Figure 1.

Phosphorylation of IRS-1 by GSK-3. (A) Recombinant IRS-1 (designated as IRS-1) or IRS-1 immunoprecipitated from CHO cells overexpressing IRS-1 (designated as IP-IRS-1) was incubated with recombinant rabbit GSK-3 in the presence of 50 mM Tris (pH 7.3), 10 mM magnesium acetate, 0.01% 2-mercaptoethanol, and 50 μM [γ-³²P]ATP (0.25 mCi/ml) in a final volume of 30 μl at 30°C for 15 min. Reactions were stopped by addition of Laemmli sample buffer, subjected to SDS/PAGE, and autoradiographed. (B) Phosphoamino acid composition of each type IRS-1 phosphorylated by GSK-3 was determined as described. The migration positions of phosphorylated tyrosine (P-Tyr), threonine (P-Thr), and serine (P-Ser) are indicated. (C) IRS-1, c-jun, and p9CREB peptide (see Materials and Methods), 10 pmol of each, were phosphorylated by GSK-3 under the conditions described in A for the indicated times. Reactions with IRS-1 or c-jun as the substrates were stopped by addition of Laemmli sample buffer, subjected to SDS/PAGE, and autoradiographed; radioactive bands were cut from the gels and counted for radioactivity. The reaction mixtures containing p9CREB was spotted on phosphocellulose paper and handled as described. Phosphate incorporation into each substrate is presented.