SGK1 Phosphorylation of IκB Kinase α and p300 Up-regulates NF-κB Activity and Increases N-Methyl-D-aspartate Receptor NR2A and NR2B Expression*

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IκB kinase α (IKKα) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKα at Thr-23 and indirectly activates IKKα at Ser-180. Furthermore, SGK1 enhanced nuclear factor κB (NF-κB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKα at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKβ, IKKβ activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-κB. In addition, SGK1 increased the acetylation of NF-κB through phosphorylation of p300 at Ser-1834, and this also leads to NF-κB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKα and NF-κB phosphorylation as well as NF-κB acetylation and NF-κB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKα-NF-κB signaling pathway, we found that transfection of the IKKα double mutant (IKKαT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

Serum and glucocorticoid-inducible kinase 1 (SGK1) 2 is a member of the serine/threonine protein kinase family that is transcriptionally induced by serum and glucocorticoids (1). SGK1 is known to regulate a variety of cellular functions, including salt homeostasis, ion channel conductance, cell proliferation, and neuronal excitability (2). In addition, SGK1 promotes cell survival and regulates cell cycle progression through phosphorylation of the forkhead transcription factor FKHRL1 (3,4). More recently, SGK1 was found to modulate the excitatory amino acid transporter function through phosphorylation of Nedd4-2 (5). SGK1 is also known to implicate several physiological functions. For example, sgk1 mRNA expression is increased in animal models of Parkinson disease, suggesting a role of SGK1 in neuroprotection (6). SGK1 increases glutamate-induced current partly by increasing GluR6 protein level in plasma membrane of Xenopus oocytes expressing rat GluR6 (7). SGK1 was also found to increase neurite outgrowth in hippocampal neurons (8,9). Moreover, SGK1 facilitates long term potentiation and spatial learning in rats (10).
SGK1 is a downstream target of phosphatidylinositol 3-kinase signaling (11). SGK1 is first phosphorylated at Ser-422 by 3-phosphoinositide-dependent PDK2, which enables SGK1 to be further phosphorylated at Thr-256 by PDK1 (12). In addition, SGK1 also receives upstream signals from cyclic AMP (13), extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) (14), p38 MAPK (15), and big mitogen-activated protein kinase 1 (16). However, with the biological functions and upstream signals of SGK1 characterized to a certain extent, the downstream targets of SGK1 are relatively less known. Because SGK1 phosphorylates substrate proteins that contain the RXRXX(S/T) motif, where X stands for any amino acid (12), in this study we investigated the SGK1 substrate and signaling pathway by using the phospho (p)-motif antibody as a tool. We also examined the expression of genes that are regulated by this signaling pathway. Our results revealed that IB kinase ␣ (IKK␣), but not IKK␤, is a novel substrate of SGK1 and that SGK1 phosphorylation of IKK␣ increases nuclear factor B (NF-B) activity and up-regulates the expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR2A and NR2B. In addition, SGK1 also phosphorylates p300 directly, and SGK1 phosphorylation of p300 increases NF-B activity and NR2A and NR2B expression mutant), IKK␤ mutant constructs (IKK␤S181A, phosphorylation site mutant; IKK␤K44M, kinase-dead mutant), p300 phosphorylation site mutant construct (p300S1834A), p65 acetylation site mutant construct (p65K221R), and IB␣ nondegradable mutant construct (IB␣S32A/S36A) were generated by using the QuickChange site-directed mutagenesis kit (Stratagene).
In some experiments, combined treatment of insulin (100 ng/ml) (Sigma) and SGK1 siRNA (20 pmol) or Akt siRNA (20 pmol) in HEK293T cells was carried out. Silencer Negative Control number 1 siRNA (Ambion, TX) was used as a control. Human SGK1 siRNA was purchased from Invitrogen and was transfected by using the Lipofectamine TM 2000 reagent. In another experiment, combined treatment of insulin-like growth factor 1 (IGF-1) (PeproTech, NJ) and SGK1 siRNA was carried out for NF-B acetylation assay.
In Vitro Kinase Assay and Coupling Kinase Assay-To identify the substrate proteins phosphorylated by SGK1 in rat hippocampus, hippocampal tissue lysate (10 g) was added with different amounts of activated SGK1 protein for kinase reaction. The kinase reaction was carried out in 20 l of reaction buffer added with 1 mM DTT, 100 nM ATP, and 20 -100 ng of activated SGK1 protein (Upstate Biotechnology, Inc.) for 10 min at 30°C. Reaction was stopped by boiling in Laemmli buffer. Proteins were separated by 6 -10% gradient SDS-PAGE and transferred to the PVDF membrane. Proteins were further characterized by immunoblot analysis with Akt-pSub antibody (Cell Signaling). To obtain the FLAG-IKK␣ fusion protein for in vitro kinase assay, HEK293T cells were transfected with FLAG-IKK␣WT or FLAG-IKK␣T23A plasmid. Twenty four hours after transfection, cell lysates prepared from cells grown in full medium or in full medium treated with 50 M LY294002 for 2 h were incubated with FLAG M2-agarose affinity gel (Sigma) at 4°C for overnight. The immune complexes on beads were then washed three times in washing buffer and twice in reaction buffer containing 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , and 0.5 mM EGTA. Kinase reaction was carried out in 20 l of reaction buffer added with 1 mM DTT, 100 nM ATP, and 60 ng of activated SGK1 protein (Upstate Biotechnology, Inc.) for 30 min at 30°C. Reactions were stopped by boiling in Laemmli buffer followed by Western blot analysis with pIKK␣/␤ Thr-23 antibody (Santa Cruz Biotechnology) and FLAG M2 antibody (Sigma) as described above. For His-IKK␣, GST-IKK␤-(1-257) (〈bnova, Taipei, Taiwan), and GST-p300 in vitro kinase assay, kinase reaction was carried out in 20 l of reaction buffer added with 500 ng of purified recombinant protein, 1 mM DTT, 6 Ci of [␥-32 P]ATP (3000 Ci/mmol), and 60 ng of activated SGK1 protein (Upstate Biotechnology, Inc.) for 30 min at 30°C. For the coupling kinase assay, the FLAG-IKK␣ IP product prepared from HEK293T cells was incubated with 60 ng of activated SGK1 protein (Upstate Biotechnology, Inc.) and 6 Ci of [␥-32 P]ATP (3000 Ci/mmol) for 30 min at 30°C as described above. After the first kinase reaction, the FLAG-IKK␣ IP product was washed twice in reaction buffer and was then incubated with 0.5 g of GST-IB␣-(1-54) protein and 6 Ci of [␥-32 P]ATP (3000 Ci/mmol) for 30 min at 30°C. Reaction was stopped by boiling in Laemmli buffer, and proteins were subjected to 8% SDS-PAGE followed by transferring onto the PVDF membrane. The membrane was exposed to x-ray film (Kodak) for visualization of protein bands.
Gene Transfection and Drug Injection to the Brain-Adult male Sprague-Dawley rats (250 -400 g) bred at the Institute of Biomedical Sciences, Academia Sinica, were used. Experimental procedures followed the Guidelines of Animal Use and Care of the National Institute of Health and were approved by the Animal Committee of the Institute of Biomedical Sciences, Academia Sinica. Animals were anesthetized with pentobarbital (40 mg/kg, intraperitoneally) and subjected to stereotaxic surgery. Two 23-gauge, stainless steel, thin wall cannulae were implanted bilaterally to the hippocampal CA1 area at the following coordinates: 3.5 mm posterior to the bregma, 2.5 mm lateral to the midline, and 3.4 mm ventral to the skull surface. After animals recovered from the surgery, the pcDNA3 vector or the constitutively active SGK, SGKS422D, was injected to the CA1 area. Before injection, plasmid DNA was diluted in 5% glucose to a stock concentration of 2.77 g/l. Branched polyethyleneimine of 25 kDa (Sigma) was diluted to 0.1 M concentration in 5% glucose and added to the DNA solution. Immediately before infusion, 0.1 M polyethyleneimine was added to reach a ratio of polyethyleneimine nitrogen per DNA phosphate equal to 10. The mixture was vortexed for 30 s and allowed to equilibrate for 15 min at room temperature (17). The volume for DNA injection was 0.5 l each side at a concentration of 1.2 g/l. The volume for IGF-1 injection was also 0.5 l each side at a concentration of 100 ng/ml. Rat SGK1 siRNA was designed according to a previous report (18). The siRNA sequences of rat SGK1 was synthesized by Ambion with the following sequences: sgk-1 sense oligonucleotide, 5Ј-GUC-CCUCUCAACAAAUCAAtt-3Ј; antisense oligonucleotide, 5Ј-UUGAUUUGUUGAGAGGGACtt-3Ј (8). The siRNA sequences for rat Akt was synthesized by MDBIO (Taipei, Taiwan) with the following sequences: Akt sense oligonucleotide, 5Ј-UGCCCUUCUACAACCAGGAtt-3Ј; antisense oligonucleotide, 5Ј-UCCUGGUUGUAGAAGGGCAtt-3Ј (19). Silencer Negative Control number 1 siRNA (Ambion) was used as a control. SGK1 siRNA and negative control siRNA (8 pmol/l each) were transfected to the hippocampus (0.5 l each side) by using the cationic polymer transfection reagent jetSI TM 10 mM (Polyplus-Transfection). The injection rate was at 0.2 l/min. Animals were sacrificed 48 h after DNA transfection or 30 min after IGF-1 injection. For combined treatment, SGK1 siRNA or Akt siRNA was given 96 h before IGF-1 treatment. The hippocampal CA1 tissue was dissected for Western blot analyses of IKK␣, pIKK␣ Thr-23, SGK1, Akt, or for real time PCR analyses of NMDA receptor subunit expression.
RNA Extraction and Quantitative Real Time PCR Analysis-Total RNA was isolated from 20 mg of hippocampal CA1 tissue or cell lines (ϳ10 6 cells, PC12 or Neuro2A) by using the RNAspin mini kit (GE Healthcare) according to the manufacturer's instructions. The RNA samples were resuspended in nuclease-free water and quantified spectrophotometrically at 260 nm. All RNA samples had an A 260 :A 280 value between 1.8 and 2.0. Total RNA (1 g) together with 0.5 g of oligo(dT) and nuclease-free water was pre-heated at 70°C for 5 min. It was then quickly chilled at 4°C for 5 min. The mixture was then added with 5ϫ reaction buffer, MgCl 2 , and Improm-II reverse transcriptase (Promega) to a total volume of 20 l. The room temperature mixture was incubated at 25°C for 5 min, 42°C for 60 min, and stopped by heating at 70°C for 15 min. The cDNA stock was then stored at Ϫ20°C. Quantitative PCRs for the endogenous control gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) and NR1 were carried out by using the TaqMan Universal PCR master mix (Applied Biosystems); for NR2A and NR2B assays, the Power SYBR Green PCR master mix (Applied Biosystems) was used. The primers and TaqMan probe of HPRT were synthesized by Applied Biosystems with the following sequences: HPRT forward primer 5Ј-GCCGAC-CGGTTCTGTCAT-3Ј and reverse primer 5Ј-TCATAACCT-GGTTCATCATCACTAATC-3Ј; TaqMan probe 5Ј-TCGAC-CCTCAGTCCCAGCGTCG-3Ј. The primers and probe of NR1, the Assays-on-Demand Gene Expression products (Rn00433800_m1, Applied Biosystems) were used. The prim-ers of NR2A and NR2B were designed according to a previous report (20). The primers of NR2A were synthesized with the following sequence: forward primer 5Ј-GACGGTCTTGG-GATCTTAAC-3Ј and reverse primer 5Ј-TGACCATGAATT-GGTGCAGG-3Ј. The primers of NR2B were synthesized with the following sequence: forward primer 5Ј-CAAGAACATG-GCCAACCT-3Ј and reverse primer 5Ј-GGTACACATTGCT-GTCCTGC-3Ј. Amplification was performed by using the 7500 Real Time PCR system (Applied Biosystems), and the reaction condition followed the manufacturer's protocols. The thermal cycler protocol used is as follows: stage 1, 50°C for 2 min; stage 2, 95°C for 10 min; stage 3, 95°C for 15 s, 60°C for 1 min (40 cycles); and stage 4 is the dissociation stage for SYBR Green system to confirm specific amplifications. The cycle threshold (C t ) values and related data were analyzed by using the 7500 System sequence detection software (Applied Biosystems). The expression of NMDA receptors subunits was normalized with that of the endogenous control gene HPRT. The relative expression levels (in fold) were determined by using the 2 Ϫ(⌬⌬Ct) method (21).
No Shift Transcription Factor Assay-A rapid and sensitive way to measure NF-B activity in hippocampal nuclear extract without isotopes or gels was also carried out by using the Noshift transcription factor assay kit with NF-B reagent (Novagen). In this assay, the biotinylated oligonucleotides with NF-B consensus sequence and NF-B in the nuclear extracts form complex first. The NF-B (p65)-specific antibody was then used to detect the NF-B-DNA complex captured on the streptavidin plate. Finally, the appropriate secondary antibody, HRP conjugate, and TMB substrate were used to develop colorimetric signal, and the result was collected by reading the absorbance at 450 nm. Tissue lysate (10 g) from rat dorsal hippocampus was treated with activated SGK1 protein (0, 20, 40, or 100 ng) and ATP (100 nM) for 10 min and subjected to kinase reaction and Western blot (WB) by using the p-motif (p-RXRXX(S/T)) antibody. Protein bands that are recognized by this antibody, and the densities that are increased by activated SGK1 are indicated by the asterisk. Actin was used as an internal control. Experiments are in triplicate.
Spatial Learning-The Morris water maze learning paradigm was used this study. The water maze was a plastic circular pool (2.0 m diameter and 0.6 m height) filled with water (25 Ϯ 2°C) to a depth of 20 cm. A circular platform (13.5 cm diameter) was placed at a specific location away from the edge of the pool. The top of the platform was submerged 1.5 cm below the water surface. Water was made cloudy by the addition of milk powder. Distinctive visual cues were set on the wall.
