Lymphocyte function-associated antigen-1-mediated T cell adhesion is impaired by low molecular weight phosphotyrosine phosphatase-dependent inhibition of FAK activity

Protein tyrosine phosphorylation is one of the earliest signaling events detected in response to lymphocyte function-associated antigen-1 (LFA-1) engagement during lymphocyte adhesion. In particular, the focal adhesion kinase p125FAK, involved in the modulation and rearrangement of the actin cytoskeleton, seems to be a crucial mediator of LFA-1 signaling. Herein, we investigate the role of a FAK tyrosine phosphatase, namely low molecular weight phosphotyrosine phosphatase (LMW-PTP), in the modulation of LFA-1-mediated T cell adhesion. Overexpression of LMW-PTP in Jurkat cells revealed an impairment of LFA-1-dependent cell-cell adhesion upon T cell receptor (TCR) stimulation. Moreover, in these conditions LMW-PTP causes FAK dephosphorylation, thus preventing the activation of FAK downstream pathways. Our results also demonstrated that, upon antigen stimulation, LMW-PTP-dependent FAK inhibition is associated to a strong reduction of LFA-1 and TCR co-clustering toward a single region of T cell surface, thus causing an impairment of receptor activity by preventing changes in their avidity state. Because co-localization of both LFA-1 and TCR is an essential event during encounters of T cells with antigen-presenting cells and immunological synapse (IS) formation, we suggest an intriguing role of LMW-PTP in IS establishment and stabilization through the negative control of FAK activity and, in turn, of cell surface receptor redistribution.     

Low M(r) phosphotyrosine protein phosphatase activity on fibroblast growth factor receptor is not associated with enzyme translocation.

Fibroblast growth factor receptor (class IV) shares a certain degree of similarity with class III members like platelet-derived growth factor and macrophage-colony-stimulating factor receptors, which, once activated, are substrates of low M(r) phosphotyrosine protein phosphatase. Up until now no phosphotyrosine phosphatase has been shown to act on this receptor in vivo. Here we demonstrate that low M(r) phosphotyrosine protein phosphatase is able to reduce receptor tyrosine phosphorylation and cell proliferation in response to basic fibroblast growth factor. Contrary to what was previously observed for platelet-derived growth factor, during cell stimulation with basic fibroblast growth factor, no enzyme redistribution among cellular compartments is observed.     

The EphA8 receptor phosphorylates and activates low molecular weight phosphotyrosine protein phosphatase in vitro

Low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) has been implicated in modulating the EphB1-mediated signaling pathway. In this study, we demonstrated that the EphA8 receptor phosphorylates LMW-PTP in vitro. In addition, we discovered that mixing these two proteins leads to EphA8 dephosphorylation in the absence of phosphatase inhibitors. Finally, we demonstrated that LMW-PTP, modified by the EphA8 autokinase activity, possesses enhanced catalytic activity in vitro. These results suggest that LMW-PTP may also participate in a feedback-control mechanism of the EphA8 receptor autokinase activity in vivo.     

Low molecular weight protein-tyrosine phosphatase controls the rate and the strength of NIH-3T3 cells adhesion through its phosphorylation on tyrosine 131 or 132

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement. Our previous results demonstrated that LMW-PTP is able to bind and dephosphorylate activated PDGF receptor, thus inhibiting cell proliferation. Recently we have shown that LMW-PTP is specifically phosphorylated by c-Src in a cytoskeleton-associated fraction in response to PDGF, and this phosphorylation increases LMW-PTP activity about 20-fold. LMW-PTP strongly influences cell adhesion, spreading, and chemotaxis induced by PDGF stimulation, by regulating the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and hence cytoskeleton rearrangement. In the present study we investigate the physiological role of the two LMW-PTP tyrosine phosphorylation sites, using LMW-PTP mutants on tyrosine 131 or 132. We demonstrate that each tyrosine residue is involved in specific LMW-PTP functions. Both of them are phosphorylated during PDGF signaling. Phosphorylation on tyrosine 131 influences mitogenesis, dephosphorylating activated PDGF-R and cytoskeleton rearrangement, acting on p190RhoGAP. Phosphorylation on tyrosine 132 leads to an increase in the strength of cell substrate adhesion, down-regulating matrix metalloproteases expression, through the inhibition of Grb2/MAPK pathway. In conclusion, LMW-PTP tyrosine phosphorylation on both Tyr(131) or Tyr(132) cooperate to determine a faster and stronger adhesion to extracellular matrix, although these two events may diverge in timing and relative amount.     

