Involvement of Hic-5 in platelet activation: integrin alphaIIbbeta3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2.
Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin alphaIIbbeta3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alphaIIbbeta3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alphaIIbbeta3-mediated outside-in signalling, through association with Pyk2.
Osada
M
M
Department of Laboratory Medicine, Yamanashi Medical University, Nakakoma, Yamanashi 409-3898, Japan.
Ohmori
T
T
Yatomi
Y
Y
Satoh
K
K
Hosogaya
S
S
Ozaki
Y
Y
D001792:Blood Platelets / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D003599:Cytoskeleton; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D005455:Fluorescent Antibody Technique; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D051419:Paxillin; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D015539:Platelet Activation / Q000502:physiology*; D010974:Platelet Aggregation / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C490351:TGFB1I1 protein, human; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D011493:Protein Kinase C