tmpBFD6 TMP

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2008/12/16/M805055200.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 7, pp. 40734089, February 13, 2009
2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
SGK1 Phosphorylation of IB Kinase and p300 Up-regulates

NF-B Activity and Increases N-Methyl-D-aspartate Receptor
NR2A and NR2B Expression*
S
Received for publication, July 2, 2008, and in revised form, November 20, 2008 Published, JBC Papers in Press, December 16, 2008, DOI 10.1074/jbc.M805055200
Derek J. C. Tai, Chia-Chen Su, Yun-Li Ma, and Eminy H. Y. Lee1

From the Graduate Institute of Life Sciences, National Defense Medical Center and Institute of Biomedical Sciences,
Academia Sinica, Taipei 115, Taiwan
Serum and glucocorticoid-inducible kinase 1 (SGK1)2 is a

member of the serine/threonine protein kinase family that is
* This work was supported by Grants NSC96-2321-B-001-001 and NSC972321-B-001-003 from the National Science Council of Taiwan. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1S4 and Table S1.
1
To whom correspondence should be addressed: 128 Academia Rd., Section
2, Nankang, Taipei 115, Taiwan. Tel.: 886-2-2789-9125; Fax: 886-2-27829224; E-mail: eminy@gate.sinica.edu.tw.
2
The abbreviations used are: SGK1, serum-and glucocorticoid-inducible
kinase 1; IKK, IB kinase ; IKK, IB kinase; NF-B, nuclear factor B; IGF-1,
insulin-like growth factor 1; NMDA, N-methyl-D-aspartate; siRNA, small
interfering RNA; MAPK, mitogen-activated protein kinase; ERK, extracellu-
FEBRUARY 13, 2009 VOLUME 284 NUMBER 7
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
lar signal-regulated kinase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene fluoride; HA, hemagglutinin; IP, immunoprecipitation; WT, wild type; GST, glutathione S-transferase; ANOVA,
analysis of variance; HPRT, hypoxanthine-guanine phosphoribosyltransferase; nt, nucleotide.
JOURNAL OF BIOLOGICAL CHEMISTRY
4073
Downloaded from www.jbc.org at LIFE SCIENCE LIBRARY, ACADEMIA SINICA, on January 26, 2011
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 IB kinase (IKK) as a
novel substrate of SGK1 by using biochemical and bioinformatic
approaches. SGK1 directly phosphorylates IKK at Thr-23 and
indirectly activates IKK at Ser-180. Furthermore, SGK1
enhanced nuclear factor B (NF-B) activity and up-regulated
N-methyl-D-aspartate receptor NR2A and NR2B expression
through activation of IKK 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 IKK,
IKK activity is still required for IKK complex activation and for
SGK1 phosphorylation and activation of NF-B. In addition,
SGK1 increased the acetylation of NF-B through phosphorylation of p300 at Ser-1834, and this also leads to NF-B activation
and NR2A and NR2B expression. Moreover, an endogenous
stimulus of SGK1, insulin, increased IKK and NF-B phosphorylation as well as NF-B acetylation and NF-B activity, but
SGK1 small interfering RNA transfection blocked these effects
of insulin. In examination of the functional significance of the
SGK1-IKK-NF-B signaling pathway, we found that transfection of the IKK double mutant (IKKT23A/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.
SGK1 Activates NF-B Signaling via IKK and p300

