Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization.
To identify the role of caveolin-1 in integrin mechanotransduction, we exposed bovine aortic endothelial cells to 10 dyn/cm2 of laminar shear stress. Caveolin-1 was acutely and transiently phosphorylated with shear, occurring downstream of beta1-integrin activation as the beta1-integrin blocking antibody JB1A was inhibitory. In manipulating Src family kinase (SFK) activity with knockdown of Csk or type 1 protein phosphatase (PP1) treatment, we observed coordinate increase and decrease in shear-induced caveolin-1 phosphorylation, respectively. Hence, shear-stimulated caveolin-1 phosphorylation is regulated by SFKs. Shear-induced recruitment and phosphorylation of caveolin-1 occurred at beta1-integrin sites in a beta1-integrin- and SFK-dependent manner. Csk, described to interact with pY14-caveolin-1 and integrins, bound to an increased pool of phosphorylated caveolin-1 after shear corresponding with elevated Csk at beta1-integrin sites. Like caveolin-1, treatment with JB1A and PP1 attenuated shear-induced Csk association with beta1-integrins. Csk function was assayed with transfection of a caveolin-1 phosphorylation domain peptide. The peptide attenuated shear-induced association of Csk at beta1-integrin sites, as well as colocalization of Csk with paxillin and phosphorylated caveolin-1. Because integrin and Csk activity regulate cytoskeletal reorganization, we evaluated the role of this mechanism in shear-induced myosin light chain (MLC) phosphorylation. Knockdown of Csk expression was sufficient to reduce MLC diphosphorylation due to shear. Disruption of Csk-integrin association by peptide treatment was also inhibitory of the MLC diphosphorylation response. Together these data indicate that integrin activation with shear stress results in SFK-regulated caveolin-1 phosphorylation that, in turn, mediates Csk association at integrin sites, where it plays a role in downstream, shear-stimulated MLC diphosphorylation.
Radel
C
C
Center for Cardiovascular Science, Albany Medical College, Albany, New York, USA.
Rizzo
V
V
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000201:enzymology*; D000081247:CSK Tyrosine-Protein Kinase; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D022001:Focal Adhesions / Q000502:physiology; D019012:Integrin beta1 / Q000378:metabolism*; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013314:Stress, Mechanical; D019061:src-Family Kinases
D000199:Actins; D051242:Caveolin 1; D022461:Caveolins; D003598:Cytoskeletal Proteins; D019012:Integrin beta1; D018994:Myosin Light Chains; D051419:Paxillin; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases