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.
Jacob
Kirsten K
KK
Department of Medicine, New York University School of Medicine, NYU Medical Center, TH 450, 550 First Avenue, New York, NY 10016, USA.
Whittaker
Jonathan
J
Stanley
Frederick M
FM
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004789:Enzyme Activation; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D007328:Insulin / Q000494:pharmacology*; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D011388:Prolactin / Q000096:biosynthesis / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
D007328:Insulin; D014443:Tyrosine; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; C410467:insulin receptor tyrosine kinase; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin