Angiotensin II stimulates serine phosphorylation of the adaptor protein Nck: physical association with the serine threonine kinases Pak1 and casein kinase I

Nck is a small adaptor protein consisting exclusively of three SH3 domains and one SH2 domain. Nck is thought to have an important role in cell signal ling by coupling receptor tyrosine kinases, via its SH3 domain, to downstre am SH3-binding effecters. We report here that angiotensin II, working: thro ugh the AT(1) receptor subtype, stimulates the phosphorylation of Nck in ra t aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserin e in quiescent cells. Treatment with angiotensin II resulted in increased p hosphorylation of these four peptides, without the appearance of new phosph opeptides, We show that Nck, via its SH3 domains, specifically binds three major phosphoproteins of 95, 82 and 66 kDa both in vitro and in intact cell s. Notably, the phosphorylation of these Nck-binding proteins was found to increase in parallel with that of Nck on stimulation by angiotensin II. One candidate for the 66 kDa phosphoprotein is the serine/threonine kinase p21 -activated kinase 1 (Pak1), which was found to form a stable complex with N ck in aortic smooth muscle cells. We have also identified the gamma 2 isofo rm of casein kinase I as another protein kinase that associates with Nck in these cells. These findings indicate that Nck is a target of G-protein-cou pled receptors and suggest a role for Pak1 and casein kinase I-gamma 2 in d ownstream signalling or regulation of the AT(1) receptor.