Molecular Mechanism of Src Regulation by Csk, SHP-2, Cbp, and Caveolin-1

Aaron T. Place, Ph.D. Department of Pharmacology University of Illinois at Chicago Chicago, Illinois (2011) Dissertation Chairperson: Richard D. Minshall Ph.D. The exact molecular details of Src family kinase activation are not yet fully understood, despite ongoing research on the family of kinases since the 1970’s. Towards this goal, this study attempted to further the current understanding of the subject by focusing on c-Src, the first member of the family discovered. First, while examining the negative regulation of c-Src by Csk, a cooperative and possibly redundant relationship was uncovered. Cav-1 and Cbp, two known Csk adapters in fibroblasts, were shown to compensate for the loss of each other in knockout and siRNA knockdown studies. When Cav-1 was knocked out in mice, Cbp expression increased. Conversely, when Cbp was knocked down by siRNA, Cav-1 phosphorylation increased. In both cases, the intrinsically cytoplasmic Csk was still recruited to the membrane where c-Src is localized. These studies also revealed that the three proteins, Cav-1, Cbp, and Csk, formed a complex under basal conditions. The loss of both adapters, or Csk, led to increased c-Src activity, consistent with what has been observed in many cancers. On the other hand, while investigating the mechanism of c-Src activation by TNF-α in normal cells, it was demonstrated that activation resulted from a shift in equilibrium between Csk and the tyrosine phosphatase, SHP-2, which both competed for the negative regulatory C-terminal tyrosine of c-Src. Knockdown of SHP-2 by siRNA in endothelial cells both reduced basal c-Src activity and blocked its activation by TNF-α. SHP-2 siRNA also increased C-terminal tyrosine phosphorylation of c-Src without affecting the phosphorylation state of the Csk binding site on Cav-1, tyrosine 14. Finally, the intrinsically cytoplasmically localized SHP-2 was also found to be present in membrane fractions under basal conditions in the Cav-1 and Csk complex, implicating Cav-1 as a SHP-2 membrane adapter or scaffold.