRNA is highly expressed by both proliferating and postmitotic cells in the cerebral cortex at embryonic day 17 and in all layers of the cortex at postnatal day 4. In contrast, RPTP gamma mRNA is expressed by postmitotic neurons in the embryo and predominantly by neurons in the superficial layers of the postnatal cortex. Several of the PTPases examined here are expressed at very high levels in the embryonic cortical plate and postnatal neocortex, including the subplate and subventricular zone. The spatial and temporal regulation of PTPase gene expression suggests that these PTPases have important roles in signal transduction during early neuronal differentiation and neural development.

Martell KJ, Kwak S, Hakes DJ, Dixon JE, Trent JM
Chromosomal localization of four human VH1-like protein-tyrosine phosphatases.
Genomics. 1994; 22: 462-4
Display abstract
Four human protein-tyrosine phosphatase (PTPase) genes of the VH1-like subclass were cloned by low-stringency screening of a genomic library. These genes were localized to their respective chromosomes by G-banding and fluorescence in situ hybridization. The genes were localized to unique regions of different chromosomes: CL100, a stress-induced PTPase, to 5q35; PAC-1, a mitogen-induced nuclear PTPase, to 2q11;hVH-3 to 10q25; and hVH-4 to 10q11.

Desai CJ, Popova E, Zinn K
A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the "HRP" carbohydrate epitope.
J Neurosci. 1994; 14: 7272-83
Display abstract
Recent studies have defined several cell surface glycoproteins expressed in the developing nervous system of insect embryos that may be involved in axon outgrowth and guidance processes. These glycoproteins include the fasciclins and a group of receptor-linked protein tyrosine phosphatases (R-PTPs). In embryos, the fasciclins are localized to axonal subsets, while the R-PTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, we have taken a biochemical approach. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted "HRP proteins") apparently bear the HRP carbohydrate epitope. We have used polyclonal anti-HRP antibodies to purify these proteins from Drosophila embryos, and have obtained protein sequences from seven HRP protein bands. These data define three major HRP proteins as neurotactin, fasciclin I, and an R-PTP, DPTP69D. Western blotting data suggest that fasciclin II, neuroglian, DPTP10D, and DPTP99A are also HRP proteins. We show that DPTP69D, like the previously characterized R-PTPs, is localized to CNS axons in the embryo. In third instar larvae, DPTP69D expression is restricted to subsets of neuronal processes in the brain, ventral nerve cord, and eye disk. In the optic lobes, DPTP69D is localized to the neuropils of the lamina and medulla, and to an array of parallel thick bundles that may be the transmedullary fibers of the developing lobula complex.

Wong C, Wu W, Obenshain SA, Diah SK, Faiola B, Kennelly PJ
High-molecular-weight polypeptide substrates for phospholysine phosphatases.
Anal Biochem. 1994; 222: 14-8
Display abstract
The detection and characterization of the phosphatases responsible for the dephosphorylation of N-phosphoryl groups, such as phospholysine, in proteins has been frustrated in large measure by a lack of suitable substrates for the assay of these enzymes. Herein we describe the preparation of phospholysine-containing amino acid homo- and heteropolymers by chemical means, and their use in a simple, nonradioactive assay for phospholysine phosphatase activity.

Sap J, Jiang YP, Friedlander D, Grumet M, Schlessinger J
Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding.
Mol Cell Biol. 1994; 14: 1-9
Display abstract
Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation.

Yakura H
The role of protein tyrosine phosphatases in lymphocyte activation and differentiation.
Crit Rev Immunol. 1994; 14: 311-36
Display abstract
Protein tyrosine phosphorylation is involved in the regulation of every facet of biological phenomena. Signal transduction mechanisms operative in lymphocyte development, activation, and differentiation have been studied intensively, and are also found to support this premise. In this review, I focus on the crucial problems surrounding CD45, a prototypic receptor-type protein tyrosine phosphatase (RPTP) of the immune system: (1) the role of CD45 for antigen receptor-initiated signaling in T and B cells, (2) the physiological relevance of CD45 isoforms, (3) potential regulatory mechanisms of CD45 PTP activity, and (4) the clinical significance of structural abnormalities in PTP. Furthermore, other PTPs that may be important to the exquisite functioning of the immune system are reviewed.