For spatial learning, animals were subjected to three trials a day with one given early in the morning, one given in the early afternoon, and another given in the late afternoon. The training procedure lasted for 4 days, and a total of 12 trials was given. For these trials, animals were placed at different starting positions spaced equally around the perimeter of the pool in a random order. Animals were given 60 s to find the platform. If an animal could not find the platform, it was guided to the platform. After mounting the platform, animals were allowed to stay there for 20 s. The time that each animal took to reach the platform was recorded as the escape latency. A probe trial of 60 s was given on day 5 to test their memory retention. Animals were placed in the pool with the platform removed, and the time the animals spent in each quadrant (target quadrant, left quadrant, opposite quadrant, and right quadrant) was recorded.

SGK1 Activates NF-B Signaling via IKK␣ and p300
sequence RXRXX(S/T), where X stands for any amino acid, we have used the p-motif antibody that recognizes phosphorylated proteins containing the RXRXX(S/T) motif to identify the substrates phosphorylated by SGK1. Cell lysates from rat hippocampus were added with different amounts of activated SGK1 protein and subjected to kinase reaction and immunoblot analysis. By doing this, several protein bands were identified as shown in Fig. 1. We have further used the protein data base motif search engine Scansite (22) to predict these proteins according to the approximate molecular weight of each band shown in Fig. 1. Based on the result of substrate prediction, the entire predicted candidate proteins were divided into three molecular weight ranges as shown in supplemental Table 1. Among these proteins, GSK-3␤ and FOXO3A are possibly two of the known candidate proteins of SGK1 (3,23), but inclusion of other proteins also at these molecular weights cannot be excluded. Furthermore, other candidate proteins are not identified yet.
SGK1 Directly Phosphorylates IKK␣ at Thr-23 and Enhances the Phosphorylation of IKK␣ at Ser-180-Based on these predictions, we have chosen IKK␣ (molecular weight around 85 kDa, marked with double asterisks in Fig. 1) as the candidate protein for the present study. To confirm the accuracy of substrate prediction and to examine whether IKK␣ phosphorylation at Thr-23 was increased in the kinase reaction, we have added different amounts of activated SGK1 protein to hippocampal lysate and examined the level of pIKK␣Thr-23 by using immunoblot analysis. Because IKK␣ is a known substrate of Akt (24), activated Akt protein was used as a positive control. Results revealed that the level of pIKK␣/␤ Thr-23 was increased in a dose-dependent manner (supplemental Fig.  S1A). Further experiment with FLAG-IKK␣WT and V5-IKK␤WT transfection to HEK293T cells and Western blot showed that pIKK␣/␤ Thr-23 antibody recognizes IKK␣ but not IKK␤ ( Fig. 2A, left). To further distinguish whether the pIKK␣/␤ Thr-23 antibody recognizes the phosphorylated IKK␣ only, recombinant His-tagged IKK␣ protein was incubated with or without activated SGK1 for in vitro kinase assay. Results revealed that this antibody only recognizes the phosphorylated IKK␣ but not the nonphosphorylated IKK␣ ( Fig.  2A, right). These results suggest that IKK␣ may be a downstream target of SGK1.
Next, we examined whether IKK␣ is a direct target of SGK1. Anti-IKK␣ and anti-Thr(P)-256 SGK1 antibodies were used to immunoprecipitate (IP) endogenous IKK␣ and SGK1 from hip-pocampal lysate. Results from co-IP experiment revealed that SGK1 forms a complex with IKK␣ (Fig. 2B). Immunoblotting for SGK1 under pSGK immunoprecipitation was not carried out because it is not distinguishable whether the band is SGK1 or the heavy chain of immunoglobulin. We then examined whether SGK1 phosphorylates IKK␣ in vitro and in vivo. When activated SGK1 was incubated with His-tagged wild-type (WT) and T23A mutant recombinant IKK␣ proteins, only IKK␣WT was phosphorylated by SGK1 but not the T23A mutant (Fig.  2C). Furthermore, when activated SGK1 was incubated with GST-tagged IKK␤, no phosphorylation signal was detected (supplemental Fig. S1B). Western blot showed that Ser-181 of IKK␤ was not phosphorylated by SGK1 either (supplemental Fig. S1B). The dot seen on the gel is still a nonspecific band even when visualized at a higher intensity (supplemental Fig. S2). These results indicated that SGK1 only phosphorylates IKK␣ but not IKK␤. Results from IP kinase assay also revealed that SGK1 phosphorylates IKK␣ at Thr-23 (supplemental Fig. S1C). Further experiments in HEK293T cells revealed that phosphorylation of IKK␣ at Thr-23 and Ser-180 was both increased after co-transfection of the constitutively active SGK1, SGKS422D, and IKK␣ (Fig. 2D). Transfection of the IKK␣T23A mutant seemed to slightly diminish the effect of SGKS422D on Ser-180 phosphorylation, but SGKS422D at the highest concentration (1.2 g) still apparently increased Ser-180 phosphorylation under IKK␣T23A (Fig. 2D). Although SGK1 enhances the phosphorylation of IKK␣ at Ser-180, further in vitro kinase assay revealed that SGK1 does not directly phosphorylate Ser-180 of IKK␣ (supplemental Fig. S1D). In another study, it is shown that SGK1 phosphorylates IKK␤ at Ser-181 (25). Because Ser-181 does not fit into the RXRXX(S/T) motif, we have re-examined this issue by co-transfection of IKK␤ and SGKS422D to HEK293T cells. Results revealed that IKK␤ Ser-181 is not phosphorylated by SGK1 in vivo (supplemental Fig.  S1E). The beads seen with IKK␣WT transfection alone in Fig. 2D and with IKK␤WT transfection alone in supplemental Fig. S1E are probably because of endogenous phosphorylation of IKK␣ and IKK␤ in HEK293 cells by endogenous kinases.
The above results demonstrated that SGK1 phosphorylates IKK␣ in vitro and in vivo, but they do not reveal whether this event also occurs under physiological conditions. This issue was examined here. IGF-1 was shown to induce SGK1 activation (11) and NF-B activation (26) and was used here as an upstream signal of SGK1. Results from Western blot showed that injection of IGF-1 (100 ng/ml) to hippocampal CA1 area  FEBRUARY 13, 2009 • VOLUME 284 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4079 significantly increased IKK␣ phosphorylation at Thr-23 (F 2,9 ϭ 26.44, p Ͻ 0.01, q ϭ 6.91, p Ͻ 0.01) without affecting the IKK␣ protein level (Fig. 2E). To verify that IGF-1-induced phosphorylation of IKK␣ is mediated through SGK1, SGK1 siRNA (4 pmol siRNA group with IGF-1 group) without affecting the IKK␣ protein level (Fig. 2E). The effectiveness of SGK1 siRNA treatment was confirmed by an apparent decrease of SGK1 protein level in the hippocampus (Fig. 2E).

SGK1 Activates NF-B Signaling via IKK␣ and p300
SGK1 Up-regulates NF-B Activity through Phosphorylation of IKK␣-Next, we examined whether SGK1 phosphorylation of IKK␣ regulates NF-B activity. In the coupling kinase assay carried out in HEK293T cells, phosphorylation of IB␣ was increased when IKK␣ was first phosphorylated by SGK1 and then incubated with recombinant IB␣ (Fig. 3A). This result suggests that SGK1 phosphorylates IKK␣ and consequently upregulates IKK complex activity. A light band of pIKK␣ and pIB without activated SGK1 is also seen here (Fig. 3A, 3rd lane). This is probably because of phosphorylation of IKK␣ by endogenous kinases in HEK293T cells, such as SGK1 and Akt. It could also be due to autophosphorylation of these proteins. Because IKK␣ is responsible for the phosphorylation of p65 at Ser-529 and Ser-536, and this phosphorylation is important for the transcriptional activity of NF-B (27), we then examined whether phosphorylation of p65 at Ser-529 and Ser-536 is regulated by SGK1. We also examined whether IB phosphorylation is regulated by SGK1 through IKK␣. Results from Western blot revealed that SGK1 increased the phosphorylation of p65 at both Ser-529 and Ser-536, and this effect is dependent on the activity of IKK␣ (Fig. 3B). In addition, phosphorylation of IKK␣ at Thr-23 and Ser-180 seems equally important in SGK1mediated NF-B phosphorylation at Ser-529, but IKK␣ phosphorylation at Ser-180 seems to play a major role in SGK1mediated NF-B phosphorylation at Ser-536 (Fig. 3B). SGK1 also increased the phosphorylation of IB that is further potentiated by co-transfection of IKK␣. Transfection of IKK␣T23A and IKK␣S180A both diminished the effect of IB phosphorylation induced by SGK1. In particular, IKK␣S180A had a more significant effect. But co-transfection of the IKK␣ double mutant (IKK␣T23A/S180A) and IKK␣ kinase-dead mutant (IKK␣K44M) both completely blocked this effect of SGK1. The IB protein level was not affected by these manipulations (Fig.  3B). Further transfection experiment showed that IKK␤ is also required for the effect of SGK1 on p65 Ser-536 phosphorylation (Fig. 3C). These results together suggest that although SGK1 does not phosphorylate IKK␤, the kinase activity of IKK␤ still has to be present for IKK complex activation. IKK␣ or IKK␤ alone is not sufficient for IKK complex activation. In the NF-B reporter assay, transfection of SGKS422D to PC12 cells markedly increased NF-B activity (one-way ANOVA and Dunnett's t test, tD ϭ 3.25, p Ͻ 0.01), and this effect was further enhanced by IKK␣WT transfection (q ϭ 5.69, p Ͻ 0.01, Newman-Keul's statistics) (Fig. 3D, upper). Transfection of IKK␣T23A only and IKK␣S180A alone both partially blocked the effect of SGKS422DϩIKK␣WT on NF-B activity, but transfection of IKK␣T23A/S180A and IKK␣K44M both abolished the effect of SGKS422D on NF-B activity (q ϭ 3.12, p Ͻ 0.05 and q ϭ 5.55, p Ͻ 0.01) (Fig. 3D, upper). Western blot for anti-HA and anti-FLAG was used for verification of plasmid transfection and expression (Fig. 3D, lower). To further confirm the effect of SGK1 on NF-B activation, we have performed an electrophoretic mobility shift assay experiment, and the result consistently showed that SGKS422D increased NF-B activity (supplemental Fig. S3). But for the purpose of quantification, we have used the luciferase reporter assay for the following experiments. This result suggests that IKK␣ phosphorylation at Thr-23 and Ser-180 plays a role in NF-B activation. Similar results were observed when SGKS422D, IKK␤WT, IKK␤S181A mutant, and IKK␤ kinase-dead mutant were transfected to PC12 cells (Fig. 3E). Transfection of SGKS422D to PC12 cells markedly increased NF-B activity (one-way ANOVA and Dunnett's t test, tD ϭ 4.44, p Ͻ 0.05), and this effect was further enhanced by IKK␤WT transfection (q ϭ 8.93, p Ͻ 0.001) (Fig.  3E, upper). Although transfection of IKK␤S181A only partially blocked the effect of SGKS422DϩIKK␤WT on NF-B activity, IKK␤ kinase-dead mutant (IKK␤K44M) completely abolished the effect of SGKS422D on NF-B activity (q ϭ 8.543, p Ͻ 0.001 and q ϭ 10.482, p Ͻ 0.001) (Fig. 3E, upper). Similarly, Western blot for anti-HA and anti-V5 was used for verification of plasmid transfection and expression (Fig. 3E, lower). This latter result supported the notion that IKK␤ kinase activity is also required for SGK1 activation of NF-B, and these results together suggest that phosphorylation of IKK␣ at Thr-23/Ser-180, IKK␣, and IKK␤ activity are all required for SGK1-mediated NF-B activation.
Next, we examined whether SGK1 is involved in NF-B activation under physiological conditions. Insulin was shown to activate both SGK1 (11) and NF-B (28) and was used here to examine this issue. Because protein kinase Akt was shown to share 50% homology to the catalytic domain of SGK1 (23), and it also up-regulates NF-B activity (29), Akt was used as a positive control here. Results from Western blot indicated that insulin treatment (100 ng/ml) to HEK293T cells increased IKK␣ phosphorylation at Thr-23 and NF-B phosphorylation at Ser-536 without affecting their protein levels (Fig. 3F). But SGK1 siRNA pretreatment (20 pmol) and Akt siRNA pretreatment (20 pmol) both blocked these effects of insulin (Fig. 3F).  6 g) 48 h before cell extraction, and cell extracts (500 g) were prepared for p65 IP assays and Western blot assay by using the acetylated lysine antibody. B, HA-SGK (1.2 g) and FLAG-p300 (0.4 g) was co-transfected to HEK293T cells 48 h before cell extraction, and the cell extracts (500 g) were subjected to co-IP assay and Western blot against FLAG-p300 and HA-SGK. C, protein extract from hippocampal tissue (1 mg) for co-IP assay was prepared from animals receiving IGF-1 injection (100 ng/ml) to the hippocampus and sacrificed 30 min later. Co-IP of SGK1 and p300 in hippocampal lysate (left) and the association is increased upon IGF-1 injection (100 ng/ml) to the hippocampus (right). D, GST-tagged p300 and GST-tagged p300S1834A fusion proteins were incubated with activated SGK1 (60 ng) and 6 Ci of [␥-32 P]ATP for 30 min for kinase reaction and Western blot by using the GST antibody. E, FLAG-p300WT (0.4 g) or FLAG-p300S1834A (0.4 g) alone or in combination with HA-SGKS422D (1.2 g) was transfected to PC12 cells 48 h before NF-B reporter assay. F, different combination of plasmids FLAG-p65 (0.4 g) alone or in combination with HA-SGKS422D (0.8 g) and FLAG-p300WT (0.4 g) or FLAG-p300S1834A (0.4 g) was transfected to HEK293T cells 48 h before protein extraction for IP-Western against FLAG and acetylated p65. G, SGK1 siRNA (50 pmol and 150 pmol) was transfected to HEK293T cells (on a 6-well plate) 48 h before insulin treatment (100 ng/ml), and NF-B acetylation assay was performed 30 min after insulin treatment. H, various combinations of IKK␣, IKK␣T23A, SGKS422D, p300, and p300S1834A plasmid was transfected to HEK293T cells to examine whether SGK1 phosphorylation of IKK␣ Thr-23 is dependent on a prior SGK1 phosphorylation of p300 (upper) and vice versa (lower). Experiments are in duplicate or triplicate. Data and statistical significance are expressed as in Fig. 3. Data are means Ϯ S.E. ##, p Ͻ 0.01 compared with the control group; *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001. FEBRUARY 13, 2009 • VOLUME 284 • NUMBER 7

SGK1 Activates NF-B Signaling via IKK␣ and p300
The effectiveness of SGK1 siRNA and Akt siRNA treatments was confirmed by an apparent reduction of SGK1 and Akt protein level in HEK293T cells, respectively (Fig. 3F).
