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Protein Kinase C Activates c-Src and Induces Podosome Formation via AFAP-110

The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, West Virginia,¹ Department of Craniofacial Biology, University of Colorado Health Sciences Center, Denver, Colorado²

Received 15 January 2004/ Returned for modification 27 February 2004/ Accepted 14 May 2004

We report that the actin filament-associated protein AFAP-110 is required to mediate protein kinase C (PKC) activation of the nonreceptor tyrosine kinase c-Src and the subsequent formation of podosomes. Immunofluorescence analysis demonstrated that activation of PKC by phorbol 12-myristate 13-acetate (PMA), or ectopic expression of constitutively activated PKC, directs AFAP-110 to colocalize with and bind to the c-Src SH3 domain, resulting in activation of the tyrosine kinase. Activation of c-Src then directs the formation of podosomes, which contain cortactin, AFAP-110, actin, and c-Src. In a cell line (CaOV3) that has very little or no detectable AFAP-110, PMA treatment was unable to activate c-Src or effect podosome formation. Ectopic expression of AFAP-110 in CaOV3 cells rescued PKC-mediated activation of c-Src and elevated tyrosine phosphorylation levels and subsequent formation of podosomes. Neither expression of activated PKC nor treatment with PMA was able to induce these changes in CAOV3 cells expressing mutant forms of AFAP-110 that are unable to bind to, or colocalize with, c-Src. We hypothesize that one major function of AFAP-110 is to relay signals from PKC that direct the activation of c-Src and the formation of podosomes.

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