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

Cirri, Paolo; Chiarugi, Paola; Taddei, Letizia; Raugei, Giovanni; Camici, Giovanni G.; Manao, Giampaolo; Ramponi, Giampietro

doi:10.1074/jbc.273.49.32522

Cited by 51 publications

(75 citation statements)

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“…It has been reported that the H 2 O 2 produced upon growth factor stimulation is really a cellular second messenger directly influencing many signal transducers such as the STAT proteins, p70 s6k, protein kinase D, and PTPs (33)(34)(35)(36). We have already demonstrated that LMW-PTP is a key regulator of PDGF-R phosphorylation upon recruitment to the membrane when cells are stimulated with the growth factor (5,6,10).…”

Section: Discussionmentioning

confidence: 90%

“…Cell Lysate Fractionations-Cell lysate fractions were obtained as already described (6). Briefly, PDGF-stimulated NIH3T3 cells were lysed in RIPA buffer, and the lysates were clarified by centrifugation for 30 min at 20,000 ϫ g. Pellets were washed twice with 1 ml of RIPA and then resuspended in complete RIPA buffer (cRIPA), which is RIPA buffer plus 0.5% sodium deoxycholate and 0.1% sodium dodecylsulfate, by shaking for 1 h at room temperature and clarifying by centrifugation at 20,000 ϫ g for 30 min.…”

Section: Site-specific Mutagenesis and Cloning Of Lmw-ptp Mutants In mentioning

confidence: 99%

“…More recently, we have found that in NIH3T3 cells LMW-PTP is constitutively localized in both cytoplasmic and cytoskeleton-associated fractions. These two LMW-PTP pools are differentially regulated because only the cytoskeleton-associated LMW-PTP fraction is specifically phosphorylated by c-Src after PDGF stimulation (6). As a consequence of its phosphorylation, LMW-PTP shows an average 20-fold increase in its in vitro catalytic activity (7,8,9).…”

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confidence: 99%

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Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

Chiarugi

Fiaschi

Taddei

et al. 2001

Journal of Biological Chemistry

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175

154

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Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H 2 O 2 or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H 2 O 2 , evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H 2 O 2 production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.Protein tyrosine phosphorylation plays a key role in the regulation of many cellular processes in eukaryotes such as cellular metabolism, proliferation, differentiation, and oncogenic transformation (1). Accumulating evidence indicates that the contribution of phosphotyrosine protein phosphatases (PTPs) 1 to the control of the cell phosphorylation state is as relevant as that of phosphotyrosine protein kinases. The PTP superfamily is composed of over 70 enzymes that, despite very limited sequence similarity, share a common CX 5 R active site motif and an identical catalytic mechanism. On the basis of their function, structure, and sequence, PTPs can be classified in four main families: 1) tyrosine-specific phosphatases, 2) VH1-like dual specificity PTPs, 3) the cdc25 phosphatases, and 4) the low molecular weight phosphatase (2).The low molecular weight protein tyrosine phosphatase (LMW-PTP) is an 18-kDa enzyme that is expressed in many mammalian tissues (3). Our previous studies on the molecular biology of LMW-PTP in NIH3T3 cells demonstrated a well defined role of this enzyme in platelet-derived growth factor (PDGF)-induced mitogenesis. The most relevant phenotypic effect of LMW-PTP overexpression is a strong reduction of the cell growth rate in response to PDGF stimulation. We ...

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Section: Discussionmentioning

confidence: 90%

Section: Site-specific Mutagenesis and Cloning Of Lmw-ptp Mutants In mentioning

confidence: 99%

mentioning

confidence: 99%

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Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

Chiarugi

Fiaschi

Taddei

et al. 2001

Journal of Biological Chemistry

Self Cite

175

154

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“…It was demonstrated previously that the cytoskeleton-associated fraction of LMW-PTP is phosphorylated by Src under PDGF stimulation in adherent cells (33). Such a phosphorylation seems fundamental for LMW-PTP association with p190Rho-GAP, which is phosphorylated only following PDGF stimulation (25).…”

Section: Lmw-ptp Did Not Affect Fes Phosphorylation (Right Panels)mentioning

confidence: 99%

Low M r Phosphotyrosine Protein Phosphatase Associates and Dephosphorylates p125 Focal Adhesion Kinase, Interfering with Cell Motility and Spreading

Rigacci¹,

Rovida

Sbarba

et al. 2002

Journal of Biological Chemistry

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Low M r phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M r phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M r phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60 src with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M r phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M r phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.Cell adhesion and motility are based on the organization and remodeling of macromolecular complexes called focal adhesions. Following integrin engagement and clustering, several proteins, such as tensin, paxillin, p190Rho-GAP, and p125 focal adhesion kinase (FAK), 1 accumulate in these specialized sites at the cytoplasmic face of plasma membrane (1-3). Autophosphorylation of FAK on Tyr-397 creates a binding site for pp60 src , which phosphorylates FAK further (4). This enables the recruitment of other structural or signaling proteins, including pp130CAS , Csk, talin, PI3-kinase, ␣-actinin, and Grb2, which triggers the activation of the extracellular signal-regulated kinase (ERK) cascade (5-7). FAK is implicated in the control of cell migration by modulating the turnover of focal adhesions and in the transmission of cell survival signals from the extracellular matrix (1-3). Cell motility and spreading are clearly affected by FAK expression as fibroblasts isolated from FAK knockout mice exhibit reduced motility and a more rounded shape with abundant focal contacts that cover much of the ventral surface (8 -10). Vice versa, overexpression of FAK stimulates cell migration in Chinese hamster ovary cells (11).The role of FAK phosphorylation in the above processes is not completely clarified yet; the catalytic activity of FAK and its autophosphorylation site are required to restore cell migration in FAK-deficient cells (12). Indeed, PTEN and PTP1B tyrosine phosphatases are negative regulators of FAK tyrosine phosphorylation, cell spreading, and migration (13,14). However, in contrast to ...

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“…Moreover, previous data have also demonstrated an involvement of LMW-PTP in the early events of PDGFr signal transduction. In fact, LMW-PTP by different mechanisms is able to specifically inhibit PDGF-induced NIH3T3 cell proliferation (14,18,19).…”

Section: Introductionmentioning

confidence: 99%

Involvement of the Tyrosine Phosphorylation on GSH Transport in NIH3T3 Fibroblasts

et al. 2003

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SummaryThis study demonstrates the involvement of phosphotyrosine phosphatases on the activity and regulation of GSH ATP-dependent transport system that we have previously identified in NIH3T3 fibroblasts. This is shown by the fact that increases of the initial rate of GSH uptake were measured in NIH3T3 overexpressing a synthetic gene coding for a low-Mr-phosphotyrosine protein phosphatase (LMW-PTP), while decreases were obtained in NIH3T3 overexpressing the phosphatase inactive mutant (LMW-C12SPTP), with respect to NIH3T3neo. Moreover, these results have been confirmed by experiments performed in the same cells by vanadate, and H 2 O 2 treatment on both GSH transport and mediated passive transport of glucose. A possible regulation of this transport system by platelet-derived growth factor receptor (PDGFr) with tyrosine kinase activity is also demonstrated. Moreover, these data show a relationship among GSH, PDGFr and phosphotyrosine phosphatase activity, and suggest a role of GSH transport systems on the cell proliferation process. IUBMB Life, 55: 159-165, 2003

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

Cited by 51 publications

References 23 publications

Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

Two Vicinal Cysteines Confer a Peculiar Redox Regulation to Low Molecular Weight Protein Tyrosine Phosphatase in Response to Platelet-derived Growth Factor Receptor Stimulation

Low M r Phosphotyrosine Protein Phosphatase Associates and Dephosphorylates p125 Focal Adhesion Kinase, Interfering with Cell Motility and Spreading

Involvement of the Tyrosine Phosphorylation on GSH Transport in NIH3T3 Fibroblasts

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