Src-induced phosphorylation of caveolin-2 on tyrosine 19. Phospho-caveolin-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.
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 hetero-oligomeric 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.
Lee
Hyangkyu
H
Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Park
David S
DS
Wang
Xiao Bo
XB
Scherer
Philipp E
PE
Schwartz
Phillip E
PE
Lisanti
Michael P
MP
D016475:3T3 Cells; D000818:Animals; D019556:COS Cells; D021941:Caveolae / Q000737:chemistry*; D051242:Caveolin 1; D051243:Caveolin 2; D022461:Caveolins / Q000737:chemistry* / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry*; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
C495541:Cav1 protein, mouse; D051242:Caveolin 1; D051243:Caveolin 2; D022461:Caveolins; D014638:Vanadates; D014443:Tyrosine; D019061:src-Family Kinases