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 signalling by coupling receptor tyrosine kinases, via its SH2 domain, to downstream SH3-binding effectors. We report here that angiotensin II, working through the AT1 receptor subtype, stimulates the phosphorylation of Nck in rat aortic smooth muscle cells. Phosphopeptide mapping analysis revealed that Nck is phosphorylated on four peptides containing exclusively phosphoserine in quiescent cells. Treatment with angiotensin II resulted in increased phosphorylation of these four peptides, without the appearance of new phosphopeptides. 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 cells. 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 Nck in aortic smooth muscle cells. We have also identified the gamma2 isoform 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-coupled receptors and suggest a role for Pak1 and casein kinase I-gamma2 in downstream signalling or regulation of the AT1 receptor.
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Centre de Recherche, Centre hospitalier de l'Université de Montréal and Department of Pharmacology, University of Montreal, Campus Hôtel-Dieu, 3850 St. Urbain, Montreal, Quebec, Canada H2W 1T8.
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D048868:Adaptor Proteins, Signal Transducing; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D047388:Casein Kinases; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000302:isolation & purification; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015398:Signal Transduction; D054462:p21-Activated Kinases; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D010768:Phosphoserine; D011494:Protein Kinases; D047388:Casein Kinases; C515906:Pak1 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases