Fig. 2.

S6K1 phosphorylates IRS-1 at Ser-1101. Serum-deprived L6 cells were incubated in an amino acid-deprived or amino acid-containing medium for 1 h and stimulated or not with 100 nM insulin for the last 30 min of incubation as indicated, and then 0.01% DMSO vehicle or 25 nM rapamycin was added during the 1-h incubation. (A) Kinase activity (by using cold ATP only) of S6K1 and S6K2 was determined by using GST-IRS-1^C as a substrate, and then the reaction product was analyzed by SDS/PAGE by using an antibody that detects IRS-1 only when phosphorylated at Ser-1101. Controls for substrate amount (GST-IRS-1^C) and immunoprecipitated kinases (S6K1 or S6K2) were analyzed by SDS/PAGE, followed by Coomassie blue staining and Western blot with specific antibodies, respectively. (B) Kinase activity of S6K1 was determined by in vitro kinase assay by using GST-IRS-1^C or GST-IRS-1^C (S1101A) as substrates. The means ± SE from three individual experiments are shown for B. *, P < 0.01 vs. corresponding S6K1 assay by using wild-type GST-IRS-1^C as a substrate.