PURIFICATION AND CHARACTERIZATION OF AN INSULIN-STIMULATED INSULIN-RECEPTOR SERINE KINASE

In cells, insulin stimulates autophosphorylation of the insulin recept or on tyrosine and its phosphorylation on serine and threonine by poor ly characterized kinases. Here we describe methods for the purificatio n of an insulin-stimulated insulin receptor serine kinase from human p lacenta and rat liver by sequential chromatography of solubilized memb ranes on wheat germ agglutinin-agarose, Mono Q, phenyl-Superose, and S uperose 12. On silver-stained SDS-polyacrylamide gels, the resulting k inase was homogeneous (human) or near-homogeneous (rat) and had an app arent M(r) of 40 000. The apparent M(r) determined by gel filtration w as also 40 000, suggesting that the kinase exists as a monomer. The ki nase could be reconstituted back to the insulin receptor stripped of t he kinase to yield a high stoichiometry of serine phosphorylation of t he insulin receptor in the presence of insulin (0.75 +/- 0.15 mol/mol of beta-subunit, mean +/- SEM, n = 3). The activity of the reconstitut ed kinase toward the insulin receptor was insulin-regulated, being sti mulated >5-fold by insulin. Insulin increased the catalytic activity o f the reconstituted kinase. The purified kinase specifically phosphory lated serine 1078 of the insulin receptor, a major site of insulin-sti mulated serine phosphorylation in vivo, showing that the purified kina se phosphorylated a physiologically relevant site on the insulin recep tor. Phosphorylation of serine 1078 of the insulin receptor to high st oichiometry by the kinase did not affect insulin-stimulated exogenous protein tyrosine kinase activity of the insulin receptor. Similarly, i nsulin receptor phosphorylated with or without the purified kinase exh ibited the same levels of tyrosine autophosphorylation and of the tyro sine kinase-activating tris-phosphorylated kinase domain species, Prop erties of the kinase distinguished it from kinases known to act on the insulin receptor and other kinases that are insulin-stimulated, indic ating that the kinase is a novel entity. The serine kinase underwent a utophosphorylation on serine and immunoprecipitated with the insulin r eceptor. The availability of the purified kinase should facilitate clo ning of the kinase, determination of the mechanism of activation of th e kinase, and study of the wider potential role of the kinase in insul in signalling, and the ability to be able to phosphorylate serine 1078 to high stoichiometry should facilitate further studies into the func tion of this serine phosphorylation site.