Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette.
Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.
Lee
H
H
Department of Molecular Pharmacology and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Volonte
D
D
Galbiati
F
F
Iyengar
P
P
Lublin
D M
DM
Bregman
D B
DB
Wilson
M T
MT
Campos-Gonzalez
R
R
Bouzahzah
B
B
Pestell
R G
RG
Scherer
P E
PE
Lisanti
M P
MP
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D051382:GRB7 Adaptor Protein; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D050356:Lipid Metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; C495955:GRB7 protein, human; C495956:Grb7 protein, mouse; D006133:Growth Substances; D007328:Insulin; D011506:Proteins; D051382:GRB7 Adaptor Protein; D014638:Vanadates; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D019061:src-Family Kinases