Résumé
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‐
P
3
) 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 [γ‐
32
P]ATP, 1151‐
P
3
decreased autophosphorylation to 60% of maximun, whereas 1151‐NP had no further effect. In both non‐activated and preactivated receptors, 1151‐
P
3
inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu
4
, 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‐
P
3
inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151‐NP and 1151‐
P
3
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‐
P
3
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‐
P
3
, 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.