Serum- And Glucocorticoid-regulated Kinase Research Articles

The Progesterone Receptor Hinge Region Regulates the Kinetics of Transcriptional Responses Through Acetylation, Phosphorylation, and Nuclear Retention

Progesterone receptors (PRs) are critical regulators of mammary gland development and contributors to breast cancer progression. Posttranslational modifications of PR have been shown to alter hormone responsiveness. Site-directed mutagenesis demonstrated that upon hormone binding, PR is acetylated at the consensus sequence, KXKK (amino acids 638-641), located within the hinge region. We created an acetylation-deficient (K-A) mutant as well as acetylation mimics (K-Q or K-T). Interestingly, similar to K-A PR, PR acetylation mimics (K-Q or K-T) displayed delayed phosphorylation and nuclear entry relative to wild-type (wt) PR-B, indicative of disruption of PR nuclear-cytoplasmic shuttling. Wt PR-B, but not K-mutant PRs, induced c-myc at 1 h of progestin treatment. However, at 6 h of treatment, c-myc induction was comparable with levels induced by wt PR-B, suggesting that the precise timing of PR phosphorylation and nuclear retention are critical for cells to rapidly initiate robust transcriptional programs. In contrast to c-myc, progestin-induced serum- and glucocorticoid-regulated kinase (SGK) expression displayed sensitivity to PR acetylation but not nuclear entry. Namely, in the presence of progestin, acetylation-deficient (K-A) mutant PR-B up-regulated SGK mRNA relative to wt PR; progesterone response element-luciferase assays confirmed this result. However, K-Q and K-T acetylation mimics only weakly induced SGK expression independently of nuclear retention. These data reveal the ability of PR acetylation to alter the magnitude of transcriptional response at selected (slow response) promoters (SGK), whereas the hinge region dictates the kinetics of the transcriptional response to hormone at other (rapid response) promoters (c-myc). In sum, the PR hinge region is multifunctional. Understanding the ability of this region to couple acetylation, phosphorylation, and nuclear entry may provide clues to mechanisms of altered hormone responsiveness.

IL-1β-induced cortisol stimulates lung fluid absorption in fetal guinea pigs via SGK-mediated Nedd4-2 inhibition

We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates distal lung fluid absorption in fetal guinea pigs via induction of serum- and glucocorticoid-regulated kinase (SGK) and inhibition of neural precursor cell expressed, developmentally downregulated protein 4-2 (Nedd4-2). IL-1beta was subcutaneously administered daily to timed-pregnant guinea pigs over 3 days. Fetuses were obtained by abdominal hysterotomy at gestation day (GD)61 and GD68 and instilled with an isosmolar 5% albumin solution into the lungs. Distal lung fluid movement was measured over 1 h from the change in distal air space protein concentration. Fetal lungs were secreting lung fluid at GD61 while absorbing lung fluid at GD68. Distal lung fluid absorption was induced at GD61 by IL-1beta but unaffected at GD68. Plasma cortisol concentrations were increased by IL-1beta at GD61 and endogenously at GD68. Distal lung fluid absorption was measured and correlated to SGK and Nedd4-2 expression and to alpha-epithelial Na channel (ENaC) expression. SGK was increased by IL-1beta and late during gestation (GD68), while Nedd4-2 was decreased by IL-1beta and late during gestation. alpha-ENaC was induced by IL-1beta at GD61 and increased late during gestation. Thus our study suggests that cortisol-stimulated fetal lung fluid absorption is mediated by increased ENaC expression and may be governed by the SGK/Nedd4-2 pathway. These observations may explain why babies delivered preterm after intrauterine inflammation have a reduced risk of developing severe respiratory distress.

