Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation.
Fluid shear stress (flow) modulates endothelial cell function via specific intracellular signaling events. Previously we showed that flow activated ERK1/2 in an integrin-dependent manner (Takahashi, M., and Berk, B. C. (1996) J. Clin. Invest. 98, 2623-2631). p130 Crk-associated substrate (Cas), a putative c-Src substrate, was originally identified as a highly phosphorylated protein that is localized to focal adhesions and acts as an adapter protein. Recent reports have shown that Cas is important in cardiovascular development and actin filament assembly. Flow (shear stress = 12 dynes/cm(2)) stimulated Cas tyrosine phosphorylation within 1 min in human umbilical vein endothelial cells. Phosphorylation peaked at 5 min (3.5 +/- 0.7-fold) and was sustained to 20 min. Tyrosine phosphorylation of Cas was functionally important because flow stimulated association of Cas with Crk in a time- and force-dependent manner. Flow-mediated activation of c-Src, phosphorylation of Cas, and association of Cas with Crk were all inhibited by calcium chelation and pretreatment with the Src family-specific tyrosine kinase inhibitor PP1. To determine the role of c-Src in flow-stimulated phosphorylation of Cas, we transduced cells with adenovirus encoding kinase-inactive Src. Expression of kinase-inactive Src prevented flow-induced Cas tyrosine phosphorylation but not ERK1/2 activation. Calcium-dependent activation of c-Src and tyrosine phosphorylation of Cas defines a new flow-stimulated signal pathway, different from ERK1/2 activation. This pathway may be involved in focal adhesion remodeling and actin filament assembly.
Okuda
M
M
Department of Medicine, Cardiology Division, University of Washington, Seattle, Washington 98195, USA.
Takahashi
M
M
Suero
J
J
Murry
C E
CE
Traub
O
O
Kawakatsu
H
H
Berk
B C
BC
D016227:Benzoquinones; D000081247:CSK Tyrosine-Protein Kinase; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047029:Lactams, Macrocyclic; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D050717:Retinoblastoma-Like Protein p130; D017828:Rifabutin / Q000031:analogs & derivatives; D013314:Stress, Mechanical; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; C493841:BCAR1 protein, human; D016227:Benzoquinones; D050719:Crk-Associated Substrate Protein; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D010750:Phosphoproteins; D011506:Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D011809:Quinones; D050717:Retinoblastoma-Like Protein p130; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium