FIG. 3.

S6K1 is sufficient to promote Rictor phosphorylation in cells. (A) Schematic representation of S6K1 constructs used in this study. Constitutively activated S6K1 (CA) consists of F5A, T389E, and R410/413/414A (R3A) mutations. The kinase-inactive allele of S6K1 (KD) consists of a K100R mutation in subdomain II of the kinase domain. (B) HEK293 cells were cotransfected with myc-tagged Rictor and constructs expressing HA-tagged wild-type (wt), constitutively activated (CA), or kinase-inactive (KD) S6K1. Cells were serum starved overnight and treated with insulin (100 nM) for 20 min prior to harvesting. The phosphorylation of Rictor was assayed in anti-myc immunoprecipitates. (C and D) As for panel B, except S6K1 CA- and Rheb-expressing cells were pretreated with rapamycin (100 nM) for 30 min prior to harvesting.