Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes.

BACKGROUND: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. METHODS: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. RESULTS: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3. CONCLUSIONS: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.

The Role of Janus Kinase 3 in the Regulation of Na⁺/K⁺ ATPase under Energy Depletion.

BACKGROUND/AIMS: Janus kinase-3 (JAK3) is activated during energy depletion. Energy-consuming pumps include the Na(+)/K(+)-ATPase. The present study explored whether JAK3 regulates Na(+)/K(+)-ATPase in dendritic cells (DCs). METHODS: Ouabain (100 µM)-sensitive (Iouabain) and K(+)-induced (Ipump) outward currents were determined by utilizing whole cell patch-clamp, Na(+)/K(+)-ATPase α1-subunit mRNA levels by RT-PCR, Na(+)/K(+)-ATPase protein abundance by flow cytometry or immunofluorescence, and cellular ATP by luciferase-assay in DCs from bone marrow of JAK3-knockout (jak3(-/-)) or wild-type mice (jak3(+/+)). Ipump was further determined by voltage clamp in Xenopus oocytes expressing JAK3, active (A568V)JAK3 or inactive (K851A)JAK3. RESULTS: Na(+)/K(+)-ATPase α1-subunit mRNA and protein levels, as well as Ipump and Iouabain were significantly higher in jak3(-/-)DCs than in jak3(+/+)DCs. Energy depletion by 4h pre-treatment with 2,4-dinitro-phenol significantly decreased Ipump in jak3(+/+) DCs but not in jak3(-/-)DCs. Cellular ATP was significantly lower in jak3(-/-)DCs than in jak3(+/+)DCs and decreased in both genotypes by 2,4-dinitro-phenol, an effect significantly more pronounced in jak3(-/-)DCs than in jak3(+/+)DCs and strongly blunted by ouabain in both jak3(+/+) and jak3(-/-)DCs. Ipump and Iouabain in oocytes were decreased by expression of JAK3 and of (A568V)JAK3 but not of (K851A)JAK3. JAK3 inhibitor WHI-P154 (4-[(3'-bromo-4'-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 22 µM) enhanced Ipump and Iouabain in JAK3 expressing oocytes. The difference between (A568V)JAK3 and (K851A)JAK3 expressing oocytes was virtually abrogated by actinomycin D (50 nM). CONCLUSIONS: JAK3 down-regulates Na(+)/K(+)-ATPase activity, an effect involving gene expression and profoundly curtailing ATP consumption.

Up-regulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB/Akt and PIKfyve.

BACKGROUND: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. METHODS: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive (T308A/S473A)PKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant (S318A)PIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. RESULTS: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (I(le)), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of (T308A/S473A)PKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of (S318A)PIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of I(le) following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. CONCLUSION: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells.

The serum and glucocorticoid inducible kinases SGK1-3 stimulate the neutral amino acid transporter SLC6A19.

The neutral amino acid transporter SLC6A19 (B(0)AT1) plays a decisive role in transport of neutral amino acids in the kidney and intestine. Recently, mutations in SLC6A19 were identified that result in severe neutral aminoaciduria known as Hartnup disorder. SLC6A19 expression and function is controlled by the brush-border angiotensin-converting enzyme 2 (ACE2). Beyond that the mechanisms regulating SLC6A19 function are unknown. The SLC6A19 sequence contains a conserved putative phosphorylation site for the serum and glucocorticoid inducible kinase isoforms SGK1-3, kinases known to regulate a variety of channels and transporters. The present study explored the role of SGK1-3 in the regulation of SLC6A19. As shown by two-electrode voltage clamp in the Xenopus oocyte expression system, leucine-induced currents in SLC6A19 expressing oocytes were activated by the protein kinases SGK1-3. The putative phosphorylation site on the transporter is not essential for SLC6A19 regulation by the kinases. As determined by quantitative immunoassay and electrophysiology, the kinases increase SLC6A19 currents by increasing the cell surface expression of the protein without altering the affinity of the carrier. Following inhibition of carrier insertion into the cell membrane by treatment with brefeldin A (BFA), the leucine-induced current declined significantly slower in Xenopus oocytes expressing SLC6A19 together with SGK1 than in oocytes expressing SLC6A19 alone, a finding pointing to SGK-mediated transporter stabilization in the plasma membrane. Coexpression of ACE2 markedly increased leucine-induced currents in SLC6A19 expressing oocytes that were further enhanced by SGK1-3 kinases. In conclusion, SGK isoforms are novel potent stimulators of SLC6A19 and may thus participate in the regulation of neutral amino acid transport in vivo.

Regulation of the glutamate transporter EAAT4 by PIKfyve.

The excitatory amino-acid transporter EAAT4 (SLC1A6), a Na(+),glutamate cotransporter expressed mainly in Purkinje cells, serves to clear glutamate from the synaptic cleft. EAAT4 activity is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent regulation of the Na(+),glucose transporter SGLT1 (SLC5A1) and the creatine transporter CreaT (SLC6A8) has recently been shown to involve the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether SGK1-dependent EAAT4-regulation similarly involves PIKfyve. In Xenopus oocytes expressing EAAT4, but not in water injected oocytes, glutamate induced a current which was significantly enhanced by coexpression of PIKfyve and SGK1. The glutamate induced current in Xenopus oocytes coexpressing EAAT4 and both, PIKfyve and SGK1, was significantly larger than the current in Xenopus oocytes expressing EAAT4 together with either kinase alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter glutamate induced current in EAAT4-expressing Xenopus oocytes and abolished the stimulation of glutamate induced current by coexpression of PIKfyve. The stimulating effect of PIKfyve was abrogated by replacement of the serine with alanine in the SGK consensus sequence ((S318A)PIKfyve). Furthermore, coexpression of (S318A)PIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.

Downregulation of angiogenin transcript levels and inhibition of colonic carcinoma by gum arabic (Acacia senegal).

Gum Arabic (GA), a nutrient from dried exudate of Acacia senegal, is widely used as emulsifier and stabilizer. It stimulates sodium and water absorption in diarrhea. This study explored the effects of GA in colonic tissue. Mice were treated with GA (10% wt/vol) in drinking water and gene array was performed. As GA modified several tumor-relevant genes, chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by 3 cycles of 3% dextrane sodium sulphate in drinking water) was induced with or without GA treatment. Within 4 days, GA treatment decreased the colonic transcript levels of the angiogenetic factors angiogenin 1, angiogenin 3, and angiogenin 4 by 78 +/- 18%, 88 +/- 15%, and 92 +/- 13%, respectively (n = 5 each), and of further genes including CD38 antigen, aquaporin4, interleukin18, vav-3-oncogene, gamma(+)-amino acid transporter, sulfatase1, ubiquitinD, and chemokine ligand5. According to Western blotting, GA treatment similarly decreased angiogenin protein expression, and according to immunohistochemistry, it decreased ss-catenin expression. Chemical cancerogenesis resulted in multiple colonic tumors within 12 wk. GA treatment (10% wt/wt) in drinking water significantly decreased the number of tumors by 70%. The observations disclose a powerful anticarcinogenic effect of GA. The nutrient could thus be used for the prophylaxis against colon carcinoma particularly in individuals at enhanced risk.

The C-terminal PDZ-binding motif in the Kv1.5 potassium channel governs its modulation by the Na+/H+ exchanger regulatory factor 2.

Kv1.5 belongs to the family of voltage-gated potassium (Kv) channels and contains a N- and a C-terminal PDZ-binding motif that might be recognized by PDZ domains on the scaffold proteins NHERF1 and NHERF2. Expression studies in Xenopus oocytes demonstrated that NHERF1 and NHERF2 activate Kv1.5, an effect requiring the C-terminal PDZ-binding motif on Kv1.5. NHERF2 enhances Kv1.5 activity and cell surface expression as determined by electrophysiology and immunoassays. NHERF2 elevates Kv1.5 abundance at the plasma membrane by decreasing channel internalization as proven by Brefeldin A experiments. Kv1.5 is stimulated by the serum and glucocorticoid inducible kinase SGK1, a kinase known to interact with the second PDZ domain of NHERF2. This study aims to identify if SGK1 and NHERF2 synergize to increase Kv1.5 currents. Expression of NHERF2 potentiated SGK1-mediated Kv1.5 activation, which was significantly attenuated by deletion of the second PDZ domain in NHERF2. Specificity of observed effects was verified by evaluating the influence of NHERFs on Kv1.3, a known SGK1 target that contains an internal PDZ binding motif. In summary, our results suggest that NHERFs might participate in the regulation of electrical excitability in part by controlling Kv1.5 surface abundance and by clustering signal transduction molecules to the channel.

Regulation of the glutamate transporter EAAT2 by PIKfyve.

