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

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 genes myc 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 Molecular Basis of the Differing Kinetic Behavior of the Two Low Molecular Mass Phosphotyrosine Protein Phosphatase Isoforms

The low molecular mass phosphotyrosine protein phosphatase is a cytosolic enzyme of 18 kDa. Mammalian species contain a single gene that codifies for two distinct isoenzymes; they are produced through alternative splicing and thus differ only in the sequence from residue 40 to residue 73. Isoenzymes differ also in substrate specificity and in the sensitivity to activity modulators. In our study, we mutated a number of residues included in the alternative 40-73 sequence by substituting the residues present in the type 2 isoenzyme with those present in type 1 and subsequently examined the kinetic properties of the purified mutated proteins. The results enabled us to identify the molecular site that determines the kinetic characteristics of each isoform; the residue in position 50 plays the main role in the determination of substrate specificity, while the residues in both positions 49 and 50 are involved in the strong activation of the type 2 low M(r) phosphotyrosine protein phosphatase isoenzyme by purine compounds such as guanosine and cGMP. The sequence 49-50 is included in a loop whose N terminus is linked to the beta 2-strand and whose C terminus is linked to the alpha 2-helix; this loop is very near the active site pocket. Our findings suggest that this loop is involved both in the regulation of the enzyme activity and in the determination of the substrate specificity of the two low M(r) phosphotyrosine protein phosphatase isoenzymes

The low M(r) protein-tyrosine phosphatase is involved in Rho-mediated cytoskeleton rearrangement after integrin and platelet-derived growth factor stimulation.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. In fact, 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 genes myc and fos in response to growth factor stimulation. Recently, we have found that LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its phosphorylation, LMW-PTP increases its catalytic activity about 20-fold. In this study, our interest was to investigate the role of LMW-PTP phosphorylation in cellular response to PDGF stimulation. To address this issue, we have transfected in NIH-3T3 cells a mutant form of LMW-PTP in which the c-Src phosphorylation sites (Tyr(131) and Tyr(132)) were mutated to alanine. We have established that LMW-PTP phosphorylation by c-Src after PDGF treatment strongly influences both cell adhesion and migration. In addition, we have discovered a new LMW-PTP substrate localized in the cytoskeleton that becomes tyrosine-phosphorylated after PDGF treatment: p190Rho-GAP. Hence, LMW-PTP plays multiple roles in PDGF receptor-mediated mitogenesis, since it can bind and dephosphorylate PDGF receptor, and, at the same time, the cytoskeleton-associated LMW-PTP, through the regulation of the p190Rho-GAP phosphorylation state, controls the cytoskeleton rearrangement in response to PDGF stimulation

Cloning of murine low molecular weight phosphotyrosine protein phosphatase cDNA: identification of a new isoform

AbstractThe low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is a 18 kDa cytosolic enzyme, involved in the negative regulation of cell proliferation. In different mammalian species LMW-PTPs are expressed in two molecular forms produced from a single primary transcript through an alternative splicing mechanism. In this paper we report the cloning, expression and characterization of mouse isoforms of LMW-PTPs (called m-IF1 and m-IF2), very similar to the corresponding rat and human isoenzymes. Moreover we have identified a third cDNA encoding a protein (m-IF2P) that presents three substitutions compared to m-IF2. This new isoform is still active on pNPP, although to a lower extent: this reduction is mainly due to the leucine to proline substitution in position 13, within the catalytic loop. The mRNA expression level of this isoform is comparable to those of m-IF1 and m-IF2. It is likely that a gene duplication process followed by mutations has generated this new gene

The Src and signal transducers and activators of transcription pathways as specific targets for low molecular weight phosphotyrosine-protein phosphatase in platelet-derived growth factor signaling.

Abstract The low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is a cytosolic phosphotyrosine-protein phosphatase specifically interacting with the activated platelet-derived growth factor (PDGF) receptor through its active site. Overexpression of the LMW-PTP results in modulation of PDGF-dependent mitogenesis. In this study we investigated the effects of this tyrosine phosphatase on the signaling pathways relevant for PDGF-dependent DNA synthesis. NIH 3T3 cells were stably transfected with active or dominant negative LMW-PTP. The effects of LMW-PTP were essentially restricted to the G1 phase of the cell cycle. Upon stimulation with PDGF, cells transfected with the dominant negative LMW-PTP showed an increased activation of Src, whereas the active LMW-PTP induced a reduced activation of this proto-oncogene. We observe that c-Src binding to PDGF receptor upon stimulation is prevented by overexpression of LMW-PTP. These effects were associated with parallel changes in myc expression. Moreover, wild-type and dominant negative LMW-PTP differentially regulated STAT1 and STAT3 activation and tyrosine phosphorylation, whereas they did not modify extracellular signal-regulated kinase activity. However, these modifications were associated with changes in fos expression despite the lack of any effect on extracellular signal-regulated kinase activation. Other independent pathways involved in PDGF-induced mitogenesis, such as phosphatidylinositol 3-kinase and phospholipase C-γ1, were not affected by LMW-PTP. These data indicate that this phosphatase selectively interferes with the Src and the STATs pathways in PDGF downstream signaling. The resulting changes in myc andfos proto-oncogene expression are likely to mediate the modifications observed in the G1 phase of the cell cycle

Insight into the role of low molecular weight phosphotyrosine phosphatase (LMW-PTP) on platelet-derived growth factor receptor (PDGF-r) signaling. LMW-PTP controls PDGF-r kinase activity through TYR-857 dephosphorylation.

Low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor-induced mitogenesis and cytoskeleton rearrangement. Our previous results demonstrated that LMW-PTP is able to bind and dephosphorylate activated platelet-derived growth factor receptor (PDGF-r), thus inhibiting cell proliferation. Here we revisit the role of LMW-PTP on activated PDGF-r dephosphorylation. We demonstrate that LMW-PTP preferentially acts on cell surface PDGF-r, excluding the internalized activated receptor pool. Many phosphotyrosine phosphatases act by site-selective dephosphorylation on several sites of PDGF-r, but until now, there has been no evidence of a direct involvement of a specific phosphotyrosine phosphatase in the dephosphorylation of the 857 kinase domain activation tyrosine. Here we report that LMW-PTP affects the kinase activity of the receptor through the binding and dephosphorylation of Tyr-857 and influences many of the signal outputs from the receptor. In particular, we demonstrate a down-regulation of phosphatidylinositol 3-kinase, Src homology phosphatase-2, and phospholipase C-gamma1 binding but not of MAPK activation. In addition, we report a slight action of LMW-PTP on Tyr-716, which directs MAPK activation through Grb2 binding. On the basis of these results, we propose a key role for LMW-PTP in PDGF-r down-regulation through the dephosphorylation of the activation loop Tyr-857, thus determining a general negative regulation of all downstream signals, with the exception of those elicited by internalized receptors