Tian SS, Zinn K
An adhesion molecule-like protein that interacts with and is a substrate for a Drosophila receptor-linked protein tyrosine phosphatase.
J Biol Chem. 1994; 269: 28478-86
Display abstract
Receptor-linked protein tyrosine phosphatases (R-PTPs) are a large and diverse group of transmembrane signaling molecules. In Drosophila, four R-PTPs are localized to central nervous system axons in the embryo and may participate in assembly of the central nervous system axon array. In this paper, we identify and characterize a transmembrane glycoprotein, gp150, that selectively interacts with the catalytic domain of the axonal R-PTP DPTP10D. gp150 does not bind to a cysteine-to-serine active site mutant, and binding is inhibited by vanadate, suggesting that it interacts with the active site. It has an extracellular domain composed of 18 leucine-rich repeats, which are found in many adhesion molecules. Its short cytoplasmic domain contains 4 tyrosine residues in sequence contexts that suggest that they could interact with SH2 domain-containing effector molecules. The overall organization of the tyrosine motifs resembles that of antigen recognition activation motif signaling elements from receptors in the vertebrate immune system. The cytoplasmic domain of gp150 is a good substrate for v-Abl tyrosine kinase, and phosphorylated gp150 can be dephosphorylated efficiently in vitro by DPTP10D. We suggest that DPTP10D may function in vivo to regulate phosphorylation of gp150 and thereby control its interactions with downstream effectors.

Ding W, Zhang WR, Sullivan K, Hashimoto N, Goldstein BJ
Identification of protein-tyrosine phosphatases prevalent in adipocytes by molecular cloning.
Biochem Biophys Res Commun. 1994; 202: 902-7
Display abstract
Protein-tyrosine phosphatases (PTPases) are among the fastest growing family of enzymes that are closely linked to signal transduction pathways involving reversible tyrosine phosphorylation. In order to identify PTPase homologs expressed in adipocytes that might regulate the action of insulin or growth factors in this tissue, we screened a rat adipocyte cDNA library at reduced stringency with a panel of candidate PTPase probes. After subcloning and sequence analysis of the positive plaques, this approach enabled us to identify the expression of LRP/RPTP-alpha, PTPase 1B, SH-PTP2/Syp, and LAR in adipocytes at an abundance of 16, 7, 6 and 3 per million, respectively. Furthermore, a sequence variant of SH-PTP2/Syp was identified that may have significance in the tissue-specific activity of this enzyme. These data provide insight into PTPase homologs that may have a physiological role in the regulation of phosphotyrosyl turnover in hormone signalling pathways in adipocytes.

Bignon JS, Siminovitch KA
Identification of PTP1C mutation as the genetic defect in motheaten and viable motheaten mice: a step toward defining the roles of protein tyrosine phosphatases in the regulation of hemopoietic cell differentiation and function.
Clin Immunol Immunopathol. 1994; 73: 168-79
Display abstract
Homozygosity for the motheaten (me) or viable motheaten (mev) mutations causes severe dysregulation of murine hematopoiesis with the consequent development of both immunodeficiency and systemic autoimmunity. Expression of this phenotype has now been linked to loss-of-function mutations in the gene encoding PTP1C, an intracellular tyrosine phosphatase predominantly expressed in cells of hemopoietic origin. As discussed in this article, the association of PTP1C mutation with the multiple hemopoietic defects found in motheaten mice indicates that this tyrosine phosphatase is critical to normal hematopoiesis and is consistent with the recognized importance of protein tyrosine phosphorylation in modulating the cell signaling pathways governing proliferation and differentiation. The motheaten mouse therefore provides a powerful model of delineating the precise function of PTP1C and thereby elucidating the specific molecular mechanisms whereby this tyrosine phosphatase participates in the control of hemopoietic cell differentiation and function.

Wang Q, Dechert U, Jirik F, Withers SG
Suicide inactivation of human prostatic acid phosphatase and a phosphotyrosine phosphatase.
Biochem Biophys Res Commun. 1994; 200: 577-83
Display abstract
4-Difluoromethylphenyl bis(cyclohexylammonium) phosphate was synthesized in 4 steps starting from dibenzyl phosphite and shown to be a time-dependent suicide inactivator of human prostatic acid phosphatase and the SHP protein tyrosine phosphatase. The inactivation of human prostatic acid phosphatase followed pseudo-first-order kinetics with inactivation constants of Ki = 1.0 mM; ki = 0.15 min-1 (t1/2 = 4.6 min at saturation). Phenyl phosphate protected the enzyme against inactivation, indicating that inactivation occurs in the active site. The inactivation of SHP also followed pseudo-first-order kinetics, with a t1/2 = approximately 15 min in the presence of 8.2 mM inhibitor. The mechanism of inactivation likely involves the enzymatic release of difluoromethyl phenol which rapidly eliminates fluoride, generating a quinone methide. This potent electrophile then reacts with residues at the active site of the enzyme. This inhibitor and peptidic derivatives thereof have excellent potential for selective inactivation and labeling of protein tyrosine phosphatases.

Iida N, Lokeshwar VB, Bourguignon LY
Mapping the fodrin binding domain in CD45, a leukocyte membrane-associated tyrosine phosphatase.
J Biol Chem. 1994; 269: 28576-83
Display abstract
CD45 belongs to a family of high molecular mass leukocyte glycoproteins. It contains both an intrinsic protein tyrosine phosphatase (PTPase) activity and a cytoskeleton binding site in its cytoplasmic domain. Certain cytoskeletal proteins, such as fodrin (a spectrin-like molecule), are known to play an important role in the regulation of CD45's PTPase activity. In this study we mapped the fodrin binding domain of CD45 by deleting various portions of the cytoplasmic region, followed by the expression of these truncated cDNAs using an in vitro transcription/translation system. The results of these experiments indicate that the CD45 fodrin binding domain resides between amino acids 825 and 939. Construction of a fusion protein encoding the region between amino acids 825 and 939 shows that this particular sequence itself is sufficient for fodrin binding. Further analyses indicate that the sequence (930EENKKKNRN939S) in CD45 has good sequence homology with the spectrin binding domain found in the MSP1 glycoprotein of the malarial parasite. Biochemical studies, using binding competition assays, and a synthetic peptide containing the sequence 930EENKKKNRN939S, support the conclusion that the sequence between amino acids 930 and 939 is a critical part of CD45's fodrin binding domain. Further analyses indicate that this sequence is also involved in the fodrin-induced up-regulation of CD45 PTPase activity. Therefore, we suggest that fodrin binding to this domain is required for the onset of CD45-mediated signal transduction and leukocyte activation.

Davis JP, Zhou MM, Van Etten RL
Kinetic and site-directed mutagenesis studies of the cysteine residues of bovine low molecular weight phosphotyrosyl protein phosphatase.
J Biol Chem. 1994; 269: 8734-40
Display abstract
The roles of the 8 conserved cysteines and 1 arginine in the low molecular weight phosphotyrosyl protein phosphatases were investigated using site-directed mutagenesis of the recombinant bovine heart enzyme. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for Cys-12, Cys-17, and Arg-18. The CD spectra of the purified proteins were effectively superimposable, consistent with the conclusion that no major structural alterations had occurred, but 1H NMR spectroscopy did reveal some spectral shifts in the aromatic region. Kinetic analysis of the mutant proteins demonstrated that only Cys-12, Cys-17, and Arg-18 had significantly altered catalytic activity toward the substrate p-nitrophenyl phosphate at pH 5. The Cys-12 and Arg-18 mutants were effectively inactive. Thus, it is concluded that Cys-12 is the catalytic nucleophile, and Arg-18 presumably serves an essential function in substrate binding. The C17S mutant had 6% residual activity compared with wild type protein, whereas the C17A mutant had 37% activity. Consistent with the observed activity of the Cys-17 mutant, a covalent phosphocysteine intermediate was trapped and identified by 31P NMR. Further kinetic analysis of C17A using several aryl phosphate monoester substrates with different leaving group pK alpha values indicated that no change in the rate-determining step of the catalytic mechanism had occurred, that is, dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. The C17A mutant had a 4-fold higher phosphate Ki and slightly higher Km values for p-nitrophenyl phosphate suggesting that Cys-17 may be important for optimal positioning of the substrate phosphate moiety.

Sage SO, Sargeant P, Jenner S, Farndale RW
Tyrosine phosphorylation and Ca2+ influx. A further mechanism for store-dependent Ca2+ entry?
Trends Pharmacol Sci. 1994; 15: 282-282

Caselli A et al.
Porcine liver low M(r) phosphotyrosine protein phosphatase: the amino acid sequence.
J Protein Chem. 1994; 13: 107-15
Display abstract
Porcine low M(r) phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH2-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH2-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low M(r) PTPases. This PTPase is strongly inhibited by pyridoxal 5'-phosphate (Ki = 21 microM) like the low M(r) PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40-73 sequence with the corresponding sequence of other low M(r) PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M(r) PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5'-phosphate inhibition has been proposed.

Serra-Pages C, Saito H, Streuli M
Mutational analysis of proprotein processing, subunit association, and shedding of the LAR transmembrane protein tyrosine phosphatase.
J Biol Chem. 1994; 269: 23632-41
Display abstract
The LAR transmembrane protein tyrosine phosphatase (PTPase) is expressed on the cell surface as a complex of two noncovalently associated subunits derived from a proprotein. The 150-kDa E-subunit contains most of the extracellular region, including the immunoglobulin-like and fibronectin type-III-like domains, whereas the 85-kDa P-subunit contains a short ectodomain, the transmembrane peptide, and the two intracellular PTPase domains. The LAR extracellular region is released from the cell surface, suggesting that shedding may be a mechanism to regulate LAR PTPase function. Functional regions necessary for LAR proprotein processing, subunit association, and shedding were determined by analyzing the effect of amino acid substitutions of residues surrounding the cleavage site and scanning the P-subunit ectodomain. Three amino acid residues were identified, two within a penta-arginine sequence and one C-terminal to the cleavage site, that are essential for efficient LAR proprotein cleavage. Several noncontiguous amino acid residues were also identified that play an essential role in LAR subunit association. LAR shedding is shown to be a consequence of proteolytic cleavage at a second site within the P-subunit ectodomain near the transmembrane peptide.

