NH 2 terminus of serum and glucocorticoid-regulated kinase 1 binds to phosphoino - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:38 |只看该作者 |倒序浏览 |打印

作者：Alan C. Pao,, James A. McCormick,, Hongyan Li, John Siu, Cedric Govaerts, Vivek Bhalla, Rama Soundararajan, and David Pearce,作者单位：1 Department of Medicine, Division of Nephrology, San Francisco General Hospital; and 2 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California


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      【摘要】
   Serum and glucocorticoid regulated kinase 1 (SGK1) has been identified as a key regulatory protein that controls a diverse set of cellular processes including sodium (Na ) homeostasis, osmoregulation, cell survival, and cell proliferation. Two other SGK isoforms, SGK2 and SGK3, have been identified, which differ most markedly from SGK1 in their NH 2 -terminal domains. We found that SGK1 and SGK3 are potent stimulators of epithelial Na channel (ENaC)-dependent Na transport, while SGK2, which has a short NH 2 terminus, is a weak stimulator of ENaC. Further characterization of the role of the SGK1 NH 2 terminus revealed that its deletion does not affect in vitro kinase activity but profoundly limits the ability of SGK1 either to stimulate ENaC-dependent Na transport or inhibit Forkhead-dependent gene transcription. The NH 2 terminus of SGK1, which shares sequence homology with the phosphoinositide 3-phosphate [PI( 3 )P] binding domain of SGK3, binds phosphoinositides in protein lipid overlay assays, interacting specifically with PI( 3 )P, PI( 4 )P, and PI( 5 )P, but not with PI( 3, 4, 5 )P 3. Moreover, a point mutation that reduces phosphoinositide binding to the NH 2 terminus also reduces SGK1 effects on Na transport and Forkhead activity. These data suggest that the NH 2 terminus, although not required for PI 3-kinase-dependent modulation of SGK1 catalytic activity, is required for multiple SGK1 functions, including stimulation of ENaC and inhibition of the proapoptotic Forkhead transcription factor. Together, these observations support the idea that the NH 2 -terminal domain acts downstream of PI 3-kinase-dependent activation to target the kinase to specific cellular compartments and/or substrates, possibly through its interactions with a subset of phosphoinositides.
      【关键词】 epithelial sodium channel Forkhead kinase activity( y# q. P- {* L
   RENAL TUBULAR CELLS must integrate a variety of hormonal and nonhormonal signals to achieve coordinated responses to changes in the environment that are appropriate within a given physiological context. One key regulatory protein that integrates multiple environmental cues in mammalian cells is the serine/threonine kinase, serum and glucocorticoid-regulated kinase 1 (SGK1). Several hormonal, growth factor, and cell stress pathways converge on SGK1 to control diverse cellular processes including sodium (Na ) homeostasis ( 14, 40 ), osmoregulation ( 45, 57 ), cell survival ( 35 ), and cell proliferation ( 13 ). In particular, the importance of SGK1 in mediating hormonal and osmotic effects on Na transport in renal collecting duct cells is well established ( 14, 40, 45, 58 ). SGK1 phosphorylates Nedd4-2 (neural precursor cell-expressed, developmentally downregulated protein 4-2) at three RXRXX(S/T) phosphorylation motifs ( 18, 46 ) and promotes an inhibitory interaction of this ubiquitin ligase with 14-3-3 proteins ( 6, 28, 36 ). Nedd4-2-mediated ubiquitination enhances removal from the plasma membrane and degradation of the epithelial Na channel (ENaC) ( 1, 29 ), while SGK1 diminishes removal, at least in part, by inhibiting Nedd4-2 ( 18, 46 ).$ T) B+ ~6 H& i- u

SGK1 belongs to the AGC family of protein kinases, which, in addition to the SGK kinases, includes the PKB/Akt kinases, p70 ribosomal S6 kinase (S6K), and atypical forms of protein kinase C (PKC). In particular, the SGK and Akt family of kinases share considerable sequence homology and a number of functional similarities, including growth factor-mediated activation via the phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway and common peptide substrates ( 33, 42 ). At the primary sequence level, their similarities are most notable in their catalytic domains and in two regulatory sequences surrounding Thr-256 and Ser-422, which lie in the activation loop and hydrophobic motif of SGK1, respectively, and are phosphorylation targets of PI3-kinase-dependent kinases. Phosphorylation of both regulatory sites is required for activation of catalytic function of these kinases and appears to be functionally indistinguishable among these kinase family members ( 33, 42 ).( ]  z0 o7 e  S4 x& G% u
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The NH 2 -terminal domains of these kinases, on the other hand, diverge markedly: PKB/Akt kinases contain an NH 2 -terminal pleckstrin homology (PH) domain, which selectively binds PI( 3, 4, 5 )P 3 and PI( 3, 4 )P 2 and directs PKB/Akt to the plasma membrane ( 2 - 4, 48, 49 ). SGK1's closest relative, SGK3, bears an NH 2 -terminal Phox homology (PX) domain that targets it to endosomal membranes primarily via interaction with PI( 3 )P ( 55, 60 ), while the NH 2 -terminal region of SGK2 is short and lacks homology to any known interaction motif. SGK1 has an NH 2 -terminal region, which is intermediate in length between SGK2 and SGK3, and contains a short sequence that bears significant homology to the SGK3 PX domain ( 41 ). Of the several subcellular compartments in which SGK1 has been detected, a role for the NH 2 -terminal region in mediating its localization has been demonstrated for plasma membrane ( 11 ), mitochondria ( 22 ), and endoplasmic reticulum ( 5, 9 ); however, whether phosphoinositides are implicated in its localization is not known. Indeed, although this region has been suggested to represent a phosphoinositide interaction motif ( 41 ), this has not been demonstrated.
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In addition to its role in directing SGK1 to various cellular compartments, the NH 2 -terminal domain has been shown to play an important role in SGK1 protein stability by triggering its ubiquitination and proteasome-mediated degradation ( 5, 9, 11, 46 ). Consequently, deletion of the NH 2 -terminal domain greatly enhances SGK1 expression ( 8, 11, 33, 39 ) and, unless the NH 2 -terminal region is implicated in another aspect of SGK1 function, would presumably increase SGK1 kinase activity and physiological functions. In the course of experiments directed at examining SGK isoform-specific functions, we compared the functional activities of SGK1, 2, and 3 in stimulating ENaC. These experiments demonstrated that SGK2 has substantially lower ENaC stimulatory activity than SGK1 or SGK3. The near lack of an NH 2 -terminal domain in SGK2, coupled with the similar NH 2 -terminal domains between SGK1 and SGK3, highlighted the importance of this region in kinase function. We therefore went on to characterize the role of this domain in SGK1 function. We found that the NH 2 -terminus is critical for the ability of SGK1 to stimulate ENaC and inhibit the proapoptotic transcription factor Forkhead, another physiological target of SGK1, but is not required for catalytic activity. Indeed, a point mutation that mimics PI 3-kinase-dependent phosphorylation confers constitutive catalytic activity to both full-length and NH 2 terminally deleted SGK1; however, such a mutant is able to stimulate ENaC or inhibit Forkhead only if the NH 2 -terminal domain is intact. We further demonstrate that the NH 2 terminus contains a phosphoinositide-binding domain that can bind to a subset of phosphoinositides that is distinct from the binding preferences of either the PH domain of PKB/Akt or the PX domain of SGK3. Taken together, these data suggest that the NH 2 -terminal domain is important for post PI 3-kinase-dependent activation events in SGK1 function, in part by interacting with a subset of phosphoinositides that are found within specific subcellular compartments.
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MATERIALS AND METHODS6 r/ O! I( G! Z- I: `5 f4 G7 ~' \
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Xenopus laevis oocyte coexpression assay. X. laevis -, -, -FLAG-ENaC psDEasy expression vectors were generously provided by Drs. B. Rossier and D. Firsov; mouse SGK1, SGK2, and SGK3 were subcloned into pMO expression vectors (gift from Dr. L. Jan). Capped RNAs (cRNAs) were synthesized as described previously ( 14 ). Stage V-VI oocytes were injected with cRNAs containing X. laevis -, -, -FLAG-ENaC (3.0 ng each), X. laevis Nedd4-2 (1.5-3.0 ng), and wild-type mouse SGK1, SGK2, or SGK3 (5.0 ng).
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To ensure uniform expression of enzymatically active SGK1 in oocytes, subsequent experiments involving mouse SGK1 were performed with a constitutively active form of SGK1 (CA-SGK1), which has an S422D mutation and does not require PI 3-kinase for activation ( 33, 42 ). Depending on the experimental condition, oocytes were injected with cRNAs (0.025-5.0 ng) containing either full-length mouse SGK1/S422D-FLAG (CA-SGK1), N-[deletion of amino acids (aa) 1-85]-SGK1/S422D-FLAG (CA-SGK1/ N), or SGK1/R31A/S422D-FLAG (CA-SGK1/R31A) in conjunction with X. laevis -, -, -FLAG-ENaC and X. laevis Nedd4-2. Based on the known differences in protein stability between wild-type SGK1 and mutants that do not associate with membrane fractions, including N-SGK1 ( 11, 34, 61 ), titrated amounts of the different CA-SGK1 constructs were injected to yield equivalent levels of expression as assessed by Western blotting. After injection, the oocytes were incubated in a low Na Barth's solution and current measurements were performed as described previously ( 14 ). The specific ENaC-mediated current ( I phe ) was defined as the difference between currents obtained in the presence or absence of 2 µM phenamil (Sigma, St. Louis, MO). Results were repeated with at least three independent experiments.; @% c; G, C  I4 w# j/ {' p; A3 r

