The Tyrosine Phosphatase 1B Regulates Linker for Activation of T-cell Phosphorylation and Platelet Aggregation upon FcγRIIa Cross-linking

Ashraf Ragab,Stéphane Bodin,Cécile Viala,Hugues Chap,Bernard Payrastre,Jeannie Ragab-Thomas

doi:10.1074/jbc.m303602200

Abstract

Human platelets express the receptor for immunoglobulin G, FcgammaRIIa, that triggers cell aggregation upon interaction with immune complexes. Here, we report that the rapid tyrosine phosphorylation of the Linker for Activation of T-cell (LAT) in human platelets stimulated by FcgammaRIIa cross-linking was followed by its complete dephosphorylation in an alphaIIb/beta3 integrin-dependent manner. Concomitant to LAT dephosphorylation, the protein tyrosine phosphatase 1B (PTP1B) was activated through a mechanism involving its proteolysis by calpains downstream of integrins. Both PTP1B and LAT were associated with the actin cytoskeleton complex formed during platelet aggregation. Moreover, phospho-LAT appeared as a good substrate of activated PTP1B in vitro and these two proteins interacted upon platelet activation by FcgammaRIIa cross-linking. The permeant substrate-trapping PTP1B (TAT-PTP1B D181A) partly inhibited LAT dephosphorylation in human platelets, strongly suggesting that this tyrosine phosphatase was involved in this regulatory pathway. Using a pharmacological inhibitor, we provide evidence that PTP1B activation and LAT dephosphorylation processes were required for irreversible platelet aggregation. Altogether, our results demonstrate that PTP1B plays an important role in the integrin-mediated dephosphorylation of LAT in human platelets and is involved in the control of irreversible aggregation upon FcgammaRIIa stimulation.

Highlights

Human platelets possess only one Fc ␥ receptor for immunoglobulin G (Fc␥RIIa), which is a 40-kDa single-chain transmembrane glycoprotein present in monocytes, neutrophils, and B lymphocytes [1]
The Rapid Tyrosine Phosphorylation of Linker for Activation of T-cell (LAT) Is Followed by an Integrin-mediated Dephosphorylation in Fc␥RIIa-stimulated Platelets—Fc␥RIIa cross-linking led to a rapid LAT phosphorylation reaching a maximum at 1 min, followed by a dephosphorylation that was complete after 5 min of stimulation (Fig. 1A)
LAT dephosphorylation is strictly dependent on platelet aggregation and does not occur when the integrin ␣IIb/␤3 is blocked by RGDS peptide

Summary

Introduction

Human platelets possess only one Fc ␥ receptor for immunoglobulin G (Fc␥RIIa), which is a 40-kDa single-chain transmembrane glycoprotein present in monocytes, neutrophils, and B lymphocytes [1]. Upon Fc␥RIIa receptor cross-linking, the tyrosine residues within the ITAM motif are rapidly phosphorylated and become docking sites for proteins containing Src homology 2 (SH2) domains [7]. In collagen-stimulated platelets, the signaling complexes recruited by tyrosine-phosphorylated LAT are essential for PLC␥2 activation [14]. SHP-2, another SH2 domain-containing PTP, is associated with the receptor PECAM 1 (CD31) in platelets and could be involved in the signaling pathway initiated by this adhesion molecule [21, 22]. When platelets are activated by thrombin, PTP1B undergoes a proteolytic cleavage in a region between its catalytic domain and its membrane-anchoring C-terminal targeting sequence This process is dependent on integrin engagement and platelet aggregation and leads to enzymatic activation of PTP1B [16]. PTP1B appears as the major PTP responsible for insulin receptor regulation [29, 30], suggesting that PTP1B inhibitors may become new drugs for type 2 diabetes treatment [31, 32]

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