We next examined whether SGK1 mediates the effect of IGF-1 on NF-B activation. Akt was also used as a positive control. Results revealed that IGF-1 treatment (100 ng/ml) significantly increased NF-B promoter activity in PC12 cells (F 3,12 ) ϭ 8.51, p Ͻ 0.01, q ϭ 9.41, p Ͻ 0.01). But transfection of SGK1 siRNA (20 pmol) and Akt siRNA (20 pmol) both completely antagonized the effect IGF-1 on NF-B promoter activity (q ϭ 8.75, p Ͻ 0.01, and q ϭ 13.9, p Ͻ 0.01, respectively) (Fig.  3G). These results together with results from Fig. 3F suggest that although both SGK1 and Akt mediate IKK␣ phosphorylation and NF-B activation; however, blockade of either SGK1 signaling or Akt signaling would prevent NF-B activation resulted from upstream stimulation.
SGK1 Promotes NF-B Acetylation through Phosphorylation of p300-Because phosphorylation and acetylation of NF-B are both important for NF-B DNA binding activity (30), and p300 activation enhances NF-B p65 acetylation (27), in this experiment we examined whether SGK1 enhances NF-B activity through the mediation of p300. We first found that SGKS422D transfection to HEK293T cells increased the acetylation level of NF-B (Fig. 4A). Because p300 contains one RXRXX(S/T) motif ( 1829 RrRmaSm 1835 ) that could be phosphorylated by SGK1, we then examined whether SGK1 may enhance NF-B acetylation through phosphorylation of p300. Co-IP experiments of overexpression of HA-SGK1 and FLAG-p300 revealed that p300 forms a complex with SGK1 in HEK293T cells (Fig. 4B). Further co-IP experiments from hippocampal tissue lysate showed that SGK1 is also associated with p300 in the hippocampus (Fig. 4C, left), and the association between SGK1 and p300 in the hippocampus is increased upon IGF-1 injection to hippocampal neurons (Fig. 4C, right). This result suggests that the association between SGK1 and p300 is up-regulated by IGF-1 physiologically. Moreover, results from in vitro kinase assay revealed that p300 is phosphorylated directly by SGK1 at Ser-1834 (Fig. 4D). Furthermore, SGKS422D and p300 both increased NF-B activity, and SGKS422D and p300 also cooperated to up-regulate NF-B activity in the reporter assay in PC12 cells (p Ͻ 0.01) (Fig. 4E). But transfection of the p300 mutant (p300S1834A) antagonized the effect of SGKS422D on NF-B activity (q ϭ 4.1, p Ͻ 0.05) (Fig. 4E). This latter result suggests that SGK1 may also regulate the acetyltransferase activity of p300. To test this hypothesis, we have transfected SGKS422D and p300 to HEK293T cells and examined the acetylation level of p65. Results revealed that p65 acetylation was increased upon transfection of p300 (q ϭ 7.01, p Ͻ 0.01). This effect was further potentiated by SGKS422D co-transfection (q ϭ 15.39, p Ͻ 0.001) but was antagonized by co-transfection of the p300 mutant (p300S1834A) with or without SGKS422D co-transfection (q ϭ 0.11 and 1.4, both p Ͼ 0.05) (Fig. 4F). IGF-1 was found to increase NF-B promoter activity (Fig. 3G), and here we examined whether insulin also increases NF-B acetylation and whether this is mediated through SGK1. Results revealed that insulin (100 ng/ml) treatment to HEK293T cells apparently increased p65 acetylation, but this effect was blocked by SGK1 siRNA pretreatment in a dose-dependent manner (Fig. 4G). SGK1 siRNA alone at a higher concentration (150 pmol) also decreased p65 acetylation (Fig. 4G). The p65 protein level was not altered by these treatments. On the other hand, insulin did not apparently increase p300 phosphorylation at Ser-1834, but SGK1 siRNA treatment decreased p300 phosphorylation (Fig. 4G). This latter result suggests that a mechanism other than SGK1 is involved in the action of insulin on p300 phosphorylation. Finally, we examined whether SGK1 phosphorylation of IKK␣ Thr-23 depends on a prior phosphorylation of p300 by SGK1 and vice versa. Results from a co-transfection experiment in HEK293T cells revealed that SGKS422D consistently increased IKK␣ phosphorylation at Thr-23, but co-transfection of p300S1834A did not alter this effect of SGKS422D (Fig. 4H, upper). Likewise, SGKS422D increased the phosphorylation of p300 at Ser-1834, but transfection of IKK␣T23A did not alter this effect of SGKS422D (Fig. 4H, lower, 2nd and 4th lanes). Together with a previous result that SGK1 activation of IKK␣ at Ser-180 is independent of SGK1 phosphorylation of IKK␣ at Thr-23 (Fig. 2D), these results suggest that SGK1 activation of IKK␣ at Thr-23, Ser-180, and SGK1 activation of p300 are independent of each other.
