Insulin receptor tyrosine kinase activity and phosphorylation of tyrosines 1162 and 1163 are required for insulin-increased prolactin gene expression.

Insulin treatment increased prolactin gene expression in GH4 cells, a rat pituitary tumor cell line, through the endogenous insulin receptor. However, insulin regulation of transfected plasmids required the expression of cotransfected insulin receptor. Prolactin-CAT expression was increased 12-fold in cells transfected with wild type insulin receptor, but insulin did not increase prolactin gene expression when a kinase negative mutant of the ATP binding site (K1030R) was expressed. Thus, receptor kinase activity was required for signaling to gene transcription. Mutation of tyrosine 1158 did not reduce insulin-increased prolactin-CAT expression while individual mutations of tyrosine 1162 and tyrosine 1163 each reduced insulin-increased prolactin-CAT expression by 50% and a triple mutant of tyrosines 1158/1162/1163 was inactive. Thus, mutation of tyrosine 1162 and 1163 was also sufficient to inactivate signaling by the insulin receptor. Insulin-stimulated auto phosphorylation occurred in all mutants in vitro except the ATP binding site mutant. However, the ability of mutant insulin receptors to mediate insulin-increased prolactin-CAT expression correlated with the substrate-specific catalytic activity of the receptors. This suggested that phosphorylation of these tyrosines was important for substrate access to the catalytic domain of the receptor.