Low Molecular Weight Protein-tyrosine Phosphatase Tyrosine Phosphorylation by c-Src during Platelet-derived Growth Factor-induced Mitogenesis Correlates with Its Subcellular Targeting (1998)

The low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. Our previous results have shown that LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular LMW-PTP is involved in pathways that regulate the transcription of the immediately early genesmyc and fos in response to growth factor stimulation. In this study we have established that, in nontransformed NIH3T3 cells, LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and to phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its tyrosine phosphorylation, LMW-PTP significantly increases its catalytic activity. After PDGF stimulation these two LMW-PTP pools act on distinct substrates, contributing in different manners to the PDGF receptor signaling. The cytoplasmic LMW-PTP fraction exerts its well known action on activated PDGF receptor. On the other hand we have now demonstrated that the cytoskeleton-associated LMW-PTP acts specifically on a few not yet identified proteins that become tyrosine-phosphorylated in response to the PDGF receptor activation. Finally, these two LMW-PTP pools markedly differ in the timing of the processes in which they are involved. The cytoplasmic LMW-PTP pool exerts its action within a few minutes from PDGF receptor activation (short term action), while tyrosine phosphorylation of cytoskeleton-associated LMW-PTP lasts for more than 40 min (long term action). In conclusion LMW-PTP is a striking example of an enzyme that exerts different functions and undergoes different regulation in consequence of its subcellular localization. The low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. Our previous results have shown that LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular LMW-PTP is involved in pathways that regulate the transcription of the immediately early genesmyc and fos in response to growth factor stimulation. In this study we have established that, in nontransformed NIH3T3 cells, LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and to phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its tyrosine phosphorylation, LMW-PTP significantly increases its catalytic activity. After PDGF stimulation these two LMW-PTP pools act on distinct substrates, contributing in different manners to the PDGF receptor signaling. The cytoplasmic LMW-PTP fraction exerts its well known action on activated PDGF receptor. On the other hand we have now demonstrated that the cytoskeleton-associated LMW-PTP acts specifically on a few not yet identified proteins that become tyrosine-phosphorylated in response to the PDGF receptor activation. Finally, these two LMW-PTP pools markedly differ in the timing of the processes in which they are involved. The cytoplasmic LMW-PTP pool exerts its action within a few minutes from PDGF receptor activation (short term action), while tyrosine phosphorylation of cytoskeleton-associated LMW-PTP lasts for more than 40 min (long term action). In conclusion LMW-PTP is a striking example of an enzyme that exerts different functions and undergoes different regulation in consequence of its subcellular localization. protein-tyrosine phosphatase low molecular weight protein-tyrosine phosphatase dominant negative LMW-PTP platelet-derived growth factor PDGF receptor wild type LMW-PTP signal transducer and activator of transcription. Signal transduction cascades driven by tyrosine phosphorylation regulate many cellular processes in eukaryotes such as cell proliferation, differentiation, and migration (1Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (165) Google Scholar, 2Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (741) Google Scholar). The extent of protein tyrosine phosphorylation is determined by the concerted activity of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs).1 In recent years the importance of PTPs in the regulation of intracellular tyrosine phosphorylation level has been widely recognized. The PTPs superfamily consists of over 70 enzymes that, despite very limited sequence similarity, have a common active site motif CX 5R and an identical catalytic mechanism. On the basis of their function, structure, and sequence, PTPs are often classified in four main families: 1) tyrosine-specific phosphatases, 2) VH1-like dual specificity PTPs, 3) the Cdc25, and 4) the low molecular weight phosphatase (3Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar). The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an 18-kDa enzyme that is expressed in a wide variety of mammalian tissues (4Ramponi G. Adv. Protein Phosphatases. 