Glucocorticoid-induced Protein Kinase Research Articles

WNK1 activates SGK1 to regulate the epithelial sodium channel

WNK (with no lysine [K]) kinases are serine-threonine protein kinases with an atypical placement of the catalytic lysine. Intronic deletions increase the expression of WNK1 in humans and cause pseudohypoaldosteronism type II, a form of hypertension. WNKs have been linked to ion carriers, but the underlying regulatory mechanisms are unknown. Here, we report a mechanism for the control of ion permeability by WNK1. We show that WNK1 activates the serum- and glucocorticoid-inducible protein kinase SGK1, leading to activation of the epithelial sodium channel. Increased channel activity induced by WNK1 depends on SGK1 and the E3 ubiquitin ligase Nedd4-2. This finding provides compelling evidence that this molecular mechanism contributes to the pathogenesis of hypertension in pseudohypoaldosteronism type II caused by WNK1 and, possibly, in other forms of hypertension.

Functional specificity of Sgk1 and Akt1 on ENaC activity

Reabsorption of sodium by the epithelial sodium channel (ENaC) is essential for maintaining the volume of the extracellular compartment and blood pressure. The function of ENaC is regulated primarily by aldosterone, antidiuretic hormone [arginine vasopressin (AVP)], and insulin, but the molecular mechanisms that increase channel activity are still poorly understood. It has been proposed that the related serine/threonine kinases serum- and glucocorticoid-induced kinase (Sgk1) and protein kinase B (Akt) mediate activation of ENaC. Here, we addressed the question of whether there is functional specificity of these kinases for the activation of ENaC in epithelial cells of the distal renal tubule. We demonstrate that Akt does not increase ENaC function under basal conditions or after stimulation with aldosterone, insulin, or AVP. In contrast, under the same experimental conditions, Sgk1 increases ENaC activity by 10-fold. The effect of Sgk1 is additive to that of aldosterone, whereas, in the presence of active Sgk1, cells do not further respond to insulin or AVP. We conclude that, in cells expressing both kinases, modulation of ENaC activity is mediated by Sgk1 but not by Akt1.

14-3-3 Proteins Modulate the Expression of Epithelial Na+ Channels by Phosphorylation-dependent Interaction with Nedd4-2 Ubiquitin Ligase

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.

Antiapoptotic Effect of Serum and Glucocorticoid-Inducible Protein Kinase Is Mediated by Novel Mechanism Activating IκB Kinase

Abstract Serum and glucocorticoid inducible protein kinase (SGK) plays a crucial role in promoting cell survival, but the mechanisms for this response are not clear. We show that SGK is involved in the regulation of apoptosis in breast cancer cells by modulating the transcriptional activity of nuclear transcription factor κB (NF-κB). High levels of SGK expression were observed in human breast cancer samples. When SGK was reduced the apoptotic rate increased, and increased SGK activity prevents serum withdrawal–induced apoptosis. SGK-induced cell survival was abolished by a dominant-negative form of IκB kinase β (IKKβ, K44A) or a null mutation of IKKβ in mouse embryonic fibroblast cells indicating involvement of the NF-κB pathway. Serum-induced SGK or increased expression of SGK activated NF-κB transcriptional activity, whereas small interference RNA to SGK blocked NF-κB activity. Coexpression of SGK and IKKβ significantly increased the activation of NF-κB (versus expression of IKKβ alone). Expression of dominant-negative IKKβ K44A, IκBα AA, and kinase-dead SGK (127KM) blocked the ability of SGK to stimulate NF-κB activity, suggesting that IKKβ is a target of SGK. We also show that SGK enhances the ability of IKKβ to phosphorylate endogenous IκBα in cells or recombinant glutathione S-transferase-IκBα in vitro and increases IκBα degradation; SGK physically associates with and activates IKKβ in MDA231 cells via phosphorylation of Ser181 in IKKβ. Taken together, we conclude that SGK acts as an oncogene in breast cancer cells through activation of the IKK-NF-κB pathway, thereby preventing apoptosis. Blocking SGK expression/activity represents a potential therapeutic approach for breast cancer treatment.

14-3-3 Protein Mediates Phosphorylation of Microtubule-associated Protein Tau by Serum- and Glucocorticoid-induced Protein Kinase 1

14-3-3 Protein Mediates Phosphorylation of Microtubule-associated Protein Tau by Serum- and Glucocorticoid-induced Protein Kinase 1

35 Clinical experiences with mTOR inhibitors

The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by PtdIns(3,4,5)P3, comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.

PDK1, the master regulator of AGC kinase signal transduction.

The interaction of insulin and growth factors with their receptors on the outside surface of a cell, leads to the activation of phosphatidylinositol 3-kinase (PI 3-kinase) and generation of the phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) second messenger at the inner surface of the cell membrane. One of the most studied signalling events controlled by PtdIns(3,4,5)P3, comprises the activation of a group of AGC family protein kinases, including isoforms of protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK) and protein kinase C (PKC), which play crucial roles in regulating physiological processes relevant to metabolism, growth, proliferation and survival. Here, we review recent biochemical, genetic and structural studies on the 3-phosphoinositide-dependent protein kinase-1 (PDK1), which phosphorylates and activates the AGC kinase members regulated by PI 3-kinase. We also discuss whether inhibitors of PDK1 might have chemotherapeutic potential in the treatment of cancers in which the PDK1-regulated AGC kinases are constitutively activated.

WNK1, the kinase mutated in an inherited high-blood-pressure syndrome, is a novel PKB (protein kinase B)/Akt substrate.

Recent evidence indicates that mutations in the gene encoding the WNK1 [with no K (lysine) protein kinase-1] results in an inherited hypertension syndrome called pseudohypoaldosteronism type II. The mechanisms by which WNK1 is regulated or the substrates it phosphorylates are currently unknown. We noticed that Thr-60 of WNK1, which lies N-terminal to the catalytic domain, is located within a PKB (protein kinase B) phosphorylation consensus sequence. We found that PKB phosphorylated WNK1 efficiently compared with known substrates, and both peptide map and mutational analysis revealed that the major PKB site of phosphorylation was Thr-60. Employing a phosphospecific Thr-60 WNK1 antibody, we demonstrated that IGF1 (insulin-like growth factor) stimulation of HEK-293 cells induced phosphorylation of endogenously expressed WNK1 at Thr-60. Consistent with PKB mediating this phosphorylation, inhibitors of PI 3-kinase (phosphoinositide 3-kinase; wortmannin and LY294002) but not inhibitors of mammalian target of rapamycin (rapamycin) or MEK1 (mitogen-activated protein kinase kinase-1) activation (PD184352), inhibited IGF1-induced phosphorylation of endogenous WNK1 at Thr-60. Moreover, IGF1-induced phosphorylation of endogenous WNK1 did not occur in PDK1-/- ES (embryonic stem) cells, in which PKB is not activated. In contrast, IGF1 still induced normal phosphorylation of WNK1 in PDK1(L155E/L155E) knock-in ES cells in which PKB, but not S6K (p70 ribosomal S6 kinase) or SGK1 (serum- and glucocorticoid-induced protein kinase 1), is activated. Our study provides strong pharmacological and genetic evidence that PKB mediates the phosphorylation of WNK1 at Thr-60 in vivo. We also performed experiments which suggest that the phosphorylation of WNK1 by PKB is not regulating its kinase activity or cellular localization directly. These results provide the first connection between the PI 3-kinase/PKB pathway and WNK1, suggesting a mechanism by which this pathway may influence blood pressure.

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

The effects of multiple stress stimuli on the cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) were examined in NMuMg mammary epithelial cells exposed to hyperosmotic stress induced by the organic osmolyte sorbitol, heat shock, ultraviolet irradiation, oxidative stress induced by hydrogen peroxide, or to dexamethasone, a synthetic glucocorticoid that represents a general class of physiological stress hormones. Each of the stress stimuli induced Sgk protein expression with differences in the kinetics and duration of induction and in subcellular localization. The environmental stresses, but not dexamethasone, stimulated Sgk expression through a p38/MAPK-dependent pathway. In each case, a hyperphosphorylated active Sgk protein was produced under conditions in which Akt, the close homolog of Sgk, remained in its non-phosphorylated state. Ectopic expression of wild type Sgk or of the T256D/S422D mutant Sgk that mimics phosphorylation conferred protection against stress-induced cell death in NMuMg cells. In contrast, expression of the T256A/S422A Sgk phosphorylation site mutant has no effect on cell survival. Sgk is known to phosphorylate and negatively regulate pro-apoptotic forkhead transcription factor FKHRL1. The environmental stress stimuli that induce Sgk, but not dexamethasone, strongly inhibited the nuclear transcriptional activity and increased the cytoplasmic retention of FKHRL1. Also, the conditional IPTG inducible expression of wild type Sgk, but not of the kinase dead T256A mutant Sgk, protected Con8 mammary epithelial tumor cells from serum starvation-induced apoptosis. Taken together, our study establishes that induction of enzymatically active Sgk functions as a key cell survival component in response to different environmental stress stimuli.

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.

The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-alpha nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-alpha with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-alpha, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-alpha to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131-141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-alpha and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-alpha as well as the PI3-kinase-dependent phosphorylation of Sgk. Our results define a new role importin-alpha in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.

Regulation of KCNE1-dependent K(+) current by the serum and glucocorticoid-inducible kinase (SGK) isoforms.

The slowly activating K(+) channel subunit KCNE1 is expressed in a variety of tissues including proximal renal tubules, cardiac myocytes and stria vascularis of inner ear. The present study has been performed to explore whether the serum- and glucocorticoid-inducible kinase family members SGK1, SGK2, or SGK3 and/or protein kinase B (PKB) influence K(+) channel activity in Xenopus oocytes expressing KCNE1. cRNA encoding KCNE1 was injected with or without cRNA encoding wild-type SGK1, constitutively active (S422D)SGK1, inactive (K127 N)SGK1, wild-type SGK2, wild-type SGK3 or constitutively active (T308D,S473D)PKB. In oocytes injected with KCNE1 cRNA but not in water-injected oocytes a depolarization from -80 mV to -10 mV led to the appearance of a slowly activating K(+) current. Coexpression of SGK1,( S422D)SGK1, SGK2, SGK3 or (T308D,S473D)PKB but not (K127 N)SGK1 significantly stimulated KCNE1-induced current. The effect did not depend on Na(+)/K(+)-ATPase activity. KCNE1-induced current was markedly upregulated by coexpression of KCNQ1 and further increased by additional expression of (S422D)SGK1, SGK2, SGK3 or (T308D,S473D)PKB. In conclusion, all three members of the SGK family of kinases SGK1-3 and protein kinase B stimulate the slowly activating K(+) channel KCNE1/KCNQ1. The kinases may thus participate in the regulation of KCNE1-dependent transport and excitability.

Ubiquitin Modification of Serum and Glucocorticoid-induced Protein Kinase-1 (SGK-1)

The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (delta60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1.

The Na +/H + exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by 3-phosphoinositide-dependent protein kinase 1

The Na +/H + exchanger regulatory factor 2 (NHERF2/TKA-1/E3KARP) contains two PSD-95/Dlg/ZO-1 (PDZ) domains which interact with the PDZ docking motif (X–(S/T)–X–(V/L)) of proteins to mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. One of the PDZ domains of NHERF2 interacts specifically with the DSLL, DSFL, and DTRL motifs present at the carboxy-termini of the 2-adrenergic receptor, the platelet-derived growth factor receptor, and the cystic fibrosis transmembrane conductance regulator, respectively. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) also carries a putative PDZ-binding motif (D–S–F–L) at its carboxy tail, implicated in the specific interaction with NHERF2. There is a 3-phosphoinositide-dependent protein kinase 1 (PDK1) interacting fragment (PIF) in the tail of NHERF2. Using pull-down assays and co-transfection experiments, we demonstrated that the DSFL tail of SGK1 interacts with the first PDZ domain of NHERF2 and the PIF of NHERF2 binds to the PIF-binding pocket of PDK1 to form an SGK1–NHERF2–PDK1 complex. Formation of the protein complex promoted the phosphorylation and activation of SGK1 by PDK1. Thus, it was suggested that NHERF2 mediates the activation and phosphorylation of SGK1 by PDK1 through its first PDZ domain and PIF motif, as a novel SGK1 activation mechanism.

Molecular Basis for the Substrate Specificity of NIMA-related Kinase-6 (NEK6): EVIDENCE THAT NEK6 DOES NOT PHOSPHORYLATE THE HYDROPHOBIC MOTIF OF RIBOSOMAL S6 PROTEIN KINASE AND SERUM- AND GLUCOCORTICOID-INDUCED PROTEIN KINASE IN VIVO

