A phosphotyrosine-dependent protein interaction screen reveals a role for phosphorylation of caveolin-1 on tyrosine 14: recruitment of C-terminal Src kinase.
Caveolin-1 is a substrate for nonreceptor tyrosine kinases including Src, Fyn, and Abl. To investigate the function of caveolin-1 phosphorylation, we modified the Gal4-based yeast two-hybrid system to screen for phosphorylation-dependent protein interactions. A cDNA library was screened using the N terminus of caveolin-1 as bait in a yeast strain expressing the catalytic domain of Abl. We identified two proteins in this screen that interact with caveolin-1 in a phosphorylation-dependent manner: tumor necrosis factor-alpha receptor-associated factor 2 (TRAF2) and C-terminal Src kinase (Csk). TRAF2 bound to nonphosphorylated caveolin-1, but this association was increased 3-fold by phosphorylation. In contrast, association of Csk with caveolin-1 was completely dependent on phosphorylation of caveolin-1, both for fusion proteins in yeast (>35-fold difference in affinity) and for endogenous proteins in tissue culture cells. Our data suggest that phosphorylation of caveolin-1 leads to Csk translocation into caveolae. This may induce a feedback loop that leads to inactivation of the Src family kinases that are highly enriched in caveolae.
Cao
Haiming
H
Department of Biochemistry, University of Nevada, Reno, Nevada 89557, USA.
Courchesne
William E
WE
Mastick
Cynthia Corley
CC
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D000081247:CSK Tyrosine-Protein Kinase; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases
C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D011506:Proteins; D047992:TNF Receptor-Associated Factor 2; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases