SRC TYROSINE KINASES, G(ALPHA) SUBUNITS, AND H-RAS SHARE A COMMON MEMBRANE-ANCHORED SCAFFOLDING PROTEIN, CAVEOLIN - CAVEOLIN BINDING NEGATIVELY REGULATES THE AUTO-ACTIVATION OF SRC TYROSINE KINASES

Caveolae are plasma membrane specializations present in most cell type s, Caveolin, a 22-kDa integral membrane protein, is a principal struct ural and regulatory component of caveolae membranes. Previous studies have demonstrated that caveolin co-purifies with lipid modified signal ing molecules, including G(alpha) subunits, H-ras c-Src, and other rel ated Src family tyrosine kinases. In addition, it has been shown that caveolin interacts directly with G(alpha) subunits and H-Ras, preferen tially recognizing the inactive conformation of these molecules. Howev er, it is not known whether caveolin interacts directly or indirectly with Src family tyrosine kinases. Here, we examine the structural and functional interaction of caveolin with Src family tyrosine kinases. C aveolin was recombinantly expressed as a glutathione S-transferase fus ion. Using an established in vitro binding assay, we find that caveoli n interacts with wild-type Src (c-Src) but does not form a stable comp lex with mutationally activated Src (v-Src). Thus, it appears that cav eolin prefers the inactive conformation of Src. Deletion mutagenesis i ndicates that the Src-interacting domain of caveolin is located within residues 82-101, a cytosolic membrane-proximal region of caveolin. A caveolin peptide derived hom this region (residues 82- 101) functional ly suppressed the auto-activation of purified recombinant c-Src tyrosi ne kinase and Fyn, a related Src family tyrosine kinase. We further an alyzed the effect of caveolin on c-Src activity in vivo by transiently co-expressing full-length caveolin and c-Src tyrosine kinase in 293T cells. Co-expression with caveolin dramatically suppressed the tyrosin e kinase activity of c-Src as measured via an immune complex kinase as say. Thus, it appears that caveolin structurally and functionally inte racts with wild-type c-Src via caveolin residues 82-101. Besides inter acting with Src family kinases, this cytosolic caveolin domain (residu es 82-101) has the following unique features. First,it is required to form multivalent homo-oligomers of caveolin. Second, it interacts with G-protein alpha-subunits and down-regulates their GTPase activity. Th ird, it binds to wild-type H-Ras. Fourth, it is membrane-proximal, sug gesting that it may be involved in other potential protein-protein int eractions. Thus, we have termed this 20-amino acid stretch of caveolin residues the caveolin scaffolding domain.