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J Biol Chem, Vol. 274, Issue 38, 26803-26809, September 17, 1999
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From the 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/cm2) 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.
Department of Medicine, Department of Pathology, University of Washington,
Seattle, Washington 98195, the ** Lung Biology Center, University of
San Francisco, San Francisco, California 94110, and the
Center for Cardiovascular Research,
University of Rochester, Rochester, New York 14642
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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