[pTyr1146][pTyr1150][pTyr1151]Insulin Receptor 1142-1153

Three peptides were synthesized corresponding to potential autophosphorylation sites of the beta subunit of the human insulin receptor. These were peptide 1150 corresponding to amino acids 1142-1153 of the pro-receptor, peptide 960 corresponding to amino acids 952-961 of the proreceptor, and peptide 1316 corresponding to amino acids 1313-1329 of the proreceptor. Peptide 1150 served as a better substrate for the insulin receptor tyrosine protein kinase than either of the other peptides or than the Src peptide (corresponding to the sequence surrounding the autophosphorylation site at Tyr-416). Microsequencing of the phosphorylated peptide 1150 indicated that Tyr-1150 rather than Tyr-1146 or Tyr-1151 was phosphorylated in the in vitro reaction. The insulin receptor was then isolated from 32P-labeled IM-9 cells that had been exposed to insulin. Tryptic digestion of the beta subunit revealed one peptide whose phosphorylation was dependent upon insulin and occurred exclusively on Tyr. This peptide was selectively immunoprecipitated by an antipeptide antibody directed to the Tyr-1150-containing sequence. We conclude that Tyr-1150 is preferentially phosphorylated by the purified receptor kinase and that one of the autophosphorylation reactions elicited by insulin in intact cells occurs in a sequence that contains this residue.

The identity of protein-tyrosine-phosphatases (PTPases) active against autophosphorylated insulin receptor was probed by using an insulin-receptor-related peptide phosphorylated on tyrosine (peptide 1142-1153). Two major peaks of PTPase activity were resolved from the particulate (Triton X-100-soluble) fraction of human placenta by chromatography on DEAE-cellulose. The two peaks were purified 1300-2300-fold; other peaks of PTPase activity (greater than 15%) were not detected. Properties of the PTPases indicated that they corresponded to subtypes 1A and 1B. Both subtypes appeared capable of catalysing dephosphorylation of all autophosphorylation sites in three domains of the insulin receptor, with no appreciable difference in the pattern of dephosphorylation detected by two-dimensional tryptic-peptide mapping. The tyrosine-1150 domain of the insulin receptor in triply phosphorylated form was found to be highly sensitive to the action of both PTPases, and was dephosphorylated at least 4 times faster than the doubly and singly phosphorylated forms of the tyrosine-1150 domain or phosphorylation sites in other domains by either PTPase. This is significant, as the level of the triphosphotyrosine-1150 species has been shown to correlate well with the capacity of the insulin-receptor tyrosine kinase to phosphorylate other proteins. Both subtypes also dephosphorylated autophosphorylated epidermal-growth-factor (EGF) receptor by greater than 95%. Placental particulate (and cytosolic) PTPase activity against either receptor distributed approximately 2:1 between subtypes 1A and 1B as assayed in the presence of EDTA. In summary, PTPases within two major subtypes have been identified as phosphotyrosyl-insulin and -EGF-receptor phosphatases in vitro. The PTPases identified exhibit high affinities for substrates and high activities in cells, which is commensurate with the PTPases being important in vivo in controlling or reversing autophosphorylation-induced regulatory or signalling events.

Synthetic peptide 1142-1153 of the insulin receptor was phosphorylated on tyrosine by the insulin receptor and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble phosphotyrosyl protein phosphatases. Apparent Km values were approximately 5 microM. Vm values (nmol phosphate removed/min per mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponds to 80% of total activity being membrane-associated, indicating that membrane-bound phosphatases are important receptor phosphatases. The phosphatase activities were distinct from acid and alkaline phosphatase. In conclusion peptide 1142-1153 provides a useful tool for the further study and characterization of phosphotyrosyl protein phosphatases.