Oncogenic activation of c-ABL by mutation within its last exon.

The c-ABL proto-oncogene is a predominantly nuclear localized tyrosine kinase. A random mutagenesis scheme was used to isolate c-ABL mutants whose expression produced a transformed phenotype in rodent fibroblast cells. An in-frame deletion within the central region of the last exon was identified in one ABL mutant. The mechanism of c-ABL oncogenic activation by mutation within the last exon differs both functionally and structurally from those of v-ABL and BCR/ABL. This class of ABL mutants shows increased tyrosine phosphorylation of cellular proteins in vivo but low levels of autophosphorylation. Last-exon ABL mutants are distinguished from v-ABL or BCR/ABL by their inability to transform primary bone marrow cells or support the growth of transformed pre-B cells. These findings define a new mechanism of oncogenic activation for the ABL kinase through mutations in the last exon which do not require amino-terminal deletions or mutations within the src homology regions.     

Insulin-stimulated hydrogen peroxide reversibly inhibits protein-tyrosine phosphatase 1b in vivo and enhances the early insulin action cascade

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins and balanced by the action of specific protein-tyrosine phosphatases (PTPases). PTPase activity, in turn, is highly regulated in vivo by oxidation/reduction reactions involving the cysteine thiol moiety required for catalysis. Here we show that insulin stimulation generates a burst of intracellular H(2)O(2) in insulin-sensitive hepatoma and adipose cells that is associated with reversible oxidative inhibition of up to 62% of overall cellular PTPase activity, as measured by a novel method using strictly anaerobic conditions. The specific activity of immunoprecipitated PTP1B, a PTPase homolog implicated in the regulation of insulin signaling, was also strongly inhibited by up to 88% following insulin stimulation. Catalase pretreatment abolished the insulin-stimulated production of H(2)O(2) as well as the inhibition of cellular PTPases, including PTP1B, and was associated with reduced insulin-stimulated tyrosine phosphorylation of its receptor and high M(r) insulin receptor substrate (IRS) proteins. These data provide compelling new evidence for a redox signal that enhances the early insulin-stimulated cascade of tyrosine phosphorylation by oxidative inactivation of PTP1B and possibly other tyrosine phosphatases.     

Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL.

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)     

BCR/ABL Recruits p53 Tumor Suppressor Protein to Induce Drug Resistance

Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoidapoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is animportant activator of apoptosis in normal cells; conversely its functional loss may causedrug resistance. The ABL oncoprotein - p53 paradigm represents the relationship between anoncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABLoncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells.Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNAdamage. In contrast, only a modest increase of p53 expression followed by activation ofcaspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15residue was associated with the accumulation of p53, and stimulation of p21Waf-1 andGADD45, resulting in G2/M delay in BCR/ABL cells after genotoxic treatment. Inhibition ofp53 by siRNA or by the temperature-sensitive mutation reduced G2/M accumulation anddrug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 proteincontributed to prolonged G2/M checkpoint activation and drug resistance in myeloid cellsexpressing the BCR/ABL oncoproteins.     

BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP(-/-) mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.     

A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.     

Transient Association of the Phosphotyrosine Phosphatase SHP-2 with TrkA Is Induced by Nerve Growth Factor

Abstract: Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an ingel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP-2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP-2 with TrkA was accompanied by the tyrosine phosphorylation of SHP-2 and an association of SHP-2 with multiple tyrosine-phosphorylated proteins. In addition, the PTPase activity in SHP-2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP-2 with TrkA is induced by NGF and that this association leads to SHP-2 activation and tyrosine phosphorylation. We conclude that SHP-2 plays a significant role in early biochemical events in TrkA-mediated signal transduction.     

SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence.

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.     

Sodium stibogluconate is a potent inhibitor of protein tyrosine phosphatases and augments cytokine responses in hemopoietic cell lines.

Using in vitro protein tyrosine phosphatase (PTPase) assays, we found that sodium stibogluconate, a drug used in treatment of leishmaniasis, is a potent inhibitor of PTPases Src homology PTPase1 (SHP-1), SHP-2, and PTP1B but not the dual-specificity phosphatase mitogen-activated protein kinase phosphatase 1. Sodium stibogluconate inhibited 99% of SHP-1 activity at 10 micrograms/ml, a therapeutic concentration of the drug for leishmaniasis. Similar degrees of inhibition of SHP-2 and PTP1B required 100 micrograms/ml sodium stibogluconate, demonstrating differential sensitivities of PTPases to the inhibitor. The drug appeared to target the SHP-1 domain because it showed similar in vitro inhibition of SHP-1 and a mutant protein containing the SHP-1 PTPase domain alone. Moreover, it forms a stable complex with the PTPase: in vitro inhibition of SHP-1 by the drug was not removed by a washing process effective in relieving the inhibition of SHP-1 by the reversible inhibitor suramin. The inhibition of cellular PTPases by the drug was suggested by its rapid induction of tyrosine phosphorylation of cellular proteins in Baf3 cells and its augmentation of IL-3-induced Janus family kinase 2/Stat5 tyrosine phosphorylation and proliferation of Baf3 cells. The augmentation of the opposite effects of GM-CSF and IFN-alpha on TF-1 cell growth by the drug indicated its broad activities in the signaling of various cytokines. These data represent the first evidence that sodium stibogluconate inhibits PTPases and augments cytokine responses. Our results provide novel insights into the pharmacological effects of the drug and suggest potential new therapeutic applications.     

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.     

ABL-fusion oncoproteins activate multi-pathway of DNA repair: role in drug resistance?

Chromosomal translocations of tyrosine kinase c-ABL gene from chromosome 9 may generate oncogenic kinases exhibiting constitutive tyrosine kinase activity. Recently, we have shown that ABL-fusion oncogenic tyrosine kinases, BCR/ABL and TEL/ABL, specific to hematopoietic malignances, induced resistance to DNA-damaging agents. To elucidate the role of DNA repair in this phenomenon we examined the capacity of murine BaF3 lymphoid cells and their TEL/ABL-transformed counterparts to repair DNA lesions caused by gamma- and UV-radiations and the anti-cancer drug, idarubicin. TEL/ABL-transformed cells displayed resistance to these DNA damaging agents as evaluated by MTT assay and the survival advantage was associated with an accelerated kinetics of DNA repair as measured by the alkaline comet assay. Deoxyribonucleosides (dNTPs) supplementation of the repair medium further stimulated DNA repair and the effect was specific to the DNA damage agent used in the experiment but only the transformed cells displayed this feature. A variety of damages induced imply the multi-pathway of DNA repair involved. We also examined the capability of BCR/ABL-fusion to modulate the repair of oxidative lesions, considered as a major side effect of various anti-cancer drugs including idarubicin and radiation. Employing the free radical scavenger alpha-phenyl-N-tert-butyl nitrone (PBN, a spin trap) and DNA repair enzymes: endonuclease III (EndoIII) that nicks DNA at sites of oxidized bases, we found that BCR/ABL-transformed cells repaired oxidative DNA lesions more effectively than control cells. Our results suggest, that oncogenic ABL-dependent stimulation of DNA repair may contribute to the cell resistance to genotoxic treatment.     

PTPH1 is a predominant protein-tyrosine phosphatase capable of interacting with and dephosphorylating the T cell receptor zeta subunit

Protein-tyrosine phosphatases (PTPases) play key roles in regulating tyrosine phosphorylation levels in cells, yet the identity of their substrates remains limited. We report here on the identification of PTPases capable of dephosphorylating the phosphorylated immune tyrosine-based activation motifs present in the T cell receptor zeta subunit. To characterize these PTPases, we purified enzyme activities directed against the phosphorylated T cell receptor zeta subunit by a combination of anion and cation chromatography procedures. A novel ELISA-based PTPase assay was developed to rapidly screen protein fractions for enzyme activity following the various chromatography steps. We present data that SHP-1 and PTPH1 are present in highly enriched protein fractions that exhibit PTPase activities toward a tyrosine-phosphorylated TCR zeta substrate (specific activity ranging from 0.23 to 40 pmol/min/microg). We also used a protein-tyrosine phosphatase substrate-trapping library comprising the catalytic domains of 47 distinct protein-tyrosine phosphatases, representing almost all the tyrosine phosphatases identified in the human genome. PTPH1 was the predominant phosphatase capable of complexing phospho-zeta. Subsequent transfection assays indicated that SHP-1 and PTPH1 are the two principal PTPases capable of regulating the phosphorylation state of the TCR zeta ITAMs, with PTPH1 directly dephosphorylating zeta. This is the first reported demonstration that PTPH1 is a candidate PTPase capable of interacting with and dephosphorylating TCR zeta.     

Protein tyrosine phosphatase regulation in fibroblasts from patients with an insulin receptor gene mutation

Tyrosine phosphorylation of the insulin receptor is the initial event following receptor binding to insulin, and it induces further tyrosine phosphorylation of various intracellular molecules. This signaling is countered by protein tyrosine phosphatases (PTPases), which reportedly are associated with insulin resistance that can be reduced by regulation of PTPases. Protein tyrosine phosphatase 1B (PTP1B) and leukocyte antigen-related PTPase (LAR) are the PTPases implicated most frequently in insulin resistance and diabetes mellitus. Here, we show that PTP1B and LAR are expressed in human fibroblasts, and we examine the regulation of PTPase activity in fibroblasts from patients with an insulin receptor gene mutation as an in vitro model of insulin resistance. Total PTPase activity was significantly lower in the cytosolic and membrane fractions of fibroblasts with mutations compared with controls (p<0.05). Insulin stimulation of fibroblasts with mutations resulted in a significantly smaller increase in PTP1B activity compared with stimulation of wild-type fibroblasts (p<0.05). This indicates that insulin receptor gene mutations blunt increases in PTPase activity in response to insulin, possibly via a negative feedback mechanism. Our data suggest that the PTPase activity in patients with insulin receptor gene mutation and severe insulin resistance may differ from that in ordinary type 2 diabetes.     

Effect of tumor necrosis factor-α on the phosphorylation of tyrosine kinase receptors is associated with dynamic alterations in specific protein-tyrosine phosphatases

Tumor necrosis factor-α (TNF-α) can modulate the signalling capacity of tyrosine kinase receptors; in particular, TNF-α has been shown to mediate the insulin resistance associated with animal models of obesity and noninsulin-dependent diabetes mellitus. In order to determine whether the effects of TNF-α might involve alterations in the expression of specific protein-tyrosine phosphatases (PTPases) that have been implicated in the regulation of growth factor receptor signalling, KRC-7 rat hepatoma cells were treated with TNF-α, and changes in overall tissue PTPase activity and the abundance of three major hepatic PTPases (LAR, PTP1B, and SH-PTP2) were measured in addition to effects of TNF-α on ligand-stimulated autophosphorylation of insulin and epidermal growth factor (EGF) receptors and insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation. TNF-α caused a dose-dependent decrease in insulin-stimulated IRS-1 phosphorylation and EGF-stimulated receptor autophosphorylation to 47–50% of control. Overall PTPase activity in the cytosol fraction did not change with TNF-α treatment, and PTPase activity in the particulate fraction was decreased by 55–66%, demonstrating that increases in total cellular PTPase activity did not account for the observed alterations in receptor signalling. However, immunoblot analysis showed that TNF-α treatment resulted in a 2.5-fold increase in the abundance of SH-PTP2, a 49% decrease in the transmembrane PTPase LAR, and no evident change in the expression of PTP1B. These data suggest that at least part of the TNF-α effect on pathways of reversible tyrosine phosphorylation may be exerted through the dynamic modulation of the expression of specific PTPases. Since SH-PTP2 has been shown to interact directly with both the EGF receptor and IRS-1, increased abundance of this PTPase may mediate the TNF-α effect to inhibit signalling through these proteins. Furthermore, decreased abundance of the LAR PTPase, which has been implicated in the regulation of insulin receptor phosphorylation, may account for the less marked effect of TNF-α on the autophosphorylation state of the insulin receptor while postreceptor actions of insulin are inhibited. J. Cell. Biochem. 64:117–127. © 1997 Wiley-Liss, Inc.     

Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.     

cDNA cloning of rat LRP, a receptor like protein tyrosine phosphatase, and evidence for its gene regulation in cultured rat mesangial cells.

Protein tyrosine phosphatases (PTPases) are a family of enzymes that play a crucial role in the regulation of signal transduction mediated by reversible protein tyrosine phosphorylation. To understand the significance of PTPases in physiological and pathophysiological processes in the kidney, we isolated three cDNA segments encoding PTPases (LAR, LRP and a novel PTPase) from rat kidney by polymerase chain reaction (PCR). Using PCR product as a probe, we isolated a full-length cDNA of rat LRP. LRP cDNA encoded a single membrane spanning protein consisted of 796 amino acids, with two tandemly located intracellular PTPase domains. By Northern analysis, a ubiquitous pattern of LRP gene expression in rat tissues was demonstrated. In cultured rat mesangial cells, LRP mRNA was detected and the mRNA level was suppressed by either interleukin-1 or interleukin-6 treatment.     

Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: purification and characterization for specificity and mechanism.

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high kcat/Km value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of 18O from 18O4 inorganic phosphate into H2(16)O at rates of approximately 1 x 10(-2) s-1. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a [32P]phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of 32P-labeled enzymes. Pulse/chase studies on the LAR 32P-enzyme showed turnover of the labeled phosphoryl group.     

Expression of a transmembrane phosphotyrosine phosphatase inhibits cellular response to platelet-derived growth factor and insulin-like growth factor-1.

Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.