Expression of SGK in X. laevis oocytes. Groups of 10-15 oocytes that underwent current measurements were transferred to Eppendorf tubes. Twenty microliters per oocyte of homogenization buffer (20 mM Tris·HCl, pH 7.6, 100 mM NaCl, 2% NP-40 with protease inhibitors) were added to the oocytes, and the oocytes were lysed by repeated vortexing and pipeting. Cellular debris was pelleted at 3,600 g for 10 min, and the supernatant was centrifuged at 3,600 g twice to remove additional debris. Floating yolk was removed with a cotton-tipped swab. All centrifugation steps were performed at 4°C.

One hundred micrograms of protein from oocyte lysates were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (Hybond-C Extra, Amersham Biosciences, Piscataway, NJ). The membranes were immunostained with polyclonal anti-SGK rabbit antiserum (raised against an SGK1 COOH-terminal epitope, Cell Signaling, Danvers, MA) diluted 1:100 for detection of SGK-immunoreactive protein. Membranes were incubated with secondary anti-rabbit IgG horseradish peroxidase conjugate (Amersham Biosciences) diluted 1:5,000 and processed as described ( 58 ).& z, @0 V4 w: x; T4 Z
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Expression of SGK in HEK 293 cells for in vitro kinase assay. HEK 293 cells were maintained at 37°C in culture medium containing DMEM, 10% fetal bovine serum, and 1% penicillin/streptomycin. Cells were plated on 6- cm dishes and were transfected with CA-SGK1, CA-SGK1/ N, or CA-SGK1/R31A on the following day. As in oocytes, amounts of transfected SGK1 for the different constructs were titrated to yield equivalent levels of expression as assessed by Western blotting.
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Protein lysates of HEK 293 cells expressing each of the different CA-SGK1 constructs were immunoprecipitated with EZview red anti-FLAG M2 affinity gel beads (Sigma). The different CA-SGK1 constructs were subsequently eluted from anti-FLAG gel beads with 3X-FLAG peptide (Sigma) at 300 µg/ml to yield free kinase. 3X-FLAG peptide was removed, and the different CA-SGK1 constructs were concentrated through the use of size exclusion spin columns (Microcon YM-30, Millipore, Billerica, MA). Equal amounts of eluted CA-SGK1 constructs were verified by Western blotting before evaluation of kinase activity. Eluted CA-SGK1 constructs were stored in aliquots at -80°C.4 Q4 a6 j4 z! {% ~3 \; u2 e
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In vitro kinase assays were performed using SGKtide (Upstate Biotechnology, Lake Placid, NY) as an SGK1-specific phosphorylation substrate in the presence of [ - 32 P] ATP, according to the manufacturer's protocol and as described ( 42 ). The reaction product was blotted on P81 phosphocellulose squares (Whatman, Clifton, NJ), washed, and quantified by scintillation counting.
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Protein lipid overlay assay. Mouse SGK3 [1-149 aa], SGK1 [1-85 aa], and SGK1/R31A [1-85 aa] NH 2 -terminal regions upstream of the respective catalytic domains were expressed in Escherichia coli BL21 as 6X-His fusion proteins using the pET 15b expression vector (Novagen, Madison, WI) and purified on nickel-charged resin (Qiagen, Valencia, CA). Protein lipid overlay assays were performed using "PIP strips" or "PIP arrays" (Echelon, Salt Lake City, UT), which are nitrocellulose membranes spotted with a diverse array of phospholipids designed to detect specific protein-phospholipid interactions. Membranes were incubated with His-tagged protein and retained protein was detected with anti-His Ab (Novagen), as described by the manufacturer's protocol and previously ( 55 ). Similar results were obtained in three independent experiments.' T( v9 o" y' r8 d
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Transient transfection luciferase assay. The Forkhead-responsive luciferase reporter plasmid (FHRE-Luc) was kindly provided by Dr. M. Greenberg (Harvard Medical School, Boston, MA). For transient transfections, low-passage HEK293 cells were seeded 24 h before transfection at a density of 2 x 10 5 cells/dish in 6-cm dishes. Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Cells were transfected with 50 ng of FHRE-Luc and depending on the experimental condition, 1,000 ng of CA-SGK1, 10-20 ng of CA-SGK1/ N, or 400-600 ng of CA-SGK1/R31A were titrated to give equivalent expression of the different mutants by immunoblot. Transfection for each experimental condition was performed in triplicate. The day after transfection, culture medium was changed and, 10 h later, was replaced with serum-free medium. Cells were harvested the following day, and protein lysates were assessed for luciferase activity using Promega Luciferin Reagent (Promega, Madison, WI) and normalized to total protein levels, which were measured by adding 100 µl of Bio-Rad protein assay dye (Bio-Rad, Hercules, CA) to 1 µl of lysate followed by measuring in a microplate reader. Similar results were obtained in three independent experiments.
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Statistics. Statistical analyses for comparisons of I phe in X. laevis oocytes and luciferase activity (normalized to total protein levels) in HEK 293 cells were performed using one-way ANOVA with Bonferroni's adjustment. Differences were considered to be significant at P values

RESULTS
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SGK1 and SGK3, but not SGK2, stimulate ENaC in X. laevis oocytes. Previous work established that SGK1 increases ENaC-mediated Na current by inhibiting the ubiquitin ligase Nedd4-2 ( 18, 46 ). Little is known about SGK isoform specificity, and our initial goal in the present study was to compare SGK isoform activities in the context of ENaC activation. Toward this end, we expressed ENaC and Nedd4-2, in combination with SGK1, 2, or 3, in X. laevis oocytes and measured ENaC-mediated Na current ( I phe ) by two-electrode voltage clamp ( Fig. 1 ). The amount of cRNA injected for each of the SGK isoforms was titrated in a series of pilot experiments to give comparable levels of protein expression (not shown). As shown previously ( 6 ), Nedd4-2 markedly inhibited ENaC current, an effect that was largely reversed by SGK1 ( Fig. 1, compare second and third bars). SGK3 also markedly increased Na current, although not to the same extent as SGK1. In contrast, SGK2 had a negligible effect on I phe when expressed at equivalent levels. At high levels of expression, SGK2 did significantly stimulate ENaC, but at all levels of expression, this effect remained substantially weaker than that of SGK1 or SGK3 (not shown). One earlier report found that SGK1, 2, and 3 all stimulate ENaC in X. laevis oocytes when equal amounts of cRNA are injected ( 23 ); however, high concentrations of cRNA were injected in this study, protein expression levels were not determined, and it seems likely that SGK2, in particular, was expressed at substantially higher levels than SGK1.
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Fig. 1. SGK1 and SGK3, but not SGK2, stimulate epithelial sodium channel (ENaC) current. SGK-stimulated Na transport was measured through a Xenopus laevis oocyte coexpression assay. Stage V-VI oocytes were injected with cRNA for ENaC subunits (3 ng each), Nedd4-2 (N4-2; 1.5 ng), and either SGK1, SGK2, or SGK3 (5 ng each), as indicated. Phenamil-sensitive Na current was recorded 48 h later by 2-electrode voltage clamp (TEVC) at a holding potential of -100 mV. Statistical significance between the different conditions was compared as indicated. * P
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The SGK isoforms share 80% homology in their kinase domains, 50% homology in their COOH-terminal domains, but ; _3 C, B) ~; ~- h( @0 E# O
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Fig. 2. NH 2 termini of SGK1 and SGK3 share primary sequence homology. A : domain comparisons of mouse SGK1, SGK2, and SGK3. The regions upstream of the more highly conserved kinase domains comprise the NH 2 -terminal domains: amino acids 1-85 for mSGK1, 1-22 for mSGK2, and 1-149 for mSGK3. The NH 2 -terminal domain of SGK3 contains a PX domain (residues 1 to 125), whereas that of SGK2 is short (residues 1 to 22). Residues 1 to 26 of SGK1 bear no resemblance to other sequences within the SGK family, while residues 27 to 85 share 36% homology with the corresponding segment in SGK3. The percent of sequence homology between SGK1 and SGK3 is indicated. B : sequence alignment between mouse SGK1 and SGK3 NH 2 -terminal domains. The first 30 residues of the kinase domains are also shown following the NH 2 -terminal domains. The numbering scheme is indicated for SGK1. The colored boxes correspond to the domains shown in A. Color coding for the amino acids is as follows: black for hydrophobic residues, green for aromatic residues, red for acidic residues, blue for basic residues, orange for glutamine or asparagine, and brown for serine and threonine. C : structure of the PX domain of SGK3 with likely position of bound phosphoinositide ( left ) and representation of the homologous region within the SGK1 NH 2 terminus that corresponds to the SGK3 PX domain ( right ). Shown on the left is a ribbon representation of the crystal structure of SGK3 (PDB id: 1XTE). The coloring follows the domain definition of A with the segment homologous to SGK1 in red. Bound PI( 3 )P is depicted here, as observed in the PX domain of P40 (PDB id: 1H6H), by superimposing the backbone atoms of the 2 PX domains. The side chain of Arg 90, corresponding to Arg 31 in SGK1 ( right ), is shown.

SGK1 effects on specific physiological endpoints require that it first be activated and then be targeted to effector substrates in appropriate subcellular compartments. SGK1 catalytic activity requires PI 3-kinase-dependent phosphorylation at Ser-422 followed by non-PI 3-kinase-dependent phosphorylation at Thr-256 ( 8, 33, 42 ). Since previous work suggested that the NH 2 -terminal domain is not required for PI 3-kinase-dependent activation of SGK1 ( 8 ), we were interested in examining the role of the NH 2 terminus in directing postactivation events in SGK stimulation of ENaC. Substitution of aspartate for serine at residue 422 (SGK1/S422D) effectively mimics PI 3-kinase-dependent phosphorylation and confers constitutive catalytic activity ( 8, 33, 42 ). To bypass this activation step and obtain uniform SGK1 activity independent of PI 3-kinase, we used the S422D mutant (CA-SGK1) for our subsequent experiments., I; b% ]" J7 O( y7 K
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We first verified that deletion of the NH 2 -terminal domain did not affect the catalytic activity of CA-SGK1. FLAG epitope-tagged full-length CA-SGK1 and NH 2 -terminal deletion derivative (CA-SGK1/ N) were expressed in HEK-293 cells and purified by immunoprecipitation from whole cell lysates. In vitro kinase assays were performed with equivalent concentrations of the two kinases using the peptide substrate SGKtide ( 42 ). As shown in Fig. 3 A, comparable amounts of labeled SGKtide were produced at all concentrations tested. We next compared the effect of CA-SGK1 and CA-SGK1/ N on ENaC activity in a X. laevis oocyte coexpression assay. Consistent with our hypothesis, CA-SGK1/ N had substantially decreased ability to stimulate ENaC current at all levels of expression ( Fig. 3 B ), in marked contrast to its preserved kinase activity. It should be noted that the lowest amount of injected cRNA for CA-SGK1/ N gave equivalent protein expression to the highest amount of injected cRNA for CA-SGK1 within oocytes, consistent with previous reports that deletion of the NH 2 terminus of SGK1 results in greater protein stability ( 11, 34, 61 ). Thus the NH 2 terminus of SGK1 harbors a domain that is required for ENaC stimulation but is not required for catalytic activity. Furthermore, this domain appears to be required for post PI 3-kinase-dependent activation events in SGK1 function.

Fig. 3. NH 2 terminus of SGK1 is required for stimulation of ENaC current. A : in vitro kinase assay showing equivalent kinase activities of CA-SGK1 (FL) and CA-SGK1/ N ( N). Kinase activity has been normalized to amount of immunoreactive protein in each kinase reaction and expressed as picomoles per minute of 32 P incorporation into SGKtide peptide substrate per µg purified kinase. Data are representative of 3 independent experiments. B : deletion of the first 85 amino acids of CA-SGK1/ N ( N) substantially decreased stimulation of ENaC current in a dose-dependent manner in a X. laevis oocyte coexpression assay. Statistical significance between the different conditions was compared as indicated. * P
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NH 2 terminus of SGK1 contains a phosphoinositide interaction domain. Closer examination of the SGK1 NH 2 terminus showed that there is a stretch of 18 amino acids within this region (residue 27-44) that shares 61% homology with the corresponding region of the SGK3 PX domain (residue 86-103), as noted previously ( 41 ). PX domains were originally identified in the P40 phox and P47 phox subunits of NADPH oxidase ( 43 ) and are found in numerous proteins involved in membrane traffic, including sorting nexins (SNX1-4, 6, 9, 15, 17) and yeast Vps proteins (Vam7p, Mvp1p) ( 44 ). The PX domain of SGK3 binds to PI( 3 )P, which localizes it to early endosomes ( 55 ). These observations suggested the possibility that the NH 2 -terminal region of SGK1, like that of SGK3, harbors a phosphoinositide binding domain.- |8 S  z2 `3 v$ q

We therefore examined directly the phosphoinositide interactions of the SGK1 NH 2 terminus and compared it with those of the SGK3 PX domain using a protein lipid overlay assay ( Fig. 4 ) ( 19 - 21 ). As shown in Fig. 4 A, the SGK3 PX domain bound specifically only to PI( 3 )P, as described previously ( 55 ). Under the same conditions, the SGK1 NH 2 terminus interacted specifically with the monophosphorylated phosphoinositide species, PI( 3 )P, PI( 4 )P, PI( 5 )P, as well as PS (phosphatidylserine). Substantially weaker signal was observed with PI( 3, 5 )P 2 and PI( 4, 5 )P 2 ( Fig. 4 B, left ). Notably, binding to PI( 3, 4, 5 )P 3, which preferentially binds to the PH domain of PKB/Akt and is thought to be the principal phosphoinositide involved in class I PI 3-kinase-dependent signaling, was undetectable. Overlay assays using membranes with a range of phosphoinositide concentrations further confirmed that the SGK1 NH 2 terminus interacted preferentially with monophosphorylated phosphoinositide species and PS but not with PI( 3, 4, 5 )P 3 ( Fig. 4 B, right ). We conclude that the NH 2 -terminal 85 residues of SGK1 contain a phosphoinositide-binding domain, which has sequence similarity and partially overlapping phosphoinositide binding specificity with the SGK3 PX domain., W" _" ^$ _6 {

Fig. 4. SGK1 NH 2 terminus binds selectively to a subset of phosphoinositides. The mSGK3 [1-149 aa], mSGK1 [1-85 aa], or mSGK1/R31A [1-85 aa] NH 2 -terminal regions upstream of the catalytic domain were expressed in E. coli BL21 as 6X-His fusion proteins and purified on nickel beads. Nitrocellulose membranes spotted with different phosphoinositide species were incubated with His-tagged proteins and retained protein was detected with anti-His Ab (Novagen). A : protein lipid overlay assay with the SGK3 PX domain incubated with a nitrocellulose strip, as described previously ( 55 ). Shown alongside is an identical membrane incubated without SGK3 protein. B : protein lipid overlay assays with the SGK1 NH 2 terminus. Left : nitrocellulose strip, as in A, which has been spotted with 15 different biologically active lipids (100 pmol/spot) and incubated with purified protein. A representative of 3 independent experiments is shown here. Right : nitrocellulose membrane that has been spotted with a concentration gradient of 8 phosphoinositides (100-1.6 pmol/spot with 2-fold dilution) and incubated with purified protein. C : protein lipid overlay assay with the SGK1/R31A NH 2 terminus resulted in poor binding to all phosphoinositide species. Overlay assays with the mSGK1/R31A NH 2 terminus were performed in parallel with the wild-type mSGK1 NH 2 terminus (as shown in B ); conditions and exposure times were identical to those in B. Similar results were obtained in 2 independent experiments. D : purified protein of wild-type and R31A mutant NH 2 -terminal domains were resolved on 10% SDS-PAGE and immunoblotted with anti-His Ab (Novagen).* ~4 x- s5 V7 Z, o0 m: K

Characterization of an NH 2 -terminal domain mutant with reduced binding to phosphoinositides. Significant sequence homology between the NH 2 terminus of SGK1 and the PX domains of SGK3 and P40 phox, whose crystal structures have been solved ( 10, 59 ), starts at residue 27 of SGK1 (residue 86 of SGK3, residue 101 of P40 phox ) (see within red box, Fig. 2 B ). In the known PX domain structures, this region (corresponding to aa 27-44 of SGK1) forms an -helix that provides the floor of the phosphoinositide-binding pocket and possesses an arginine (R90 of SGK3, equivalent to R31 of SGK1) involved in the coordination of bound ligand ( 10 ). In the case of P40 phox, the analogous residue (R105) interacts with the 4'-OH group of the inositol ring of the phosphoinositide head group of PI( 3 )P ( 10 ). Mutations of this residue [e.g., R105A in P40 phox ( 10 ), R90L in P47 phox ( 30 ), or R90A in SGK3 ( 60 )] markedly reduce phosphoinositide binding.% {$ V6 W! K8 `  t" L

Modeling of the SGK1 structure surrounding R31 based on the solved structures of the P40 phox and SGK3 PX domains suggested that R31 can also potentially form a H-bond with the 4'-OH group of the inositol ring of PI( 3 )P ( Fig. 2 C ). We therefore tested the ability of the SGK1 NH 2 terminus, mutated at this position (SGK1 [1-85 aa]/R31A), to bind to phosphoinositides ( Fig. 4 C ). Consistent with its predicted role in phosphoinositide binding, SGK1 [1-85 aa]/R31A showed decreased binding to all phosphoinositide species. Notably, however, significant residual binding was detected, particularly to PI( 3 )P ( Fig. 4 C ). This was more apparent with longer film exposure times (not shown).

SGK1/R31A has reduced ENaC-stimulatory activity. We next examined the functional consequences of the R31A mutation on SGK1 regulation of ENaC. First, we assessed whether CA-SGK1 and CA-SGK1/R31A have similar kinase activities. Indeed, CA-SGK1 and CA-SGK1/R31A showed equivalent kinase activities at equivalent molar input levels, indicating that, as with deletion of the NH 2 -terminal domain, the R31A mutation does not affect SGK1 in vitro kinase activity ( Fig. 5 A ). Next, we examined the ability of SGK1/R31A to stimulate ENaC. X. laevis oocytes were coinjected with ENaC and Nedd4-2 cRNA, in combination with CA-SGK1 or CA-SGK1/R31A, and ENaC current was determined ( Fig. 5 B ). cRNA for CA-SGK1 and CA-SGK1/R31A was titrated to give equivalent levels of expression. Under these conditions, CA-SGK1/R31A had decreased ability to stimulate ENaC current, which was particularly apparent at low levels of expression ( Fig. 5 B ); at high levels of expression, the ENaC-stimulatory activity of CA-SGK1/R31A approached that of CA-SGK1. Thus this mutation significantly impaired the ENaC-stimulatory activity of CA-SGK1, although not as profoundly as did complete deletion of the NH 2 terminus.8 f  `1 C3 N; Y/ l
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Fig. 5. Intact NH 2 terminus within SGK1 is required for full stimulation of ENaC current. A : in vitro kinase assay showing equivalent kinase activities between CA-SGK1 and CA-SGK1/R31A. Kinase activity has been normalized to amount of immunoreactive protein in each kinase reaction and expressed as picomoles per minute of 32 P incorporation into SGKtide peptide substrate per µg purified kinase (as in Fig. 3 A ). Data are representative of 3 independent experiments. B : R31A mutant of CA-SGK1 (R31A) stimulated ENaC current less potently than CA-SGK1 (FL) in a X. laevis oocyte coexpression assay. Statistical significance between the different conditions was compared as indicated. * P # C: l6 F; y' s; z1 V7 [

