Figure 4.

ULK1 reduces S6K1 phosphorylation and activity. (A) HEK293 cells were transfected with HA-S6K1 with or without V5-ULK1, serum-starved overnight and stimulated with 100 nM insulin for 30 min prior to lysis where indicated. HA-S6K1 was immunoprecipitated and used in an in vitro kinase assay against recombinant rpS6 peptide. [³²P]-radiolabel incorporation into rpS6 was determined by autoradiography. S6K1 phosphorylation was determined using phospho-Thr389 antibody. The densitometry of the phospho-rpS6 autorads from the three experiments is shown in the graph (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.01. (B) A S6K1 assay was performed as in (A) but in the presence or absence of Flag-Rheb expression instead of insulin stimulation. S6K1 activity was determined by Thr389 phosphorylation of S6K1 and [³²P]-radiolabel incorporation into rpS6. The densitometry of the [³²P]-radiolabeled rpS6 autorads from the three experiments is shown in the graph (mean ± SD). Statistical significance was analyzed using a student's t-test, *p < 0.05.