Open AccessCatalysis of serine and tyrosine autophosphorylation by the human insulin receptor.
Author(s) -
Kurt Baltensperger,
Robert E. Lewis,
Chee-Wai Woon,
Prabhakar Vissavajjhala,
Alonzo H. Ross,
Michael P. Czech
Publication year - 1992
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.89.17.7885
Subject(s) - autophosphorylation , insulin receptor , biochemistry , tyrosine phosphorylation , phosphorylation , insulin receptor substrate , receptor tyrosine kinase , irs2 , tyrosine , biology , microbiology and biotechnology , chemistry , protein kinase a , insulin , endocrinology , insulin resistance
The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.