through enhanced acetylation of NF-B. These results suggest
that SGK1 phosphorylation of IKK and p300 regulates neuronal plasticity.
4074 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 284 NUMBER 7 FEBRUARY 13, 2009
EXPERIMENTAL PROCEDURES
Cell Culture Preparation, DNA Transfection, and Plasmid
ConstructionHEK293T were maintained in Dulbeccos modified Eagles medium containing 10% fetal calf serum. PC12 cells
were maintained in Dulbeccos modified Eagles medium containing 10% equine serum and 5% fetal calf serum and incubated at 37 C in a humidified atmosphere with 5% CO2. Transfection was made by using the LipofectamineTM 2000 reagent
(Invitrogen) in 12-well culture plates according to the manufacturers instructions. For NF-B reporter assay, PC12 cells were
maintained in the medium for 24 h after transfection and then
maintained in serum-free medium for 18 h. After serum starvation, PC12 cells were changed to normal culture medium for
6 h with serum stimulation. Cell lysate was then collected by
using passive lysis buffer (Promega, WI) and was ready for the
luciferase assay. The sgk gene has different isoforms, and this
study we have examined SGK1. For construction of the hemagglutinin (HA) epitope-tagged DNA plasmid (HA-SGK), fulllength SGK1 was cloned by amplifying the rat hippocampal
SGK1 cDNA with primers 5-CGGAATTCACCGTCAAAACCGAGGCTCG-3 and 5-GCTCTAGATCAGAGGAAGGAGTCCATAGG-3. The PCR product was subcloned into the
mammalian expression vector pcDNA3-HA. For the SGK1
deletion construct, N-SGKS422D, the N-terminal 159
amino acids of SGK1 were deleted to reduce its degradation and
increase its constitutive activity. For construction of the FLAGtagged DNA plasmid (FLAG-IKK, FLAG-IB, FLAG-p300,
and FLAG-p65), full-length IKK was cloned by amplifying the
rat hippocampal IKK cDNA with primers 5-GCTCCCGCCCCATGGAGCG-3 and 5-TCGAGTCATTCTGCTAACCAAC-3. The full-length p300 cDNA clone was purchased
from Addgene (Cambridge, MA) and was subcloned by amplifying with primers 5-ATCAAGCTTATGGCCGAGAATGTGGTGG-3 and 5-GGGGAGCTCGCTAGCAAGCTGAGAAAGC-3. The full-length IB was cloned by amplifying the
rat hippocampal IB cDNA with primers 5-ATCAAGCTTAATGTTTCAGCCAGCTGGGC-3 and 5-CGACCTCGAGTTATAACGTCAGACGCTGG-3. The full-length p65
was cloned by amplifying the rat hippocampal p65 cDNA with
primers 5-GGCGGATCCCATGGACCCCGAGAAC-3 and
5-ATCGAATTCTCAGGTCTGGGGACT-3. The PCR product of IKK, IB, p300, and p65 was then subcloned into the
mammalian expression vector pCMVTag2A (Stratagene). For
construction of the V5-tagged DNA plasmid (V5-IKK), fulllength IKK was cloned by amplifying the rat hippocampal
IKK cDNA with primers 5-ATTTGCGGCCGCCACCATGAGCTGGTCACCTTCTCTC-3 and 5-TAATCTAGACTGTCACAGGCCTGCTCCAG-3. The PCR product of IKK
was then subcloned into the mammalian expression vector
pcDNA3.1/V5-His-TOPO (Invitrogen). SGK1 mutant constructs (SGKS422D, constitutively active mutant; SGKK127M,
kinase-dead mutant), IKK mutant constructs (IKK23,
phosphorylation site mutant; IKKS180A, phosphorylation
site mutant; IKKT23A/S180A, IKKK44M, kinase-dead
mutant), IKK mutant constructs (IKKS181A, phosphorylation site mutant; IKKK44M, kinase-dead mutant), p300
phosphorylation site mutant construct (p300S1834A), p65
acetylation site mutant construct (p65K221R), and IB
nondegradable mutant construct (IBS32A/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 LipofectamineTM 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.
Whole-cell Lysate and Hippocampal Lysate Preparation
Animals were killed by decapitation, and their hippocampal
tissue was dissected out. Rat hippocampal tissue was lysed by
brief sonication in lysis buffer containing 50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 2 mM EDTA, 1% IGEPAL CA-630, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 20 g/ml pepstatin A,
20 g/ml leupeptin, 20 g/ml aprotinin, 50 mM NaF, and 1 mM
Na3VO4. The HEK293T cell lysate was prepared in 1 ml of
lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1
mM MgCl2, 1% IGEPAL CA-630, 10% glycerol, 1 mM dithiothreitol (DTT), 50 mM -glycerophosphate, 50 mM NaF, 10
g/ml PMSF, 4 g/ml aprotinin, 4 g/ml leupeptin, and 4
g/ml pepstatin.
Immunoprecipitation (IP) and Western Blot AnalysisFor IP
reaction, the specific primary antibodies were unconjugated to
agarose beads, and rabbit or mouse IgG was used in the control
group. For IP assay of SGK1 and IKK, the clarified hippocampal lysate (1 mg) was immunoprecipitated with 4 l of antiphospho-Thr-256-SGK1 antibody (Upstate Biotechnology,
Inc.) or anti-IKK antibody (M-280, Santa Cruz Biotechnology) at 4 C for 2 h. For IP assay of SGK1 and p300, the clarified
hippocampal lysate (1 mg) was immunoprecipitated with 4 l
of anti-phospho-Thr-256-SGK1 antibody (Upstate Biotechnology, Inc.) or anti-p300 antibody (Upstate Biotechnology, Inc.)
at 4 C overnight. The protein G-agarose beads (100 l, 50%
slurry; GE Healthcare) were added to the IP reaction product to
catch the immune complexes at 4 C for 2 h. The immune complexes on beads were then washed three times with washing
buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM
EDTA, 1% IGEPAL CA-630, 1 mM DTT, 50 mM -glycerophosphate, 50 mM NaF, 10 g/ml PMSF, 4 g/ml aprotinin, 4 g/ml
leupeptin, and 4 g/ml pepstatin and boiled in Laemmli sample
buffer. The immunoprecipitated products were subjected to 8%
SDS-PAGE followed by transferring onto the polyvinylidene
fluoride (PVDF) membrane (Millipore). The membranes were
immunoblotted with anti-phospho-Thr-256 SGK1 antibody,
anti-IKK antibody, and anti-p300 antibody, respectively. For
IP-Western blot assay of acetylated p65, HEK293T cells were
treated with trichostatin A (400 nM; Calbiochem), a histone
deacetylase inhibitor, for 30 min and then added with fresh
medium as a stimulation for 1 h before protein extraction for IP
of p65. For anti-p65, anti-FLAG, and anti-HA IP assay, 500 g
In Vitro Kinase Assay and Coupling Kinase AssayTo 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-IKKWT or FLAG-IKKT23A 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
MgCl2, 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, GSTIKK-(1257) (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 [-32P]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 [-32P]ATP (3000 Ci/mmol) for 30 min at 30 C as
described above. After the first kinase reaction, the FLAGIKK IP product was washed twice in reaction buffer and was
then incubated with 0.5 g of GST-IB-(154) protein and 6
Ci of [-32P]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 BrainAdult
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
4075
of lysate from cell lines or hippocampal tissue was incubated

