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 ␣IIb3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. ␣IIb3 interacts directly with tyrosine kinases c-Src and Syk
In a Chinese hamster ovary cell model system, adhesion to fibrinogen caused green fluorescent protein-PKCI to associate with ␣IIb3 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 ␣IIb3 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 ␣IIb3
By using human and mouse platelets and a CHO cell model system, the results show that one particular PKC isoform, PKC, inducibly associates with ␣IIb3 in response to fibrinogen binding to cells
Summary
Studies with inhibitors have implicated protein kinase C (PKC) in the adhesive functions of integrin ␣IIb3 in platelets, but the responsible PKC isoforms and mechanisms are unknown. ␣IIb3 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 ␣IIb3 in response to fibrinogen binding to cells.
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