An intact NH 2 -terminal domain within SGK1 is required for full inhibition of Forkhead-dependent transcription. SGK1 is a multifunctional kinase; in addition to its well-characterized effects on Na transport, it is also known to have effects on cell survival ( 35 ), apoptosis ( 39 ), and cell cycle progression ( 13, 26 ), in part by phosphorylating and inactivating the proapoptotic Forkhead box transcription factor FKHRL1 ( 12 ). To evaluate the potential role of the NH 2 terminus in mediating SGK1 regulation of Forkhead-dependent gene transcription, we cotransfected HEK-293 cells with a Forkhead response element luciferase reporter gene [FHRE-Luc ( 12 )] and either CA-SGK1 or one of the phosphoinositide binding-deficient CA-SGK1 mutants. Similar to our X. laevis oocyte coexpression experiments, amounts of transfected DNA were titrated to give equivalent levels of protein expression. As shown in Fig. 6 A, CA-SGK1 resulted in 70% inhibition of Forkhead reporter activity. In contrast, the CA-SGK1/ N mutant failed to inhibit reporter activity ( Fig. 6 A ), whereas the CA-SGK1/R31A mutant had intermediate activity ( Fig. 6 B ), similar to what was observed for ENaC regulation. Taken together, these results strongly suggest that the NH 2 terminus is an important determinant of SGK1 action in Forkhead inhibition as well as in ENaC stimulation, potentially by serving as a site for phosphoinositide binding.) n! f3 N. S! s" {# @
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Fig. 6. Intact NH 2 terminus within SGK1 is required for full inhibition of FKHRL1-driven gene transcription. A : in HEK293 cells, activity of a reporter containing a Forkhead-binding site driving luciferase (FHRE-Luc) was stimulated by endogenous FKHRL1, and the effects of transfected CA-SGK1 (FL) or CA-SGK1/ N ( N) on FKHRL1-mediated stimulation of FHRE-Luc were determined. Luciferase activity has been normalized to total protein levels and expressed relative to activity of the FHRE-Luc control group. Statistical significance between the different conditions was compared as indicated. * P

DISCUSSION% i1 T8 u' n7 G9 e! W

NH 2 terminus as a determinant of SGK1 activities and of isoform specificity. The three SGK isoforms have substantial sequence homology in their catalytic and COOH-terminal domains, common substrate preferences in vitro, and similar PI 3-kinase dependence for catalytic activity. SGK1 and SGK3 also share significant homology in a short stretch of amino acids (residue 27-44 for SGK1, residue 86-103 for SGK3) within their NH 2 termini, which in SGK3 comprises part of the PI( 3 )P binding PX domain ( 55 ). The SGK2 NH 2 terminus, in contrast, is short and bears no resemblance to any known interaction module. Initial experiments showing that SGK1 and SGK3 can stimulate ENaC when expressed at comparable levels in the presence of Nedd4-2 ( Fig. 1 ) suggested the possibility that the NH 2 -terminal regions of these two kinases contain functionally important selectivity determinants that are not shared by SGK2. It is notable that in a prior report, all three SGK isoforms stimulated ENaC when expressed at high levels in X. laevis oocytes in the absence of Nedd4-2 ( 23 ). Our present data show that under conditions of lower levels of kinase expression in the presence of Nedd4-2, SGK2 is substantially less efficacious than SGK1 or SGK3 at stimulating ENaC current ( Fig. 1 ). At higher expression levels, SGK2 did significantly stimulate ENaC, likely because normal determinants of isoform specificity were overridden.
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The foregoing data pointed to the NH 2 terminus as an important determinant of SGK function. By using a constitutively active form of SGK1, we examined the role of the NH 2 terminus in influencing events post-PI 3-kinase activation, an approach used previously for assessment of phosphorylation of physiological SGK1 substrates, as well as for substrates of other SGK and PKB/Akt kinases ( 6, 12, 38 ). Under these conditions, the in vitro kinase activities of CA-SGK1 and CA-SGK1/ N were indistinguishable ( Figs. 3 A and 5 A ), and yet the kinases differed markedly in their abilities either to stimulate ENaC or inhibit FKHRL1 ( Figs. 3 B, 5B, and 6 ). While two previous studies examined the effect of NH 2 -terminal deletion on SGK1-mediated ENaC activity ( 27 ) and anti-apoptosis ( 39 ), this is the first study to separate the effects of NH 2 -terminal deletion on SGK1 kinase activity from effects on its function. Our data strongly support the conclusion that the NH 2 terminus is a functionally important domain, which acts downstream of PI 3-kinase-dependent activation to target the activated kinase to a distinct set of protein substrates involved in the regulation of ENaC, FKHRL1, and possibly other cellular processes.- i# C/ r/ v$ @- J( b* I$ ?
6 a9 b+ X; R# f  H
Role of phosphoinositide binding to the NH 2 terminus in determining SGK1 action. One potential mechanism for how the NH 2 terminus might control targeting is through its ability to bind to phosphoinositides. Phosphoinositides serve as second messengers and integrators of signal transduction by helping to ensure that a specific action (such as phosphorylation of a particular substrate by a protein kinase) occurs in the appropriate cellular compartment and under the appropriate cellular conditions ( 44 ). The NH 2 -terminal 85 amino acids of SGK1, which contain a region with high homology to the PX domain, bound specifically to PI( 3 )P, PI( 4 )P, PI( 5 )P, and PS with approximately equal affinity and to PI( 3, 5 )P 2 and PI( 4, 5 )P 2 with lower affinity. It notably does not bind to PI( 3, 4, 5 )P 3, the principal phosphoinositide implicated in PI 3-kinase-dependent activation of Akt. The limited sequence similarity to PX domains and shared binding to PI( 3 )P ( Fig. 4 ) suggested the SGK1 NH 2 terminus is "Phox-like" with some significant differences. Most notably, the far NH 2 terminus of SGK1 diverges markedly from the PX domain consensus, lacking residues known to comprise -pleated sheets and an -helix implicated in PX domain-phosphoinositide binding ( 10, 31, 59 ). Instead, the SGK1 NH 2 terminus has a distinct 27 amino acid stretch, which is relatively rich in basic amino acids and could in principle form an amphipathic -helix that contributes to SGK1's distinct pattern of phosphoinositide binding (orange box, Fig. 2 B ).9 a3 F1 v) k: a5 m( O( }( g8 q
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The marked defect in both ENaC stimulation and Forkhead inhibition by NH 2 terminally deleted SGK1 strongly supports the importance of this domain in SGK1 function. The possibility that the phosphoinositide-binding capacity of this domain is functionally important is supported by the observation that a specific point mutation R31A, which disrupts lipid binding to the NH 2 terminus ( Fig. 4 ), significantly decreased modulation of both ENaC-mediated Na transport and Forkhead-driven gene transcription by a constitutively active form of SGK1 ( Figs. 5 and 6 ). These results support the interesting idea that phosphoinositides influence processes downstream of PI 3-kinase-dependent activation, perhaps by controlling SGK1 targeting to subcellular compartments that contain SGK1 substrates such as Nedd4-2 or FKHRL1. It is notable that the R31A mutation did not appear to affect ENaC-stimulatory activity as profoundly as it did the ability of SGK1 to bind phosphoinositides ( Figs. 4 C and 5 B ), nor did it have as profound an effect on ENaC-stimulatory activity as deletion of the NH 2 terminus. Although this may have been due to residual phosphoinositide binding by the R31A mutant, it is possible that the NH 2 terminus of SGK1 contains additional motifs, which are important for both ENaC stimulation and FKHRL1 inhibition. More work examining the function of refined deletion mutants and additional point mutants will be required to fully address this question.

