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

Hyangkyu Lee, David S. Park, Xiao Bo Wang, Philipp E. Scherer, Phillip E. Schwartz, Michael P. Lisanti

Research output: Contribution to journalArticle

70 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)34556-34567
Number of pages12
JournalJournal of Biological Chemistry
Volume277
Issue number37
DOIs
Publication statusPublished - 2002 Sep 13

Fingerprint

Caveolin 2
Caveolin 1
Caveolae
Phosphorylation
Focal Adhesions
Molecular mass
Oligomers
Tyrosine
Adhesion
Lipids
NIH 3T3 Cells
ras GTPase-Activating Proteins
src Homology Domains
Integrins
Ligation
2-tyrosine
Phospho-Specific Antibodies
Caveolins
Signal transduction
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "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",
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 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.",
author = "Hyangkyu Lee and Park, {David S.} and Wang, {Xiao Bo} and Scherer, {Philipp E.} and Schwartz, {Phillip E.} and Lisanti, {Michael P.}",
year = "2002",
month = "9",
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language = "English",
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pages = "34556--34567",
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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. / Lee, Hyangkyu; Park, David S.; Wang, Xiao Bo; Scherer, Philipp E.; Schwartz, Phillip E.; Lisanti, Michael P.

In: Journal of Biological Chemistry, Vol. 277, No. 37, 13.09.2002, p. 34556-34567.

Research output: Contribution to journalArticle

TY - JOUR

T1 - 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

AU - Lee, Hyangkyu

AU - Park, David S.

AU - Wang, Xiao Bo

AU - Scherer, Philipp E.

AU - Schwartz, Phillip E.

AU - Lisanti, Michael P.

PY - 2002/9/13

Y1 - 2002/9/13

N2 - 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.

AB - 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.

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