In the present study, we assessed the cooperative roles of Csk Binding Protein (Cbp) and Caveolin-1 (Cav-1) in the mechanism of Src family tyrosine kinase (SFK) inhibition by Csk. SFKs are inactivated by phosphorylation of their C-terminal tyrosine by Csk. While SFKs are membrane-associated, Csk is a cytoplasmic protein and therefore requires membrane adaptors such as Cbp or Cav-1 for recruitment to the plasma membrane to mediate SFK inhibition. To determine the specific role of Cav-1 and Cbp in SFK inhibition, we measured c-Src activity in the absence of each membrane adaptor. Interestingly, in lungs and fibroblasts from Cav-1-/- mice, we observed increased expression of Cbp compared to WT controls. However, both c-Src activity and Csk localization at the membrane were similar between Cav-1-/- fibroblasts and WT cells. Similarly, Cbp depletion by siRNA treatment of WT cells had no effect on basal c-Src activity, but it increased the phosphorylation state of Cav-1. Immunoprecipitation then confirmed increased association of Csk with phospho-mimicking Cav-1. Knockdown of Cbp by siRNA in Cav-1-/- cells revealed increased basal c-Src activity, and re-expression of WT Cav-1 in the same cells reduced basal c-Src activity. Taken together, these results indicate that Cav-1 and Cbp cooperatively regulate c-Src activity by recruiting Csk to the membrane where it phosphorylates c-Src inhibitory tyrosine 529. Furthermore, when either Cav-1 or Cbp expression is reduced or absent, there is a compensatory increase in the phosphorylation state or expression level of the other membrane-associated Csk adaptor to maintain SFK inhibition.
- Received June 3, 2011.
- Revision received July 19, 2011.
- Accepted July 21, 2011.
- The American Society for Pharmacology and Experimental Therapeutics