Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a)

Abstract

Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.

FIG. 1

SGK phosphorylates FKHRL1 in vitro. (A) HEK 293 cells were transfected with WT SGK, a KN mutant of SGK (K127Q), or a CA mutant of SGK (S422D) and stimulated with insulin or IGF-I for 15 min. SGK was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 (upper panels). The total level of GST-FKHRL1 was analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panels). (B) HEK 293 cells were transfected with a CA mutant of Akt. Akt was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (upper panel). Purified CA SGK was obtained from Upstate Biotechnology and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (middle panel). The total levels of GST-FKHRL1 were analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panel). CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). The numbers represent the percentage of phosphorylation of FKHRL1 mutants compared to wild-type FKHRL1. (C) Extracts obtained for panel B with WT-FKHRL1 were analyzed by immunoblotting with antibodies directed against phospho-T32, phospho-S253, and phospho-S315 or with the antibody directed against total FKHRL1.

FIG. 1

SGK phosphorylates FKHRL1 in vitro. (A) HEK 293 cells were transfected with WT SGK, a KN mutant of SGK (K127Q), or a CA mutant of SGK (S422D) and stimulated with insulin or IGF-I for 15 min. SGK was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 (upper panels). The total level of GST-FKHRL1 was analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panels). (B) HEK 293 cells were transfected with a CA mutant of Akt. Akt was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (upper panel). Purified CA SGK was obtained from Upstate Biotechnology and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (middle panel). The total levels of GST-FKHRL1 were analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panel). CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). The numbers represent the percentage of phosphorylation of FKHRL1 mutants compared to wild-type FKHRL1. (C) Extracts obtained for panel B with WT-FKHRL1 were analyzed by immunoblotting with antibodies directed against phospho-T32, phospho-S253, and phospho-S315 or with the antibody directed against total FKHRL1.

FIG. 1

SGK phosphorylates FKHRL1 in vitro. (A) HEK 293 cells were transfected with WT SGK, a KN mutant of SGK (K127Q), or a CA mutant of SGK (S422D) and stimulated with insulin or IGF-I for 15 min. SGK was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 (upper panels). The total level of GST-FKHRL1 was analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panels). (B) HEK 293 cells were transfected with a CA mutant of Akt. Akt was immunoprecipitated from cell lysates with the anti-HA antibody and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (upper panel). Purified CA SGK was obtained from Upstate Biotechnology and incubated in the presence of [γ-³²P]ATP with WT GST-FKHRL1 or a series of phosphorylation mutants of FKHRL1 (middle panel). The total levels of GST-FKHRL1 were analyzed by immunoblotting with an antibody directed against total FKHRL1 (lower panel). CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). The numbers represent the percentage of phosphorylation of FKHRL1 mutants compared to wild-type FKHRL1. (C) Extracts obtained for panel B with WT-FKHRL1 were analyzed by immunoblotting with antibodies directed against phospho-T32, phospho-S253, and phospho-S315 or with the antibody directed against total FKHRL1.

FIG. 2

SGK induces FKHRL1 phosphorylation in vivo. (A) CCL39 fibroblasts or HEK 293T cells were cotransfected with a construct encoding WT HA-FKHRL1 and either a control empty vector (CTL) or constructs encoding CA mutants of Akt (Akt c.a.) or SGK (SGK c.a.). Small amounts of the Akt plasmid were transfected in order to obtain a level of expression of Akt that is close to the low level of expression of SGK. Cell lysates were analyzed by immunoblotting with antibodies directed against phospho-T32, phospho-S253, or phospho-S315 FKHRL1 or with the antibody directed against the HA epitope (for HA-FKHRL1, HA-Akt, and HA-SGK). In fibroblasts, the levels of SGK expression are too low to be detected by the anti-HA antibody. (B) Primary cultures of cerebellar granule neurons were incubated in the presence of 10% serum, treated with 10 μM LY for 1 h, or stimulated with 100 ng of IGF-I/ml for 20 min. Cell lysates were analyzed by immunoblotting with antibodies directed against SGK1, phospho-T32, or phospho-S315 or with the antibody directed against total FKHRL1. ∗, phosphorylated form of FKHRL1.

FIG. 3

Effects of various protein kinases on FKHRL1 phosphorylation. CCL39 fibroblasts were cotransfected with a construct encoding WT HA-FKHRL1 and constructs encoding CA forms of various protein kinases. Immunoblot analyses were performed as described for Fig. 2.

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.

FIG. 5

SGK plays a critical role in promoting FKHRL1 exclusion from the nucleus in response to growth factors. (A) CCL39 fibroblasts were cotransfected with a WT M2-tagged FKHRL1 construct and either an empty vector (CTL) or plasmids encoding WT SGK or CA SGK (S422D) (SGK c.a.); then they were serum starved for 8 h and incubated in the presence of 20 μM LY for the last hour. FKHRL1 was detected by immunolocalization with an anti-M2 antibody. Quantification of a representative experiment is shown. (B) CCL39 fibroblasts were cotransfected with a WT M2-tagged FKHRL1 construct and either an empty vector (CTL) or plasmids encoding WT SGK or KN SGK (T256A/S422A) and were incubated with 10% FCS. FKHRL1 was detected by immunolocalization with the anti-M2 antibody. Quantification of a representative experiment is shown. (C) CCL39 fibroblasts were transiently transfected with an HA-tagged FKHRL1 construct (WT or the different phosphorylation site mutants) and were incubated in the presence of 10% FCS. FKHRL1 was detected by immunolocalization with the anti-HA antibody. Quantification of a representative experiment is shown. TM, triple mutant of FKHRL1 (T32A/S253A/S315A).

FIG. 6

SGK inhibits FKHRL1-dependent transcription. (A) CCL39 fibroblasts were transiently cotransfected with an empty vector (CTL) or a vector encoding WT FKHRL1 together with WT SGK, CA SGK (S422D) (SGK c.a.), or CA mutants of various protein kinases and the FHRE-Luc reporter construct. The day after transfection, cells were starved for 24 h and luciferase activity was assayed. Data are the means and variances for two independent experiments conducted in duplicate. (B) CCL39 fibroblasts were transiently cotransfected with an empty vector (CTL), a vector encoding CA SGK (S422D) (SGK c.a.), or a vector encoding CA Akt (Akt c.a.), together with vectors encoding WT FKHRL1 or the different FKHRL1 phosphorylation site mutants and the FHRE-Luc reporter construct. Luciferase assays were performed as described for panel A. Data are the means and variances for two independent experiments conducted in duplicate. (C) CCL39 fibroblasts were transiently cotransfected with an empty vector (CTL) or vectors encoding WT FKHRL1 or the different FKHRL1 phosphorylation site mutants and the FHRE-Luc reporter construct. The day after transfection, cells were incubated in the presence of IGF-I for 24 h and luciferase activity was assayed. Data are the means and variances of two independent experiments conducted in duplicate. Expression of the different mutants of FKHRL1 was monitored by immunoblotting with the anti-HA antibody. TM, triple mutant of FKHRL1 (T32A/S253A/S315A) .

FIG. 6

SGK inhibits FKHRL1-dependent transcription. (A) CCL39 fibroblasts were transiently cotransfected with an empty vector (CTL) or a vector encoding WT FKHRL1 together with WT SGK, CA SGK (S422D) (SGK c.a.), or CA mutants of various protein kinases and the FHRE-Luc reporter construct. The day after transfection, cells were starved for 24 h and luciferase activity was assayed. Data are the means and variances for two independent experiments conducted in duplicate. (B) CCL39 fibroblasts were transiently cotransfected with an empty vector (CTL), a vector encoding CA SGK (S422D) (SGK c.a.), or a vector encoding CA Akt (Akt c.a.), together with vectors encoding WT FKHRL1 or the different FKHRL1 phosphorylation site mutants and the FHRE-Luc reporter construct. Luciferase assays were performed as described for panel A. Data are the means and variances for two independent experiments conducted in duplicate. (C) CCL39 fibroblasts were transiently cotransfected with an empty vector (CTL) or vectors encoding WT FKHRL1 or the different FKHRL1 phosphorylation site mutants and the FHRE-Luc reporter construct. The day after transfection, cells were incubated in the presence of IGF-I for 24 h and luciferase activity was assayed. Data are the means and variances of two independent experiments conducted in duplicate. Expression of the different mutants of FKHRL1 was monitored by immunoblotting with the anti-HA antibody. TM, triple mutant of FKHRL1 (T32A/S253A/S315A) .

FIG. 7

The phosphorylation of FKHRL1 at the three regulatory sites is required to prevent FKHRL1-induced cell cycle arrest and apoptosis. (A) CCL39 fibroblasts were transfected with the various phosphorylation site mutants of FKHRL1, starved for 24 h, and stimulated for 24 h with 20% FCS in the presence of BrdU. Transfected cells and the incorporation of BrdU were identified by immunofluorescence with an anti-FKHRL1 antibody and an anti-BrdU antibody, respectively. Data are the means and variances of two independent experiments. CTL, control; TM, triple mutant of FKHRL1 (T32A/S253A/S315A). (B) HEK 293T cells were transfected with the various FKHRL1 phosphorylation site mutants and stimulated for 24 h with IGF-I. Immunoblot analyses were performed with an antibody directed against the cleaved product of PARP, a control antibody to a protein whose levels remain constant (MKK1), and an antibody to HA. TM, triple mutant of FKHRL1 (T32A/S253A/S315A) .

FIG. 8

SGK plays a role in cell survival regulation. (A) Cerebellar granule cells were transiently cotransfected with a vector encoding GFP and either an empty vector (CTL), vectors encoding WT or CA Akt (Akt c.a.), or vectors encoding WT SGK, CA SGK (S422D) (SGK c.a.), or KN SGK (T256A/S422A) (SGK k.n.). Cells were either left in culture medium, incubated with IGF-I for 24 h, or deprived of all survival factors for 8 h. Transfected cells were followed by detection of GFP fluorescence, and DNA was stained with the Hoechst compound. The experiments were performed in duplicate, and 100 to 200 cells per coverslip were counted in a blinded fashion. Data in the left panel are the means and variances of two independent experiments conducted in duplicate; data in the middle and right panels are the means and standard errors of the means of three independent experiments conducted in duplicate. The results comparing the effects of the KN and CA mutants of SGK are statistically significant according to analysis of variance, with a P value of <0.05 (∗) or <0.01 (∗∗). (B) HEK 293T cells were transiently transfected with a vector alone (CTL), a vector encoding CA Akt or SGK (S422D), or a vector encoding KN Akt (K197M) or SGK (T256A/S422A). At 24 h after transfection, cells were either starved or incubated in the presence of 50 ng of IGF-I/ml for 24 h. Similar amounts of total proteins were resolved by SDS-PAGE. Immunoblot analyses were performed using an antibody directed against the cleaved product of PARP, an antibody to a control protein (MKK1), and an antibody to HA.