The AGC family of protein kinases, which includes isoforms of protein kinase B (also known as Akt), ribosomal S6 protein kinase (S6K), and serum- and glucocorticoid-induced protein kinase (SGK) are activated in response to many extracellular signals and play key roles in regulating diverse cellular processes. They are activated by the phosphorylation of the T loop of their kinase domain by the 3-phosphoinositide-dependent protein kinase-1 and by phosphorylation of a residue located C-terminal to the kinase domain in a region termed the hydrophobic motif. Recent work has implicated the NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) as the enzyme that phosphorylates the hydrophobic motif of S6K1 in vivo. Here we demonstrate that in addition to phosphorylating S6K1 and SGK1 at their hydrophobic motif, NEK6 also phosphorylates S6K1 at two other sites and phosphorylates SGK1 at one other site in vitro. Employing the Jerini pepSTAR method in combination with kinetic analysis of phosphorylation of variant peptides, we establish the key substrate specificity determinants for NEK6. Our analysis indicates that NEK6 has a strong preference for Leu 3 residues N-terminal to the site of phosphorylation. Its mutation to either Ile or Val severely reduced the efficacy with which NEK6-phosphorylated peptide substrates, and moreover, mutation of the equivalent Leu residue in S6K1 or SGK1 prevented phosphorylation of their hydrophobic motifs by NEK6 in vitro. However, these mutants of S6K1 or SGK1 still became phosphorylated at their hydrophobic motif following insulin-like growth factor-1 stimulation of transfected 293 cells. This study provides the first description of the basis for the substrate specificity of NEK6 and indicates that NEK6 is unlikely to be responsible for the IGF1-induced phosphorylation of the hydrophobic motif of S6K, SGK, and protein kinase B isoforms in vivo.

Pkh1 and Pkh2 differentially phosphorylate and activate Ypk1 and Ykr2 and define protein kinase modules required for maintenance of cell wall integrity.

Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of a ypk1Delta strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Delta ypk1Delta, pkh2Delta ypk1Delta, pkh1Delta ykr2Delta, and pkh2Delta ykr2Delta) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature, ypk1-1(ts) ykr2Delta cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of the PKC1 pathway) was lethal in ypk1-1(ts) ykr2Delta cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that acts in parallel with the Pkc1-dependent pathway.

The Phosphatidylinositol 3-Kinase (PI3K)-Akt Pathway Suppresses Bax Translocation to Mitochondria

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria in response to apoptotic stimuli, where it induces cytochrome c release. In this study, we show that the phosphatidylinositol 3-OH kinase (PI3K)-Akt pathway plays an important role in the regulation of Bax subcellular localization. We found that LY294002, a PI3K inhibitor, blocked the effects of serum to prevent Bax translocation to mitochondria and that expression of an active form of PI3K suppressed staurosporine-induced Bax translocation, suggesting that PI3K activity is essential for retaining Bax in the cytoplasm. In contrast, both U0126, a MEK inhibitor, and active MEK had little effect on Bax localization. In respect to downstream effectors of PI3K, we found that expression of active Akt, but not serum and glucocorticoid-induced protein kinase (SGK), suppressed staurosporine-induced translocation of Bax, whereas dominant negative Akt moderately promoted Bax translocation. Expression of Akt did not alter the levels of Bax, Bcl-2, Bcl-X(L), or phosphorylated JNK under the conditions used, suggesting that there were alternative mechanisms for Akt in the suppression of Bax translocation. Collectively, these results suggest that the PI3K-Akt pathway inhibits Bax translocation from cytoplasm to mitochondria and have revealed a novel mechanism by which the PI3K-Akt pathway promotes survival.

Glucocorticoid Activation of Na+/H+Exchanger Isoform 3 Revisited: THE ROLES OF SGK1 AND NHERF2

The stimulative effect of glucocorticoids on intestinal salt and water absorption has been known for more than two decades. However, molecular mechanisms underlying this activation remain elusive. Previous studies showed that methylprednisolone specifically increased Na(+)/H(+) exchanger isoform (NHE) 3 mRNA in ileum and kidney without affecting NHE1 mRNA levels. These results suggest that glucocorticoids activate NHE3 activity by induction of NHE3 transcripts. We recently found in PS120 and opossum kidney cells that chronic incubation with dexamethasone activated NHE3 independent of gene induction, indicating that the transcriptional activation may not be the only determining factor in the NHE3 activation. Furthermore, dexamethasone activated NHE3 activity only in the presence of a NHE3 regulatory protein, NHERF2, which was previously shown to confer cAMP-dependent inhibition of NHE3. This activation of NHE3 could not be duplicated by NHERF1. We identified serum- and glucocorticoid-induced protein kinase, SGK1, as the protein interacting with PDZ domains of NHERF2 to regulate NHE3 activity. The expression of SGK1 enhanced NHE3 transport in PS120 fibroblasts. In addition, the "kinase-dead" SGK1 blocked activation of NHE3 by dexamethasone in opossum kidney cells. These data demonstrated that glucocorticoid activation of NHE3 requires the activation of SGK1 and the presence of NHERF2 acting as a scaffold protein.