Ostman A, Yang Q, Tonks NK
Expression of DEP-1, a receptor-like protein-tyrosine-phosphatase, is enhanced with increasing cell density.
Proc Natl Acad Sci U S A. 1994; 91: 9680-4
Display abstract
cDNA encoding a receptor-like protein-tyrosine-phosphatase (PTP) termed DEP-1 was isolated from a HeLa cell library. The cDNA predicts an enzyme consisting of an extracellular segment containing eight fibronectin type III repeats, a single transmembrane segment, and a single intracellular PTP domain. Following expression of DEP-1 cDNA in COS cells a glycoprotein of 180 kDa was detected and PTP activity was demonstrated in immunocomplexes with a C-terminal peptide antiserum. Endogenous DEP-1 was detected in WI-38 human embryonic lung fibroblasts by immunoblotting and immunocomplex PTP assays. Immunoblot analysis of DEP-1 expression in WI-38 cells revealed dramatically increased levels and activity of the PTP in dense cultures relative to sparse cultures. Also, DEP-1 activity, detected in PTP assays of immunocomplexes, was increased in dense cell cultures. In contrast, the expression levels of PTP-1B did not change with cell density. This enhancement of DEP-1 expression with increasing cell density was also observed in another fibroblast cell line, AG1518. The increase in DEP-1 occurs gradually with increasing cell contact and is initiated before saturation cell density is reached. These observations suggest that DEP-1 may contribute to the mechanism of contact inhibition of cell growth.

Hiraga A, Hata K, Suzuki Y, Tsuiki S
Identification of a rat liver protein-tyrosine phosphatase similar to human placental PTPase-1B using quantitatively phosphorylated protein substrates.
J Biochem (Tokyo). 1993; 113: 180-8
Display abstract
Erythrocyte Band 3 protein (Band 3), brain microtubule associated protein 2 (MAP2), and tubulin were phosphorylated to high stoichiometries (1-6 mol Pi/mol protein) on tyrosine residues using a rat spleen protein-tyrosine kinase in the presence of polylysine. After total removal of polylysine, the quantitatively phosphorylated proteins as well as tyrosine-glutamate copolymer [Poly(Glu4, Tyr1)], which was also phosphorylated (1.5 mol/mol) by the kinase, were employed to assay rat liver protein-tyrosine phosphatases (PTPases). Of the four partially purified PTPases termed L1, L2, L3, and L4, PTPase L1 was previously purified to homogeneity and demonstrated to be a novel enzyme with sequence similarity to src-homology region 2 [Hiraga, A. et al. (1992) Eur. J. Biochem. 209, 195-206]. In the present work PTPase L2 was purified to near homogeneity by a procedure involving chromatography on DEAE-cellulose, Blue Sepharose CL-6B, hydroxylapatite, Phenyl Sepharose CL-4B, and TSKgel Heparin-5PW. PTPase L2 was purified 20,000-fold with a recovery of 0.9% from the extract and 0.005 mg was isolated from 300 g of liver. The highly purified PTPase L2 showed a major protein band of 36 kDa on SDS/polyacrylamide gel electrophoresis. PTPase L2 had a specific activity of about 6,000 nmol of P1 released min-1.mg-1 toward either Band 3 or poly(Glu4, Tyr1), the values being within the range of those obtained for PTPases purified thus far. PTPase L2 dephosphorylated Band-3 9-fold and 5-fold faster than tubulin and MAP2, respectively, under the assay conditions employed.(ABSTRACT TRUNCATED AT 250 WORDS)

Walton KM, Martell KJ, Kwak SP, Dixon JE, Largent BL
A novel receptor-type protein tyrosine phosphatase is expressed during neurogenesis in the olfactory neuroepithelium.
Neuron. 1993; 11: 387-400
Display abstract
Tyrosine phosphorylation plays a central role in the control of neuronal cell development and function. Yet, few neuronal protein tyrosine phosphatases (PTPs) have been identified. We examined rat olfactory neuroepithelium for expression of novel PTPs potentially important in neuronal development and regeneration. Using the polymerase chain reaction with degenerate DNA oligomers directed to the conserved tyrosine phosphatase domain, we identified 6 novel tyrosine phosphatases. One of these, PTP NE-3, is a receptor-type PTP expressed selectively in both rat brain and olfactory neuroepithelium. In the olfactory neuroepithelium, PTP NE-3 expression is restricted to neurons and describes a novel pattern of expression with a high level in the immature neurons and a lower level in mature olfactory sensory neurons.

Woodford-Thomas T, Thomas ML
The leukocyte common antigen, CD45 and other protein tyrosine phosphatases in hematopoietic cells.
Semin Cell Biol. 1993; 4: 409-18
Display abstract
Numerous protein tyrosine phosphatases have been characterized that are expressed by hematopoietic-derived cells. CD45, a transmembrane protein tyrosine phosphatase expressed by all nucleated cells of hematopoietic origin, has been a prototype for studying the function of these enzymes. In T lymphocytes, CD45 serves to dephosphorylate and activate members of the Src-tyrosine kinase family. Without CD45 function, T cells cannot respond to antigen. Therefore, CD45 functions positively to regulate lymphocyte activation. The characteristics of other protein tyrosine phosphatases found in hematopoietic-derived cells suggests that they are also essential in regulating cell differentiation and activation.

Takeda A
Sphingolipid-like molecule linked to CD45, a protein tyrosine phosphatase.
Adv Lipid Res. 1993; 26: 293-317

Sahin M, Hockfield S
Protein tyrosine phosphatases expressed in the developing rat brain.
J Neurosci. 1993; 13: 4968-78
Display abstract
Previous studies of the developing nervous system have shown that cell-cell and cell-matrix interactions are involved in a variety of processes such as the proliferation, migration, and differentiation of neurons. While many cell-surface molecules have been identified, the signal transduction mechanisms through which they modify cellular responses are poorly understood. Recent studies have described a new and large family of enzymes, protein tyrosine phosphatases (PTPases), that may play a key role in transduction of cell surface events. Opposing the actions of protein tyrosine kinases (PTKs), PTPases can determine the state of tyrosine phosphorylation of a protein and regulate its function. Within the family of PTPases, two subgroups have been characterized: low-molecular-weight cytoplasmic (nonreceptor) PTPases and high-molecular-weight transmembrane (receptor) PTPases. Many receptor PTPases have fibronectin type III and/or Ig-like domains in their extracellular domains, suggesting that they have dual functions: cell adhesion and signal transduction. Such molecules may play a role in cellular recognition events that mediate the accurate assembly of the nervous system. Using polymerase chain reaction with degenerate primers and a neonatal rat cortex cDNA library, we have identified a number of putative PTPase domains expressed in brain. Three are characterized here. These three sequences are most abundantly expressed in the developing cortex and so are named cortex-enriched protein tyrosine phosphatases (CPTPs) 1, 2, and 3. CPTP1 and CPTP3 show sequence homology to receptor PTPases and detect multiple high-molecular-weight mRNAs that are expressed preferentially in the developing CNS. Analysis of a longer cDNA indicates that CPTP1 and CPTP3 are the first and second phosphatase domains of a single receptor PTPase. CPTP2 identifies a single, smaller mRNA species with sequence homology to nonreceptor PTPases. Within the CNS, mRNAs detected by all three CPTPs are expressed at highest levels during prenatal and early postnatal days and are downregulated in the adult. In situ hybridization demonstrates that the CPTPs are expressed by progenitor cells and developing neurons. The spatial and temporal regulation of CPTPs suggests that they may play a role in neuronal development.

Sridhar TS, Swarup G, Khar A
Downregulation of phospho-tyrosine phosphatases in a macrophage tumor.
FEBS Lett. 1993; 326: 75-9
Display abstract
We provide evidence for the downregulation of phospho-tyrosine phosphatases (PTPases) in malignancy. AK-5, a rat macrophage tumor, shows the downregulation of the transcripts of two non-receptor-type PTPases, PTP-1 and PTP-S. Though downregulated fourfold, the genomic organization of PTP-S is unaltered. There is no gross alteration of the PTPase activity in AK-5 as compared to macrophages. Immunoblot analysis reveals no significant change in the total phospho-tyrosine levels in AK-5, but there is a qualitative difference in the pattern between AK-5 and macrophages. Our results lend credence to the conjecture that PTPases also might be involved in malignancy.

Martin BL, Graves DJ
Hydrolysis of trifluoroethyl phosphate as evidence that the serine and tyrosine phosphatase activities of calcineurin share the same specificity determinant.
Biochem Biophys Res Commun. 1993; 194: 150-6
Display abstract
Calcineurin is shown to hydrolyze 2,2,2-trifluoroethyl phosphate. The time course of hydrolysis showed a lag period. The lag was present at all substrate concentrations used and was dependent on substrate concentration. Evaluation of the kinetic constants from the steady state portion of the enzymatic reaction indicated that hydrolysis of this compound fit the Bronsted relationship determined previously for Vmax/Km term for the hydrolysis of aromatic phosphate esters. Moreover, a Bronsted relationship for the Vmax was developed for the substrates containing leaving groups that are more basic than 2,3,4,5-tetrafluorotyrosine. The hydrolysis of 2,3,4,5-tetrafluorotyrosine phosphate did not fit this relationship implying a change in the rate limiting step with this substrate. The significance of these data to the serine and tyrosine phosphatase activities of calcineurin is discussed. A rationale for understanding the determinant of both activities of calcineurin is presented.