RNAi Screen for Sensitivity to Chemotherapeutics

Inappropriate cell death or cell survival can lead to various diseases, such as neurodegenerative disorders (excessive cell death) and cancer (excessive cell survival). MacKeigan et al. performed large-scale RNA interference (RNAi) screens using small interfering RNAs (siRNAs) transfected into cultured human cell lines to identify kinases and phosphatases involved in cell survival. The authors identified 73 kinases (11% of the kinases in HeLa cells) and 72 phosphatases (32% of the phosphatases in HeLa cells) as positively contributing to cell survival, based on an increase in markers for apoptotic cell death upon the knockdown of these proteins. The phosphatase siRNA library was also used to screen for phosphatases that attenuate cell death. HeLa cells were transfected with the phosphatase siRNAs and then also exposed to apoptosis-inducing pharmaceuticals (cisplatin, Taxol, etopside), and 12 phosphatases were identified that, when knocked down, conferred resistance to apoptosis. The RNAi screens were also used to identify kinases that, when knocked down, conferred an increased sensitivity to apoptosis-inducing drugs. Using this approach, it is possible to predict what combinations of chemotherapeutic agents may be most effective. The authors demonstrated that Taxol combined with the siRNA for SGK (serum and glucocorticoid-regulated kinase), the siRNA for the tyrosine kinase FER, the siRNA for CDK8 (cyclin-dependent kinase 8), or the siRNA for mTOR all increased cell death compared with the siRNA alone or the drug alone. This type of screen may lead to new combinations of therapies for cancers and can be applied to cell lines or cancers with different cell survival signaling pathways, thus allowing tailoring of the chemotherapy to the cancer. For example, the authors also tested for sensitization to apoptosis-inducing drugs in the breast carcinoma cell line BT474, which exhibits less sensitivity to survival kinase knockdown than do the HeLa cells. Indeed, Taxol synergized with siRNA for FER, JIK (JNK inhibitory kinase, also known as Ste20-like kinase), or PLK2 (Polo-like kinase 2) to promote cell death. Thus, this type of global genomic screen may serve to point to novel chemotherapeutic combination therapies with existing drugs, as well as to identify novel targets for regulating cell death. J. P. MacKeigan, L. O. Murphy, J. Blenis, Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat. Cell Biol. 7 , 591-600 (2005). [PubMed]

Novel Small Molecule Inhibitors of 3-Phosphoinositide-dependent Kinase-1

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.

Nedd4-2 Phosphorylation Induces Serum and Glucocorticoid-regulated Kinase (SGK) Ubiquitination and Degradation

Serum and glucocorticoid-regulated kinase (SGK) plays a key role in the regulation of epithelial Na+ transport. SGK phosphorylates Nedd4-2, an E3 ubiquitin-protein ligase that targets the epithelial Na+ channel (ENaC) for degradation. Phosphorylation increases Na+ transport by reducing Nedd4-2 binding to ENaC, which increases ENaC expression at the cell surface. Thus, SGK expression must be tightly controlled to maintain Na+ homeostasis. This occurs in part by regulation of SGK transcription; a variety of signals including steroid hormones (aldosterone and glucocorticoids) increase SGK levels by inducing transcription of SGK. However, SGK has a short half-life, suggesting that SGK levels might also be controlled by regulation of SGK degradation. Here we found that SGK degradation is mediated in part by Nedd4-2. Consistent with this model, overexpression of Nedd4-2 decreased steady-state levels of SGK in a dose-dependent manner by increasing SGK ubiquitination and degradation in the 26S proteasome. Conversely, silencing of Nedd4-2 by RNA interference stabilized SGK. Nedd4-2 phosphorylation potentiates SGK degradation; degradation was reduced by Nedd4-2 and SGK mutations that disrupt phosphorylation or by inhibition of SGK kinase activity. Together with previous work, the data support a model in which SGK and Nedd4-2 regulate one another in a reciprocal manner. SGK phosphorylates Nedd4-2, which reduces Nedd4-2 binding and inhibition of ENaC. Conversely, phosphorylation increases Nedd4-2-mediated degradation of SGK. Thus, by phosphorylating Nedd4-2, SGK induces its own degradation. This feedback inhibition may fine-tune the regulation of epithelial Na+ absorption.

Targeted disruption of the protein kinase SGK3/CISK impairs postnatal hair follicle development.

Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of growth factor and hormone signaling that, like their close relatives in the Akt family, are regulated by lipid products of phosphatidylinositol-3-kinase. SGK3 has been implicated in the control of cell survival and regulation of ion channel activity in cultured cells. To begin to dissect the in vivo functions of SGK3, we generated and characterized Sgk3 null mice. These mice are viable and fertile, and in contrast to mice lacking SGK1 or Akt2, respectively, display normal sodium handling and glucose tolerance. However, although normal at birth, by postpartum day 4 they have begun to display an unexpected defect in hair follicle morphogenesis. The abnormality in hair follicle development is preceded by a defect in proliferation and nuclear accumulation of beta-catenin in hair bulb keratinocytes. Furthermore, in cultured keratinocytes, heterologous expression of SGK3 potently modulates activation of beta-catenin/Lef-1-mediated gene transcription. These data establish a role for SGK3 in normal postnatal hair follicle development, possibly involving effects on beta-catenin/Lef-1-mediated gene transcription.

Gene expression profiling in the midbrain of striatal 6-hydroxydopamine-injected mice.

