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

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin α IIb β 3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. α IIb β 3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether α IIb β 3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with α IIb β 3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to α IIb β 3 was accompanied by a 9-fold increase in PKC activity in α IIb β 3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with α IIb β 3 , but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to α IIb β 3 and the spread of platelets on fibrinogen. Similarly, mouse platelets that are genetically deficient in PKCβ 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-PKCβI to associate with α IIb β 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 β 3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β 3 . These studies demonstrate that the interaction of α IIb β 3 with activated PKCβ is regulated by integrin occupancy and can be mediated by RACK1 and that the interaction is required for platelet spreading triggered through α IIb β 3 . Furthermore, the studies extend the concept of α IIb β 3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.