A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: Recruitment of C-terminal Src kinase

Caveolae are plasma membrane invaginations that have been implicated in the physical organization of the components of multiple signaling pathways. The major structural protein of caveolae, caveolin-1, is phosphorylated on tyrosine 14 by Src family kinases (SFKs) in response to several stimuli including insulin, cellular stress, endothelin-1, and angiotensin II. To investigate the functions of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A 3T3-L1 adipocyte cDNA library was screened using the amino terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified c-terminal Src kinase (Csk) as a protein that binds specifically to phosphocaveolin. We proposed a model in which phosphorylation of caveolin by Src family kinases would create a feedback inhibition loop through the recruitment of Csk. This feedback mechanism was supported by experimental results that were consistent with a number of predictions that can be made based on this model. Multiple stimuli that activate caveolin phosphorylation increase caveolin-Csk binding. Csk activity increases concomitantly with the increase of caveolin phosphorylation, consistent with the recruitment and activation of Csk by phosphocaveolin. As predicted in a feedback inhibition loop, caveolin phosphorylation is self-limiting by a mechanism requiring Csk. In response to cellular stress, caveolin, phosphocaveolin, and Csk translocate to a pellet fraction enriched in paxillin, indicating that this feedback loop acts at focal adhesions. The localization of phosphocaveolin to focal adhesions was confirmed by immunofluorescence microscopy. Significantly, in cells attaching and spreading on fibronectin, caveolin was only transiently phosphorylated in newly formed focal adhesions. Many lines of evidence link caveolin to the actin cytoskeleton. We propose that this feedback loop is involved in the transient activation of Src kinases at points of contact of the actin cytoskeleton with the plasma membrane. This transient activation may be necessary for controlled actin cytoskeleton remodeling.