PMA-directed activation of PKCα will induce the cSrc binding partner AFAP-110 to colocalize with and activate cSrc. The ability of AFAP-110 to colocalize with cSrc was dependent upon the integrity of its’ amino terminal Pleckstrin Homology (PH1) domain, while the ability to activate cSrc was dependent upon the integrity of its’ SH3 binding motif, which engages the cSrc SH3 domain. The result of AFAP-110-directed cSrc activation is the formation of podosomes. In this work, we sought to address how AFAP-110 is able to colocalize with cSrc in response to PMA and where colocalization occurred. As the integrity of the PH1 domain was required for PMA induced colocalization between AFAP-110 and cSrc, and PH domains are known to interact with phosphoinositide-3-kinase (PI3K) generated lipid products, we sought to determine whether PI3K signaling played a role in PMA induced colocalization between AFAP-110 and cSrc. Treatment of cells with a PI3K inhibitor, LY294002, blocked PMA-directed colocalization between AFAP-110 and cSrc and subsequent cSrc activation. PMA was unable to induce colocalization or cSrc activation in cells that lacked the p85α and β regulatory subunits of PI3K. In normal mouse embryo fibroblasts, PMA was able to induce activation of PI3K and the PH1 domain of AFAP-110 was able to bind to PI3K generated lipid products, in vitro. These data indicate that PI3K activity is required for PMA induced colocalization between AFAP-110 and cSrc and subsequent cSrc activation. Interestingly, PMA directed colocalization between AFAP-110 and cSrc occurred in the perinuclear region of the cell. As inactive cSrc is known to exist on perinuclear vesicles, we propose a signaling cascade where PMA directs activation of PKCα and PI3K, which in turn directs AFAP-110 to subsequently colocalize with and activate cSrc in the perinuclear region of the cell. We hypothesize that this signaling cascade may play a role in the trafficking of activated cSrc to the membrane. This work is supported by NIH grants CA60731 and RR16440 (to DCF), and CA60731-MS1 (to VGW).
[Proc Amer Assoc Cancer Res, Volume 47, 2006]