1994; 8: 1-25Google Scholar). In our previous studies on the physiologic functions of LMW-PTP, we have expressed in NIH3T3 cells a catalytically inactive Cys12 to Ser LMW-PTP mutant, which has maintained its capacity of substrate binding (5Chiarugi P. Marzocchini R. Raugei G. Pazzagli C. Berti A. Camici G. Manao G. Cappugi G. Ramponi G. FEBS Lett. 1992; 310: 9-12Crossref PubMed Scopus (31) Google Scholar, 6Cirri P. Chiarugi P. Camici G. Manao G. Raugei G. Ramponi G. Eur. J. Biochem. 1993; 214: 647-657Crossref PubMed Scopus (99) Google Scholar). Overexpression of this LMW-PTP mutant form caused enhanced cell proliferation and serum-induced mitogenesis, indicating that the C12S mutant is a dominant negative LMW-PTP (dnLMW-PTP) (7Chiarugi P. Cirri P. Raugei G. Camici G. Dolfi F. Berti A. Ramponi G. FEBS Lett. 1995; 372: 49-53Crossref PubMed Scopus (104) Google Scholar). Analysis of dnLMW-PTP overexpressing clones led to the identification of PDGF-R as a specific in vivosubstrate of LMW-PTP and to the demonstration that this phosphatase is most probably involved in the control of one or more signaling pathways triggered by PDGF-R activation (8Chiarugi P. Cirri P. Marra F. Raugei G. Fiaschi T. Camici G. Manao G. Romanelli R.G. Ramponi G. J. Biol. Chem. 1998; 273: 6776-6785Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The action of LMW-PTP is essentially restricted to G1 phase of the cell cycle and influences myc and fos gene induction driven by PDGF-R activation. In particular, LMW-PTP appears to be involved in the regulation of myc expression interfering with Src pathway and in the regulation of fos activation through an extracellular signal-regulated kinase-independent pathway mediated by the STAT proteins (8Chiarugi P. Cirri P. Marra F. Raugei G. Fiaschi T. Camici G. Manao G. Romanelli R.G. Ramponi G. J. Biol. Chem. 1998; 273: 6776-6785Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Previous results (9Rigacci S. Degl'Innocenti D. Bucciantini M. Cirri P. Berti A. Ramponi G. J. Biol. Chem. 1996; 271: 1278-1281Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) have shown that LMW-PTP is tyrosine-phosphorylated in unstimulated v-Src overexpressing cells, although no direct association was detected between v-Src and LMW-PTP. More recently, it has been reported that in resting Jurkat cells LMW-PTP is tyrosine-phosphorylated, apparently by Lck kinase, and rapidly dephosphorylated upon T cell receptor stimulation (10Tailor P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5374Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The same paper reported that LMW-PTP phosphorylation occurs in Tyr131 and partially in Tyr132, leading to a 2-fold enhancement of LMW-PTP enzymatic activity. Src tyrosine kinase associates with activated PDGF receptor, becomes tyrosine-phosphorylated, slightly increases its catalytic activity, and translocates to the cell periphery by an actin-dependent process (11Fincham V.J. Unlu M. Brunton V.G. Pitts J.D. Wyke J.A. Frame M.C. J. Cell Biol. 1996; 135: 1551-1564Crossref PubMed Scopus (155) Google Scholar). Here Src becomes associated with pp125FAK, and this fact represents an early and critical event in the assembly of focal adhesion complex (12Schaller M.D. Hilldebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1121) Google Scholar). It has been demonstrated that Src/FAK association enhances Src activity, and this is a prerequisite for tyrosine phosphorylation of other focal adhesion or cytoskeleton-associated proteins (13Parsons J.T. Parsons S.J. Curr. Opin. Cell Biol. 1997; 9: 187-192Crossref PubMed Scopus (355) Google Scholar). These considerations led us to examine the role of the LMW-PTP tyrosine phosphorylation in relation to its subcellular targeting upon PDGF-induced mitogenesis in NIH3T3 cells. We find that in NIH3T3 cells LMW-PTP is localized in both cytoplasmic and cytoskeleton-associated fractions also in the absence of PDGF treatment. These two different LMW-PTP pools are differentially regulated, since only the cytoskeleton-associated LMW-PTP fraction is specifically phosphorylated by c-Src after PDGF stimulation. As a consequence of this phosphorylation, LMW-PTP greatly increases its catalytic activity probably toward cytoskeleton-associated Tyr-phosphorylated substrate(s). Furthermore we show that these two intracellular LMW-PTP pools exert their actions at different times starting from PDGF-R stimulation. Altogether these data are consistent with the hypothesis that LMW-PTP is subjected to different regulatory mechanisms and has different substrate specificity in consequence of its subcellular localization. Unless specified all reagents were obtained from Sigma. NIH3T3 cells were purchased from ATCC; human recombinant platelet-derived growth factor BB (PDGF-BB) was from Peprotech; enhanced chemiluminescence kit was from Amersham Pharmacia Biotech; Src kinase assay kit and anti-Src antibodies were from Upstate Biotechnology Inc.; anti-PDGF receptor antibodies and PY20 and PY99 anti-phosphotyrosine antibodies were from Santa Cruz. NIH3T3 cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in 5% CO2 humidified atmosphere. 10 μg of pSVT7PTPC12S (7Chiarugi P. Cirri P. Raugei G. Camici G. Dolfi F. Berti A. Ramponi G. FEBS Lett. 1995; 372: 49-53Crossref PubMed Scopus (104) Google Scholar) or pSVT7PTP (14Ramponi G. Ruggiero M. Raugei G. Berti A. Modesti A. Degl'Innocenti D. Magnelli L. Pazzagli C. Chiarugi V.P. Camici G. Int. J. Cancer. 1992; 51: 652-656Crossref PubMed Scopus (59) Google Scholar) and 0.5 μg of pSV2neo, conferring neomycin resistance, were cotransfected in NIH3T3 cells using the calcium phosphate method. Stable transfected clonal cell lines were isolated by selection with G418 (400 μg/ml). Control cell lines were obtained by transfecting 2 μg of pSV2neo alone. The clonal lines were screened for expression of the transfected genes by (a) Northern blot analysis and (b) enzyme-linked immunosorbent assay polyclonal anti-LMW-PTP rabbit antibodies, which do not cross-react with murine endogenous LMW-PTP. 10 μg of pSGT vector-base constructs overexpressing dominant negative c-Src (SrcK−) (kindly provided by Dr. S. Courtneidge) and 0.5 μg of pBABEpuro, conferring puromycin resistance were cotransfected in NIH3T3 cells or in wtLMW-PTP overexpressing cells, using the calcium phosphate method. Stable transfected clonal cell lines were isolated by selection with puromycin (1 mg/liter). The clonal lines were screened for expression of the transfected genes by anti-p60 Src monoclonal antibodies (Santa Cruz). 1 × 106 cells were seeded in 10-cm plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were serum-starved for 24 h before receiving 30 ng/ml PDGF-BB. Freshly made pervanadate solution (50 mm sodium vanadate and 50 mm H2O2) was added to the cells at a final concentration of 0.1 mm 30 min before the cells were lysed. Cells were then lysed for 20 min on ice in 500 μl of RIPA lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 2 mm EGTA, 1 mm sodium orthovanadate, 1 mmphenylmethanesulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin). Complete RIPA lysis buffer (cRIPA) is RIPA plus 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate. Lysates were clarified by centrifugation and immunoprecipitated for 4 h at 4 °C with 1 μg of the specific antibodies. Immune complexes were collected on protein A-Sepharose (Amersham Pharmacia Biotech), separated by SDS-PAGE, and transferred onto were in serum 10 mm pH 7.5, 1 mm and 0.1% for 1 h at with specific antibodies and then with antibodies with and with the enhanced chemiluminescence kit (Amersham Pharmacia NIH3T3 were lysed in RIPA and the were clarified by centrifugation at × for 30 were with 1 of RIPA and then in buffer by for 1 h at and clarified by centrifugation at × for 30 RIPA or fractions were then for localization of many such as protein kinase is by extracellular and phase is a S. J. Cell Biol. 1998; PubMed Scopus Google Scholar, J. Cell Biol. 1997; PubMed Scopus Google Scholar). RIPA buffer is a lysis buffer such as or Nonidet It is that the fraction cytosolic and many of the focal and cytoskeleton-associated to be lysed. are by while cytoskeleton-associated are using such as deoxycholate or J. Cell Biol. 1997; PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). We have demonstrated that LMW-PTP is an enzyme that upon PDGF stimulation with the activated PDGF receptor (7Chiarugi P. Cirri P. Raugei G. Camici G. Dolfi F. Berti A. Ramponi G. FEBS Lett. 1995; 372: 49-53Crossref PubMed Scopus (104) Google Scholar). LMW-PTP action is restricted to cytosolic fraction or we have the LMW-PTP in RIPA and fractions after PDGF stimulation. NIH3T3 cells overexpressing LMW-PTP were with 30 ng/ml of PDGF for and the were as RIPA and fractions were in with anti-LMW-PTP antibodies. blot 1 that LMW-PTP in both fractions and that its is not on growth factor We reported that LMW-PTP is not tyrosine-phosphorylated, in the resting activation of the receptor transduction pathway (7Chiarugi P. Cirri P. Raugei G. Camici G. Dolfi F. Berti A. Ramponi G. FEBS Lett. 1995; 372: 49-53Crossref PubMed Scopus (104) Google Scholar). the LMW-PTP in the fraction not become tyrosine-phosphorylated in response to PDGF stimulation 1 we that in the same the LMW-PTP associated to the fraction becomes tyrosine-phosphorylated upon PDGF stimulation as by the same blot with anti-phosphotyrosine antibodies 1 We demonstrated that LMW-PTP associates with the activated to PDGF-R and specifically the Src and the STAT pathways of the signaling (8Chiarugi P. Cirri P. Marra F. Raugei G. Fiaschi T. Camici G. Manao G. Romanelli R.G. Ramponi G. J. Biol. Chem. 1998; 273: 6776-6785Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In 2 we the analysis of the association between PDGF-R and LMW-PTP after subcellular with RIPA and In this we have NIH3T3 overexpressing dnLMW-PTP to the between the PDGF-R and the which occurs the LMW-PTP catalytic site (7Chiarugi P. Cirri P. Raugei G. Camici G. Dolfi F. Berti A. Ramponi G. FEBS Lett. 1995; 372: 49-53Crossref PubMed Scopus (104) Google Scholar). Cells were for Lysates were then as and in anti-LMW-PTP The 2 that PDGF-R with LMW-PTP only in the RIPA while it not in the fraction. The same were then for LMW-PTP tyrosine phosphorylation 2 that the of LMW-PTP with the activated PDGF-R is not by LMW-PTP tyrosine phosphorylation, since this is restricted to the cytoskeleton-associated fraction. to the results of the previous the RIPA and pools of LMW-PTP are separated within the cell and exert different functions in cell We have the of both association and the LMW-PTP tyrosine NIH3T3 cells overexpressing dnLMW-PTP were serum-starved for 24 h and then with 30 ng/ml of PDGF for or 20 the association analysis cells were lysed using RIPA buffer and immunoprecipitated with antibodies. The anti-LMW-PTP that the association between the two is a min after PDGF and then rapidly In the phosphorylation analysis cells were lysed in and the were immunoprecipitated using anti-LMW-PTP antibodies. The anti-phosphotyrosine in that LMW-PTP tyrosine phosphorylation is a since it a 10 min after PDGF stimulation and then Previous that LMW-PTP is phosphorylated on tyrosine in cells overexpressing v-Src and in resting Jurkat (9Rigacci S. Degl'Innocenti D. Bucciantini M. Cirri P. Berti A. Ramponi G. J. Biol. Chem. 1996; 271: 1278-1281Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, P. Gilman J. Williams S. Couture C. Mustelin T. J. Biol. Chem. 1997; 272: 5371-5374Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). study the role of c-Src tyrosine kinase in LMW-PTP phosphorylation PDGF signal we to cells overexpressing both the dominant negative form of c-Src (SrcK−) and the After PDGF stimulation, we have the LMW-PTP tyrosine phosphorylation level in overexpressing cells with control cells overexpressing wtLMW-PTP alone. the tyrosine phosphorylation level of LMW-PTP, both cell lines were with 1 mm pervanadate for 30 min before PDGF stimulation to phosphotyrosine phosphatases activity. is a specific and of PTPs action to of tyrosine phosphorylation level of many intracellular proteins M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Cells were lysed with and immunoprecipitated with anti-LMW-PTP antibodies. of the anti-phosphotyrosine are shown in 4 A. After PDGF stimulation, in overexpressing cells we a LMW-PTP phosphorylation level with to cells overexpressing wtLMW-PTP both in and cells. In 4 we as the LMW-PTP in the same In a recent we reported that expression not PDGF-R as by the fact that, in these cells, the level of phosphorylation of PDGF-R is with to not transfected cells P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. 1997; PubMed Scopus Google Scholar). overexpressing cells a active PDGF-R while a Src kinase activity. these data a specific role of Src tyrosine kinase and not of PDGF-R in LMW-PTP tyrosine phosphorylation PDGF stimulation. LMW-PTP phosphorylation is by c-Src or by a tyrosine kinase, we to the association between LMW-PTP and Src by of analysis both in RIPA and wtLMW-PTP and overexpressing cells were serum-starved for 24 h and with PDGF for the anti-LMW-PTP of the anti-Src of the Our results that is an association between LMW-PTP and c-Src both in and wtLMW-PTP overexpressing cells, after PDGF stimulation. The association is restricted to the fraction in with our previous the of between LMW-PTP and c-Src in the cytosolic fraction PDGF stimulation (8Chiarugi P. Cirri P. Marra F. Raugei G. Fiaschi T. Camici G. Manao G. Romanelli R.G. Ramponi G. J. Biol. Chem. 1998; 273: 6776-6785Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). was cells with The results shown in that is from pervanadate thus that the association between the two is not mediated by LMW-PTP catalytic PDGF-R is a substrate of LMW-PTP only in the RIPA since is not association between these two proteins in the fraction find of LMW-PTP in the we the tyrosine phosphorylation level of the cytoskeleton-associated proteins upon PDGF stimulation. Analysis of was by of anti-phosphotyrosine and anti-phosphotyrosine in cells overexpressing and cells. in led us to the identification of a differentially tyrosine-phosphorylated with an molecular of and two of and These proteins LMW-PTP in the fraction. in cells the tyrosine-phosphorylated protein in the is PDGF-R we the with antibodies with negative these data that LMW-PTP have different in distinct subcellular localization. Our previous studies the role of LMW-PTP in the PDGF-induced mitogenesis. We have shown that LMW-PTP is a in the early of PDGF-R signal it to activated receptor and the activation of Src tyrosine kinase and the phosphorylation of STAT We have also that LMW-PTP action on PDGF signal transduction is since this enzyme influences only these two signaling In recent data from our have shown that in NIH3T3 cells, c-Src to an of both and the of LMW-PTP on STAT pathway be a consequence of the action of LMW-PTP on Src activity P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. 1997; PubMed Scopus Google Scholar). the of LMW-PTP with the Src and the pathways myc and fos two for G1 (8Chiarugi P. Cirri P. Marra F. Raugei G. Fiaschi T. Camici G. Manao G. Romanelli R.G. Ramponi G. J. Biol. Chem. 1998; 273: 6776-6785Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In this study we have by of subcellular two different and LMW-PTP The that we RIPA the and many of the The the and such as focal adhesion We have established that the LMW-PTP associated with fraction is tyrosine-phosphorylated upon PDGF stimulation, while cytosolic LMW-PTP is not The LMW-PTP pool is the only one that with the activated as by our data in assay in RIPA and fractions In no tyrosine phosphorylation is in LMW-PTP, thus that LMW-PTP phosphorylation is not by In with our reported results (7Chiarugi P. Cirri P. Raugei G. Camici G. Dolfi F. Berti A. Ramponi G. FEBS Lett. 1995; 372: 49-53Crossref PubMed Scopus (104) Google these data that tyrosine kinase be in this We have now shown that LMW-PTP tyrosine phosphorylation is by Src tyrosine kinase on the basis of two in cells overexpressing the dominant negative form of which is catalytically inactive able to bind its substrates, we a of LMW-PTP phosphorylation in response to PDGF with to control cells c-Src with LMW-PTP, as by this association is restricted to the LMW-PTP, and it is not in the RIPA to the that LMW-PTP is phosphorylated only in the cytoskeleton-associated fraction On the other we demonstrated that the of PDGF-R is not in cells, as by tyrosine phosphorylation level of PDGF-R in cells in with NIH3T3 control cells P. Chiarugi P. Marra F. Raugei G. Camici G. Manao G. Ramponi G. Biochem. 