A casein kinase I activity is constitutively associated with Nck.
Nck is a 47-kDa cytosolic protein devoid of intrinsic catalytic activity and consisting of Src homology 2 and 3 (SH2 and SH3) domains organized as follows: SH3-SH3-SH3-SH2. Nck is believed to act as an adaptor protein mediating signal transduction initiated by receptor tyrosine kinases (RTKs). Through its SH2 domain, Nck recognizes a specific phosphotyrosine residue on RTKs or on protein substrates of RTKs like insulin receptor substrate-1, the major substrate of the insulin receptor, and through its SH3 domains it interacts with poorly characterized effector molecules. To identify novel proteins that might interact with Nck, we have used the amino-terminal segment of Nck encompassing its three SH3 domains in the yeast two-hybrid system. Among the polypeptides that associate with Nck, we have identified the gamma2 isoform of the serine/threonine casein kinase I (CKI-gamma2). In transformed rat hepatocytes overexpressing the insulin receptor (HTC-IR cells), serine/threonine protein kinase activity coimmunoprecipitates with Nck, an interaction mediated mainly by the third SH3 domain of Nck. This kinase activity is not apparently modulated by insulin, nor is it sensitive to staurosporine or heparin, and it does not use GTP as a phosphate donor. However the kinase activity coimmunoprecipitated with Nck is completely abolished by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inhibitor of casein kinase I. In an in vitro renaturation gel kinase assay, a protein kinase of 70-75 kDa was detected associated with the SH3 domains of Nck. Far Western analysis demonstrated that the SH3 domains of Nck bound directly to a cytosolic protein of 70-75 kDa. A rabbit polyclonal antibody raised against the C-terminal region of CKI-gamma2 protein kinase immunoprecipitated a single specific protein of 70-75 kDa from HTC-IR cell lysates and detected CKI-gamma2 among the proteins coimmunoprecipitated with Nck. These results support an in vivo interaction between Nck and CKI-gamma2 and suggest that CKI-gamma2 could be involved in signaling pathways downstream of RTKs.
Lussier
G
G
Polypeptide Laboratory, Division of Endocrinology, Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada.
Larose
L
L
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001665:Binding Sites; D047388:Casein Kinases; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D005982:Glutathione Transferase; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D015513:Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D007328:Insulin; C078332:Nck protein; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases; D011972:Receptor, Insulin; D047388:Casein Kinases