with 10 l of anti-p65 antibody (C-20, Santa Cruz Biotechnology), 10 l of anti-FLAG M2-agarose affinity gel (50% slurry)
(Sigma), or anti-HA agarose affinity gel (50% slurry) (Sigma) at
4 C for 2 h, respectively. The immune complexes of p65 were
precipitated by using the protein G-agarose beads (50 l, 50%
slurry; GE Healthcare) at 4 C for 2 h. Antibodies used in this
study included Akt-pSub (Cell Signaling), pSGK1 Thr-256,
IKK, IKK (Upstate Biotechnology, Inc.), pIKK/ Thr-23
(Santa Cruz Biotechnology), pIKK/ Ser-180/181 (Cell Signaling), p65 (C-20, Santa Cruz Biotechnology), acetylated
lysine (Cell Signaling), NR1 (Upstate Biotechnology, Inc.),
NR2A (Chemicon), NR2B (Chemicon), V5 (Serotec), FLAG M2
(Sigma), and -actin (Chemicon) antibodies. After washing,
blots were incubated with horseradish peroxidase-conjugated
secondary antibodies (Chemicon) and were exposed to x-ray
film (Eastman Kodak Co.) or LAS-3000 imaging system (Fujifilm, Tokyo, Japan) for visualization of protein bands by
enhanced chemiluminescence (ECL PLUS, GE Healthcare).
The protein bands were quantified by using the NIH Image J
software.
Luciferase Reporter AssayLuciferase assay was performed
in PC12 cells transfected with SGKS422D, IKK constructs,
IKK constructs, p300 constructs, pNF-B-Luc (Stratagene),
phRG-TK (Promega), NR2A promoter construct, NR2B promoter construct, and pGL4.10 (Promega) according to the
manufacturers protocols (Promega). The relative activity was
normalized by Renilla luciferase activity.
GST and His6 Fusion Protein Purification-GST-IB-(154)
For construction of the GST-p300 (1714 1883) plasmid, the
p300 fragment (p300 (1714 1883) containing residue 1829
1835 of the p300 protein) was subcloned into the pGEX-4T-1
vector (pGEX-p300). The mutant p300 plasmid (pGEXp300S1834A) was generated by using the QuickChange sitedirected mutagenesis kit (Stratagene). The GST-IB-(154)
plasmid containing residues 154 of the IB protein was a gift
from Dr. Shu Chien. For construction of the His-IKK, HisIKKT23A, and His-IKKS180A plasmids, full-length IKK
was subcloned into the pRSET-A vector (pRSET-IKK). The
mutant IKK plasmids (pRSET-IKK23 and pRSETIKKS180A) were generated by using the QuickChange sitedirected mutagenesis kit (Stratagene). Bacteria culture (200 ml)
of Rosetta-gami 2(DE3)pLysS (Novagen, WI) transformed with
any of these expression plasmids was grown until A600 0.2 0.3
before isopropyl D-thiogalactopyranoside was added to reach a
final concentration of 1 mM. After further growth for 2 h at
37 C, cells were resuspended in Bugbuster protein extraction
reagent (Novagen) for protein extraction according to the
instruction manual. GST fusion proteins were precipitated with
0.5 ml of 80% glutathione-Sepharose beads (GE Healthcare)
and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl
(pH 8.0), 20% glycerol, 1 mM DTT, 1 mM PMSF, and protease
inhibitors. Various His fusion proteins were purified by using
the nickel-nitrilotriacetic acid His-Bind resin (Novagen). The
eluted fusion proteins were further dialyzed in 1 liter of dialysis
buffer containing 20 mM Tris (pH 8.0), 0.2 mM EDTA, 100 mM
KCl, 20% glycerol, 0.5 mM DTT, and 0.2 mM PMSF.
FIGURE 1. Putative substrate proteins of SGK1. 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.
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 5-GACGGTCTTGGGATCTTAAC-3 and reverse primer 5-TGACCATGAATTGGTGCAGG-3. The primers of NR2B were synthesized with
the following sequence: forward primer 5-CAAGAACATGGCCAACCT-3 and reverse primer 5-GGTACACATTGCTGTCCTGC-3. Amplification was performed by using the 7500
Real Time PCR system (Applied Biosystems), and the reaction
condition followed the manufacturers 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
(Ct) 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(Ct) method (21).
No Shift Transcription Factor AssayA 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.
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, 5-GUCCCUCUCAACAAAUCAAtt-3; antisense oligonucleotide, 5UUGAUUUGUUGAGAGGGACtt-3 (8). The siRNA sequences for rat Akt was synthesized by MDBIO (Taipei,
Taiwan) with the following sequences: Akt sense oligonucleotide, 5-UGCCCUUCUACAACCAGGAtt-3; antisense oligonucleotide, 5-UCCUGGUUGUAGAAGGGCAtt-3 (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
jetSITM 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 (106 cells, PC12 or Neuro2A) by using the
RNAspin mini kit (GE Healthcare) according to the manufacturers instructions. The RNA samples were resuspended in
nuclease-free water and quantified spectrophotometrically at
260 nm. All RNA samples had an A260:A280 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 5 reaction buffer, MgCl2, 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 20 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 5-GCCGACCGGTTCTGTCAT-3 and reverse primer 5-TCATAACCTGGTTCATCATCACTAATC-3; TaqMan probe 5-TCGACCCTCAGTCCCAGCGTCG-3. The primers and probe of
NR1, the Assays-on-Demand Gene Expression products
(Rn00433800_m1, Applied Biosystems) were used. The prim-
genate was centrifuged at 1,000 g for 5 min at 4 C. The

supernatant was removed, and the pellet was resuspended in
Identification of SGK1 Substrate ProteinsBecause SGK1

and Akt both phosphorylate substrates at the consensus
4077
300 400 l of extraction buffer

containing 20 mM HEPES (pH 7.9),
1.5 mM MgCl2, 300 mM NaCl, 0.2
mM EDTA, 20% glycerol, 1 mM
EGTA, 1 mM DTT, 0.5 mM PMSF, 5
g/ml aprotinin, and 2.5 g/ml leupeptin. The suspension was incubated on ice for 30 min for high salt
extraction followed by centrifugation at 15,000 g for 10 min at 4 C.
The resulting supernatant, which
contains DNA-binding proteins,
was carefully removed and stored at
80 C until further use.
Spatial LearningThe 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 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
FIGURE 2. SGK1 phosphorylates IKK but not IKK. A, FLAG-IKKWT (1.6 g) or V5-IKKWT (1.6 g) was days, and a total of 12 trials was
transfected to HEK293T cells 48 h before protein extraction. Western blot with the pIKK/ Thr-23 antibody given. For these trials, animals were
was carried out for recognition of IKK. Western blot with the V5 and FLAG antibodies was also carried out for
verification of transfection (left). His-tagged IKK fusion protein (500 ng) was incubated with or without acti- placed at different starting positions
vated SGK1 (60 ng) and ATP (100 nM) for 30 min for kinase reaction and Western blot. pIKK/ Thr-23 antibody spaced equally around the perimeand His tag antibody were used for Western blot. The pIKK/ Thr-23 antibody recognizes phosphorylated ter of the pool in a random order.
IKK only (right). B, hippocampal tissue lysate (1 mg) immunoprecipitated (IP) with anti-Thr(P)-256-SGK1 antibody or anti-IKK antibody was separated by 8% SDS-PAGE and subjected to Western blot by using the Animals were given 60 s to find the
Thr(P)-256-SGK1 antibody or IKK antibody. C, His-tagged IKKWT and IKKT23A recombinant proteins were platform. If an animal could not find
incubated with activated SGK1 (60 ng) and 6 Ci of [-32P] ATP for 30 min for kinase reaction and Western blot the platform, it was guided to the
by using the anti-His Tag antibody. D, FLAG-IKKWT (0.4 g) or FLAG-IKKT23A (0.4 g) was co-transfected
with HA-SGKS422D (0, 0.4, or 1.2 g) to HEK293T cells 48 h before protein extraction. Western blot was carried platform. After mounting the platout for pIKK Thr-23, HA, actin (upper), and pIKK Ser-180, FLAG (lower). E, IGF-1 (100 ng/ml) was injected to rat form, animals were allowed to stay
hippocampal neurons, and animals were sacrificed 30 min later for Western blot against pIKK Thr-23, IKK,
p422SGK, and SGK1. SGK1 siRNA (4 pmol) was transfected to rat hippocampal neurons 96 h before IGF-1 there for 20 s. The time that each
injection (100 ng/ml), and animals were sacrificed 30 min after IGF-1 injection. Protein extract from hippocam- animal took to reach the platform
pal tissue (20 g) was subjected to Western blot for pIKK, IKK, SGK1, and actin. Experiments are in duplicate. was recorded as the escape latency.
Data are means S.E. ##, p 0.01 compared with the control group; **, p 0.01.
A probe trial of 60 s was given on day
5 to test their memory retention.
Nuclear Extract PreparationBriefly, 90 120 mg of hip- Animals were placed in the pool with the platform removed,
pocampal tissue was homogenized with a Dounce tissue and the time the animals spent in each quadrant (target quadhomogenizer (Wheaton, NJ) in a homogenization buffer (10% rant, left quadrant, opposite quadrant, and right quadrant) was
w/v) containing 20 mM HEPES (pH 7.9), 1 mM MgCl2, 0.5 mM recorded.
EDTA, 1% IGEPAL CA-630, 1 mM EGTA, 1 mM DTT, 0.5 mM
PMSF, 5 g/ml aprotinin, and 2.5 g/ml leupeptin. The homo- RESULTS

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 IKKWT
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 IKKT23A 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 IKKT23A (Fig. 2D). Although SGK1 enhances the
phosphorylation of IKK at Ser-180, further in vitro kinase
assay revealed that SGK1 does not directly phosphorylate Ser180 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 Ser181 is not phosphorylated by SGK1 in vivo (supplemental Fig.
S1E). The beads seen with IKKWT transfection alone in
Fig. 2D and with IKKWT 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
FIGURE 3. SGK1 enhances NF-B activity through IKK and IKK. A, FLAG-IKKWT (1.6 g) was transfected to HEK293T cells 48 h before the coupling kinase
assay. The IP product of FLAG-IKKWT was first incubated with activated SGK1 (60 ng) and 6 Ci of [-32P]ATP for 30 min and then incubated with GST-IB(154) fusion protein (0.5 g) for 30 min. SGK1 increases IKK complex activity. B, FLAG-p65 (0.4 g) was co-transfected with HA-SGKS422D (0.8 g) and
FLAG-IKKWT (0.4 g), FLAG-IKKT23A (0.4 g), FLAG-IKKS180A (0.4 g), FLAG-IKKT23A/S180A (0.4 g), or FLAG-IKKK44M (0.4 g) to HEK293T cells 48 h
before protein extraction. Western blot for Ser(P)-536 p65, Ser(P)-529 p65, pIB, IB, FLAG-IKK, FLAG-65, HA, and FLAG was carried out. C, FLAG-p65 (0.4 g)
was co-transfected with HA-SGKS422D (0.8 g) and V5-IKKWT (0.4 g) or V5-IKKK44M (0.4 g) to HEK293T cells 48 h before protein extraction. Western blot
for Ser(P)-536 p65, V5-IKK, FLAG-p65 and HA was carried out. D, HA-SGKS422D (1.2 g) was co-transfected with FLAG-IKKWT (0.4 g), FLAG-IKKT23A (0.4
g), FLAG-IKKS180A (0.4 g), FLAG-IKKT23A/S180A (0.4 g), or FLAG-IKKK44M (0.4 g) to PC12 cells 48 h before NF-B reporter assay (n 57 each group).
Western blot for HA and FLAG was carried out to confirm transfection and expression. E, HA-SGKS422D (1.2 g) was co-transfected with V5-IKKWT (0.4 g),
V5-IKKS181A (0.4 g), or V5-IKKK44M (0.4 g) to PC12 cells 48 h before NF-B reporter assay (n 57 each group). F, SGK1 siRNA (20 pmol) (left) or Akt siRNA
(20 pmol) (right) was transfected to HEK293T cells 60 h before insulin treatment (100 ng/ml), and protein extraction was performed 30 min after insulin
treatment. Western blot against pIKK Thr-23, IKK, Ser(P)-536 p65, p65, SGK1 (left), or Akt (right) and actin was carried out. G, SGK1 siRNA (20 pmol) and Akt
siRNA (20 pmol) was transfected to PC12 cells 48 h before IGF-1 treatment (100 ng/ml), and NF-B reporter assay was carried out 6 h after IGF-1 treatment.
Experiments are in duplicate or triplicate. Data are mean S.E. *, p 0.05; **, p 0.01; ***, p 0.001; #, p 0.05; ##, p 0.01; ###, p 0.001 compared with
the control group.
4079
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-180Based 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 pIKKThr-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-IKKWT and V5IKKWT 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-

significantly increased IKK phosphorylation at Thr-23 (F2,9
26.44, p 0.01, q 6.91, p 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) was administered to hippocampal CA1 area 96 h before

IGF-1 injection. Results revealed that SGK1 siRNA pretreatment blocked the effect of IGF-1 on IKK phosphorylation at
Thr-23 (q 5.26, p 0.01 when comparing IGF-1SGK1

by IKKWT transfection (q 5.69, p 0.01, Newman-Keuls
statistics) (Fig. 3D, upper). Transfection of IKKT23A only and
IKKS180A alone both partially blocked the effect of
SGKS422DIKKWT on NF-B activity, but transfection of
IKKT23A/S180A and IKKK44M both abolished the effect
of SGKS422D on NF-B activity (q 3.12, p 0.05 and q
5.55, p 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, IKKWT, IKKS181A
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
Dunnetts t test, tD 4.44, p 0.05), and this effect was further
enhanced by IKKWT transfection (q 8.93, p 0.001) (Fig.
3E, upper). Although transfection of IKKS181A only partially
blocked the effect of SGKS422DIKKWT on NF-B activity,
IKK kinase-dead mutant (IKKK44M) completely abolished
the effect of SGKS422D on NF-B activity (q 8.543, p 0.001
and q 10.482, p 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/Ser180, 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).
FIGURE 4. SGK1 promotes NF-B acetylation through phosphorylation of p300. A, HEK293T cells were transfected with pcDNA3 or SGKS422D (1.6 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 [- 32P]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, IKKT23A, 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 S.E. ##, p 0.01 compared with the control group; *, p 0.05; **, p 0.01; and ***, p 0.001.
4081
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 Up-regulates NF-B Activity through Phosphorylation
of IKKNext, 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 IKKT23A
and IKKS180A both diminished the effect of IB phosphorylation induced by SGK1. In particular, IKKS180A had a more
significant effect. But co-transfection of the IKK double
mutant (IKKT23A/S180A) and IKK kinase-dead mutant
(IKKK44M) 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 Dunnetts
t test, tD 3.25, p 0.01), and this effect was further enhanced
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 IKKT23A 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 MediationAfter
identification of SGK1-IKK-NF-B signaling and SGK1p300-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 (t1,8
4.839, 4.855, and 4.753, respectively; all p 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 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
(t1,8 4.23, 3.99, and 5.56, respectively, all p 0.01) (Fig. 5E).
We further examined whether SGK1 siRNA affects NR1,
NR2A, and NR2B protein level in the hippocampus. SGK1
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
(F3,12) 8.51, p 0.01, q 9.41, p 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 8.75, p 0.01, and q 13.9, p 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 p300Because 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 (1829RrRmaSm1835) 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 FLAGp300 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 0.01) (Fig. 4E).
But transfection of the p300 mutant (p300S1834A) antagonized
the effect of SGKS422D on NF-B activity (q 4.1, p 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 7.01,
p 0.01). This effect was further potentiated by SGKS422D
co-transfection (q 15.39, p 0.001) but was antagonized by
co-transfection of the p300 mutant (p300S1834A) with or without SGKS422D co-transfection (q 0.11 and 1.4, both p
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
4083
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 S.E. ***, p 0.001; #, p 0.05; ##, p 0.01; ###, p 0.001 compared with the control group.
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 (t1,10 1.84 and
1.14, both p 0.05), and it only significantly decreased NR2B mRNA
expression (t1,10 3.53, p 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
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 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 (1.2 g), IKKWT (0.4 g), IKKT23A (0.4 g), IKKS180A
(0.4 g), IKK double mutant (0.4 g), and combined SGKS422D various IKK mutant plasmids were transfected to Neuro2A cells 48 h before RNA extraction. Real time PCR was performed to determine NR2A and NR2B
mRNA expressions. D, SGKS422D (1.2 g), p300 WT (0.4 g), p300 S1834A (0.4 g), and the combined treatment of SGKS422Dp300S1834A were transfected to Neuro2A cells 48 h before RNA extraction. Real time PCR
was performed to determine NR2A and NR2B mRNA levels. E, SGKS422D (1.2 g), p65 (0.4 g), the p65 acetylation mutant p65K221R (0.4 g), and combined SGKS422Dp65K221R plasmid were transfected to Neuro2A
cells 48 h before RNA extraction. Real time PCR was performed to determine NR2A and NR2B mRNA levels.
Experiments are in triplicate. Data and statistical significance are expressed as in Fig. 3. Data are means S.E.
#, p 0.05; ###, p 0.001 compared with the control group. **, p 0.01; ***, p 0.001.
ined by real time PCR. Results

