CrkII associates with BCAR3 in response to endothelin-1 in human glomerular mesangial cells

Endothelin-1 (ET-1) effects in human glomerular mesangial cells (GMC) include proliferation, contraction, and extracellular matrix synthesis. Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human glomerulae. Working in concert with Pyk2, adaptor protein CrkII and a recently discovered guanidine exchange factor for certain small GTPases BCAR3 can be involved in ET-1 signaling in the kidney. Signaling through CrkII and BCAR3 might be critical in some proliferative kidney pathologies. The current study was designed to determine the possibility of CrkII and BCAR3 interaction in response to ET-1 in human GMC and the role of Pyk2 in the association of these proteins. Using adenovirus-mediated transfer of genes encoding either green fluorescent protein (control) or dominant interfering Pyk2 construct, we demonstrated that CrkII and BCAR3 can be coprecipitated from unstimulated and ET-1 stimulated GMC; ET-1 treatment time-dependently increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to a significant decrease in CrkII/BCAR3 association both basal and stimulated.