Phosphorylation of Caveolin by Src Tyrosine Kinases

Abstract

Caveolae are flask-shaped plasma membrane specializations that are thought to exist in most cell types. A 22-kDa protein, caveolin, is an integral membrane component of caveolae membranes in vivo. Previous studies have demonstrated that caveolin is phosphorylated on tyrosine by oncogenic viral Src (v-Src) and that caveolin is physically associated as a hetero-oligomeric complex with normal cellular Src (c-Src) and other Src family tyrosine kinases. Caveolin contains eight conserved tyrosine residues that may serve as potential substrates for Src. Here, we have begun to study the phosphorylation of caveolin by Src family tyrosine kinases both in vitro and in vivo. Using purified recombinant components, we first reconstituted the phosphorylation of caveolin by Src kinase in vitro. Microsequencing of Src-phosphorylated caveolin revealed that phosphorylation occurs within the extreme N-terminal region of full-length caveolin between residues 6 and 26. This region contains three tyrosine residues at positions 6, 14, and 25. Deletion mutagenesis demonstrates that caveolin residues 1-21 are sufficient to support this phosphorylation event, implicating tyrosine 6 and/or 14. In vitro phosphorylation of caveolin-derived synthetic peptides and site-directed mutagenesis directly show that tyrosine 14 is the principal substrate for Src kinase. In support of these observations, tyrosine 14 is the only tyrosine residue within caveolin that bears any resemblance to the known recognition motifs for Src family tyrosine kinases. In order to confirm or refute the relevance of these in vitro studies, we next analyzed the tyrosine phosphorylation of endogenous caveolin in v-Src transformed NIH 3T3 cells. In vivo, two isoforms of caveolin are known to exist: alpha-caveolin contains residues 1-178 and beta-caveolin contains residues 32-178. Only alpha-caveolin underwent tyrosine phosphorylation in v-Src transformed NIH 3T3 cells, although beta-caveolin is well expressed in these cells. As beta-caveolin lacks residues 1-31 (and therefore tyrosine 14), these in vivo studies directly demonstrate the validity of our in vitro studies. Because alpha- and beta-caveolin are known to assume a distinct but overlapping subcellular distribution within a single cell, v-Src phosphorylation of alpha-caveolin may only affect a subpopulation of caveolae that contain alpha-caveolin.