STUDIES ON AN INSULIN-STIMULATED INSULIN-RECEPTOR SERINE KINASE-ACTIVITY - SEPARATION OF THE KINASE-ACTIVITY FROM THE INSULIN-RECEPTOR AND ITS RECONSTITUTION BACK TO THE INSULIN-RECEPTOR

In cells insulin stimulates autophosphorylation of the insulin recepto r on tyrosine and its phosphorylation on serine and threonine by poorl y characterized kinases. Recently we have achieved co-purification of the insulin receptor with insulin-stimulated insulin receptor serine k inase activity. We now show that the co-purified serine kinase activit y can be removed by NaCl washing and reconstituted by adding back the NaCl eluate. Reconstitution enabled higher serine phosphorylation than achieved with the co-purified preparation. Myelin basic protein was d iscovered to be a potent substrate for insulin-stimulated serine phosp horylation by the co-purified preparation, with the activity responsib le having similar properties to the serine kinase activity towards the receptor. Myelin basic protein was also phosphorylated on serine by t he NaCl eluate. Myelin basic protein phosphorylated by the co-purified preparation or the NaCl eluate gave the same set of phosphoserine pep tides. The major myelin basic protein serine kinase activity in the Na Cl eluate co-purified exactly on Mono Q with the activity that restore d insulin-stimulated insulin receptor serine phosphorylation. These re sults provide strong evidence for the true separation of the serine ki nase from the insulin receptor and the distinctiveness of the serine k inase activity from the insulin receptor tyrosine kinase and mitogen-a ctivated protein kinases. The procedures developed for the isolation o f the serine kinase and the establishment of an effective in vitro sub strate should allow purification of the kinase. The protocols also pro vide flexible systems for identifying the functions of the insulin-sti mulated serine phosphorylations and the respective kinase(s).