Pyk2 mediates endothelin-1 signaling via p130Cas/BCAR3 cascade and regulates human glomerular mesangial cell adhesion and spreading

Calcium-regulated non-receptor proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of endothelin-1 (ET-1) signaling in human glomerular mesangial cells (GMC). We aimed to identify which small G-protein is acting downstream of Pyk2. Dominant interfering Pyk2 construct, termed calcium regulated non kinase (CRNK) or green fluorescent protein (control) were expressed in GMC using adenovirus-mediated gene transfer. ET-1 stimulation resulted in a significant increase of Pyk2 phosphorylation accompanied by GTP-loading of Rap1 and RhoA. CRNK expression inhibited ET-1-induced autophosphorylation of endogenous Pyk2 and diminished Rap1, but not RhoA, activation. The mechanism linking Pyk2 and Rap1 included (1) increased autophosphorylation of Pyk2 associated with p130Cas, (2) augmented p130Cas Y165 and Y249 phosphorylation, and (3) enhanced p130Cas-BCAR3 complex formation. CRNK expression prevented p130Cas phosphorylation and attenuated p130Cas association with BCAR3. Downregulation of endogenous BCAR3 protein expression using an siRNA technique led to a significant decrease in Rap1 activation in response to ET-1. We observed that endogenous Pyk2 was important for GMC adhesion and spreading. Our data suggest that ET-1 stimulated the GTPase Rap1 (but neither RhoA nor Ras) by a mechanism involving Pyk2 activation and recruitment of the p130Cas/BCAR3 complex in GMC.