1997; PubMed Scopus Google Scholar). these that the in LMW-PTP tyrosine phosphorylation be to c-Src kinase activity. In the association between these two is of a of These data the hypothesis that the between LMW-PTP and c-Src is not mediated by LMW-PTP catalytic On the we reported that in the of the activated in the RIPA the of the this association P. Cirri P. Raugei G. Manao G. L. Ramponi G. Biochem. 1996; PubMed Scopus (31) Google Scholar). The c-Src has been to in both cellular adhesion and within the growth factor stimulation D. M. Sci. S. A. PubMed Scopus Google although its v-Src has been in adhesion in cells (11Fincham V.J. Unlu M. Brunton V.G. Pitts J.D. Wyke J.A. Frame M.C. J. Cell Biol. 1996; 135: 1551-1564Crossref PubMed Scopus (155) Google Scholar, Cell. Full Text PDF PubMed Scopus Google Scholar). fact for the reported LMW-PTP phosphorylation (9Rigacci S. Degl'Innocenti D. Bucciantini M. Cirri P. Berti A. Ramponi G. J. Biol. Chem. 1996; 271: 1278-1281Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In this we show that the LMW-PTP tyrosine phosphorylation is a not restricted to cells, occurs also in cells in response to PDGF stimulation. Src tyrosine kinase has many different one of the tyrosine kinase, is active in focal of cell and growth factor to a in tyrosine phosphorylation of and of focal proteins such as and J. Cell Sci. 1995; PubMed Google Scholar). The of a kinase between and c-Src is an early and critical event in the of focal adhesion complexes the activation of growth and signaling that the association of Src and enhances Src activity, and this is a prerequisite for cytoskeleton-associated Src activation leading to the phosphorylation of proteins in focal adhesion (13Parsons J.T. Parsons S.J. Curr. Opin. Cell Biol. 1997; 9: 187-192Crossref PubMed Scopus (355) Google Scholar). LMW-PTP is these phosphorylated We have that the phosphorylation of LMW-PTP in by c-Src to an of in the LMW-PTP enzymatic activity. P. L. G. G. G. and G. for after PDGF stimulation the two LMW-PTP pools very in enzymatic activity that distinct in signal we the of the two in which LMW-PTP is involved. LMW-PTP with restricted to the RIPA is and a min after PDGF stimulation, while LMW-PTP phosphorylation in cytoskeleton-associated fraction is a and only after 40 min These and between the two LMW-PTP pools distinct substrate specificity and different or of the two LMW-PTP pools in different subcellular localization. Finally, we have for tyrosine-phosphorylated proteins that LMW-PTP substrate in the cytoskeleton-associated fraction. Our results with wtLMW-PTP or dnLMW-PTP overexpressing cells in fraction show a protein of which is differentially phosphorylated in these cell lines We have that this is not we were not able to this protein that be a cytoskeleton-associated substrate of LMW-PTP. proteins 1) have a molecular of 2) in the and 3) become tyrosine-phosphorylated in response to and a yet Eur. J. Biochem. 1996; PubMed Scopus Google Scholar) are to be a LMW-PTP substrate in the fraction. In the role of LMW-PTP the transduction of the PDGF signaling appears more complex than the regulation of PDGF-R LMW-PTP is constitutively in distinct intracellular most probably with different The cytosolic LMW-PTP fraction is to the activated which is a is a event that not to LMW-PTP tyrosine On the other in response to PDGF stimulation, the cytoskeleton-associated LMW-PTP pool becomes tyrosine-phosphorylated by event is a which to an in LMW-PTP catalytic activity and be for the of cytoskeleton-associated proteins such as In the two LMW-PTP distinct subcellular localization and different to PDGF stimulation, in cell