A novel redox-based switch: LMW-PTP oxidation enhances Grb2 binding and leads to ERK activation

Low molecular weight-PTP has been reported as a redox-sensitive protein during both platelet-derived growth factor and integrin signalling. In response to oxidation the phosphatase undergoes a reversible inactivation, which in turn leads to the increase in tyrosine phosphorylation of its substrates and the properly executed anchorage-dependent proliferation program. Here, we report that an exogenous oxidative stress enhances LMW-PTP tyrosine phosphorylation, through oxidation/inactivation of the enzyme, thus preventing its auto-dephosphorylation activity. In particular, we observed a selective hyper-phosphorylation of Tyr132, that acts as a docking site for the adaptor protein Grb2. The redox-dependent enhancement of Grb2 recruitment to LMW-PTP ultimately leads to an improvement of ERK activation, likely triggering a prosurvival signal against the oxidant environment.     

Modulation of STAT5 interaction with LMW-PTP during early megakaryocyte differentiation

STATs are involved in a variety of cellular processes, including cell proliferation and differentiation. They are activated through tyrosine phosphorylation, which promotes their dimerization via SH2 domains. We have demonstrated previously that in DAMI megakaryoblastic cells LMW-PTP dephosphorylates STAT5, interacting with an essential sequence of nine amino acids in its C-terminal region. Here we characterize STAT5 tyrosine phosphorylation and its interaction with LMW-PTP during early phorbol-12-myristate-13-acetate-induced megakaryocyte differentiation; these processes show clear dependence on STAT5 threonine phosphorylation. Since protein kinase C inhibition prevents phorbol-12-myristate-13-acetate-induced STAT5 threonine phosphorylation and association with LMW-PTP, it follows that these processes depend on protein kinase C activity. By using a Thr757/Val mutant of STAT5 we also demonstrate that the 757 serine/threonine conserved residue, which is in the STAT5A region involved in the interaction with LMW-PTP, is essential for such an association, though its phosphorylation is not necessary.     

Low molecular weight protein tyrosine phosphatase (LMW-PTP) and its possible physiological functions of redox signaling in the eye lens

Low molecular weight protein tyrosine phosphatase (LMW-PTP) was cloned from human lens epithelial B3 cells (HLE B3) and the recombinant enzyme was purified to homogeneity. The pure enzyme reacted positively with anti-LMW-PTP antibody, displayed tyrosine-specific phosphatase activity and was extremely sensitive to H(2)O(2). The inactivated LMW-PTP could be regenerated by thioltransferase (TTase)/GSH system as demonstrated by both activity assay and by mass spectrometry (MS). The MS study also showed that an intramolecular disulfide bond was formed between C13 and C18 at the active site, and was reduced by the TTase/GSH system. The putative role of LMW-PTP in regulating platelet derived growth factor (PDGF)-stimulated cell signaling was demonstrated in wild type mouse lens epithelial cells (LEC) in which LMW-PTP was transiently inactivated, corroborated with the transient phosphorylation of Tyr857 at the active site of PDGF receptor and the downstream signaling components of Akt and ERK1/2. In contrast, LMW-PTP activity in PDGF-stimulated LEC from TTase(-/-) mice was progressively lost, concomitant with the high basal and sustained high phosphorylation levels at Tyr857, Akt and ERK1/2. We conclude that the reversible LMW-PTP activity regulated by ROS-mediated oxidation and TTase/GSH reduction is the likely mechanism of redox signaling in lens epithelial cells.     

Insulin receptor protein-tyrosine phosphatases. Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain.

A number of protein-tyrosine phosphatase(s) (PTPases) have been shown to dephosphorylate the insulin receptor in vitro; however, it is not known whether any individual PTPase has specificity for certain phosphotyrosine residues of the receptor that regulate its intrinsic tyrosine kinase activity. We evaluated the deactivation of the insulin receptor kinase by three candidate enzymes that are expressed in insulin-sensitive rat tissues, including the receptor-like PTPases LAR and LRP, and the intracellular enzyme, PTPase1B. Purified insulin receptors were activated by insulin and receptor dephosphorylation, and kinase activity was quantitated after incubation with recombinant PTPases from an Escherichia coli expression system. When related to the level of overall receptor dephosphorylation, LAR deactivated the receptor kinase 3.1 and 2.1 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.03). To assess whether these effects were associated with preferential dephosphorylation of the regulatory (Tyr-1150) domain of the receptor beta-subunit, we performed tryptic mapping of the insulin receptor beta-subunit after dephosphorylation by PTPases. Relative to the rate of initial loss of 32P from receptor C-terminal sites, LAR dephosphorylated the Tris-phosphorylated Tyr-1150 domain 3.5 and 3.7 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.01). The accelerated deactivation of the insulin receptor kinase by LAR and its relative preference for regulatory phosphotyrosine residues further support a potential role for this transmembrane PTPase in the physiological regulation of insulin receptors in intact cells.     

