THE INSULIN-DEPENDENT TYROSINE PROTEIN KINASE SUBSTRATE SPECIFICITY AND REGULATION BY CYCLIC-AMP

Abstract

Insulin stimulates the phosphorylation of the {dollar}\beta{dollar} subunit of its receptor in intact cells and in cell free preparations. In cells, serine and tyrosine residues are phosphorylated whereas in vitro, phosphorylation occurs exclusively on tyrosine residues. In addition to autophosphorylation, the insulin-dependent tyrosine kinase activity of the purified human placental insulin receptor was shown to catalyze the phosphorylation of exogenous protein and peptide substrates. Tyrosine-containing peptide substrates include a src-related peptide derived from the sequence of the autophosphorylation site of pp60{dollar}\sp{lcub}\rm src{rcub}{dollar}, Angiotensin II, gastrin, and three synthetic peptides corresponding to potential autophosphorylation sites of the {dollar}\beta{dollar} subunit of the human insulin receptor. These latter peptides were designated peptide 1150 (amino acids 1142-1153 of the proreceptor), peptide 960 (amino acids 952-961 of the proreceptor); and peptide 1316 (amino acids 1313-1329 of the proreceptor). Peptide 1150 (TRDIYETDYYRK) served as a better substrate for the insulin receptor tyrosine protein kinase than either of the other peptides or than the src-related peptide. Microsequencing of the phosphorylated peptide 1150 indicated that the Tyr-1150 rather than Tyr-1146 or Tyr 1151 was phosphorylated in the in vitro reaction. In addition, one of the autophosphorylation reactions elicited by insulin in intact IM-9 cells occurs within a sequence that contains Tyr 1150. This was established by isolating the insulin receptor from {dollar}\sp{lcub}32{rcub}{dollar}P-labelled cells exposed to insulin and subjecting it to trypsin digestion and selective immunoprecipitation with an antipeptide antibody directed to the Tyr-1150-containing sequence. The results suggest that the two adjacent tyrosines and nearby acidic amino acid residues are important features of the substrate specificity of the insulin receptor kinase.;To study the relationship between the serine and tyrosine phosphorylation seen in vivo, the effect of 8-Br-cAMP and forskolin on insulin receptor phosphorylation was studied in cultured IM-9 lymphoblasts. The addition of 8-Br-cAMP or forskolin, agents that raise intracellular cAMP, increased the phosphorylation of the insulin receptor on serine and threonine residues, reduced insulin-mediated receptor phosphorylation on tyrosine, serine and threonine residues and inhibited the insulin-dependent tyrosine protein kinase activity of the receptor. Thus, cAMP may attenuate insulin action by altering the state of phosphorylation of the insulin receptor.