revealed that SGKS422D transfection increased the expression of
NR2A and NR2B (both p 0.01);
this effect was partially antagonized
by IKKT23A and IKKS180A
transfection, but it was completely
antagonized by IKKT23A/S180A
transfection (p 0.05 comparing
SGKS422DIKK T23A/S180A
group with SGKS422D group) (Fig.
6C). IKKWT transfection alone
also increased NR2A and NR2B
expression (both p 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 0.001 and p
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
0.05 and p 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 0.001), but these effects were
antagonized by p65K221R co-transfection (p 0.05 compared with controls) (Fig. 6E).
SGK1 Enhances NR2A and NR2B
Promoter ActivityBecause 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 1 to 3000; 2500 bp, nt
500 to 3000; 2000 bp, nt 1000
to 3000; 1500 bp, nt 1480 to
4085
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 1480 and 2020 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 0.001), but SGK1 siRNA
transfection markedly decreased
NR2B promoter activity (p 0.01)
(Fig. 7D).
SGK1 Facilitates Spatial Memory
Formation through Phosphorylation
of IKKUpon 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
FIGURE 7. SGK1 increases NR2A and NR2B promoter activity. A, NR2A promoter construct (0.8 g) in the maze performance (31), we have
length of 0.5K with or without () the NF-B-binding site was transfected to Neuro2A cells, and NR2A promoter used the water maze learning task to
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, examine the possible involvement
and NR2A promoter activity was determined 48 h later by luciferase reporter assay. C, different lengths of the of this pathway in spatial memory
NR2B promoter construct (0.5K, 1.0K, 1K, 1.5K, 2K, 2.5K, and 3K; where K means thousand base pairs of promoter
formation. We first found that
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 water maze training significantly
length NR2B promoter construct to Neuro2A cells, and NR2B promoter activity was determined 48 h later by increased IKK phosphorylation at
luciferase reporter assay. Data and statistical significance are expressed as in Fig. 3. Data are means S.E. ##,
Thr-23 and Ser-180 in rat hipp 0.01; ###, p 0.01 compared with the control group.
pocampus (t 4.85 and 3.93, both
3000; 1000 bp, nt 2020 to 3000; 500 bp, nt 2510 to p 0.01, Students t test) (Fig. 8, A and B). Water maze training
3000). Similarly, we have cloned the rat NR2A promoter con- also significantly increased NF-B activity in the hippocampus
struct (0.5K, nt 1 to 500, see Fig. 5A) and the rat NR2A (t1,8 3.5, p 0.05) (Fig. 8C). We then examined whether
promoter construct with deleted NF-B-binding sites () to IKK phosphorylation mediates the effect of SGK1 on spatial
examine whether NR2A promoter activity is regulated by SGK1 memory formation. Results revealed that SGKS422D transfecthrough NF-B. These promoter constructs were then trans- tion to hippocampal CA1 area markedly enhanced acquisition
fected to Neuro2A cells alone or in combination with performance (F3,31 3.12, p 0.05; q 3.71, p 0.05). TransSGKS422D. The results revealed that transfection of the NR2A fection of the IKK double mutant alone did not have a signifpromoter construct alone increased NR2A promoter activity icant effect on spatial learning (q 0.89, p 0.05), but it comabout 2.8-fold, but this effect was reversed when the NR2A pletely antagonized the effect of SGKS422D on spatial learning
promoter construct containing deleted NF-B-binding sites (q 3.94, p 0.05 comparing S422DIKKT23A/S180A
was transfected (Fig. 7A). Furthermore, co-transfection of group with the S422D group) (Fig. 8D, left). Transfection and
SGKS422D and NR2A promoter constructs markedly in- expression of these plasmids was confirmed by immunoprecreased NR2A promoter activity (q 6.01, p 0.001), but cipitation and Western blot (Fig. 8D, right). In analyzing the
this effect was reversed by co-transfection of the NR2A pro- probe trial performance of these animals, we found that
moter construct with deleted NF-B-binding site NR2A pro- SGKS422D significantly increased the time that animals spent
moter (p 0.05 compared with control) (Fig. 7B). We next in the target quadrant (F3,31 3.02, p 0.05, tD 2.41, p
examined NR2B promoter activity. Different lengths of the 0.05), but this effect was blocked by IKK double mutant coNR2B promoter constructs were transfected to Neuro2A cells. transfection (tD 2.92, p 0.01) (Fig. 8E). We next examined
Results revealed that when the 1500-bp-long promoter con- the effect of knockdown of SGK1 on spatial learning. The effect

of knockdown of Akt was also examined here. Results showed
that SGK1 siRNA transfection to CA1 area significantly
impaired spatial learning (F2,24 18.15, p 0.001; q 4.44, p
0.01), but Akt siRNA markedly enhanced spatial learning (q

4.73, p 0.01) (Fig. 8F). SGK1 siRNA also markedly decreased
the time that animals spent in the target quadrant for the probe

trial test (F2,24 7.11, p 0.01; tD 2.03, p 0.05), but Akt
siRNA significantly increased the time that animals spent in the
target quadrant (tD 2.33, p 0.05) (Fig. 8G). The effectiveness of SGK1 siRNA transfection and Akt siRNA transfection
was confirmed by Western blot (Fig. 8H).
FIGURE 8. SGK1 enhances spatial memory formation through the mediation of IKK. A, naive rats were subjected to water maze training for 12 trials (60
s each trial), and their hippocampal tissue containing the CA1 area was dissected out at the end of training and subjected to Western blot analysis of pIKK
Thr-23, pIKK Ser-180, and IKK. B, quantification of the intensity of the IKK Thr-23 and IKK Ser-180 bands seen in A. **, p 0.01 compared with non-trained
group. C, naive rats were subjected to water maze training for 12 trials, and their hippocampal tissue containing the CA1 area was dissected out at the end of
training and subjected to NF-B activity assay determined by no shift assay. *, p 0.05 compared with non-trained group. D, SGKS422D (0.6 g) was
co-transfected with IKK double mutant (0.6 g) at 48 h before the first trial of the water maze training and at 72 h after the first transfection (twice in all). A total
of 12 trials over 4 training days were given. The escape latency was recorded for the acquisition performance. SGKS422D transfection significantly facilitates acquisition
(p 0.05), but this effect is completely blocked by IKK double mutant co-transfection (p 0.05 compared with control) (left). Immunoprecipitations followed by
immunoblotting experiments were conducted for verification of plasmid transfection and expression in rat hippocampus (right). E, same animals were subjected a
probe trial test (60 s) on day 5, and the time that animals spent in each quadrant was recorded as the retention measure. T, target quadrant; L, left of target quadrant;
R, right of target quadrant; O, opposite of target quadrant. F, SGK1 siRNA (4 pmol) or Akt siRNA (4 pmol) was transfected to rat hippocampal CA1 area at the time points
described in D. Water maze learning was also conducted as that described in D. SGK1 siRNA impairs whereas Akt siRNA facilitates acquisition (both p 0.01). G, same
animals were subjected to the probe trial test as described in E. H, effectiveness of SGK1 siRNA transfection and Akt siRNA transfection were confirmed by
Western blot for SGK1 and Akt. Data are expressed as mean S.E. Statistical significance for acquisition performance was done by two-way ANOVA followed
by post-hoc Newman-Kuels comparison. Statistical significance for comparisons between two groups was evaluated by Students t test, and that for the probe
trial test was performed by one-way ANOVA followed by Dunnetts t test. Data are means S.E. *, p 0.05; **, p 0.01.
4087
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 (361RvRtlS366).
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 1480 to 2020. However, it also seems that
the promoter sequence between nt 1000 and 1480 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 SGK1IKK-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
AcknowledgmentWe thank S. J. Fan for technical help.
REFERENCES
1. Webster, M. K., Goya, L., Ge, Y., Maiyar, A. C., and Firestone, G. L. (1993)
Mol. Cell. Biol. 13, 20312040
2. Lang, F., Bohmer, C., Palmada, M., Seebohm, G., Strutz-Seebohm, N., and
Vallon, V. (2006) Physiol. Rev. 86, 11511178
3. Brunet, A., Park, J., Tran, H., Hu, L. S., Hemmings, B. A., and Greenberg,
M. E. (2001) Mol. Cell. Biol. 21, 952965
4. Leong, M. L., Maiyar, A. C., Kim, B., OKeeffe, B. A., and Firestone, G. L.
(2003) J. Biol. Chem. 278, 58715882
5. Rajamanickam, J., Palmada, M., Lang, F., and Boehmer, C. (2007) J. Neurochem. 102, 858 866
6. Schoenebeck, B., Bader, V., Zhu, X. R., Schmitz, B., Lubbert, H., and
Stichel, C. C. (2005) Mol. Cell. Neurosci. 30, 249 264
7. Strutz-Seebohm, N., Seebohm, G., Shumilina, E., Mack, A. F., Wagner,
H. J., Lampert, A., Grahammer, F., Henke, G., Just, L., Skutella, T., Hollmann, M., and Lang, F. (2005) J. Physiol. (Lond.) 565, 391 401
8. Yang, Y. C., Lin, C. H., and Lee, E. H. (2006) Mol. Cell. Biol. 26, 8357 8370
9. Ma, Y. L., Tsai, M. C., Hsu, W. L., and Lee, E. H. (2006) Learn. Mem. (Cold
Spring Harbor) 13, 114 118
10. Tsai, K. J., Chen, S. K., Ma, Y. L., Hsu, W. L., and Lee, E. H. (2002) Proc.
Natl. Acad. Sci. U. S. A. 99, 3990 3995
11. Park, J., Leong, M. L., Buse, P., Maiyar, A. C., Firestone, G. L., and Hemmings, B. A. (1999) EMBO J. 18, 3024 3033
12. Kobayashi, T., Deak, M., Morrice, N., and Cohen, P. (1999) Biochem. J.
344, 189 197
13. Perrotti, N., He, R. A., Phillips, S. A., Haft, C. R., and Taylor, S. I. (2001)
J. Biol. Chem. 276, 9406 9412
14. Lee, C. T., Tyan, S. W., Ma, Y. L., Tsai, M. C., Yang, Y. C., and Lee, E. H.
(2006) Eur. J. Neurosci. 23, 13111320
15. Meng, F., Yamagiwa, Y., Taffetani, S., Han, J., and Patel, T. (2005) Am. J.
FIGURE 9. A schematic diagram showing that SGK1 phosphorylation of

IKK and p300 both increase NF-B activity and up-regulate NMDA
receptor NR2A and NR2B expression. A stimulus, such as IGF-1 or insulin,
would activate SGK1. SGK1 activation leads to the phosphorylation of IKK at
Thr-23 and Ser-180 that results in the phosphorylation and activation of
NF-B. SGK1 activation also phosphorylates p300 at Ser-1834 that results in
the acetylation and activation of NF-B. NF-B activation resulted from these
signaling events leads to up-regulation of the expression of NMDA receptor
NR2A and NR2B.
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).
30. Chen, L. F., Williams, S. A., Mu, Y., Nakano, H., Duerr, J. M., Buckbinder,
L., and Greene, W. C. (2005) Mol. Cell. Biol. 25, 7966 7975
31. Meffert, M. K., Chang, J. M., Wiltgen, B. J., Fanselow, M. S., and Baltimore,
D. (2003) Nat. Neurosci. 6, 10721078
32. Tsien, J. Z., Huerta, P. T., and Tonegawa, S. (1996) Cell 87, 13271338
33. Liu, A., Hoffman, P. W., Lu, W., and Bai, G. (2004) J. Biol. Chem. 279,
17449 17458
34. Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M.
(1997) Cell 90, 373383
35. Nemoto, S., DiDonato, J. A., and Lin, A. (1998) Mol. Cell. Biol. 18,
7336 7343
36. Yamamoto, Y., Verma, U. N., Prajapati, S., Kwak, Y. T., and Gaynor, R. B.
(2003) Nature 423, 655 659
37. Huang, W. C., and Chen, C. C. (2005) Mol. Cell. Biol. 25, 6592 6602
38. Hoberg, J. E., Popko, A. E., Ramsey, C. S., and Mayo, M. W. (2006) Mol.
Cell. Biol. 26, 457 471
39. Liu, B. H., Wang, X., Ma, Y. X., and Wang, S. (2004) Gene Ther. 11, 52 60
40. Tang, Y. P., Shimizu, E., Dube, G. R., Rampon, C., Kerchner, G. A., Zhuo,
M., Liu, G., and Tsien, J. Z. (1999) Nature 401, 63 69
41. Oliveira, A. M., Wood, M. A., McDonough, C. B., and Abel, T. (2007)
Learn. Mem. (Cold Spring Harbor) 14, 564 572
42. Kaltschmidt, B., Ndiaye, D., Korte, M., Pothion, S., Arbibe, L., Prullage, M.,
Pfeiffer, J., Lindecke, A., Staiger, V., Israel, A., Kaltschmidt, C., and Memet,
S. (2006) Mol. Cell. Biol. 26, 2936 2946
43. Chao, C. C., Ma, Y. L., and Lee, E. H. (2007) J. Neurosci. 27, 6243 6248
4089
Physiol. 289, C971C981

