The Low M r Protein-tyrosine Phosphatase Is Involved in Rho-mediated Cytoskeleton Rearrangement after Integrin and Platelet-derived Growth Factor Stimulation

Abstract

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.Many cellular processes such as cell migration, adhesion, and proliferation require the collaborative interaction between growth factors and extracellular matrix (ECM) 1 stimuli (1-3).Cell adhesion on ECM results in clustering of integrins in focal adhesions that contain both cytoskeletal and signaling proteins. Formation of focal adhesions as well as the closely associated actin stress fibers requires activation of the small GTPbinding protein Rho (4). Rho, a member of the Ras superfamily of GTP-binding proteins, cycles between a GDP-bound inactive form and a GTP-bound active state. Rho is regulated primarily by two groups of proteins: guanine nucleotide exchange factors that catalyze exchange of GDP for GTP and GTPase-activating proteins (GAPs) that stimulate the hydrolysis of GTP to GDP. Upon binding to GTP, Rho interacts with and activates proteins such as Rho kinase and phosphatidylinositol 4-phosphate 5-kinase (5). p190Rho-GAP is a GTPase-activating protein for Rho. During growth factor stimulation p190Rho-GAP becomes tyrosinephosphorylated by c-Src in Tyr 1105 (6, 7) and undergoes transient redistribution into perinuclear concentric arcs that coincide with epidermal growth factor-mediated focal adhesion assembly and reassembly (6). p190Rho-GAP tyrosine phosphorylation correlates with rapid disassembly of...

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

“…We have previously demonstrated that LMW-PTP exists in two different cellular pools: a cytosolic pool, which is recruited to the membrane upon PDGF stimulation acting directly on PDGF-R, and a second pool anchored to the cytoskeleton that acts on a different substrate, p190Rho-GAP (10). LMW-PTP is thus able to influence cell growth through PDGF-R dephosphorylation and cytoskeleton rearrangement through Rho regulation.…”

“…Cytoskeleton-associated LMW-PTP influences cell adhesion, spreading, and migration, controlling the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and, consequently, cytoskeleton rearrangement in response to PDGF stimulation. Hence, LMW-PTP is able to perform multiple roles in PDGF-induced mitogenesis: while cytosolic LMW-PTP binds and dephosphorylates PDGF-R (4), thus modulating part of its signaling cascade, cytoskeleton-associated LMW-PTP acts on phosphorylated p190Rho-GAP, consequently play-ing a role in PDGF-mediated cytoskeleton rearrangement (10).…”

The protein-tyrosine phosphatase DEP-1 modulates growth factor-stimulated cell migration and cell–matrix adhesion

“…The assay was performed as described elsewhere (Chiarugi et al, 2000). In brief, 96-well plates were coated with 30 mg/ml collagen type 1 (PromoCell, Heidelberg) at 41C overnight and washed twice with PBS.…”

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

“…This reversible phosphorylation/dephosphorylation process is catalysed by opposing reactions of protein kinases and protein phosphatases (1,2). It has been well studied that in eukaryotes protein phosphorylation on tyrosine residues plays a key role in the regulatory mechanism of various cellular processes including growth, differentiation, cell cycle regulation and cytoskeletal function (3,4), and as well as controls many diseases (5,6) including infectious diseases (7,8). In comparison to eukaryotes, the occurrence of protein tyrosine kinases (PTKs)/protein tyrosine phosphatases (PTPs) in bacteria including photosynthetic cyanobacteria, was suggested much later (9 15).…”