Recombinant human insulin receptor substrate-1 protein. Tyrosine phosphorylation and in vitro binding of insulin receptor kinase.
Insulin receptor substrate-1 (IRS-1) is a major endogenous substrate of the insulin receptor. To study the interaction of the insulin receptor with IRS-1 in vitro, we expressed in Escherichia coli the amino acids 516-777 of human IRS-1 (hIRS-p30) covering five potential tyrosine phosphorylation sites within YXXM motifs. Kinetic data for tyrosine phosphorylation of hIRS-p30 by partially purified insulin receptor and insulin-like growth factor I receptor and by baculovirus-expressed insulin receptor kinase domain were determined. Native insulin receptor demonstrated the highest affinity to hIRS-p30 (Km = 6.8 +/- 0.6 microM), followed by the insulin-like growth factor I receptor (Km = 9.9 +/- 1.0 microM). We used the soluble recombinant insulin receptor kinase domain, which phosphorylated hIRS-p30 with high affinity (Km = 11.9 +/- 0.8 microM), and affinity columns prepared by coupling hIRS-p30 to NHS-activated Sepharose for binding assays. The insulin receptor kinase domain phosphorylated the hIRS-p30 on the column, was bound by the immobilized hIRS-p30, and was eluted with high salt buffer. Autophosphorylated and EDTA-inactivated insulin receptor kinase domain was bound only by immobilized hIRS-p30 protein that has been prephosphorylated. Our results indicate that the recombinant hIRS-p30 protein is a high affinity substrate for insulin receptor and insulin-like growth factor I receptor in vitro. Moreover, we show that only tyrosine-phosphorylated hIRS-p30 is able to bind to the insulin receptor.
Siemeister
G
G
Klinik II und Poliklinik für Innere Medizin, University of Cologne, Federal Republic of Germany.
al-Hasani
H
H
Klein
H W
HW
Kellner
S
S
Streicher
R
R
Krone
W
W
Müller-Wieland
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011972:Receptor, Insulin