The Na(+),glutamate cotransporter EAAT2 is expressed in astrocytes and clears glutamate from the synaptic cleft. EAAT2 dependent currrent is stimulated by the serum and glucocorticoid inducible kinase SGK1. Phosphorylation targets of SGK1 include the human phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). Nothing is known, however, on the role of PIKfyve in the regulation of EAAT2. The present experiments thus explored, whether PIKfyve expression modifies EAAT2 dependent currrent and protein abundance in the cell membrane. In Xenopus oocytes expressing EAAT2 but not in water injected oocytes application of glutamate (2 mM) induced an inward current (I(glu)). Coexpression of either, SGK1 or PIKfyve, significantly enhanced I(glu) in EAAT2 expressing oocytes. I(glu) was significantly higher in Xenopus oocytes coexpressing EAAT2, SGK1 and PIKfyve than in Xenopus oocytes expressing EAAT2 and either, SGK1 or PIKfyve, alone. Additional coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K127N)SGK1 did not significantly alter I(glu) in EAAT2 expressing oocytes and significantly decreased I(glu) in oocytes coexpressing EAAT2 together with PIKfyve. The stimulating effect of PIKfyve on I(glu) was abrogated by replacement of the serine in the SGK consensus sequence by alanine ((S318A)PIKfyve). Furthermore, additional coexpression of (S318A)PIKfyve virtually abolished I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT2. Confocal microscopy reveals that PIKfyve enhances the EAAT2 protein abundance in the cell membrane. The observations disclose that PIKfyve indeed participates in the regulation of EAAT2.

Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination.

The serum and glucocorticoid inducible kinase SGK1 is involved in dexamethasone-induced inhibition of insulin secretion by increasing voltage-gated potassium channel (Kv) activity. SGK1 upregulates the Kv1.5 channel but the precise mechanism underlying the SGK1 dependent regulation of Kv1.5 has not been defined yet. The present study explored the signal transduction processes involved. Expression studies in Xenopus oocytes revealed that SGK1 promotes channel activity by interfering with the Nedd4-2 ubiquitination pathway, irrespective of the presence of putative SGK1 phosphorylation sites on Kv1.5. Expression of the ubiquitin ligase Nedd4-2 declined Kv1.5 currents by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression. Increasing concentrations of SGK1 gradually compensated the inhibiting effect of Nedd4-2 on Kv1.5. Enhanced Kv1.5 surface abundance by SGK1 reflects decreased channel internalization as indicated by Brefeldin A experiments. In conclusion, Kv1.5 upregulation by SGK1 involves inhibition of channel ubiquitination by Nedd4-2 that leads to Kv1.5 stabilization in the plasma membrane. Our results suggest that the kinase might participate in the regulation of insulin secretion in part by controlling Kv1.5 surface abundance.

The peptide transporter PEPT2 is targeted by the protein kinase SGK1 and the scaffold protein NHERF2.

PEPT1 and PEPT2 are members of the family of proton-dependent oligopeptide transporters that mediate electrogenic uphill transport of small peptides and peptidomimetics into a variety of cells. Kinetic properties and substrate recognition sites of those transporters have been well defined previously. Little is known, however, about regulation of those transporters. Both PEPT isoforms contain putative phosphorylation sites for the serum and glucocorticoid inducible kinase SGK1 and a C-terminal PDZ binding motif that might be recognized by PDZ domains of the Na(+)/H(+) exchanger regulatory factors NHERF1 and NHERF2. Thus, the present study attempted to clarify the role of SGK1 and NHERFs in the modulation of PEPT isoforms. Expression studies in Xenopus oocytes with subsequent electrophysiology and immunoassays revealed that SGK1 and NHERF2, but not the NHERF1 isoform specifically enhance PEPT2 function and surface abundance. The kinase is effective through phosphorylation of (185)Ser within the SGK1 consensus site, since disruption of this site prevented transporter modulation by the kinase. NHERF2 failed to regulate the C-terminal deletion mutant (PEPT2DeltaC) indicating that the C-terminal PDZ-binding motif in PEPT2 governs transport modulation by NHERF2. Coexpression of NHE3 stimulates PEPT2 activity to a similar extent as coexpression of NHERF2. Dynasore experiments demonstrated that SGK1 and NHERF2 activate PEPT2 by stabilizing the transporter at the cell surface. In conclusion, the present results reveal two novel PEPT2 posttranslational modulators, SGK1 and NHERF2, which might regulate transport of oligopeptides and peptidomimetic drugs.

Role and regulation of the serum- and glucocorticoid-regulated kinase 1 in fertile and infertile human endometrium.