Although our present data do not address the role of the SGK1 NH 2 terminus in steps before PI 3-kinase activation, previous evidence supports the idea that, in contrast to PKB/Akt, SGK1 binds directly to the "PIF" binding pocket of PDK1 and is subsequently activated without itself binding to phosphoinositides ( 8 ). As noted above, together with our present data, this suggests that phosphoinositide binding to SGK1 is only required for SGK1 action downstream of PI 3-kinase-dependent activation and that phosphoinositides generated by PI 3-kinase may not be implicated in this phase of SGK1 function.4 {0 v2 L" \9 g4 d2 O! N
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Role of phosphoinositides in SGK1 regulation of diverse cellular processes. In view of the functional similarities between SGK1 and SGK3, as well as their shared interactions with PI( 3 )P, our present view is that PI( 3 )P binding to SGK1 is important for SGK1 regulation of ENaC. It is notable that PI( 3 )P directs various proteins, including SGK3, to the early endosome ( 16, 17, 44, 55 ); similarly, it may direct SGK1 to this compartment. Previous reports support a role for SGK1 and Nedd4-2 in reciprocally modulating ENaC trafficking events in the endocytic pathway ( 7, 18, 32 ), although localization of SGK1 along this pathway remains to be demonstrated.8 _& P/ o, I8 _- M
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SGK1 has been found in multiple subcellular compartments, including nucleus ( 13, 35, 37, 42 ), cytoplasm ( 11, 13, 35, 42 ), plasma membrane ( 11 ), endoplasmic reticulum ( 5, 9 ), and mitochondria ( 22 ), which likely reflect the multiple functions SGK1 can perform within a given physiological context. SGK1 has a nuclear localization sequence (NLS) located within its kinase domain, yet our findings demonstrate that disruption of the NH 2 -terminal lipid binding domain weakens the suppressive effect of SGK1 on Forkhead-driven gene transcription ( Fig. 6 ). This suggests that the NLS is not sufficient for directing SGK1 repression of FKHRL1 and is consistent with the possibility that nuclear phosphoinositides, including PI( 3 )P ( 24 ), PI( 4 )P ( 15 ), or PI( 5 )P ( 15, 25 ), are implicated in SGK1 nuclear localization under the appropriate conditions. With regard to SGK1 localization to endoplasmic reticulum, recent studies have implicated a stretch of hydrophobic amino acids within the NH 2 -terminus [residues 19-24 ( 9 ) or 18-30 ( 5 )] that is important for targeting SGK1 to this compartment and for ubiquitination and proteasomal degradation. Another recent study identified overlapping residues ( 17 - 32 ) as important for targeting SGK1 to the mitochondria and for proteasomal degradation ( 22 ). While the mechanisms by which the NH 2 terminus directs SGK1 to the endoplasmic reticulum or mitochondria remain unclear, it is notable that the residues implicated in SGK1 subcellular targeting overlap with the lipid binding domain; protein-lipid interactions provide a compelling mechanism for SGK1 recruitment to these various compartments. For instance, in yeast and mammalian cells, the major site for synthesis of PS is the endoplasmic reticulum; after its synthesis, PS can be transported to the plasma membrane, mitochondria, Golgi apparatus, and endosomes ( 47, 50 - 54, 56 ). We propose that the more promiscuous phosphoinositide binding profile of the SGK1 NH 2 terminus might contribute to its regulation of diverse cellular processes by directing it to these multiple compartments in a context-dependent fashion.5 R7 p- b$ l# a9 I3 n
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GRANTS

This work was supported by grants from National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases [K08-DK-073487 (to A. C. Pao), P30-DK-63720 (to D. Pearce), and R01-DK-56695 (to D. Pearce)] and from the American Heart Association Western States Postdoctoral Fellowship (0225080Y to J. A. McCormick).
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Present address of J. A. McCormick: Div. of Nephrology and Hypertension, Dept. of Medicine, Oregon Health and Science University, Portland, OR 97239.

ACKNOWLEDGMENTS& p  |# n& F& Z* Q

We are grateful to K. Ashrafi for helpful comments on the manuscript.
      【参考文献】. l$ ]) N+ G4 L) r6 _3 y2 D. `
Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, Staub O. Defective regulation of the epithelial Na channel by Nedd4 in Liddle's syndrome. J Clin Invest 103: 667-673, 1999.  r: |( Z7 O5 R

/ ^" F" K! b* P) I, s
Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15: 6541-6551, 1996." I& E, S) O, @3 I
8 ^% j3 o+ N, _9 Z

+ i) y6 X: A  G+ C+ {% q
Alessi DR, Deak M, Casamayor A, Caudwell FB, Morrice N, Norman DG, Gaffney P, Reese CB, MacDougall CN, Harbison D, Ashworth A, Bownes M. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol 7: 776-789, 1997.5 Q7 I* `/ d4 m5 M& Y* u- i3 W
4 c1 d5 G( v3 F8 c

Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, Cohen P. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7: 261-269, 1997.0 {1 S5 h1 q, I; h) h/ g# U, o
! a/ C9 v. y6 t1 X- P5 f* o

. l* T& k1 |0 i+ h9 c/ s
Arteaga MF, Wang L, Ravid T, Hochstrasser M, Canessa CM. An amphipathic helix targets serum and glucocorticoid-induced kinase 1 to the endoplasmic reticulum-associated ubiquitin-conjugation machinery. Proc Natl Acad Sci USA 103: 11178-11183, 2006.
6 x# J& U8 _+ h- m$ X

Bhalla V, Daidie D, Li H, Pao AC, LaGrange LP, Wang J, Vandewalle A, Stockand JD, Staub O, Pearce D. Serum- and glucocorticoid-regulated kinase 1 regulates ubiquitin ligase neural precursor cell-expressed, developmentally downregulated protein 4-2 by inducing interaction with 14-3-3. Mol Endocrinol 19: 3073-3084, 2005.# q3 i2 g/ I- p5 |- Q2 K- O

0 F% o0 w/ y/ ?
! q: r  Y, k+ E- O
Bhalla V, Soundararajan R, Pao AC, Li H, Pearce D. Disinhibitory pathways for control of sodium transport: regulation of ENaC by SGK1 and GILZ. Am J Physiol Renal Physiol In press.9 z) S+ j4 d+ r6 ?5 C. [
! X, _7 o& I) M+ u7 o* M# d0 `

Biondi RM, Kieloch A, Currie RA, Deak M, Alessi DR. The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J 20: 4380-4390, 2001.

7 \# [8 d( p" \8 D* o) p3 X0 }. i
Bogusz AM, Brickley DR, Pew T, Conzen SD. A novel N-terminal hydrophobic motif mediates constitutive degradation of serum- and glucocorticoid-induced kinase-1 by the ubiquitin-proteasome pathway. FEBS J 273: 2913-2928, 2006.$ x3 X2 Z5 O! L( `; a

% K# H1 t! S" p9 _
Bravo J, Karathanassis D, Pacold CM, Pacold ME, Ellson CD, Anderson KE, Butler PJ, Lavenir I, Perisic O, Hawkins PT, Stephens L, Williams RL. The crystal structure of the PX domain from p40(phox) bound to phosphatidylinositol 3-phosphate. Mol Cell 8: 829-839, 2001.

( m' X" P8 ~3 }' Z; _3 C

Brickley DR, Mikosz CA, Hagan CR, Conzen SD. Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1). J Biol Chem 277: 43064-43070, 2002.! K) o% n4 B# @% `. F& @& G( {

  T4 w/ ~; m" |1 [5 Y2 w

Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21: 952-965, 2001.

