INSULIN-RECEPTOR SERINE KINASE ACTIVATION BY CASEIN KINASE-2 AND A MEMBRANE TYROSINE KINASE

The insulin receptor (IR) tyrosine kinase can apparently directly phos phorylate and activate one or more serine kinases. The identities of s uch serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor seri ne (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphor ylation of the membrane glucoproteins by casein kinase-1, casein kinas e-2, or casein kinase-3 (Biochem Biophys Res Commun 171: 75-83,1990). In this study, IRS kinase was further characterized. The presence of v anadate or phosphotyrosine in reaction mixtures was required for activ ation to be observed. Phosphoserine and phosphothreonine are only abou t 25% as effective as phosphotyrosine, whereas sodium fluoride and mol ybdate were ineffective in supporting activation. Vanadate and phospho tyrosine support IRS kinase activation by apparently inhibiting phosph otyrosine protein phosphatases present among the membrane glycoprotein s. IR beta-subunit, myelin basic protein, and microtubule-associated p rotein-2 are good substrates for IRS kinase. The kinase prefers Mn2+ ( K(a) = 1.3mM) as a metal cofactor. Mg2+ (K(a) = 3.3mM) is only 30% as effective as Mn2+. The kinase activity is stimulated by basic polypept ides, with greater than 30-fold activation achieved with polylysine an d protamine. Our results suggest that both serine/threonine and tyrosi ne phosphorylation are required for activation of IRS kinase. Serine p hosphorylation is catalyzed by one of the casein kinases, whereas tyro sine phosphorylation is catalyzed by a membrane tyrosine kinase. possi bly IR tyrosine kinase.