16. Hayashi, M., Tapping, R. I., Chao, T. H., Lo, J. F., King, C. C., Yang, Y., and
Lee, J. D. (2001) J. Biol. Chem. 276, 8631 8634
17. Abdallah, B., Hassan, A., Benoist, C., Goula, D., Behr, J. P., and Demeneix,
B. A. (1996) Hum. Gene Ther. 7, 19471954
18. Wu, W., Chaudhuri, S., Brickley, D. R., Pang, D., Karrison, T., and Conzen,
S. D. (2004) Cancer Res. 64, 17571764
19. Katome, T., Obata, T., Matsushima, R., Masuyama, N., Cantley, L. C.,
Gotoh, Y., Kishi, K., Shiota, H., and Ebina, Y. (2003) J. Biol. Chem. 278,
2831228323
20. Sun, L., Shipley, M. T., and Lidow, M. S. (2000) Synapse 35, 212221
21. Livak, K. J., and Schmittgen, T. D. (2001) Methods (San Diego) 25,
402 408
22. Yaffe, M. B., Leparc, G. G., Lai, J., Obata, T., Volinia, S., and Cantley, L. C.
(2001) Nat. Biotechnol. 19, 348 353
23. Kobayashi, T., and Cohen, P. (1999) Biochem. J. 339, 319 328
24. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and
Donner, D. B. (1999) Nature 401, 82 85
25. Zhang, L., Cui, R., Cheng, X., and Du, J. (2005) Cancer Res. 65, 457 464
26. Chetty, A., Cao, G. J., and Nielsen, H. C. (2006) Pediatr. Res. 60, 389 394
27. Hayden, M. S., and Ghosh, S. (2004) Genes Dev. 18, 21952224
28. Benoliel, A. M., Kahn-Perles, B., Imbert, J., and Verrando, P. (1997) J. Cell
Sci. 110, 2089 2097
29. Kane, L. P., Shapiro, V. S., Stokoe, D., and Weiss, A. (1999) Curr. Biol. 9,
601 604

tmpBFD6 TMP

Transféré par

Informations du document

Description originale:

Titre original

Copyright

Formats disponibles

Partager ce document

Partager ou intégrer le document

Options de partage

Avez-vous trouvé ce document utile ?

Ce contenu est-il inapproprié ?

Droits d'auteur :

Formats disponibles

tmpBFD6 TMP

Transféré par

Droits d'auteur :

Formats disponibles

Supplemental Material can be found at:

SGK1 Phosphorylation of IB Kinase and p300 Up-regulates

Derek J. C. Tai, Chia-Chen Su, Yun-Li Ma, and Eminy H. Y. Lee1

Serum and glucocorticoid-inducible kinase 1 (SGK1)2 is a

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

transcriptionally induced by serum and glucocorticoids (1).

JOURNAL OF BIOLOGICAL CHEMISTRY

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a

SGK1 Activates NF-B Signaling via IKK and p300

4074 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 284 NUMBER 7 FEBRUARY 13, 2009

SGK1 Activates NF-B Signaling via IKK and p300

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

of lysate from cell lines or hippocampal tissue was incubated

SGK1 Activates NF-B Signaling via IKK and p300

4076 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 1. Putative substrate proteins of SGK1. Tissue lysate (10 g) from

ers of NR2A and NR2B were designed according to a previous

CA1 area. Before injection, plasmid DNA was diluted in 5%

SGK1 Activates NF-B Signaling via IKK and p300

genate was centrifuged at 1,000 g for 5 min at 4 C. The

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

Identification of SGK1 Substrate ProteinsBecause SGK1

300 400 l of extraction buffer

SGK1 Activates NF-B Signaling via IKK and p300

4078 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 284 NUMBER 7 FEBRUARY 13, 2009

SGK1 Activates NF-B Signaling via IKK and p300

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

JOURNAL OF BIOLOGICAL CHEMISTRY

sequence RXRXX(S/T), where X stands for any amino acid, we

SGK1 Activates NF-B Signaling via IKK and p300

pmol) was administered to hippocampal CA1 area 96 h before

4080 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 284 NUMBER 7 FEBRUARY 13, 2009

SGK1 Activates NF-B Signaling via IKK and p300

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

JOURNAL OF BIOLOGICAL CHEMISTRY

siRNA group with IGF-1 group) without affecting the IKK

SGK1 Activates NF-B Signaling via IKK and p300

4082 JOURNAL OF BIOLOGICAL CHEMISTRY

dose-dependent manner (Fig. 4G). SGK1 siRNA alone at a

The effectiveness of SGK1 siRNA and Akt siRNA treatments

SGK1 Activates NF-B Signaling via IKK and p300

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

JOURNAL OF BIOLOGICAL CHEMISTRY

siRNA (4 pmol) was transfected to

SGK1 Activates NF-B Signaling via IKK and p300

4084 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 284 NUMBER 7 FEBRUARY 13, 2009

ined by real time PCR. Results

SGK1 Activates NF-B Signaling via IKK and p300

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

JOURNAL OF BIOLOGICAL CHEMISTRY

struct was transfected, there was a

SGK1 Activates NF-B Signaling via IKK and p300

0.01), but Akt siRNA markedly enhanced spatial learning (q

4086 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 284 NUMBER 7 FEBRUARY 13, 2009

SGK1 Activates NF-B Signaling via IKK and p300

FEBRUARY 13, 2009 VOLUME 284 NUMBER 7

JOURNAL OF BIOLOGICAL CHEMISTRY