Hadari YR et al.
Hepatic tyrosine-phosphorylated proteins identified and localized following in vivo inhibition of protein tyrosine phosphatases: effects of H2O2 and vanadate administration into rat livers.
Mol Cell Endocrinol. 1993; 97: 9-17
Display abstract
Injection of a combination of H2O2 and vanadate (H/V) into the portal vein of rat livers resulted in inhibition of protein tyrosine phosphatase activity and led to a dramatic enhanced in vivo protein tyrosine phosphorylation. Some of the phosphorylated proteins were identified as the beta-subunit of the insulin receptor, the insulin receptor substrate 1 (pp185), PLC-gamma (pp145), and a 100 kDa PLC-gamma-associated protein. Immunofluorescense and immune electron microscopy of frozen liver sections with anti-P-Tyr antibodies revealed that most of the tyrosine-phosphorylated proteins are localized in close proximity to the plasma membrane in intercellular adherence junctions and tight junction regions. This close in vivo association between membranal protein tyrosine kinases, their target proteins, and cytoskeletal elements could enable formation of 'signaling complexes' which may play a role in transmembrane signal transduction. By affinity chromatography over immobilized anti-P-Tyr antibodies, a large number of these tyrosine-phosphorylated proteins were partially purified.

Kaneko Y et al.
Identification of protein tyrosine phosphatases expressed in murine male germ cells.
Biochem Biophys Res Commun. 1993; 197: 625-31
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We cloned five cDNAs encoding putative protein tyrosine phosphatases (PTPs; protein-tyrosine-phosphatase phosphohydrolase, EC 3.1.3.48) from the murine testis by using degenerate primers and the polymerase chain reaction cloning technique. Two of them were identical to the mouse cytoplasmic PTP and PTP-1B. The remaining three were likely to represent the murine counterparts of human PTP delta, human PTP epsilon, and rat striatum-enriched PTP. The cells expressing these genes were determined either by in situ hybridization histochemistry or by Northern blot hybridization. Moreover, Northern blot hybridization revealed that the transcripts of PTP-1B, mouse cytoplasmic PTP and the murine homologue of human PTP delta were quantitatively and/or structurally regulated during germ cell development, suggesting their roles in spermatogenesis.

Dawicki DD, Steiner M
Identification of a protein-tyrosine phosphatase from human platelet membranes by an immobilon-based solid phase assay.
Anal Biochem. 1993; 213: 245-55
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The present investigations show that a 53-kDa platelet-membrane protein is a protein tyrosine phosphatase. Identification involved a novel methodology in which membrane proteins are resolved by polyacrylamide gel electrophoresis and then transblotted to a polyvinylidene difluoride membrane surface-labeled with [32P-Tyr](Glu4.Tyr1)n. Phosphatase activity appears as clear areas in the autoradiograph of the renatured 32P Western blot. Studies using this new solid-phase system indicate that a 53-kDa platelet-membrane protein dephosphorylates [32P-Tyr](Glu4.Tyr1)n. Dephosphorylation is both time- and dose-dependent. The protein-tyrosine phosphatase antagonists orthovanadate and molybdate block dephosphorylation in a concentration-dependent manner. Inhibitors of protein serine/threonine phosphatases and of acid and alkaline phosphatases do not significantly affect enzymatic activity. The enzyme is active toward phosphotyrosyl proteins but not phosphoseryl proteins. Results from the solid-phase phosphatase assays correlate with data from standard liquid-phase studies. A Lineweaver-Burk plot gives an apparent Km of 4.3 microM and an apparent Vmax = 168 nmol P(i) transferred/min/mg for the membrane enzyme towards the 32P-labeled polymer in a liquid system. Platelet lysate was shown by the solid-phase assay to possess a protein-tyrosine phosphatase of approximately equal to 50 kDa. The tyrosine phosphatase activities associated with a placental preparation, pure recombinant protein-tyrosine phosphatase 1B, and YOPR, the product of the YOP51 gene of Yersinia enterocolitica, which contains the C235R mutation, were also visualized by this technique. However, the catalytic activity of domain 1 of the transmembrane leukocyte antigen-related protein-tyrosine phosphatase was not detected using the solid-phase assay.

Shekels LL, Smith AJ, Van Etten RL, Bernlohr DA
Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.
Protein Sci. 1992; 1: 710-21
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We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.

Zhang ZY et al.
Expression, purification, and physicochemical characterization of a recombinant Yersinia protein tyrosine phosphatase.
J Biol Chem. 1992; 267: 23759-66
Display abstract
The Yersinia protein tyrosine phosphatase (PTPase) Yop51, a C235R point mutation (Yop51*), and a protein lacking the first 162 amino acids at the NH2 terminus (Yop51*delta 162) have been overexpressed in Escherichia coli and purified to homogeneity through the use of CM Sephadex C25 cation exchange chromatography followed by Sephadex G-100 gel filtration. Greater than 50 mg of homogeneous Yop51* and Yop51*delta 162 can be obtained from a single liter of bacterial culture, whereas the same procedure yields only 5 mg of pure Yop51. Large, diffraction-quality crystals have been obtained for Yop51*delta 162. Size exclusion chromatography, sedimentation equilibrium, and enzyme concentration dependence experiments have established that the Yersinia PTPases exist and function as monomers in solution. Yop51 and Yop51* display identical UV, CD, and fluorescence spectra and have identical kinetic and structural stability properties. These full-length Yersinia PTPases have 31% alpha-helix, an emission maximum of 342 nm, a turn-over number of 1200 s-1 at pH 5.0, 30 degrees C, and an unfolding delta G value of 6 kcal/mol at 25 degrees C. Yop51*delta 162 has very similar kinetic and fluorescence characteristics to the full-length molecules, whereas its CD and UV spectra show noticeable differences due to the elimination of 162 NH2-terminal residues. The Yersinia PTPases are by far the most active PTPases known, and their kinetic parameters are extremely sensitive to the ionic strength of reaction medium.

