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Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

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...

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Autores principales: Zimnicka, Adriana M., Husain, Yawer S., Shajahan, Ayesha N., Sverdlov, Maria, Chaga, Oleg, Chen, Zhenlong, Toth, Peter T., Klomp, Jennifer, Karginov, Andrei V., Tiruppathi, Chinnaswamy, Malik, Asrar B., Minshall, Richard D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Cell Biology 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927282/
https://www.ncbi.nlm.nih.gov/pubmed/27170175
http://dx.doi.org/10.1091/mbc.E15-11-0756
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author Zimnicka, Adriana M.
Husain, Yawer S.
Shajahan, Ayesha N.
Sverdlov, Maria
Chaga, Oleg
Chen, Zhenlong
Toth, Peter T.
Klomp, Jennifer
Karginov, Andrei V.
Tiruppathi, Chinnaswamy
Malik, Asrar B.
Minshall, Richard D.
author_facet Zimnicka, Adriana M.
Husain, Yawer S.
Shajahan, Ayesha N.
Sverdlov, Maria
Chaga, Oleg
Chen, Zhenlong
Toth, Peter T.
Klomp, Jennifer
Karginov, Andrei V.
Tiruppathi, Chinnaswamy
Malik, Asrar B.
Minshall, Richard D.
author_sort Zimnicka, Adriana M.
collection PubMed
description 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.
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spelling pubmed-49272822016-09-16 Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae Zimnicka, Adriana M. Husain, Yawer S. Shajahan, Ayesha N. Sverdlov, Maria Chaga, Oleg Chen, Zhenlong Toth, Peter T. Klomp, Jennifer Karginov, Andrei V. Tiruppathi, Chinnaswamy Malik, Asrar B. Minshall, Richard D. Mol Biol Cell Articles 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. The American Society for Cell Biology 2016-07-01 /pmc/articles/PMC4927282/ /pubmed/27170175 http://dx.doi.org/10.1091/mbc.E15-11-0756 Text en © 2016 Zimnicka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.
spellingShingle Articles
Zimnicka, Adriana M.
Husain, Yawer S.
Shajahan, Ayesha N.
Sverdlov, Maria
Chaga, Oleg
Chen, Zhenlong
Toth, Peter T.
Klomp, Jennifer
Karginov, Andrei V.
Tiruppathi, Chinnaswamy
Malik, Asrar B.
Minshall, Richard D.
Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
title Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
title_full Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
title_fullStr Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
title_full_unstemmed Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
title_short Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
title_sort src-dependent phosphorylation of caveolin-1 tyr-14 promotes swelling and release of caveolae
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4927282/
https://www.ncbi.nlm.nih.gov/pubmed/27170175
http://dx.doi.org/10.1091/mbc.E15-11-0756
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