Using cDNA microarray analysis, we identified SGK1 (serum- and glucocorticoid-regulated kinase 1) as a gene aberrantly expressed in midsecretory endometrium of women with unexplained infertility. SGK1 is a serine/threonine kinase involved primarily in epithelial ion transport and cell survival responses. Real-time quantitative PCR analysis of a larger, independent sample set timed to coincide with the period of uterine receptivity confirmed increased expression of SGK1 transcripts in infertile women compared with fertile controls. We further demonstrate that SGK1 expression is regulated by progesterone in human endometrium in vivo as well as in explant cultures. During the midsecretory phase of the cycle, SGK1 mRNA and protein were predominantly but not exclusively expressed in the luminal epithelium, and expression in this cellular compartment was higher in infertile women. In the stromal compartment, SGK1 expression was largely confined to decidualizing cells adjacent to the luminal epithelium. In primary culture, SGK1 was induced and phosphorylated upon decidualization of endometrial stromal cells in response to 8-bromo-cAMP and progestin treatment. Moreover, overexpression of SGK1 in decidualizing cells enhanced phosphorylation and cytoplasmic translocation of the forkhead transcription factor FOXO1 and inhibited the expression of PRL, a major decidual marker gene. Conversely, knockdown of endogenous SGK1 by small interfering RNA increased nuclear FOXO1 levels and enhanced PRL expression. The observation that SGK1 targets FOXO1 in differentiating human endometrium, together with its distinct temporal and spatial expression pattern and increased expression in infertile patients, suggest a major role for this kinase in early pregnancy events.

SGK1 kinase upregulates GLUT1 activity and plasma membrane expression.

Phosphatidylinositol 3-kinase (PI3 kinase) inhibition disrupts the ability of insulin to stimulate GLUT1 and GLUT4 translocation into the cell membrane and thus glucose transport. The effect on GLUT4 but not on GLUT1 is mediated by activation of protein kinase B (PKB). The serum- and glucocorticoid-inducible kinase SGK1, a further kinase downstream of PI3 kinase, regulates several transporters by enhancing their plasma membrane abundance. GLUT1 contains a consensus site ((95)Ser) for phosphorylation by SGK1. Thus, the present study investigated whether GLUT1 is regulated by the kinase. Tracer-flux studies in Xenopus oocytes and HEK-293 cells demonstrated that GLUT1 transport is enhanced by constitutively active (S422D)SGK1. The effect requires the kinase catalytical activity since the inactive mutant (K127N)SGK1 failed to modulate GLUT1. GLUT1 stimulation by (S422D)SGK1 is not due to de novo protein synthesis but rather to an increase of the transporter's abundance in the plasma membrane. Kinetic analysis revealed that SGK1 enhances maximal transport rate without altering GLUT1 substrate affinity. These observations suggest that SGK1 regulates GLUT1 and may contribute to or account for the PI3 kinase-dependent but PKB-independent stimulation of GLUT1 by insulin.

Serum- and glucocorticoid-inducible kinase 1 mediates salt sensitivity of glucose tolerance.

Excess salt intake decreases peripheral glucose uptake, thus impairing glucose tolerance. Stimulation of cellular glucose uptake involves phosphatidylinositide-3-kinase (PI-3K)-dependent activation of protein kinase B/Akt. A further kinase downstream of PI-3K is serum- and glucocorticoid-inducible kinase (SGK)1, which is upregulated by mineralocorticoids and, thus, downregulated by salt intake. To explore the role of SGK1 in salt-dependent glucose uptake, SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)) were allowed free access to either tap water (control) or 1% saline (high salt). According to Western blotting, high salt decreased and deoxycorticosterone acetate (DOCA; 35 mg/kg body wt) increased SGK1 protein abundance in skeletal muscle and fat tissue of sgk1(+/+) mice. Intraperitoneal injection of glucose (3 g/kg body wt) into sgk1(+/+) mice transiently increased plasma glucose concentration approaching significantly higher values ([glucose]p,max) in high salt (281 +/- 39 mg/dl) than in control (164 +/- 23 mg/dl) animals. DOCA did not significantly modify [glucose]p,max in control sgk1(+/+) mice but significantly decreased [glucose]p,max in high-salt sgk1(+/+) mice, an effect reversed by spironolactone (50 mg/kg body wt). [Glucose]p,max was in sgk1(-/-) mice insensitive to high salt and significantly higher than in control sgk1(+/+) mice. Uptake of 2-deoxy-d-[1,2-(3)H]glucose into skeletal muscle and fat tissue was significantly smaller in sgk1(-/-) mice than in sgk1(+/+) mice and decreased by high salt in sgk1(+/+) mice. Transfection of HEK-293 cells with active (S422D)SGK1, but not inactive (K127N)SGK, stimulated phloretin-sensitive glucose uptake. In conclusion, high salt decreases SGK1-dependent cellular glucose uptake. SGK1 thus participates in the link between salt intake and glucose tolerance.

Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A.

The serum and glucocorticoid inducible kinase SGK1 and its isoform SGK3 are both expressed in cardiac tissue. One of the functions of SGK1 is the phosphorylation and inactivation of the ubiquitin ligase Nedd4-2, which in turn could be shown to downregulate the voltage-gated Na+ channel SCN5A (hH1). The present study has been performed to test for a role of SGK1 and SGK3 in the regulation of SCN5A. To this end cRNA encoding the human Na+ channel SCN5A was injected into Xenopus laevis oocytes with or without cRNA encoding the wild-type kinases SGK1, the constitutively active kinase (S422D)SGK1, the inactive form K127NSGK1 or the wild-type SGK3. SCN5A currents were activated by coexpression of either wild-type SGK1 or SGK3 or the constitutively active S422DSGK1. In contrast, the inactive mutant K127NSGK1 significantly decreased the currents. Moreover, coexpression of SGK3 significantly altered SCN5A gating, i.e. it hyperpolarized the activation threshold and depolarized the prepotential required for 50% availability of the channel. Opposite shifts of gating properties were elicited by mutation of serine to alanine (S483ASCN5A and S663ASCN5A) in the SGK consensus sequences of SCN5A. The present observations disclose a role of the kinases SGK1 and SGK3 in the regulation of cardiac Na+ channels. As SGK1 is upregulated by glucocorticoids, mineralocorticoids and a variety of inflammatory mediators and both kinases are activated by insulin and IGF1, the kinases could mediate effects of those hormones and mediators on cardiac function.

Retinal colocalization and in vitro interaction of the glutamate transporter EAAT3 and the serum- and glucocorticoid-inducible kinase SGK1 [correction].

PURPOSE: The serum- and glucocorticoid-inducible kinase SGK1 regulates several epithelial channels and transporters, the related protein kinase B (PKB) regulates glucose transport. SGK1 is expressed in the brain and could thus regulate glial and/or neuronal transport processes. The present study explores whether SGK1 is expressed in the retina and whether it regulates EAAT3, a Na(+)-coupled glutamate transporter. EAAT3 is expressed in retinal ganglion cells and accomplishes the clearance of glutamate from synaptic clefts. METHODS: Immunohistochemistry was performed to test for retinal SGK1 expression. For functional analysis, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without additional injection of wild-type SGK1, constitutively active (S422D)SGK1, inactive (K127N)SGK1, and/or constitutively active (T308D,S473D)PKB. Glutamate induced current (I(GLU)) was taken as a measure for transport. RESULTS: SGK1 is indeed expressed in several retinal cells including retinal ganglion cells where it is colocalized with EAAT3. In EAAT3-expressing Xenopus oocytes, glutamate-induced current was stimulated by coexpression of wild-type SGK1, constitutively active (S422D)SGK1, and constitutively active (T308D,S473D)PKB, but not by inactive (K127N)SGK1. CONCLUSIONS: SGK1 and EAAT3 are coexpressed in retinal neurons, and SGK1 serves to stimulate EAAT3. This function is shared by protein kinase B (PKB). The experiments reveal a novel mechanism regulating EAAT3, which may be essential for the function of the retinal ganglion cells.

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.

Acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs.

Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.

Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve.

The Na(+), glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end,EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase.

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.

Role of SGK1 kinase in regulating glucose transport via glucose transporter GLUT4.

Insulin stimulates glucose transport into muscle and fat cells by enhancing GLUT4 abundance in the plasma membrane through activation of phosphatidylinositol 3-kinase (PI3K). Protein kinase B (PKB) and PKCzeta are known PI3K downstream targets in the regulation of GLUT4. The serum- and glucocorticoid-inducible kinase SGK1 is similarly activated by insulin and capable to regulate cell surface expression of several metabolite transporters. In this study, we evaluated the putative role of SGK1 in the modulation of GLUT4. Coexpression of the kinase along with GLUT4 in Xenopus oocytes stimulated glucose transport. The enhanced GLUT4 activity was paralleled by increased transporter abundance in the plasma membrane. Disruption of the SGK1 phosphorylation site on GLUT4 ((S274A)GLUT4) abrogated the stimulating effect of SGK1. In summary, SGK1 promotes glucose transporter membrane abundance via GLUT4 phosphorylation at Ser274. Thus, SGK1 may contribute to the insulin and GLUT4-dependent regulation of cellular glucose uptake.