Lee JP, Cho H, Bannwarth W, Kitas EA, Walsh CT
NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase.
Protein Sci. 1992; 1: 1353-62
Display abstract
An autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142-1,153 of the insulin receptor was prepared and incubated with the 40-kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H-NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri-, di-, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.

Ottilie S, Chernoff J, Hannig G, Hoffman CS, Erikson RL
The fission yeast genes pyp1+ and pyp2+ encode protein tyrosine phosphatases that negatively regulate mitosis.
Mol Cell Biol. 1992; 12: 5571-80
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We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway.

Manao G et al.
Rat liver low M(r) phosphotyrosine protein phosphatase isoenzymes: purification and amino acid sequences.
J Protein Chem. 1992; 11: 333-45
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Two low M(r) phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as low M(r) acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40-73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.

Matthews RJ, Bowne DB, Flores E, Thomas ML
Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences.
Mol Cell Biol. 1992; 12: 2396-405
Display abstract
Protein tyrosine phosphatases (PTPases) are a family of enzymes important in cellular regulation. Characterization of two cDNAs encoding intracellular PTPases expressed primarily in hematopoietic tissues and cell lines has revealed proteins that are potential regulators of signal transduction. One of these, SHP (Src homology region 2 [SH2]-domain phosphatase), possesses two tandem SH2 domains at the amino terminus of the molecule. SH2 domains have previously been described in proteins implicated in signal transduction, and SHP may be one of a family of nonreceptor PTPases that can act as direct antagonists to the nonreceptor protein tyrosine kinases. The SH2 domains of SHP preferentially bind a 15,000-Mr protein expressed by LSTRA cells. LSTRA cells were shown to express SHP protein by immunoprecipitation, thus demonstrating a potential physiological interaction. The other PTPase, PEP (proline-, glutamic acid-, serine-, and threonine-rich [PEST]-domain phosphatase), is distinguished by virtue of a large carboxy-terminal domain of approximately 500 amino acids that is rich in PEST residues. PEST sequences are found in proteins that are rapidly degraded. Both proteins have been expressed by in vitro transcription and translation and in bacterial expression systems, and both have been demonstrated to have PTPase activity. These two additional members of the PTPase family accentuate the variety of PTPase structures and indicate the potential diversity of function for intracellular tyrosine phosphatases.

Saito H, Streuli M
Molecular characterization of protein tyrosine phosphatases.
Cell Growth Differ. 1991; 2: 59-65

Gordon JA
Use of vanadate as protein-phosphotyrosine phosphatase inhibitor.
Methods Enzymol. 1991; 201: 477-82

Clemens JC, Guan K, Bliska JB, Falkow S, Dixon JE
Microbial pathogenesis and tyrosine dephosphorylation: surprising 'bedfellows'.
Mol Microbiol. 1991; 5: 2617-20
Display abstract
The importance of parasite-directed phosphatases in such diseases as smallpox and the bubonic plague emphasizes the need to understand the molecular events associated with the normal function of protein tyrosine phosphatase in eukaryotic cells.

Tonks NK, Diltz CD, Fischer EH
Purification of protein-tyrosine phosphatases from human placenta.
Methods Enzymol. 1991; 201: 427-42

Wang Y, Pallen CJ
The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities.
EMBO J. 1991; 10: 3231-7
Display abstract
Cloning and expression of the homologous domains of the receptor-like tyrosine phosphatase HPTP alpha shows that both domain 1 (D1) and domain 2 (D2) are enzymatically active. The two domains display different substrate specificities with D1 preferentially dephosphorylating MBP approximately RR-src greater than PNPP while D2 favours PNPP much much greater than RR-src and is inactive towards MBP. Each domain has lower activity than an expressed protein containing both domains. Analysis of chimaeric D1/2 proteins suggests that no particular region of D2 is responsible for the low activity of D2 on RR-src and that the specificity differences of D1 and D2 reflect overall sequence dissimilarities. Activities of D1 and D2 are inhibited by zinc, vanadate and EDTA and differentially susceptible to inhibition by heparin and poly(Glu4:Tyr1). Unusually, the activity of the protein containing both domains is stimulated by these polyanions. Regions amino-terminal to each domain are important for catalysis since deletion of these sequences abolishes phosphatase activity. Activity of the double domain polypeptide was also lost upon deletion of the sequence amino-terminal to D1, indicating that inactivation of D1 may suppress D2 activity. Differences in substrate specificity and responses to effectors and the interdependence between the two domains are likely important properties in the function of this PTPase in signal transduction.