Inactivation of platelet-derived growth factor receptor by the tumor suppressor PTEN provides a novel mechanism of action of the phosphatase

PTEN, mutated in a variety of human cancers, is a dual specificity protein phosphatase and also possesses D3-phosphoinositide phosphatase activity on phosphatidylinositol 3,4,5-tris-phosphate (PIP(3)), a product of phosphatidylinositol 3-kinase. This PIP(3) phosphatase activity of PTEN contributes to its tumor suppressor function by inhibition of Akt kinase, a direct target of PIP(3). We have recently shown that Akt regulates PDGF-induced DNA synthesis in mesangial cells. In this study, we demonstrate that expression of PTEN in mesangial cells inhibits PDGF-induced Akt activation leading to reduction in PDGF-induced DNA synthesis. As a potential mechanism, we show that PTEN inhibits PDGF-induced protein tyrosine phosphorylation with concomitant dephosphorylation and inactivation of tyrosine phosphorylated and activated PDGF receptor. Recombinant as well as immunopurified PTEN dephosphorylates autophosphorylated PDGF receptor in vitro. Expression of phosphatase deficient mutant of PTEN does not dephosphorylate PDGF-induced tyrosine phosphorylated PDGF receptor. Rather its expression increases tyrosine phosphorylation of PDGF receptor. Furthermore, expression of PTEN attenuated PDGF-induced signal transduction including phosphatidylinositol 3-kinase and Erk1/2 MAPK activities. Our data provide the first evidence that PTEN is physically associated with platelet-derived growth factor (PDGF) receptor and that PDGF causes its dissociation from the receptor. Finally, we show that both the C2 and tail domains of PTEN contribute to binding to the PDGF receptor. These data demonstrate a novel aspect of PTEN function where it acts as an effector for the PDGF receptor function and negatively regulates PDGF receptor activation.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

Inactivation of mitogen-activated protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7

Mitogen-activated protein kinase (MAPK) is inactivated through dephosphorylation of tyrosyl and threonyl regulatory sites. In yeast, both dual-specificity and tyrosine-specific phosphatases are involved in dephosphorylation. In mammals, however, no tyrosine-specific phosphatase has been identified molecularly to dephosphorylate MAPK in vivo. Recently, we and others have cloned a murine tyrosine-specific phosphatase, PTPBR7/PTP-SL, which is expressed predominantly in the brain. Here we report inactivation of the extracellular signal-regulated kinase (ERK) family MAPK by PTPBR7. PTPBR7 made complexes with ERK1/ERK2 in vivo and dephosphorylated ERK1 in vitro. When overexpressed in mammalian cells, wild-type PTPBR7 suppressed the phosphorylation and activation of ERK by epidermal growth factor (EGF), nerve growth factor (NGF), and constitutively active MEK1, a mutant MAPK kinase. In contrast, catalytically inactive and ERK-binding-deficient mutants revealed little inhibition on the ERK cascade. These results indicate that PTPBR7 suppresses MAPK directly in vivo.     

A-Raf associates with and regulates platelet-derived growth factor receptor signalling

Raf kinases are important intermediates in epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) mediated activation of the mitogen-activated protein kinase (MAPK) pathway. In this report, we show that the A-Raf kinase is associated with activated EGF receptor complexes and with PDGF receptor (PDGFR) complexes independent of prior PDGF treatment. The ability of A-Raf to associate with receptor tyrosine kinases could provide a Ras-GTP-independent mechanism for the membrane localization of A-Raf. Expression of a partially activated A-Raf mutant resulted in decreased tyrosine phosphorylation of the PDGFR, specifically on Y857 (autophosphorylation site) and Y1021 (phospholipase Cgamma1 (PLCgamma1) binding site), but not the binding sites for other signalling proteins (Nck, phosphatidylinositol 3'-kinase (PI3K), RasGAP, Grb2, SHP). Activated A-Raf expression also altered the activation of PLCgamma1, and p85-associated PI3K. Thus, A-Raf can regulate PLCgamma1 signalling via a PDGFR-dependent mechanism and may also regulate PI3K signalling via a PDGFR-independent mechanism.     