( d6 ?3 J0 C% Y9 ~% u
Buse P, Tran SH, Luther E, Phu PT, Aponte GW, Firestone GL. Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways. J Biol Chem 274: 7253-7263, 1999.
) s$ p# L' u& o# X) ]9 }
/ `- |7 }7 b; m& @& H" D
; D6 X0 y  e, m9 L" z
Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, Pearce D. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514-2519, 1999." ~; ]8 ]7 B. U, H9 }
* W/ {  k/ c. X* S
1 T# Z4 y1 y5 K9 J  J9 q" |( ?
, a& Y: ], y7 X$ F
Clarke JH, Letcher AJ, D'Santos CS, Halstead JR, Irvine RF, Divecha N. Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells. Biochem J 357: 905-910, 2001.0 B" Z( N" T& e  h8 P5 C5 @
. b4 M* h& u; E. d. j

1 j4 ?* o$ _4 c5 l5 A6 z( r8 k
Czech MP. Dynamics of phosphoinositides in membrane retrieval and insertion. Annu Rev Physiol 65: 791-815, 2003., J2 O" _6 F$ t; K. z, [

De Matteis MA, Godi A. PI-loting membrane traffic. Nat Cell Biol 6: 487-492, 2004., R  m: m2 V6 `2 T
4 ]5 ~2 h6 n: J4 F* i0 A
' @6 |9 W, R# Q
+ n+ M8 O' R2 U# ^+ U/ V* |3 d. Z
Debonneville C, Flores SY, Kamynina E, Plant PJ, Tauxe C, Thomas MA, Munster C, Chraibi A, Pratt JH, Horisberger JD, Pearce D, Loffing J, Staub O. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na channel cell surface expression. EMBO J 20: 7052-7059, 2001.! }3 k5 `! e- I( F, a* u
2 K! j2 \4 b) Q" A6 I3 h- r8 B7 \
. v- O8 e; h: E) x

Dowler S, Currie RA, Campbell DG, Deak M, Kular G, Downes CP, Alessi DR. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities. Biochem J 351: 19-31, 2000.2 J* _7 a6 J/ [8 G& g- m( l
% o( g4 W/ i4 j- k, ^& M" h* |
- r1 \5 r6 C3 a

Dowler S, Currie RA, Downes CP, Alessi DR. DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides. Biochem J 342: 7-12, 1999.% Z: E! W0 z, r

/ h3 @# N, t! v# @
Dowler S, Kular G, Alessi DR. Protein lipid overlay assay. Sci STKE 2002: PL6, 2002.1 f8 N0 F+ _6 X: L
2 H( O. f) j6 n7 C8 ?4 c6 Q, p

9 S- R* ]: ^- Q4 ]$ u" O
Engelsberg A, Kobelt F, Kuhl D. The N-terminus of the serum- and glucocorticoid-inducible kinase Sgk1 specifies mitochondrial localization and rapid turnover. Biochem J 399: 69-76, 2006.

$ z5 T# M6 z- n5 L* O1 x! j  W
2 m% [& N# c. e5 [
Friedrich B, Feng Y, Cohen P, Risler T, Vandewalle A, Broer S, Wang J, Pearce D, Lang F. The serine/threonine kinases SGK2 and SGK3 are potent stimulators of the epithelial Na channel alpha,beta,gamma-ENaC. Pflügers Arch 445: 693-696, 2003.) h6 |) K8 N8 u) s$ x6 \9 K! M
1 s; K- ?, a. }2 X, D; O/ _6 \
# Q7 g  F% V+ D3 b$ L$ E

Gillooly DJ, Morrow IC, Lindsay M, Gould R, Bryant NJ, Gaullier JM, Parton RG, Stenmark H. Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J 19: 4577-4588, 2000.

Gozani O, Karuman P, Jones DR, Ivanov D, Cha J, Lugovskoy AA, Baird CL, Zhu H, Field SJ, Lessnick SL, Villasenor J, Mehrotra B, Chen J, Rao VR, Brugge JS, Ferguson CG, Payrastre B, Myszka DG, Cantley LC, Wagner G, Divecha N, Prestwich GD, Yuan J. The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 114: 99-111, 2003.

$ X: m# Z9 b4 j
; E& G$ V7 U% r3 _  y
Hayashi M, Tapping RI, Chao TH, Lo JF, King CC, Yang Y, Lee JD. BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase. J Biol Chem 276: 8631-8634, 2001.
/ H* o4 ^8 @. n

8 ~, ^+ b8 W9 [1 v2 C
Helms MN, Fejes-Toth G, Naray-Fejes-Toth A. Hormone-regulated transepithelial Na transport in mammalian CCD cells requires SGK1 expression. Am J Physiol Renal Physiol 284: F480-F487, 2003.) m" a7 {  O, Z, h
8 Y2 F8 B5 b" g

Ichimura T, Yamamura H, Sasamoto K, Tominaga Y, Taoka M, Kakiuchi K, Shinkawa T, Takahashi N, Shimada S, Isobe T. 14-3-3 Proteins modulate the expression of epithelial Na channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin ligase. J Biol Chem 280: 13187-13194, 2005.
) w; ]9 f8 p4 m: ^
- d" O& e4 W! u$ h( C+ z; `
% q: {4 ?9 f' |
Kamynina E, Debonneville C, Bens M, Vandewalle A, Staub O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na channel. FASEB J 15: 204-214, 2001.

" I: g! y+ U# |0 M1 s* N! c' D

Kanai F, Liu H, Field SJ, Akbary H, Matsuo T, Brown GE, Cantley LC, Yaffe MB. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol 3: 675-678, 2001.. W2 H8 R+ D9 T- i9 f& M
# ]4 q3 c5 p' m" j
; D& p# B' c) e: z" [

Karathanassis D, Stahelin RV, Bravo J, Perisic O, Pacold CM, Cho W, Williams RL. Binding of the PX domain of p47(phox) to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction. EMBO J 21: 5057-5068, 2002.0 ^& M  N) h6 l  O% w8 z' l8 y

9 O$ p5 ~2 l5 v7 M1 I

Knight KK, Olson DR, Zhou R, Snyder PM. Liddle's syndrome mutations increase Na transport through dual effects on epithelial Na channel surface expression and proteolytic cleavage. Proc Natl Acad Sci USA 103: 2805-2808, 2006.
9 v3 s8 a7 G* d8 C' {/ v5 }
7 J7 q3 ?- L3 A3 b1 i

Kobayashi T, Cohen P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J 339: 319-328, 1999.
5 }3 E" v4 s2 V
$ q* s- G0 G4 d4 L: ~( T
1 @6 ?3 f' u+ J9 E& p2 R5 P4 M9 i
Kobayashi T, Deak M, Morrice N, Cohen P. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem J 344: 189-197, 1999.
0 g7 ~' ]; n- f& F0 Z8 P
) G% R- l3 k0 F% A" `
9 E" G9 K- L; p+ Z; X
Leong ML, Maiyar AC, Kim B, O'Keeffe BA, Firestone GL. Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells. J Biol Chem 278: 5871-5882, 2003.2 P  T. a  |5 F- A6 h7 ?

