Differential Regulation of Protein-tyrosine Phosphatases by Integrin αIIbβ3 through Cytoskeletal Reorganization and Tyrosine Phosphorylation in Human Platelets

Abstract

The major platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) has been implicated in the regulation of tyrosine phosphorylation and dephosphorylation in activated platelets. To investigate the mechanisms of the alpha IIb beta 3-dependent tyrosine dephosphorylation, normal platelets or thrombasthenic platelets lacking alpha IIb beta 3 were stimulated with thrombin and fractionated into Triton X-100-soluble or -insoluble subcellular matrices. We then examined the kinetics of the tyrosine-phosphorylated proteins and distribution of protein-tyrosine phosphatases in these fractions and whole cell lysates. First, alpha IIb beta 3-dependent tyrosine dephosphorylation was recovered mainly in the cytoskeleton with similar kinetics to the whole cell lysate. Second, protein-tyrosine phosphatase (PTP) 1B and its cleaved 42-kDa form were associated with the cytoskeleton in an aggregation-dependent manner, whereas association of PTP1C with the cytoskeleton was regulated differentially both by thrombin stimulation and by alpha IIb beta 3-mediated aggregation. Several calpain inhibitors did not affect either tyrosine phosphorylation and dephosphorylation or relocation of PTP1B, but they did inhibit cleavage of PTP1B. Cytochalasin D blocked relocation of both PTP1B and PTP1C but not PTP1B cleavage. SH-PTP2 was distributed in the other fractions than the cytoskeleton and showed no relocation on thrombin stimulation. Finally, the cytoskeleton-associated PTP1C became tyrosine-phosphorylated in an alpha IIb beta 3-mediated aggregation-dependent manner. Thus, integrin alpha IIb beta 3 was involved differentially in the regulation of PTP1B and PTP1C.

Turn‐off, drop‐out: Functional state switching of cadherins

“…The carboxy terminus of PTP1B directs its localization to the cytosolic face of the endoplasmic reticulum (Frangioni et al, 1992). In platelets and activated T cells, proteolytic cleavage in the ER targeting domain results in translocation of PTP1B to the cytoskeletal/membrane fraction (Frangioni et al, 1993;Ezumi et al, 1995;Rock et al, 1997). This cleavage is dependent on integrin engagement, resulting in increased Ca 2ϩ levels and, consequently, activation of calpain.…”

Thrombopoietin, c‐Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists‐induced aggregation in platelets in vitro

“…For preparation of whole platelet lysates, the extracts were centrifuged at 10,000 x g for 30 rain, diluted with an equal volume of 2 x Laemmli SDS sample buffer, and boiled for 5 min. Immunoprecipitation for JAKs and STATs was done as previously described [20], using 5 ¢tg/ml antibodies specific for each protein and rabbit anti-mouse IgG as a control.…”

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c‐Src in human platelets

“…A Cell lysate ti ~n [23,24]. The migration in SDS-PAGE of PTP1C corresponded to the migration of the prominent tyrosine protein of 64 kDa [29].…”

“…In platelets, it is not clearly established whether this phosphatase plays a role in an early signalling event or in an aggregationdependent mechanism. Indeed, PTP1C phosphorylation occurs within the first seconds of stimulation [23,24]. However, depending on the reports, this phosphorylation follows platelet aggregation [23] or does not [24].…”

“…Indeed, PTP1C phosphorylation occurs within the first seconds of stimulation [23,24]. However, depending on the reports, this phosphorylation follows platelet aggregation [23] or does not [24]. Moreover, translocation of c-Src and PTP1C to the platelet cytoskeleton has been described, but up to now there is no evidence of any interaction between the two molecules.…”