title: Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae.
abstract: Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Adriana M
authors.initials: AM
authors.lastname: Zimnicka
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Yawer S
authors.initials: YS
authors.lastname: Husain
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Ayesha N
authors.initials: AN
authors.lastname: Shajahan
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Maria
authors.initials: M
authors.lastname: Sverdlov
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Oleg
authors.initials: O
authors.lastname: Chaga
authors.affiliation: Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Zhenlong
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Peter T
authors.initials: PT
authors.lastname: Toth
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Klomp
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Andrei V
authors.initials: AV
authors.lastname: Karginov
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Tiruppathi
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 Center for Lung and Vascular Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612.
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL 60612 Center for Lung and Vascular Biology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612 rminsh@uic.edu.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Minshall
mesh_terms: D000818:Animals; D001692:Biological Transport; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000235:genetics* / Q000378:metabolism*; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D021381:Protein Transport; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D051242:Caveolin 1; D019061:src-Family Kinases