Liang X, Peters KW, Butterworth MB, Frizzell RA. 14-3-3 Isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels. J Biol Chem 281: 16323-16332, 2006.6 m; c% Q* p% M, L  i
' D0 f- }' n$ L

  e2 A* }1 ~8 m8 g  ~
Maiyar AC, Leong ML, Firestone GL. Importin-alpha mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain. Mol Biol Cell 14: 1221-1239, 2003., |; `1 u# k* T5 ^( ^9 V
8 M$ m/ ?- X7 A3 n9 ^
8 X/ M5 A6 M- v; }2 Y

McCormick JA, Feng Y, Dawson K, Behne MJ, Yu B, Wang J, Wyatt AW, Henke G, Grahammer F, Mauro TM, Lang F, Pearce D. Targeted disruption of the protein kinase SGK3/CISK impairs postnatal hair follicle development. Mol Biol Cell 15: 4278-4288, 2004.

- D' l- E9 `) ^! i7 o9 B9 R

Mikosz CA, Brickley DR, Sharkey MS, Moran TW, Conzen SD. Glucocorticoid receptor-mediated protection from apoptosis is associated with induction of the serine/threonine survival kinase gene, sgk-1. J Biol Chem 276: 16649-16654, 2001.
. m; E5 E3 q# ]- r! l8 ]( }

Naray-Fejes-Toth A, Canessa C, Cleaveland ES, Aldrich G, Fejes-Toth G. sgk Is an aldosterone-induced kinase in the renal collecting duct. Effects on epithelial Na channels. J Biol Chem 274: 16973-16978, 1999.: \. d! }; g0 p  a6 o1 h7 f

" O, k: v' Y1 y# V# p
Naray-Fejes-Toth A, Helms MN, Stokes JB, Fejes-Toth G. Regulation of sodium transport in mammalian collecting duct cells by aldosterone-induced kinase, SGK1: structure/function studies. Mol Cell Endocrinol 217: 197-202, 2004.
9 o' s# d2 C  P7 H  R
( [$ A1 ?- l% z9 ?$ p

Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, Hemmings BA. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18: 3024-3033, 1999.
1 \/ b$ f* m1 Z
+ \( O$ H- F5 y
* C4 L4 D. s1 ?' w" w# t- F
Ponting CP. Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? Protein Sci 5: 2353-2357, 1996.; W5 F7 c4 @$ s+ P1 @$ T4 R9 i# M5 h

& T1 E2 V+ r% ~2 p- @  y

Roth MG. Phosphoinositides in constitutive membrane traffic. Physiol Rev 84: 699-730, 2004.8 A! ]) B% h& _" K3 T

& Y& U) u6 Z5 C

Rozansky DJ, Wang J, Doan N, Purdy T, Faulk T, Bhargava A, Dawson K, Pearce D. Hypotonic induction of SGK1 and Na transport in A6 cells. Am J Physiol Renal Physiol 283: F105-F113, 2002.( l8 U0 {7 I/ ~& t' V6 g
; r% a* r/ P# z4 }8 d, A/ A; @) I

Snyder PM, Olson DR, Thomas BC. Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na channel. J Biol Chem 277: 5-8, 2002." J5 _. l3 z' E+ l/ u

& u, e  N, b6 x6 d" {0 t. l8 E% o

Steenbergen R, Nanowski TS, Beigneux A, Kulinski A, Young SG, Vance JE. Disruption of the phosphatidylserine decarboxylase gene in mice causes embryonic lethality and mitochondrial defects. J Biol Chem 280: 40032-40040, 2005.

Stephens L, Anderson K, Stokoe D, Erdjument-Bromage H, Painter GF, Holmes AB, Gaffney PR, Reese CB, McCormick F, Tempst P, Coadwell J, Hawkins PT. Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279: 710-714, 1998.) @+ G, \2 _6 w8 e$ o2 e

. ~& B1 a% w/ d- R
Stokoe D, Stephens LR, Copeland T, Gaffney PR, Reese CB, Painter GF, Holmes AB, McCormick F, Hawkins PT. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277: 567-570, 1997.) d3 y2 K0 U3 A3 u, H$ g( Y

3 B) x+ f* N: [8 b* c
; |( f! ^; T* ]
Trotter PJ, Pedretti J, Voelker DR. Phosphatidylserine decarboxylase from Saccharomyces cerevisiae. Isolation of mutants, cloning of the gene, and creation of a null allele. J Biol Chem 268: 21416-21424, 1993.% h: _, c6 X- h0 Y
: E; J$ {- @2 @9 I" R- W9 E3 \6 |* W

Trotter PJ, Pedretti J, Yates R, Voelker DR. Phosphatidylserine decarboxylase 2 of Saccharomyces cerevisiae. Cloning and mapping of the gene, heterologous expression, and creation of the null allele. J Biol Chem 270: 6071-6080, 1995.; M7 _; l+ K  k3 M
% C, Z$ y: l. V% ]) B0 ?
: j, x( n& ~, P" ]  w2 r7 Z! F3 [

Trotter PJ, Voelker DR. Identification of a non-mitochondrial phosphatidylserine decarboxylase activity (PSD2) in the yeast Saccharomyces cerevisiae. J Biol Chem 270: 6062-6070, 1995.
% `: e" n  T, L
4 I. F  n1 u, ?3 ~6 F# o

Vance JE. Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum. J Biol Chem 266: 89-97, 1991.$ P7 K; {4 f, I! V% b$ x7 `- X' ?

Vance JE. Phospholipid synthesis in a membrane fraction associated with mitochondria. J Biol Chem 265: 7248-7256, 1990./ J/ v7 Z- ^( m7 g. K$ a

) @  |9 h6 t$ i! y. R

Virbasius JV, Song X, Pomerleau DP, Zhan Y, Zhou GW, Czech MP. Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate. Proc Natl Acad Sci USA 98: 12908-12913, 2001.
& J+ o. j0 q- j6 M

) {' X  X7 Q: g
Voelker DR. Protein and lipid motifs regulate phosphatidylserine traffic in yeast. Biochem Soc Trans 33: 1141-1145, 2005.7 a9 }2 u3 H  B' U# G0 R

" J  P' [$ L2 F" q6 [2 R

Waldegger S, Barth P, Forrest JN Jr, Greger R, Lang F. Cloning of sgk serine-threonine protein kinase from shark rectal gland-a gene induced by hypertonicity and secretagogues. Pflügers Arch 436: 575-580, 1998.
" v7 O% o- C0 u, [( Y# l
( `# Q* U! h+ Q1 p7 c/ N

Wang J, Barbry P, Maiyar AC, Rozansky DJ, Bhargava A, Leong M, Firestone GL, Pearce D. SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am J Physiol Renal Physiol 280: F303-F313, 2001.

0 B- V% \6 y2 |. x. {1 U
% C+ Y8 l, ~# s. k
Xing Y, Liu D, Zhang R, Joachimiak A, Songyang Z, Xu W. Structural basis of membrane targeting by the Phox homology domain of cytokine-independent survival kinase (CISK-PX). J Biol Chem 279: 30662-30669, 2004.
7 I+ M1 M5 ~3 g! J) N
) w. @  }' A/ N" a9 f! m& Z

Xu J, Liu D, Gill G, Songyang Z. Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides. J Cell Biol 154: 699-705, 2001.
7 i/ C0 F1 n  \% ?; X
9 \8 M1 g& t4 A
9 e* B( D& ]# f
Zhou R, Snyder PM. Nedd4-2 phosphorylation induces serum and glucocorticoid-regulated kinase (SGK) ubiquitination and degradation. J Biol Chem 280: 4518-4523, 2005.

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