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.
Highlights
Differential Regulation of Protein-tyrosine Phosphatases by Integrin o:11bPa through Cytoskeletal Reorganization and Tyrosine Phosphorylation in Human Platelets*
Protein-tyrosine Phosphorylation and Dephosphorylation in the Subcellular Fractions of Thrombin-activated Platelets-To investigate the involvement of integrin cxllbf33 in protein-tyrosine phosphorylation and dephosphorylation, we first examined tyrosine-phosphorylated proteins by immunoblot assay in whole lysates of platelets from normal donors and from patients with Glanzmann's thrombasthenia
Our previous work has shown for the first time that integrin allbJ33 is involved in protein-tyrosine dephosphorylation in activated platelets [33]
Summary
Materials-Anti-phosphotyrosine monoclonal antibodies 4G10 and PY20 were purchased from Upstate Biotechnology, Inc. Lmmunoprecipitation-Washed platelets (1 X 109 cells/ml) were stimulated with thrombin for appropriate periods and lysed for 1 h in 0.5 volume of 3 X radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 15 mM EGTA, 3% Triton X-100, 3% sodium deoxycholate, 0.3% SDS, 3 mM PMSF, 3 mM Na3V04 , 60 1-'g/mlleupeptin, 60 1-'g/ml aprotinin, and 50 mM Tris-HCI, pH 7.4). This and all subsequent steps were carried out at 4 oc. Immunoprecipitated proteins (from 4 X 108 platelets/lane) were eluted from protein A-Sepharose beads in 1 X SDS sample buffer, boiled for 5 min, resolved on SDS-PAGE, and analyzed by immunoblotting as described above
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