Phosphorylation of Caveolin by Src Tyrosine Kinases THE a-ISOFORM OF CAVEOLIN IS SELECTIVELY PHOSPHORYLATED BY v-Src IN VIVO*

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 sitedirected 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: a-caveolin contains residues 1–178 and b-caveolin contains residues 32–178. Only a-caveolin underwent tyrosine phosphorylation in v-Src transformed NIH 3T3 cells, although b-caveolin is well expressed in these cells. As b-caveolin lacks residues 1–31 (and therefore tyrosine 14), these in vivo studies directly demonstrate the validity of our in vitro studies. Because aand b-caveolin are known to assume a distinct but overlapping subcellular distribution within a single cell, v-Src phosphorylation of a-caveolin may only affect a subpopulation of caveolae that contain a-caveolin. Caveolae are specialized domains of the plasma membrane (1, 2). They are most numerous in endothelial cells, fibroblasts, adipocytes, and smooth muscle cells, although they appear to exist in most cells (3). Their exact function remains unknown, although they have recently been implicated in certain transmembrane signaling events, including G protein-coupled signaling (1, 4–13). Caveolin, a 21–24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo (14). Several independent lines of evidence suggest that caveolin may act as a scaffolding protein within caveolae membranes. In support of this assertion: (i) both the N-terminal and C-terminal domains of caveolin face the cytoplasm (15); (ii) caveolin exists within caveolae membranes as a high molecular mass homo-oligomer (16, 17); (iii) these caveolin homo-oligomers have the capacity to self-associate in vitro to form larger structures that resemble caveolae (16); (iv) caveolin co-purifies with cytoplasmic signaling molecules including trimeric G proteins, Src family tyrosine kinases, and Ras-related GTPases (4–6); (v) recombinant caveolin interacts directly with hetero-trimeric G proteins (10); and (vi) caveolin binding can functionally suppress the GTPase activity and GTP binding of purified trimeric G proteins, holding the G protein in the inactive conformation (10). Thus, caveolin may serve as an oligomeric docking site for organizing and concentrating inactive signaling molecules within caveolae membranes (16). Modification and/or inactivation of caveolin may be a common feature of the transformed phenotype (11). Historically, caveolin was first identified as a major v-Src substrate in Rous sarcoma virus-transformed cells (18). Caveolin is one of only a few known transformation-dependent v-Src substrates; both cell transformation and tyrosine phosphorylation of caveolin by v-Src are critically dependent on membrane attachment of Src via N-terminal myristoylation (19). Based on these observations, Glenney and co-workers have proposed that caveolin may represent a critical target during cellular transformation. In direct support of this notion, caveolin expression and caveolae are dramatically reduced or absent in cells transformed by activated oncogenes other than v-Src (v-Abl, Bcr-Abl, and activated Ras) (11). The functional consequences of the phosphorylation of caveolin on tyrosine are not yet known. At steady state, caveolin is not phosphorylated on tyrosine (12, 20). This is in contrast to v-Src-transformed cells where caveolin is constitutively phosphorylated on tyrosine (19). However, caveolin tyrosine phosphorylation also occurs in normal cells but in a tightly regulated fashion (12). If 3T3-L1 adipocytes are serum-starved and rapidly stimulated with insulin, caveolin transiently undergoes phosphorylation on tyrosine in a timeand dose-dependent manner (12). It has been suggested that insulin-stimulated tyrosine phosphorylation of caveolin occurs indirectly via an endogenous Src family tyrosine kinase, rather than directly via * This work was supported in part by a grant from the W. M. Keck Foundation to the Whitehead Fellows Program (to M. P. L.), National Institutes of Health FIRST Award GM-50443 (to M. P. L.), and a grant from the Elsa U. Pardee Foundation (to M. P. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed: Whitehead Inst. for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142-1479. Tel. : 617-258-5225; Fax: 617-258-9872; E-mail: lisanti@wi.mit. edu. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 7, Issue of February 16, pp. 3863–3868, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.