Src-induced Phosphorylation of Caveolin-2 on Tyrosine 19

Abstract

Caveolin-2 is the least well studied member of the caveolin gene family. It is believed that caveolin-2 is an "accessory protein" that functions in conjunction with caveolin-1. At the level of the ER, caveolin-2 interacts with caveolin-1 to form a high molecular mass heterooligomeric complex that is targeted to lipid rafts and drives the formation of caveolae. However, caveolin-2 is not required for caveolae formation, implying that it may fulfill some unknown regulatory role. Here, we present the first evidence that caveolin-2 is a phosphoprotein. We show that caveolin-2 undergoes Src-induced phosphorylation on tyrosine 19. To study this phosphorylation event in vivo, we generated a novel phospho-specific antibody probe that only recognizes phosphocaveolin-2 (Tyr(P) 19 ). We then used NIH-3T3 cells stably overexpressing c-Src to examine the localization and biochemical properties of phosphocaveolin-2 (Tyr(P) 19 ). Our results indicate that phosphocaveolin-2 (Tyr(P) 19 ) is localized near focal adhesions, remains associated with lipid rafts/caveolae, but no longer forms a high molecular mass hetero-oligomer with caveolin-1. Instead, phosphocaveolin-2 (Tyr(P) 19 ) behaves as a monomer/dimer in velocity gradients. Thus, we conclude that the tyrosine phosphorylation of caveolin-2 (Tyr(P) 19 ) may function as a signal that is recognized by the cellular machinery to induce the dissociation of caveolin-2 from caveolin-1 oligomers. We also demonstrate that (i) insulin-stimulation of adipocytes and (ii) integrin ligation of endothelial cells can both induce the tyrosine phosphorylation of caveolin-2 (Tyr(P) 19 ). During integrin ligation, phosphocaveolin-2 (Tyr(P) 19 ) co-localizes with activated FAK at focal adhesions. Thus, phosphocaveolin-2 (Tyr(P) 19 ) may function as a docking site for Src homology domain-2 (SH2) domain containing proteins during signal transduction. In support of this notion, we identify several SH2 domain containing proteins, namely c-Src, NCK, and Ras-GAP, that interact with caveolin-2 in a phosphorylation-dependent manner. Furthermore, our co-immunoprecipitation experiments show that caveolin-2 and Ras-GAP are constitutively associated in c-Src expressing NIH-3T3 cells, but not in untransfected NIH-3T3 cells.Receptor tyrosine kinases (RTKs) 1 contain one or more autophosphorylation sites. Upon ligand binding, a specific receptor is activated and undergoes autophosphorylation on specific tyrosine residues. One known function for tyrosine phosphorylation is to confer docking sites for Src homology domain-2 (SH2)-containing proteins. Numerous downstream signaling molecules possessing either intrinsic kinase activity or an adapter function are then recruited to the plasma membrane and utilize their SH2 domains to form hetero-oligomeric signaling complexes with activated RTKs.Numerous RTKs, such as epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), and insulin receptor (Ins-R), as well as non-receptor tyrosine kinases (NRTKs; Src-family tyrosine kinases) are ...

Characterization of the Stomatin Domain Involved in Homo-oligomerization and Lipid Raft Association

“…These data resemble the behavior of two forms of phosphorylated caveolin-2, Cav-2(pTyr-19) and Cav-2(pTyr-27), that are targeted to DRMs but are monomeric or dimeric. Moreover, phosphorylation of Tyr-27 alters the binding of caveolin-2 to SH2 domain-containing proteins (48,49). Although we have identified stomatin Tyr-251 as essential for oligomerization, it is unlikely because of the lack of a consensus (50) that this residue is phosphorylated.…”

Lipid-dependent Recruitment of Neuronal Src to Lipid Rafts in the Brain

“…It was found previously that caveolin-2 tyrosine phosphorylation by endogenous tyrosine kinases present in purified caveolin-rich domains from lung is inhibited by a synthetic myristoylated NH 2 -terminal Src peptide but not by a nonmyristoylated Src peptide or by either myristoylated or nonmyristoylated peptides from Yes (45). This may indicate that the specific myristoylated NH 2 terminus of Src (but not Yes) displaces the main caveolin-2 kinase (possibly endogenous Src) from these caveolae preparations.…”

“…Some studies used "panSrc" antibody, or antibodies against the phosphorylated activation loop tyrosine of Src, which do not distinguish Src from other SFKs (44,45). However, Src monoclonal antibodies have been used to show ligand-stimulated Src recruitment to EphrinB1-containing microdomains in neurons (46), and specific antibodies show the coexistence of Src, Fyn, Lyn, and Yes in rafts from neuroblastoma cells (47,48).…”

Microtubules and Actin Microfilaments Regulate Lipid Raft/Caveolae Localization of Adenylyl Cyclase Signaling Components

“…4 and 8). Although the role of phosphorylated caveolins in signal transduction is not well understood, we hypothesize that such phosphorylation is important for maintaining caveolin hetero-and homo-oligomers (29,52) and for scaffolding and recruitment of signaling components (53,54) to caveolin-associated complexes, perhaps by stabilizing multiprotein complexes between caveolar resident and cytoskeletal proteins (i.e. tubulin and filamin), thereby optimizing cell signaling.…”