Yi T, Cleveland JL, Ihle JN
Identification of novel protein tyrosine phosphatases of hematopoietic cells by polymerase chain reaction amplification.
Blood. 1991; 78: 2222-8
Display abstract
Polymerase chain reaction (PCR) conditions were used to amplify cDNAs that encode putative protein tyrosine phosphatases (PTPs) from a murine interleukin-3-dependent myeloid cell line. Primers for the reactions were based on conserved sequences of the catalytic domain that are shared among all known PTPs. Sequencing of 100 PCR-amplified cDNA clones identified seven different cDNA sequences. Two of these sequences were identical to the murine PTP genes Ly5/CD45/LCA and LRP/R-PTP-alpha. Two of the cDNA sequences were 95% identical to human PTP epsilon (HPTP epsilon) and rat brain PTP (PTP1B), respectively, and are likely to represent their murine homologs. Three of the cDNA sequences encoded novel potential PTPs. One of the putative PTPs was ubiquitously expressed while a second was predominantly expressed in brain, kidney, and liver and at much lower levels in a variety of other cell tkpes and tissues. The third novel putative phosphatase was expressed primarily in hematopoietic cells and tissues in a pattern that was comparable with Ly5/CD45/LCA. Further characterization of these novel PTPs will provide insights into the growth regulation of hematopoietic cells.

Tonks NK, Diltz CD, Fischer EH
CD45, an integral membrane protein tyrosine phosphatase. Characterization of enzyme activity.
J Biol Chem. 1990; 265: 10674-80
Display abstract
Although CD45 resembles the low Mr protein tyrosine phosphatases (PTPases) from human placenta in its specificity for phosphotyrosyl residues and absolute dependence on sulfhydryl compounds for activity, it also exhibits a number of distinguishing features. Most notably, it displayed substrate specificity in vitro, preferentially dephosphorylating myelin basic protein, over the other substrates tested, with high specific activity. Limited trypsinization of CD45 generated active fragments of approximately 65 kDa that were apparently derived exclusively from the intracellular segment of the molecule. These retained high activity against myelin basic protein, suggesting that this is an intrinsic feature of the PTPase domains and not the result of secondary interactions between the substrate and the putative ligand binding structure. With reduced carboxamidomethylated and maleylated lysozyme as substrate, CD45 was stimulated up to 12-fold by basic compounds such as spermine; divalent metal ions were also stimulatory, most notably Zn2+, which was previously identified as a potent inhibitor of the low Mr PTPases. CD45 was phosphorylated to high stoichiometry by casein kinase-2 (up to 1.5 mol/mol) and also by glycogen synthase kinase 3 (approximately 0.3 mol/mol) and protein kinase C (approximately 0.1 mol/mol); in all cases, no alteration in enzyme activity was detected following these modifications. Autophosphorylated preparations of epidermal growth factor receptor, insulin receptor, and p56lck protein tyrosine kinases were also substrates for CD45 in vitro.

Lau KH, Farley JR, Baylink DJ
Phosphotyrosyl protein phosphatases.
Biochem J. 1989; 257: 23-36

Tonks NK, Diltz CD, Fischer EH
Characterization of the major protein-tyrosine-phosphatases of human placenta.
J Biol Chem. 1988; 263: 6731-7
Display abstract
In the preceding article (Tonks, N. K., Diltz, C. D., and Fischer, E. H. (1988) J. Biol. Chem. 263, 6722-6730), the purification of the major protein-tyrosine-phosphatases from human placenta, some to apparent homogeneity, was described. This report compares the characteristics of these enzymes and clearly identifies at least two distinct protein-tyrosine-phosphatase catalytic subunits. All were absolutely specific for phosphotyrosyl residues and showed no activity on any of the phosphoseryl/phosphothreonyl-containing proteins tested; they exhibited a high affinity for substrate with Km values in the submicromolar range. All were absolutely dependent on sulfhydryl compounds and appeared to contain at least one highly reactive cysteinyl residue essential for activity. Subtypes 1A and 1B could be distinguished by their response to polyanionic and polycationic compounds. The 1B enzymes were activated by EDTA, spermine, spermidine, and myelin basic protein to a greater extent than the 1A subtypes. Furthermore, they were inhibited by approximately 2 orders of magnitude lower concentrations of heparin (IC50 approximately 20 nM) and 1:1 or 4:1 poly (glutamate/tyrosine) (IC50 approximately 50 nM) than the 1A subtypes. Surprisingly, inhibition by the glutamate/tyrosine copolymers was strictly noncompetitive. Peptide mapping following digestion with Achromobacter protease I or Staphylococcus aureus V8 protease supported the view that, whereas protein-tyrosine-phosphatase subtypes 1A and 1B are different, their soluble and particulate counterparts are closely related structurally and are distinct from serine/threonine phosphatases 1 and 2A.