title: Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.
abstract: We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.
authors.affiliation: Centre de Recherche de Biochimie Macromoléculaire, Institut National de la Santé et de la Recherche Médicale Unité 249, Montpellier, France.
authors.forename: A
authors.initials: A
authors.lastname: Chavanieu
authors.forename: B
authors.initials: B
authors.lastname: Calas
authors.forename: P
authors.initials: P
authors.lastname: Vaglio
authors.forename: F
authors.initials: F
authors.lastname: Grigorescu
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010750:Phosphoproteins / Q000737:chemistry / Q000494:pharmacology*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014638:Vanadates; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin