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

Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin αIIbβ3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. αIIbβ3 interacts directly with tyrosine kinases c-Src and Syk. Therefore, we asked whether αIIbβ3 might also interact with PKC. Of the several PKC isoforms expressed in platelets, only PKCβ co-immunoprecipitated with αIIbβ3 in response to the interaction of platelets with soluble or immobilized fibrinogen. PKCβ recruitment to αIIbβ3 was accompanied by a 9-fold increase in PKC activity in αIIbβ3 immunoprecipitates. RACK1, an intracellular adapter for activated PKCβ, also co-immunoprecipitated with αIIbβ3, but in this case, the interaction was constitutive. Broad spectrum PKC inhibitors blocked both PKCβ recruitment to αIIbβ3 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 αIIbβ3 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 β3 cytoplasmic tail or by co-expression of a RACK1 mutant incapable of binding to β3. These studies demonstrate that the interaction of αIIbβ3 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 αIIbβ3. Furthermore, the studies extend the concept of αIIbβ3 as a scaffold for multiple protein kinases that regulate the platelet actin cytoskeleton.