Protein kinase Calpha activates c-Src and induces podosome formation via AFAP-110.
We report that the actin filament-associated protein AFAP-110 is required to mediate protein kinase Calpha (PKCalpha) activation of the nonreceptor tyrosine kinase c-Src and the subsequent formation of podosomes. Immunofluorescence analysis demonstrated that activation of PKCalpha by phorbol 12-myristate 13-acetate (PMA), or ectopic expression of constitutively activated PKCalpha, 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 PKCalpha-mediated activation of c-Src and elevated tyrosine phosphorylation levels and subsequent formation of podosomes. Neither expression of activated PKCalpha 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 PKCalpha that direct the activation of c-Src and the formation of podosomes.
The Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology, and Cell Biology, West Virginia University, Morgantown, WV 26506-9300, USA.
Amanda
A
Gatesman
Valerie G
VG
Walker
Joseph M
JM
Baisden
Scott A
SA
Weed
Daniel C
DC
Flynn
D000199:Actins / Q000378:metabolism; D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D002460:Cell Line; D022081:Cell Surface Extensions / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000378:metabolism; D008301:Maleimides / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
C084510:AFAP 110; D000199:Actins; D045163:Antibodies, Phospho-Specific; D007211:Indoles; D008301:Maleimides; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D016392:Proto-Oncogene Proteins pp60(c-src); C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate