ACTIVATION OF MITOGEN-ACTIVATED PROTEIN (MAP) KINASES BY VANADATE IS INDEPENDENT OF INSULIN-RECEPTOR AUTOPHOSPHORYLATION

Treatment of Chinese hamster ovary (CHO) cells over-expressing the hum an insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadat e, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyl phosphory lation of the beta-subunit of the insulin receptor. By using myelin ba sic protein (MBP) as the substrate to measure mitogen-activated protei n (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyr osyl phosphorylation of p42 and p44 was associated with a dose- and ti me-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column fo llowed by immunoblotting with a specific antibody to MAP kinases demon strated that vanadate treatment increased the tyrosyl phosphorylation of both p44(mapk) and p42(mapk) by Several folds, as compared to contr ols, in concert with MAP kinase activation. In addition, retardation i n gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44(mapk) and p42(mapk) in CHO-HIRc. A similar eff ect of vanadate on MAP kinase tyrosyl phosphorylation and activation w as also observed in CHO cells over-expressing a protein tyrosine kinas e-deficient insulin receptor (CHO-1018). These results demonstrate tha t the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediate d tyrosyl phosphorylation and activation of MAP kinases.