Palmitoylation of caveolin-1 at a single site (Cys-156) controls its coupling to the c-Src tyrosine kinase - Targeting of dually acylated molecules (GPI-linked, transmembrane, or cytoplasmic) to caveolae effectively uncouples c-Src and caveolin-1 (Tyr-14)

Caveolin-1 was initially identified as a phosphoprotein in Rous sarcoma vir us-transformed cells. Previous studies have shown that caveolin-1 is phosph orylated on tyrosine 14 by c-Src and that lipid modification of c-Src is re quired for this phosphorylation event to occur in vivo. Phosphocaveolin-1 ( Tyr(P)-14) localizes within caveolae near focal adhesions and, through its interaction with Grb7, augments anchorage-independent growth and epidermal growth factor-stimulated cell migration. However, the cellular factors that govern the coupling of caveolin-1 to the c-Src tyrosine kinase remain larg ely unknown. Here, we show that palmitoylation of caveolin-1 at a single si te (Cys-156) is required for coupling caveolin-1 to the c-Src tyrosine kina se. Furthermore, upon evaluating a battery of nonreceptor and receptor tyro sine kinases, we demonstrate that the tyrosine phosphorylation of caveolin- 1 by c-Src is a highly selective event. We show that Src-induced tyrosine p hosphorylation of caveolin-1 can be inhibited or uncoupled by targeting dua lly acylated proteins (namely carcinoembryonic antigen (CEA), CD36, and the NH2-terminal domain of G alpha (i1)) to the exoplasmic, transmembrane, and cytoplasmic regions of the caveolae membrane, respectively. Conversely, wh en these proteins are not properly targeted or lipid-modified, the ability of c-Src to phosphorylate caveolin-1 remains unaffected. In addition, when purified caveolae preparations are preincubated with a myristoylated peptid e derived from the extreme N terminus of c-Src, the tyrosine phosphorylatio n of caveolin-1 is abrogated; the same peptide lacking myristoylation has n o inhibitory activity. However, an analogous myristoylated peptide derived from c-Yes also has no inhibitory activity. Thus, the inhibitory effects of the myristoylated c-Src peptide are both myristoylation-dependent and sequ ence-specific. Finally, we investigated whether phosphocaveolin-1 (Tyr(P)-1 4) interacts with the Src homology 2 and/or phosphotyrosine binding domains of Grb7, the only characterized downstream mediator of its function. Taken together, our data identify a series of novel lipid-lipid-based interactio ns as important regulatory factors for coupling caveolin-1 to the c-Src tyr osine kinase in vivo.