In order to clarify mechanisms underlying dopaminergic neuronal death in Parkinson's disease (PD), a gene expression profiling study was performed in a rodent model of PD. In this model, mice are intrastriatally injected with 6-hydroxydopamine (6-OHDA) and dopaminergic neurons in the substantia nigra (SN) gradually die by retrograde degeneration. The SN were removed 2 h, 24 h, or 14 days after 6-OHDA administration. Levels of mRNAs related to cell death or survival were quantified using adaptor-tagged competitive PCR (ATAC-PCR). The cyclin D1 gene showed an immediate increase in mRNA expression. After 24 h, when dopaminergic neurons were under intense degeneration, levels of caspase 8 mRNA and p53 apoptosis effecter related to pmp 22 (PERP) mRNA increased and, conversely, FAS mRNA decreased. After 14 days, when the degeneration was attenuated, levels of PERP mRNA and serum- and glucocorticoid-regulated kinase (SGK) mRNA still increased. SGK has a similarity to AKT, which is an important molecule involved in nerve growth factor signal transduction. AKT mRNA levels are low in dopaminergic neurons. These results suggest that an increase in cyclin D1 mRNA triggers dopaminergic neurons to enter an abnormal cell cycle, which leads to neuronal degeneration and cell death, possibly induced by PERP and caspase 8. In addition to cell death-related genes, several survival-related genes are activated. SGK might function as a key enzyme for the survival of dopaminergic neurons.

Differing Roles of Akt and Serum- and Glucocorticoid-regulated Kinase in Glucose Metabolism, DNA Synthesis, and Oncogenic Activity

Serum- and glucocorticoid-regulated kinase (SGK) is a serine kinase that has a catalytic domain homologous to that of Akt, but lacks the pleckstrin homology domain present in Akt. Akt reportedly plays a key role in various cellular actions, including glucose transport, glycogen synthesis, DNA synthesis, anti-apoptotic activity, and cell proliferation. In this study, we attempted to reveal the different roles of SGK and Akt by overexpressing active mutants of Akt and SGK. We found that adenovirus-mediated overexpression of myristoylated (myr-) forms of Akt resulted in high glucose transport activity in 3T3-L1 adipocytes, phosphorylated glycogen synthase kinase-3 (GSK3) and enhanced glycogen synthase activity in hepatocytes, and the promotion of DNA synthesis in interleukin-3-dependent 32D cells. In addition, stable transfection of myr-Akt in NIH3T3 cells induced an oncogenic transformation in soft agar assays. The active mutant of SGK (D-SGK, substitution of Ser422 with Asp) and myr-SGK were shown to phosphorylate GSK3 and to enhance glycogen synthase activity in hepatocytes in a manner very similar to that observed for myr-Akt. However, despite the comparable degree of GSK3 phosphorylation between myr-Akt and d-SGK or myr-SGK, d-SGK and myr-SGK failed to enhance glucose transport activity in 3T3-L1 cells, DNA synthesis in 32D cells, and oncogenic transformation in NIH3T3 cells. Therefore, the different roles of SGK and Akt cannot be attributed to ability or inability to translocate to the membrane thorough the pleckstrin homology domain, but rather must be attributable to differences in the relatively narrow substrate specificities of these kinases. In addition, our observations strongly suggest that phosphorylation of GSK3 is either not involved in or not sufficient for GLUT4 translocation, DNA synthesis, or oncogenic transformation. Thus, the identification of substrates selectively phosphorylated by Akt, but by not SGK, may provide clues to clarifying the pathway leading from Akt activation to these cellular activities.

Serum- and glucocorticoid-regulated kinase isoform-1 and epithelial sodium channel subunits in human ocular ciliary epithelium.

In peripheral sodium-transporting tissues, the serum- and glucocorticoid-regulated kinase (SGK) isoform-1 is an early corticosteroid target gene in the activation of epithelial sodium channels (ENaCs). Sodium transport across the human ocular nonpigmented and pigmented ciliary epithelial bilayer (NPE-PE) is essential for aqueous humor production, but the expression of SGK1 and ENaC subunits remain to be defined. SGK1 and ENaC subunits were evaluated by in situ hybridization and RT-PCR analysis on human NPE-PE sections and an NPE cell line (ODM-2). Northern blot analyses were conducted on ODM-2 cells incubated with dexamethasone (DEX) or aldosterone (ALDO) and RU38486 (a glucocorticoid receptor [GR] antagonist) or RU26752 (a mineralocorticoid receptor [MR] antagonist) or both inhibitors. The affinity of the GRs and MRs for DEX and ALDO was assessed by radioligand-binding assays. Expression of SGK1 and ENaC subunits was confirmed in NPE-PE tissues and ODM-2 cells. Dose-dependent induction of SGK1 mRNA in the ODM-2 cells was demonstrated after incubation with DEX or ALDO. While response to DEX was not inhibited by RU38486 or RU26752, there was a moderate reduction in induction by ALDO in the presence of RU26752 that was completely abolished in the presence of both inhibitors. Specific binding of (3)[H]DEX and (3)[H]ALDO was established, revealing greater expression of GRs than MRs. The expression of ENaCs within the NPE-PE and corticosteroid regulation of SGK1 through the GR and MR, indicate that this mechanism may be a feature of sodium transport in the human ocular ciliary epithelium.