SGK1 Up-regulates NMDA Receptor NR2A and NR2B Expression through IKK␣, p300, and NF-B Mediation-After identification of SGK1-IKK␣-NF-B signaling and SGK1-p300-NF-B signaling, we next examined gene expressions that are regulated by these signaling pathways. Because SGK1 (10), NF-B (31), and the NMDA receptors (32) all play an important role in learning and memory function, and the promoters of NMDA receptor NR1 and NR2A may contain the NF-B binding elements based on an earlier study of the NR1 promoter (33) and the transcription element search system (Fig. 5A), we therefore examined whether these signaling pathways may regulate NMDA receptor subunit expression. SGKS422D was first transfected to rat hippocampal neurons, and the expression of different NMDA receptor subtypes was examined by real time PCR. Results revealed that SGKS422D transfection markedly increased the expression of NR1, NR2A, and NR2B (t 1,8 ϭ 4.839, 4.855, and 4.753, respectively; all p Ͻ 0.001) (Fig. 5B, left). Immunoprecipitation of HA followed by immunoblotting with anti-SGK1 antibody confirmed the expression of SGKS422D in hippocampal neurons (Fig. 5B, right). Different cell lines were then used for the following experiments for gene expression analyses because NR1, NR2A, and NR2B are differentially expressed in different cells. Results obtained in PC12 cells and Neuro2A cells revealed that SGKS422D increased the expression of NR1 (Fig. 5C), NR2A, and NR2B (Fig. 5D). But this effect is blocked by transfection of the kinase-dead SGK1, SGKK127M (p Ͼ 0.05 compared with controls) (Fig. 5, C and  D). To further examine the role of SGK1 on NR1, NR2A, and NR2B expression, we have transfected SGK1 siRNA (20 pmol) to PC12 cells (for NR1) and Neuro2A cells (for NR2A and NR2B) and examined NR1, NR2A, and NR2B mRNA level by using real time PCR. Results showed that SGK1 siRNA significantly decreased the mRNA level of NR1, NR2A, and NR2B (t 1,8 ϭ 4.23, 3.99, and 5.56, respectively, all p Ͻ 0.01) (Fig. 5E). We further examined whether SGK1 siRNA affects NR1, NR2A, and NR2B protein level in the hippocampus. SGK1

SGK1 Activates NF-B Signaling via IKK␣ and p300
siRNA (4 pmol) was transfected to hippocampal CA1 area, and Western blot was carried out. Results showed that SGK1 siRNA apparently decreased NR1, NR2A, and NR2B protein level in the hippocampus (Fig. 5F). The effectiveness of SGK1 siRNA transfection was confirmed by decreased SGK1 protein level. We also examined the effect of Akt siRNA on NR1, NR2A, and NR2B mRNA expression. Results showed that Akt siRNA (20 pmol) had a marginal effect on NR1 mRNA expression (t 1,10 ϭ 1.84 and 1.14, both p Ͼ 0.05), and it only significantly decreased NR2B mRNA expression (t 1,10 ϭ 3.53, p Ͻ 0.01) (Fig. 5G). Akt siRNA at a higher concentration (50 pmol) did not further decrease NR2A and NR2B mRNA levels (supplemental Fig.  S4). We then examined whether IGF-1 may up-regulate NR1, NR2A, and NR2B mRNA expression, and whether this effect is mediated through SGK1. Results revealed that IGF-1 (100 ng/ml) administration to PC12 cells and Neuro2A cells did not alter NR1, NR2A, and NR2B mRNA expression at all (all p Ͼ 0.05; Fig. 5H). Therefore, we did not perform a further SGK1 siRNA and IGF-1 interaction study.
Next, we examined whether SGK1 regulates NR1, NR2A, and NR2B expression through NF-B. SGKS422D was transfected to PC12 cells and Neuro2A cells with or without SN50, an NF-B inhibitor. Results revealed that SN50 did not alter the effect of SGKS422D on NR1 (Fig. 6A), but it blocked the effect of SGKS422D on NR2A and NR2B expression (Fig. 6B). The lack of an effect of SN50 in blocking SGKS422D effect on NR1 expression could be due to the possibility that the NF-B-binding site on NR1 promoter is not functionally activated. We next examined whether NR2A and NR2B expression is also regulated by SGK1 phosphorylation of IKK␣ and p300. Various combinations of SGKS422D and IKK␣ mutant plasmids were transfected to Neuro2A cells, and the expression of NR2A and NR2B was exam-FIGURE 5. SGK1 increases NMDA receptor NR1, NR2A, and NR2B expression. A, analyses of the promoter sequence of the NR1 and NR2A for NF-B-binding element. Numbers indicate positions of residues relative to the transcription start codon of each gene. B, quantitative real time PCR was performed to analyze mRNA expression of various NMDA receptor subunits (NR1, NR2A, and NR2B) 48 h after SGKS422D (0.75 g) transfection to hippocampal CA1 area (left). Immunoprecipitation of HA followed by Western blot against SGK1 was performed for verification of SGKS422D transfection and expression in the hippocampus (right). C, different concentrations of SGKS422D (0, 0.4, 0.8, or 1.6 g), ⌬N-SGKS422D (1.6 g) and SGKK127M (1.6 g) was transfected to PC12 cells 48 h before RNA extraction. Real time PCR was performed to determine NR1 mRNA level. D, SGKS422D (0, 0.4, or 1.6 g), ⌬N-SGKS422D (1.6 g), and SGKK127M (1.6 g) were transfected to Neuro2A cells 48 h before RNA extraction. Real time PCR was performed to determine NR2A and NR2B mRNA levels. E, SGK1 siRNA (20 pmol) was transfected to PC12 cells (for NR1) or Neuro2A cells (for NR2A and NR2B), and quantitative real time PCR was performed 48 h later for NR1, NR2A, and NR2B mRNA expression. F, same treatment was given to rat hippocampus, and Western blot was conducted for determination of NR1, NR2A, and NR2B protein levels. Western blot for SGK1 was also carried out to confirm the effectiveness of SGK1 siRNA transfection. G, Akt siRNA (20 pmol) was transfected to PC12 cells (for NR1) or Neuro2A cells (for NR2A and NR2B), and quantitative real time PCR was performed 48 h later for NR1, NR2A, and NR2B mRNA expression. H, IGF-1 (100 ng/ml) was given to PC12 cells, and Neuro2A cells and real time PCR was performed for NR1, NR2A, and NR2B mRNA determination 30 min later. Experiments are in duplicate or triplicate. Data and statistical significance are expressed as in Fig. 3. Data are means Ϯ S.E. ***, p Ͻ 0.001; #, p Ͻ 0.05; ##, p Ͻ 0.01; ###, p Ͻ 0.001 compared with the control group. FEBRUARY 13, 2009 • VOLUME 284 • NUMBER 7 ined by real time PCR. Results revealed that SGKS422D transfection increased the expression of NR2A and NR2B (both p Ͻ 0.01); this effect was partially antagonized by IKK␣T23A and IKK␣S180A transfection, but it was completely antagonized by IKK␣T23A/S180A transfection (p Ͼ 0.05 comparing SGKS422DϩIKK␣T23A/S180A group with SGKS422D group) (Fig.  6C). IKK␣WT transfection alone also increased NR2A and NR2B expression (both p Ͻ 0.05) (Fig. 6C). In another experiment, the relationship between SGKS422D and p300 on NR2A and NR2B expression was examined. Results showed that SGKS422D similarly increased the expression of NR2A and NR2B in Neuro2A cells (p Ͻ 0.001 and p Ͻ 0.05), and this effect was reversed by co-transfection of the p300 mutant (Fig. 6D). Transfection of p300 WT alone also enhanced NR2A and NR2B expression (p Ͻ 0.05 and p Ͻ 0.001) (Fig. 6D). Because p300 interacts with several transcription factors that may lead to down-regulation of NR2A and NR2B expression, to clarify whether SGK1 indeed increases NR2A and NR2B expression through p65 acetylation, the acetylation mutant of p65, p65K221R, was co-transfected with SGKS422D to Neuro2A cells. Results revealed that both SGKS422D and p65 increased NR2A and NR2B expression (both p Ͻ 0.001), but these effects were antagonized by p65K221R co-transfection (p Ͼ 0.05 compared with controls) (Fig. 6E).

SGK1 Activates NF-B Signaling via IKK␣ and p300
SGK1 Enhances NR2A and NR2B Promoter Activity-Because SGK1 increased the expression of NR2A and NR2B through activation of NF-B, there is no known NF-Bbinding site on the NR2B promoter. We have therefore cloned different lengths of the NR2B promoter constructs and examined whether NR2B promoter activity is regulated by SGK1. The rat NR2B promoter constructs are in different lengths (3000 bp, nt ϩ1 to Ϫ3000; 2500 bp, nt Ϫ500 to Ϫ3000; 2000 bp, nt Ϫ1000 to Ϫ3000; 1500 bp, nt Ϫ1480 to FIGURE 6. SGK1 up-regulates NMDA receptor NR2A and NR2B expression through IKK␣, NF-B, and p300 mediation. A, SGKS422D (1.6 g) was transfected to PC12 cells 48 h before RNA extraction, and SN50 (an NF-B inhibitor, 50 g/ml; Calbiochem), alone or in combination with HA-SGKS422D, was added to the same PC12 cells 1 h before RNA extraction. Real time PCR was performed to determine NR1 mRNA level. B, SGKS422D (1.6 g) was transfected to Neuro2A cells 48 h before RNA extraction, and SN50 (50 g/ml), alone or in combination with SGKS422D, was added to the same cells 1 h before RNA extraction. Real time PCR was performed to determine NR2A and NR2B mRNA levels. C, SGKS422D (1. Ϫ3000; 1000 bp, nt Ϫ2020 to Ϫ3000; 500 bp, nt Ϫ2510 to Ϫ3000). Similarly, we have cloned the rat NR2A promoter construct (0.5K, nt ϩ1 to Ϫ500, see Fig. 5A) and the rat NR2A promoter construct with deleted NF-B-binding sites (⌬) to examine whether NR2A promoter activity is regulated by SGK1 through NF-B. These promoter constructs were then transfected to Neuro2A cells alone or in combination with SGKS422D. The results revealed that transfection of the NR2A promoter construct alone increased NR2A promoter activity about 2.8-fold, but this effect was reversed when the NR2A promoter construct containing deleted NF-B-binding sites was transfected (Fig. 7A). Furthermore, co-transfection of SGKS422D and NR2A promoter constructs markedly increased NR2A promoter activity (q ϭ 6.01, p Ͻ 0.001), but this effect was reversed by co-transfection of the NR2A promoter construct with deleted NF-B-binding site NR2A promoter (p Ͼ 0.05 compared with control) (Fig. 7B). We next examined NR2B promoter activity. Different lengths of the NR2B promoter constructs were transfected to Neuro2A cells. Results revealed that when the 1500-bp-long promoter con-struct was transfected, there was a 5.5-fold increase in NR2B promoter activity (Fig. 7C). Other promoter constructs are without any effect (Fig. 7C). These results suggest that the NR2B promoter sequence between nt Ϫ1480 and Ϫ2020 contains NF-B-binding site(s). To further examine whether NR2B promoter activity is regulated by SGK1, we have co-transfected the 1500bp-long NR2B promoter construct with SGKS422D or SGK1 siRNA. Results revealed that SGKS422D transfection significantly increased NR2B promoter activity for about 6-fold (p Ͻ 0.001), but SGK1 siRNA transfection markedly decreased NR2B promoter activity (p Ͻ 0.01) (Fig. 7D).
SGK1 Facilitates Spatial Memory Formation through Phosphorylation of IKK␣-Upon identification of the SGK1-IKK␣-NF-B pathway, we then assessed the functional significance of this pathway. Because SGK1 plays an important role in spatial learning (10) and p65 knockout mice show impaired radial arm maze performance (31), we have used the water maze learning task to examine the possible involvement of this pathway in spatial memory formation. We first found that water maze training significantly increased IKK␣ phosphorylation at Thr-23 and Ser-180 in rat hippocampus (t ϭ 4.85 and 3.93, both p Ͻ 0.01, Student's t test) (Fig. 8, A and B). Water maze training also significantly increased NF-B activity in the hippocampus (t 1,8 ϭ 3.5, p Ͻ 0.05) (Fig. 8C). We then examined whether IKK␣ phosphorylation mediates the effect of SGK1 on spatial memory formation. Results revealed that SGKS422D transfection to hippocampal CA1 area markedly enhanced acquisition performance (F 3,31 ϭ 3.12, p Ͻ 0.05; q ϭ 3.71, p Ͻ 0.05). Transfection of the IKK␣ double mutant alone did not have a significant effect on spatial learning (q ϭ 0.89, p Ͼ 0.05), but it completely antagonized the effect of SGKS422D on spatial learning (q ϭ 3.94, p Ͻ 0.05 comparing S422DϩIKK␣T23A/S180A group with the S422D group) (Fig. 8D, left). Transfection and expression of these plasmids was confirmed by immunoprecipitation and Western blot (Fig. 8D, right). In analyzing the probe trial performance of these animals, we found that SGKS422D significantly increased the time that animals spent in the target quadrant (F 3,31 ϭ 3.02, p Ͻ 0.05, tD ϭ 2.41, p Ͻ 0.05), but this effect was blocked by IKK␣ double mutant cotransfection (tD ϭ 2.92, p Ͻ 0.01) (Fig. 8E). We next examined the effect of knockdown of SGK1 on spatial learning. The effect FIGURE 7. SGK1 increases NR2A and NR2B promoter activity. A, NR2A promoter construct (0.8 g) in the length of 0.5K with or without (⌬) the NF-B-binding site was transfected to Neuro2A cells, and NR2A promoter activity was determined by luciferase reporter assay 48 h after transfection. B, SGKS422D (0.8 g) was cotransfected with the NR2A promoter construct with or without the NF-B-binding site (0.6 g) to Neuro2A cells, and NR2A promoter activity was determined 48 h later by luciferase reporter assay. C, different lengths of the NR2B promoter construct (0.5K, 1.0K, 1K, 1.5K, 2K, 2.5K, and 3K; where K means thousand base pairs of promoter length) (0.8 g each) was transfected to Neuro2A cells, and NR2B promoter activity was determined by luciferase reporter assay 48 h later. D, SGKS422D (0.8 g) or SGK1 siRNA (20 pmol) was co-transfected with the 1.5K length NR2B promoter construct to Neuro2A cells, and NR2B promoter activity was determined 48 h later by luciferase reporter assay. Data and statistical significance are expressed as in Fig. 3. Data are means Ϯ S.E. ##, p Ͻ 0.01; ###, p Ͻ 0.01 compared with the control group. FEBRUARY 13, 2009 • VOLUME 284 • NUMBER 7 of knockdown of Akt was also examined here. Results showed that SGK1 siRNA transfection to CA1 area significantly impaired spatial learning (F 2,24 ϭ 18.15, p Ͻ 0.001; q ϭ 4.44, p Ͻ 0.01), but Akt siRNA markedly enhanced spatial learning (q ϭ 4.73, p Ͻ 0.01) (Fig. 8F). SGK1 siRNA also markedly decreased the time that animals spent in the target quadrant for the probe

DISCUSSION
In this study, we have identified IKK␣ as a novel substrate of SGK1, and SGK1 directly phosphorylates IKK␣ at Thr-23. In another study, IKK␣ phosphorylation at Thr-23 was also shown to be important for NF-B activation after TNF␣ stimulation (24). In addition, we have found that SGK1 also indirectly increased IKK␣ phosphorylation at Ser-180, and that IKK␣ phosphorylation at Ser-180 is equally important as IKK␣ phosphorylation at Thr-23 in SGK1-mediated NF-B phosphorylation, NF-B activation, and NR2A and NR2B expression. Although the kinase that is regulated by SGK1 and phosphorylates IKK␣ at Ser-180 is not known yet, this result is consistent with the report that Ser-180 of IKK␣ is an important residue in regulation of IKK␣ activity (27). In speculation of the kinase that phosphorylates IKK␣ at Ser-180, few candidate proteins could be considered, for example NF-B-inducing kinase and ERK/MAPK kinase kinase (MEKK1), because both proteins are known to phosphorylate IKK␣ at Ser-180 (34,35). In addition to the role of IKK␣ involved in the effect of SGK1 on NF-B activation, our results showed that IKK␣ also mediates the effect of SGK1 on NF-B phosphorylation, whereas NF-B phosphorylation was shown to enhance the acetylation and the transcriptional activity of NF-B (30). On the other hand, our results suggest that SGK1 does not phosphorylate IKK␤, which is inconsistent with the report showing that SGK1 phosphorylation of IKK␤ at Ser-177/Ser-181 mediates the anti-apoptotic effect of SGK1 through NF-B (25). But our results do suggest that IKK␤ kinase activity is still required in mediating the effect of SGK1 on NF-B phosphorylation and activation. These results do not conflict each other because although IKK␤ was considered as the major subunit in the IKK complex to activate the NF-B pathway, IKK␣ was shown to also regulate IKK␤ kinase activity (36). Thus, SGK1 phosphorylation of IKK␣ would also activate IKK␤ indirectly and consequently activate the IKK complex and the NF-B pathway. In addition, although we have presently identified IKK␣ as a substrate protein of SGK1, we cannot exclude the possible involvement of other substrate proteins at a similar molecular weight. For example, the elongation factor eEF-2K has a molecular mass around 82 kDa, and it also contains the RXRXX(S/T) motif ( 361 RvRtlS 366 ). The identification of this protein and perhaps other proteins also as substrate of SGK1 requires further investigation.
In addition to the identified SGK1-IKK␣-NF-B pathway, we have also found that SGK1 increases the acetylation of NF-B through phosphorylation of p300, and this pathway similarly up-regulates the expression of NR2A and NR2B. The acetylation of NF-B is also important for NF-B activation, and it is different from the typical IKK-mediated NF-B activation. In this study, we have identified Ser-1834 of p300 that is phosphorylated by SGK1, and p300 phosphorylation at Ser-1834 is important for p300 acetyltransferase activity and NF-B acetylation. This result is consistent with another report showing that p300 is phosphorylated by Akt at Ser-1834, and this phosphorylation is essential for p300 histone acetyltransferase activity (37). In another study, Hoberg et al. (38) have found that IKK␣ could enhance the acetylation of RelA/p65 also through p300. But these studies did not address the functional significance of these regulations. In this study, although we have found that SGK1 phosphorylates both IKK␣ and p300, we do not know whether these events occur in the cytoplasm or in the nucleus because total cell lysate was used for the experiments. But in another study, a nuclear role of IKK␣ is identified to be responsible for histone H3 phosphorylation and NF-B activation (36). In addition, the kinase that is regulated by SGK1 and phosphorylates IKK␣ at Ser-180 is not known yet and also needs to be identified.
The glutamate NMDA receptor is known to play an important role in mammalian learning and memory (32). In addition, the NR2A subunit is essential for the induction of long term potentiation (39), and the NR2B transgenic mice show improved long term memory (40). How the NMDA receptor subunit has been regulated is not known. In this study, we have found that SGK1 phosphorylation of IKK␣ and p300 both upregulates NMDA receptor NR2A and NR2B expression through NF-B. Although SGK1 also increases the expression of NR1, another subunit of the NMDA receptor, our results showed that it is not regulated through NF-B. In addition, although the promoter of the NR2B gene does not contain the known NF-B-binding site, results from promoter activity assay revealed that there is probably an NF-B-binding site located within nt Ϫ1480 to Ϫ2020. However, it also seems that the promoter sequence between nt Ϫ1000 and Ϫ1480 contains another binding element that inhibits NR2B gene expression. Future experiments are required to identify the exact location of the NF-B-binding site on the NR2B promoter. Moreover, our result that SGK1 phosphorylation of p300 up-regulates NR2A and NR2B expression is also congruent with the report that p300 mutant mice show impaired memory performance (41).
In examination of the functional significance of the SGK1-IKK␣-NF-B signaling pathway, we have found that both IKK␣ phosphorylation and NF-B activity are increased in animals subjected to water maze training and that IKK␣ double mutant transfection antagonized the facilitating effect of SGK1 on spatial memory formation. These results are consistent with the findings that both SGK1 and NF-B are important for spatial learning (10,31). They are also congruent with the reports that the IB␣ double mutant and p65 knock-out mice both show impaired spatial learning and memory (31,42). On the other hand, Akt was shown to also phosphorylate IKK␣ and p300 (24,37) and up-regulate NF-B activity (29) as does SGK1. It seems that SGK1 or Akt alone is sufficient to activate IKK␣ and NF-B in terms of phosphatidylinositol 3-kinase signaling, but Akt only had a small effect in regulating NR1, NR2A, and NR2B mRNA expression. In addition, Akt siRNA treatment significantly enhanced, rather than impaired, spatial memory formation. One explanation for the opposite effect of Sgk1 siRNA and Akt siRNA on spatial memory is probably because Akt would also activate other signaling pathways that may down-regulate NR1, NR2A, and NR2B expression. Alternatively, Akt may regulate the expression of other genes, and the expression of these genes impairs spatial memory formation. However, the present results are consistent with our earlier finding that Akt transfection to the hippocampus impairs spatial learning in rats (43). The molecular mechanism underlying Akt-mediated spatial memory impairment requires further investigation.
By using an endogenous stimulus of SGK1, we have found that IGF-1 increased IKK␣ phosphorylation and NF-B activity, and these effects are blocked by SGK1 siRNA treatment; yet IGF-1 did not increase NR1, NR2A, and NR2B mRNA expression. The reason for the discrepancy between these results is not known. It is possible that IGF-1 would also activate other signaling pathways that lead to the inhibition of NMDA receptor expression. In future experiments, it is worth studying whether IGF-1 also facilitates spatial memory formation and whether this effect is mediated through SGK1.
In summary, our results together suggest a novel SGK1 signaling pathway that is involved in neuronal plasticity. SGK1 directly phosphorylates IKK␣ at Thr-23 and indirectly activates IKK␣ at Ser-180. SGK1 phosphorylation of IKK␣ results in the phosphorylation and activation of NF-B that consequently up-regulates NR2A and NR2B expression. In addition, SGK1 also phosphorylates p300 and that results in the acetylation, and therefore the activation, of NF-B and enhanced expression of NR2A and NR2B (Fig. 9).