Fyn is required for oxidative- and hyperosmotic-stress-induced tyrosine phosphorylation of caveolin-1.
Caveolin-1 is phosphorylated on Tyr(14) in response to both oxidative and hyperosmotic stress. In the present paper, we show that this phosphorylation requires activation of the Src family kinase Fyn. Stress-induced caveolin phosphorylation was abolished by three Src kinase inhibitors, SU6656, PP2 and PD180970, and was not observed in fibroblasts derived from a Src, Yes and Fyn triple-knockout mouse (SYF-/-). Using cell lines derived from single-kinase-knockout mice (Src-/-, Yes-/- and Fyn-/-), we show that expression of Fyn, but not Src or Yes, is required for stress-induced caveolin phosphorylation. Heterologous expression of Fyn in the SYF-/- and Fyn-/- cells was sufficient to reconstitute stress-induced caveolin phosphorylation, and overexpression of Fyn in wild-type cells induced hyperphosphorylation of caveolin. Fyn was autophosphorylated following oxidative stress, verifying activation of this kinase. Interestingly, there was a concomitant increase in the phosphorylation of Fyn on its Csk (C-terminal Src kinase) site, indicating feedback inhibition. Csk binds to phosphocaveolin [Cao, Courchesne and Mastick (2002) J. Biol. Chem. 277, 8771-8774] and should phosphorylate any co-localized Src-family kinases. Oxidative-stress-induced phosphorylation of caveolin-1 also requires expression of Abl [Sanguinetti and Mastick (2003) Cell Signal. 15, 289-298]. Using inhibitors and cells derived from knockout mice, we verified a requirement for both Abl and Fyn in stress-induced caveolin phosphorylation in a single cell type. Our data suggest a novel mechanism for attenuation of Src-kinase activity by Abl: stable tyrosine phosphorylation of a scaffolding protein, caveolin, and recruitment of Csk. Paxillin, a substrate of both Abl and Src, organizes a similar regulatory complex.
Sanguinetti
Amy R
AR
Department of Biochemistry, College of Agriculture, Biotechnology and Natural Resources, and School of Medicine, University of Nevada, Reno, NV 89557, USA.
Cao
Haiming
H
Corley Mastick
Cynthia
C
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009997:Osmotic Pressure; D018384:Oxidative Stress; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
C495540:CAV1 protein, human; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C494957:FYN protein, human; C494960:Fyn protein, mouse; D016315:Proto-Oncogene Proteins c-abl; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; C000631346:CSK protein, human