Regulation and physiological roles of serum- and glucocorticoid-induced protein kinase isoforms.

Serum- and glucocorticoid-induced protein kinase 1 (SGK1) was identified in 1993 as an immediate early gene whose mRNA levels increase dramatically within 30 minutes when cells are exposed to serum or glucocorticoids, or both. Subsequently, many other agonists, acting through a variety of signal transduction pathways, have been shown to induce SGK1 gene transcription in cells and tissues. SGK1 is a member of the "AGC" subfamily, which includes protein kinases A, G, and C, and its catalytic domain is most similar to protein kinase B (PKB). Like PKB, SGK1 is activated by phosphorylation in response to signals that stimulate phosphatidylinositol 3-kinase, and this is mediated by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and another protein kinase that has yet to be identified. Thus, SGK1 is remarkable in being activated at both the transcriptional and posttranslational levels by a huge number of extracellular signals. In contrast, little is known about the transcriptional regulation of the two closely related isoforms SGK2 and SGK3, although they can be activated by phosphorylation. The substrate specificity of SGK isoforms superficially resembles that of PKB in that serine and threonine residues lying in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr sequences (where Xaa is a variable amino acid) are phosphorylated. However, although they may have some substrates in common, evidence is emerging that SGK1 and PKB phosphorylate distinct proteins and have different functions in vivo. In particular, SGK1 plays an important role in activating certain potassium, sodium, and chloride channels, suggesting an involvement in the regulation of processes such as cell survival, neuronal excitability, and renal sodium excretion. Moreover, sustained high levels of SGK1 protein and activity may contribute to conditions such as hypertension and diabetic nephropathy. This raises the possibility that specific inhibitors of SGK1 may have therapeutic potential for the treatment of several diseases.

Regulation and Physiological Roles of Serum- and Glucocorticoid-Induced Protein Kinase Isoforms

Serum- and glucocorticoid-induced protein kinase 1 (SGK1) was identified in 1993 as an immediate early gene whose mRNA levels increase dramatically within 30 minutes when cells are exposed to serum or glucocorticoids, or both. Subsequently, many other agonists, acting through a variety of signal transduction pathways, have been shown to induce SGK1 gene transcription in cells and tissues. SGK1 is a member of the "AGC" subfamily, which includes protein kinases A, G, and C, and its catalytic domain is most similar to protein kinase B (PKB). Like PKB, SGK1 is activated by phosphorylation in response to signals that stimulate phosphatidylinositol 3-kinase, and this is mediated by 3-phosphoinositide-dependent protein kinase 1 (PDK1) and another protein kinase that has yet to be identified. Thus, SGK1 is remarkable in being activated at both the transcriptional and posttranslational levels by a huge number of extracellular signals. In contrast, little is known about the transcriptional regulation of the two closely related isoforms SGK2 and SGK3, although they can be activated by phosphorylation. The substrate specificity of SGK isoforms superficially resembles that of PKB in that serine and threonine residues lying in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr sequences (where Xaa is a variable amino acid) are phosphorylated. However, although they may have some substrates in common, evidence is emerging that SGK1 and PKB phosphorylate distinct proteins and have different functions in vivo. In particular, SGK1 plays an important role in activating certain potassium, sodium, and chloride channels, suggesting an involvement in the regulation of processes such as cell survival, neuronal excitability, and renal sodium excretion. Moreover, sustained high levels of SGK1 protein and activity may contribute to conditions such as hypertension and diabetic nephropathy. This raises the possibility that specific inhibitors of SGK1 may have therapeutic potential for the treatment of several diseases.

Tissue-specific expression of the transcriptionally regulated serum and glucocorticoid-inducible protein kinase (Sgk) during mouse embryogenesis.

In situ hybridization of mouse embryo whole mounts and sagittal sections revealed a tissue- and stage-specific expression pattern of the transcriptionally regulated serum and glucocorticoid-inducible protein kinase (sgk) during embryogenesis. Sgk expression is first observed at embryonic day 8.5 (E8.5) in the decidua and yolk sac, and then during developmental stages E9.5 through E12.5 this kinase is highly localized in the heart chamber, otic vesicle, blood vessels surrounding the somites, and lung buds. At the later stages of mouse embryogenesis, E13.5 through E16.5, sgk expression becomes highly concentrated in brain (choroid plexus), distal epithelium and the terminal bronchi/bronchioles, adrenal gland, liver, thymus and intestines, remains high in heart tissue, and is expressed at a low level in the other embryonic tissues.