Regulation of Outside-in Signaling in Platelets by Integrin-associated Protein Kinase Cβ

Achim Obergfell,Alessandra Soriani,Koji Eto,Toshiaki Kawakami,William B. Kiosses,Sanford J. Shattil,Elena G. Arias-Salgado,Charito S. Buensuceso

doi:10.1074/jbc.m410229200

Abstract

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin alpha(IIb)beta(3) in platelets, but the responsible PKC isoforms and mechanisms are unknown. Alpha(IIb)beta(3) interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether alpha(IIb)beta(3) might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKC beta co-immunoprecipitated with alpha(IIb)beta(3) in response to the interaction of platelets with soluble or immobilized fibrinogen. PKC beta recruitment to alpha(IIb)beta(3) was accompanied by a 9-fold increase in PKC activity in alpha(IIb)beta(3) immunoprecipitates. RACK1, an intracellular adapter for activated PKC beta, also co-immunoprecipitated with alpha(IIb)beta(3), but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKC beta recruitment to alpha(IIb)beta(3) and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKC beta spread poorly on fibrinogen, despite normal agonist-induced fibrinogen binding. In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC beta I to associate with alpha(IIb)beta(3) and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the beta(3) cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to beta(3). These studies demonstrate that the interaction of alpha(IIb)beta(3) with activated PKC beta is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through alpha(IIb)beta(3). Furthermore, the studies extend the concept of alpha(IIb)beta(3) as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.

Highlights

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin ␣IIb␤3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. ␣IIb␤3 interacts directly with tyrosine kinases c-Src and Syk
In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKC␤I to associate with ␣IIb␤3 and to co-localize with it at lamellipodial edges. These responses, as well as Chinese hamster ovary cell migration on fibrinogen, were blocked by the deletion of the ␤3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to ␤3. These studies demonstrate that the interaction of ␣IIb␤3 with activated PKC␤ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through ␣IIb␤3
By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKC␤, inducibly associates with ␣IIb␤3 in response to fibrinogen binding to cells

Summary

Introduction

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin ␣IIb␤3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. ␣IIb␤3 interacts directly with tyrosine kinases c-Src and Syk. By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKC␤, inducibly associates with ␣IIb␤3 in response to fibrinogen binding to cells.

Results

Conclusion