INSULIN ACTIVATES A P21-ACTIVATED KINASE IN MUSCLE-CELLS VIA PHOSPHATIDYLINOSITOL 3-KINASE

Insulin activates rapidly a complex cascade of lipid and protein kinas es leading to stimulation of mitogenic and metabolic events. Mere we d escribe a renaturable kinase of 65 kDa (PK65) that becomes rapidly act ivated by insulin in differentiated L6 muscle cells (myotubes) and can phosphorylate histones immobilized in polyacrylamide gels. Insulin ac tivation of PK65 was abolished by the tyrosine kinase inhibitor erbsta tin and by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor w ortmannin, but was unaffected by inhibitors of protein kinase C or of the activation of p70(S6K). Recently, a number of protein kinases have been described which become activated through interaction with the sm all GTP-binding proteins Rac and Cdc42 (p21-activated kinases, or PAKs ) and lead to activation of the stress-induced mitogen-activated prote in kinase (MAPK) p38 MAPK. Two different polyclonal antibodies recogni zing the carboxyl-terminal or the Rac-binding domain of a 65-kDa PAK ( PAK65) immunoprecipitated the myotube PK65. The insulin-induced activa tion of PK65 in myotubes was detectable following immunoprecipitation of the kinase, Furthermore, PK65 associated with and became activated by glutathione S-transferase-Cdc42Hs in the presence of GTP gamma S (g uanosine 5'-3-O-(thio)triphosphate). In myotubes insulin also induced tyrosine phosphorylation of p38 MAPK. However, this phosphorylation wa s insensitive to wortmannin, indicating that p38 MAPK is not activated by PK65 in insulin-stimulated cells. The results suggest that insulin activates in muscle cells a renaturable kinase (PK65) closely related to PAK65. Tyrosine kinases and PI 3-kinase act upstream of PK65 in th e insulin signaling cascade, Insulin activates p38 MAPK in myotubes, b ut this occurs by a pathway independent of PI 3-kinase and PK65.