In this report we describe experiments demonstrating that the PI3K-regulated kinase SGK1 phosphorylates the FOXO transcription factor FKHRL1, thereby inducing the exit of FKHRL1 from the nucleus and the repression of FKHRL1-dependent transcription. Our experiments identify FKHRL1 as a physiological substrate of SGK and suggest a biological role for this protein kinase in the regulation of cell survival and cell cycle progression. Our findings suggest that SGK functions as a critical link between growth factor activation of the PI3K and the regulation of gene expression events that are critical for biological responses such as cell survival and cell cycle reentry.
SGK and Akt may cooperate in promoting a variety of biological responses by phosphorylating common substrates at overlapping but different regulatory sites. SGK and Akt differ with respect to the efficacy with which they phosphorylate specific sites on FKHRL1, since SGK preferentially phosphorylates Ser-315 whereas Akt favors Ser-253. The coordinated phosphorylation of FKHRL1 at three sites by SGK and Akt could involve the sequential phosphorylation of the different sites of FKHRL1. In a recent study of the FKHRL1 family member FKHR, the site equivalent to FKHRL1 Ser-253 was suggested to act as a “gatekeeper,” since the phosphorylation of Ser-253 was found to be required to release a negative constraint on the FKHR molecule so that FKHR could then be phosphorylated at additional sites (37
). Nakae and colleagues suggested that while Akt is responsible for phosphorylating FKHR at Ser-253, other unidentified protein kinases that are distinct from Akt may phosphorylate the two other sites on FKHR (37
). Given the findings in the present study, it is tempting to speculate that Akt may first phosphorylate FKHRL1 at Ser-253 and that this phosphorylation event may induce a conformational change in FKHRL1 that allows SGK to phosphorylate FKHRL1 at Ser-315. Alternatively, the phosphorylation of FKHRL1 at Ser-253 by Akt may allow the binding to FKHRL1 of 14-3-3 proteins, which then act as cofactors that promote the phosphorylation of FKHRL1 by SGK. A role for 14-3-3 proteins as cofactors that promote sequential protein phosphorylation was recently demonstrated for another Akt target, the Bcl-2 family member BAD (18
The efficient phosphorylation of the three regulatory sites of FKHRL1 appears to be required to ensure the effective translocation of FKHRL1 from the nucleus to the cytoplasm and the effective repression of FKHRL1-dependent transcription. The phosphorylation of FKHRL1 at each of the regulatory sites may involve different mechanisms that cooperate to promote the efficient exclusion of FKHRL1 from the nucleus. FKHRL1 Ser-315, which is the phosphorylation site favored by SGK, is located close to the nuclear export signal (NES) of FOXOs (6
). One possibility is that SGK phosphorylation of Ser-315 unmasks the NES of FKHRL1, thereby rendering the NES accessible to the nuclear export machinery. Thus, SGK phosphorylation of Ser-315 may play a primary role in exporting nuclear FKHRL1 to the cytoplasm upon activation of the PI3K-SGK signaling pathway. The phosphorylation of the other two sites on FKHRL1, Thr-32 and Ser-253, is catalyzed more effectively by Akt and is required for FKHRL1 binding to 14-3-3 proteins (7
). When bound to FKHRL1, 14-3-3 proteins may participate in the nuclear export of FKHRL1, since the 14-3-3 proteins have been shown to contain a NES and to be involved in the coexport of their binding partners (33
). 14-3-3 protein binding to FKHRL1 may also ensure the retention of FKHRL1 in the cytoplasm. As FKHRL1 Ser-253 is located within the nuclear localization signal of FKHRL1, Akt phosphorylation of Ser-253 may also inhibit the function of the nuclear localization signal of FKHRL1, thereby preventing the reentry of cytoplasmic FKHRL1 into the nucleus.
The regulation of FKHRL1 subcellular localization by phosphorylation is reminiscent of the regulation of the yeast transcription factor Pho4 (27
). Pho4 is phosphorylated at several regulatory sites by different kinases and the multiple phosphorylation events cooperate to promote the nuclear export of Pho4 and to prevent the nuclear import of Pho4, thus resulting in the exclusion of this transcription factor from the nucleus (29
). The sequential phosphorylation of transcription factors, such as Pho4 or FKHRL1, at several sites that have overlapping but distinct roles in regulating nuclear export may therefore provide a general and efficient mechanism that ensures the effective translocation of these transcription factors from the nucleus to the cytoplasm.
The primary consequence of FKHRL1 phosphorylation at its regulatory sites appears to be the translocation of FKHRL1 from the nucleus to the cytoplasm and the inhibition of FKHRL1-dependent transcription. However, a question that remains to be answered is that of whether there are kinase-dependent mechanisms in addition to the regulation of the subcellular localization of FKHRL1 that contribute to the control of FKHRL1 function. Given that Ser-315, the amino acid residue that is preferentially phosphorylated by SGK, is located within the transactivation domain of FKHRL1, it is possible that the phosphorylation of Ser-315 may also regulate the transactivation function of FKHLR1. In addition, phosphorylation of FKHRL1 at different phosphorylation sites may facilitate FKHRL1 binding to distinct protein partners that have yet to be identified but could prove to be important for FKHRL1 function.
It is not yet clear why there are two families of PI3K-regulated kinases that phosphorylate FKHRL1. One possibility is that when expressed at physiological levels, SGK and Akt display a strict preference for one FKHRL1 site over the others, so that SGK exclusively phosphorylates FKHRL1 at Ser-315 while Akt selectively phosphorylates FKHRL1 at Ser-253. This is consistent with the observation that when expressed at low levels, SGK selectively phosphorylated Ser-315 while Akt was more effective at phosphorylating Ser-253. Another possibility is suggested by the observation that Akt and SGK are differentially expressed in cells in response to environmental stimuli. Whereas Akt is activated within seconds upon growth factor addition and may be involved in the phosphorylation of FKHRL1 at early times after growth factor stimulation, SGK protein is present only at very low levels in cells in the absence of growth factor stimulation (8
). The expression of SGK is induced within hours of exposure of the cells to growth factors (8
). Thus, active SGK could phosphorylate FKHRL1 at later times after growth factor stimulation and may prolong the effects of growth factor stimulation on gene expression.
The overexpression of KN mutants of SGK or Akt in cells reduces the phosphorylation of FKHRL1 and inhibits FKHRL1 cytoplasmic relocalization in response to growth factors, suggesting that endogenous SGK and Akt may both be required for the phosphorylation of FKHRL1 and for FKHRL1 cytoplasmic retention. In addition, overexpression of KN mutants of SGK or Akt in cells promotes apoptosis, raising the possibility that endogenous SGK and Akt may both play an important role in mediating PI3K-dependent cell survival. However, a potential limitation to the use of the dominant-interfering SGK and Akt mutants to selectively inhibit their endogenous counterparts is that SGK and Akt are both activated by the same upstream kinase, PDK1 (28
). Thus, the expression of high levels of dominant-interfering mutants of SGK may lead to inhibition of PDK1 and consequently may prevent the activation of both SGK and Akt (48
). Conversely, it is possible that the overexpression of dominant-negative mutants of Akt may interfere with endogenous SGK function. Therefore, many of the data that had been generated in the past few years using dominant-negative mutants of Akt to block endogenous Akt function may have to be reexamined, as the results obtained may reflect the inhibition of both endogenous Akt and SGK. The identification of chemical inhibitors that specifically block SGK or Akt will be particularly useful for resolving this issue.
In our present study, we have defined a new function for SGK in mediating survival in response to survival factors. It will be important to determine whether the survival function of SGK can be generalized to cells that have been challenged with a range of apoptotic stimuli, such as DNA damage, matrix detachment, or cell cycle discordance. The transcription factor FKHRL1 is also likely just one of a number of SGK substrates that mediate the survival function of SGK. Other previously characterized targets of Akt that had been implicated in the regulation of cell survival, such as BAD and caspase 9 (9
), may also be found to be phosphorylated and regulated by SGK.
The physiological roles of SGK are not likely to be restricted to the promotion of cell survival. Previous studies have indicated that SGK is present in the nucleus during the S phase of the cell cycle, suggesting that SGK plays an active role in cell cycle progression (8
). Importantly, FOXO transcription factors have recently been shown to promote cell cycle arrest by inducing the expression of the cell cycle inhibitor p27KIP1 (36
). Consistent with a role for SGK phosphorylation of FKHRL1 in cell cycle progression, we find that mutating any one of the three phosphorylation sites of FKHRL1 generates a form of FKHRL1 that promotes cell cycle arrest. These results suggest that SGK and Akt may together promote cell cycle progression by phosphorylating and inactivating FOXOs, thereby decreasing p27KIP1 levels.
Given the importance of SGK and Akt for cell survival and proliferation, mutations in these kinases are likely to have an impact on cancer development in vertebrates. CA versions of Akt and PI3K are transduced by oncogenic retroviruses and can trigger cell transformation (5
). It will be interesting to determine whether the CA versions of SGK will also be found to be tumorigenic. The ability of CA forms of Akt and SGK to induce cell transformation may be a consequence of the inappropriate suppression of FOXO-dependent transcription, due to SGK and/or Akt phosphorylation of FOXO transcription factors. One attractive model is that when cells are exposed to deleterious conditions that inhibit PI3K signaling, FOXOs will translocate to the nucleus and induce the expression of cell death or cell cycle arrest genes. CA SGK and Akt may shut off the expression of these cell death and arrest genes by excluding FOXOs from the nucleus, thereby triggering abnormal proliferation and survival and ultimately cell transformation.
Members of the PI3K pathway are conserved throughout evolution. In particular, SGK homologues are present in yeast, nematodes, flies, and mammals. In the nematode Caenorhabditis elegans
, the PI3K-Akt pathway plays an important role in the regulation of insulin metabolism and organismal aging by suppressing the activity of a Forkhead transcription factor termed Daf16 (39
). Since in addition to Akt, there exists an SGK counterpart in the nematode, it will be interesting to determine whether the nematode SGK contributes to the suppression of Daf16 function and participates in the control of insulin metabolism and organismal aging.
In summary, the protein kinase SGK appears to integrate the effects of different extracellular stimuli and to regulate a variety of biological functions by phosphorylating key substrates including the FOXO transcription factor FKHRL1. The identification of the array of biological functions and substrates of SGK will be of critical importance in understanding how the PI3K signaling pathway orchestrates cellular responses to various extracellular cues.