Involvement of the tyrosine phosphorylation on GSH transport in NIH3T3 fibroblasts

This 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 H2O2 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.     

Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins

We have identified two platelet-derived growth factor (PDGF)-dependent autophosphorylation sites in the beta subunit of the human PDGF receptor (PDGF-R). The major site of phosphorylation (Tyr-857) corresponds to the major autophosphorylation site in many other tyrosine kinases. Tyr-751, which lies within the kinase insert region, is a second in vivo site and the major in vitro site. Immunoprecipitates of wild-type PDGF-Rs prepared from PDGF-treated cells contained a phosphatidylinositol (PI) 3 kinase activity and three specific polypeptides as well as the PDGF-R. Mutation of Tyr-751 to Phe or Gly, or mutation of the catalytic domain to abolish kinase activity, blocked association of the PDGF-R with the PI kinase and the three proteins. These results suggest that autophosphorylation in the kinase insert region triggers the binding of the activated PDGF-R to specific cellular proteins, including a PI kinase whose activity is known to be stimulated by PDGF. Thus autophosphorylation may play a novel role in signal transduction via the PDGF-R.     

LMW-PTP modulates glucose metabolism in cancer cells

Background

Low Molecular Weight Phosphotyrosine Protein Phosphatase (LMW-PTP) is an enzyme involved not only in tumor onset and progression but also in type 2 diabetes. A recent review shows that LMW-PTP acts on several RTK (receptor tyrosine kinase) such as PDGFR, EGFR, EphA2, Insulin receptor. It is well described also its interaction with cSrc. It is noteworthy that most of these conclusions are based on the use of cell lines expressing low levels of LMW-PTP. The aim of the present study was to discover new LMW-PTP substrates in aggressive human tumors where the over-expression of this phosphatase is a common feature.

The in vivo tyrosine phosphorylation level of yeast immunophilin Fpr3 is influenced by the LMW-PTP Ltp1

Tyr-phosphorylation in Saccharomyces cerevisiae is essential in controlling the activity of MAP kinase regulating mating, pseudohyphal growth, and cell wall biosynthesis. Yeast serves as a model system for studying the biological function of many protein kinases and PTPs. Two LMW-PTP from yeast have been cloned, namely, Ltp1 from S. cerevisiae and Stp1 from Schizosaccharomyces pombe. The sequences of both enzymes are relatively similar to those of the mammalian LMW-PTP. Recently we showed that the yeast immunophilin Fpr3 interacts with Stp1 and its dephosphorylated state induces a growth defective phenotype. Here we show the phosphatase activity of Ltp1 on Fpr3 and we demonstrated that Tyr 184 is the residue phosphorylated on in vivo Fpr3. We also described the marked activation of Ltp1 by adenine in S. cerevisiae proteome and determined in vivo the influence of tyrosine phosphorylation on Fpr3 localization.     

Low-molecular-weight protein tyrosine phosphatase is a positive component of the fibroblast growth factor receptor signaling pathway

Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) has been implicated in the regulation of cell growth and actin rearrangement mediated by several receptor tyrosine kinases, including platelet-derived growth factor and epidermal growth factor. Here we identify the Xenopus laevis homolog of LMW-PTP1 (XLPTP1) as an additional positive regulator in the fibroblast growth factor (FGF) signaling pathway during Xenopus development. XLPTP1 has an expression pattern that displays substantial overlap with FGF receptor 1 (FGFR1) during Xenopus development. Using morpholino antisense technology, we show that inhibition of endogenous XLPTP1 expression dramatically restricts anterior and posterior structure development and inhibits mesoderm formation. In ectodermal explants, loss of XLPTP1 expression dramatically blocks the induction of the early mesoderm gene, Xbrachyury (Xbra), by FGF and partially blocks Xbra induction by Activin. Moreover, FGF-induced activation of mitogen-activated protein (MAP) kinase is also inhibited by XLPTP1 morpholino antisense oligonucleotides; however, introduction of RNA encoding XLPTP1 is able to rescue morphological and biochemical effects of antisense inhibition. Inhibition of FGF-induced MAP kinase activity due to loss of XLPTP1 is also rescued by an active Ras, implying that XLPTP1 may act upstream of or parallel to Ras. Finally, XLPTP1 physically associates only with an activated FGFR1, and this interaction requires the presence of SNT1/FRS-2 (FGFR substrate 2). Although LMW-PTP1 has been shown to participate in other receptor systems, the data presented here also reveal XLPTP1 as a new and important component of the FGF signaling pathway.