Serum and Glucocorticoid-regulated Kinase Modulates Nedd4-2-mediated Inhibition of the Epithelial Na+Channel

The epithelial Na+ channel (ENaC) forms the pathway for Na+ absorption across epithelia, including the kidney collecting duct, where it plays a critical role in Na+ homeostasis and blood pressure control. Na+ absorption is regulated in part by mechanisms that control the expression of ENaC at the apical cell surface. Nedd4 family members (e.g. Nedd4, Nedd4-2) bind to the channel and decrease its surface expression by catalyzing its ubiquitination and degradation. Conversely, serum and glucocorticoid-regulated kinase (SGK), a downstream mediator of aldosterone, increases the expression of ENaC at the cell surface. Here we show that SGK and human Nedd4-2 (hNedd4-2) converge in a common pathway to regulate epithelial Na+ absorption. Consistent with this model, we found that SGK bound to hNedd4-2 and hNedd4. A PY motif in SGK mediated the interaction and was required for SGK to stimulate ENaC. SGK phosphorylated hNedd4-2 (but not hNedd4), altering hNedd4-2 function; phosphorylation reduced the binding of hNedd4-2 to alphaENaC, and hence, the hNedd4-2-mediated inhibition of Na+ absorption. These data suggest that SGK regulates epithelial Na+ absorption in part by modulating the function of hNedd4-2.

Mineralocorticoids and cardiac fibrosis: the decade in review.

1. Over the past decade, aldosterone has been shown to have direct extra-epithelial actions and substantial (patho)physiological roles in the cardiovascular system in the context of inappropriate salt status. In experimental studies on uninephrectomized rats given 0.9% NaCl solution to drink, these include blood pressure elevation via activation of circumventricular mineralocorticoid receptors in the central nervous system and production of pressure-independent cardiac hypertrophy and fibrosis by a direct effect on the heart. 2. In the Randomized Aldactone Evaluation Study (RALES) trial, patients with severe congestive heart failure (CHF) were continued on their current therapy (angiotensin-converting enzyme inhibitor, diuretic etc.) and given either placebo or spironolactone at an average dose of 26 mg/day. Mineralocorticoid receptor inhibition was accompanied by a 30% improvement in mortality and 35% less hospitalization, striking confirmation of a pathophysiological role for aldosterone in CHF. 3. Although the current basic and clinical studies are conflicting, there is evidence both for aldosterone synthesis by the failing human heart and for substantial cardiac metabolism of aldosterone. The extent to which this potential paracrine source for aldosterone may be involved in cardiac hypertrophy and cardiac fibrosis remains to be established. 4. Belatedly, aldosterone-induced proteins (e.g. serum and glucocorticoid-regulated kinase (SGK)) have been identified in epithelial mineralocorticoid target tissue. Studies are currently in progress on the cellular and molecular mechanisms involved in the coronary vasculitis provoked early in the mineralocorticoid/salt model, which, in turn, appears to trigger the subsequent perivascular and interstitial fibrotic response.

Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate.

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

SRC family kinases mediate epithelial Na+ channel inhibition by endothelin.

The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.

The Serum- and Glucocorticoid-Induced Kinase Is a Physiological Mediator of Aldosterone Action*

Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to Xenopus A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 microg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 microg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.

Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.

Aldosterone controls sodium reabsorption and potassium secretion in the aldosterone-sensitive distal nephron (ASDN). Although clearance measurements have shown that aldosterone induces these transports within 30--60 min, no early effects have been demonstrated in vivo at the level of the apical epithelial sodium channel (ENaC), the main effector of this regulation. Here we show by real-time RT-PCR and immunofluorescence that an aldosterone injection in adrenalectomized rats induces alpha-ENaC subunit expression along the entire ASDN within 2 h, whereas beta- and gamma-ENaC are constitutively expressed. In the proximal ASDN portions only, ENaC is shifted toward the apical cellular pole and the apical plasma membrane within 2 and 4 h, respectively. To address the question of whether the early aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) might mediate this apical shift of ENaC, we analyzed SGK induction in vivo. Two hours after aldosterone, SGK was highly induced in all segment-specific cells of the ASDN, and its level decreased thereafter. In Xenopus laevis oocytes, SGK induced ENaC activation and surface expression by a kinase activity-dependent mechanism. In conclusion, the rapid in vivo accumulation of SGK and alpha-ENaC after aldosterone injection takes place along the entire ASDN, whereas the translocation of alpha,beta,gamma-ENaC to the apical plasma membrane is restricted to its proximal portions. Results from oocyte experiments suggest the hypothesis that a localized activation of SGK may play a role in the mediation of ENaC translocation.