Protein kinase C regulates αvβ5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation

Abstract

Integrins αvβ3 and αvβ5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin αvβ3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas αvβ5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of β3 or β5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that αvβ5 induced colocalization of talin, α- actinin, tensin, and actin very weakly relative to αvβ3. αvβ5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to αvβ5-expressing cells induced spreading, increased colocalization of α-actinin, tensin, vinculin, p130(cas) and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, α-actinin, tensin, and actin for both αvβ3 and αvβ5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by αvβ5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.