FIG. 4.

Endogenous SGK is required for FKHRL1 phosphorylation induced by growth factors. (A) HEK 293T cells were starved for 6 h and then treated with 10 μM LY for 1 h or treated for the indicated periods of time (in minutes) with a combination of 20% FCS and 10⁻⁴ M dexamethasone (D) to induce SGK1 mRNA. RT-PCRs were performed on total RNA using two different pairs of primers that are specific for the human SGK1 isoform (hSGK1-1–hSGK1-2 and hSGK1-3–hSGK1-4). (B) HEK 293T cells were transfected with an empty vector (CTL) or constructs encoding the KN Akt (K197M) mutant (Akt k.n.) or the KN SGK (T256A/S422A) mutant (SGK k.n.). Cells were incubated in the presence of 10% FCS, and extracts were resolved by SDS-PAGE. Phosphorylation of endogenous FKHRL1 was detected by immunoblot analysis with antibodies directed against phospho-T32, phospho-S253, or phospho-S315 FKHRL1. Mobility shift of endogenous FKHRL1 was assessed by immunoblot analysis with the antibody directed against total FKHRL1. ∗, phosphorylated form of FKHRL1.