Purification and Characterization of an Insulin-Stimulated Insulin Receptor Serine Kinase

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Abstract
In cells, insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Here we describe methods for the purification of an insulin-stimulated insulin receptor serine kinase from human placenta and rat liver by sequential chromatography of solubilized membranes on wheat germ agglutinin−agarose, Mono Q, phenyl-Superose, and Superose 12. On silver-stained SDS−polyacrylamide gels, the resulting kinase was homogeneous (human) or near-homogeneous (rat) and had an apparent Mr of 40 000. The apparent Mr determined by gel filtration was also 40 000, suggesting that the kinase exists as a monomer. The kinase could be reconstituted back to the insulin receptor stripped of the kinase to yield a high stoichiometry of serine phosphorylation of the insulin receptor in the presence of insulin (0.75 ± 0.15 mol/mol of β-subunit, mean ± SEM, n = 3). The activity of the reconstituted kinase toward the insulin receptor was insulin-regulated, being stimulated >5-fold by insulin. Insulin increased the catalytic activity of the reconstituted kinase. The purified kinase specifically phosphorylated serine 1078 of the insulin receptor, a major site of insulin-stimulated serine phosphorylation in vivo, showing that the purified kinase phosphorylated a physiologically relevant site on the insulin receptor. Phosphorylation of serine 1078 of the insulin receptor to high stoichiometry by the kinase did not affect insulin-stimulated exogenous protein tyrosine kinase activity of the insulin receptor. Similarly, insulin receptor phosphorylated with or without the purified kinase exhibited the same levels of tyrosine autophosphorylation and of the tyrosine kinase-activating tris-phosphorylated kinase domain species. Properties of the kinase distinguished it from kinases known to act on the insulin receptor and other kinases that are insulin-stimulated, indicating that the kinase is a novel entity. The serine kinase underwent autophosphorylation on serine and immunoprecipitated with the insulin receptor. The availability of the purified kinase should facilitate cloning of the kinase, determination of the mechanism of activation of the kinase, and study of the wider potential role of the kinase in insulin signalling, and the ability to be able to phosphorylate serine 1078 to high stoichiometry should facilitate further studies into the function of this serine phosphorylation site.

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