c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Insulin resistance in adult cardiomyocytes undergoing dedifferentiation: role of GLUT4 expression and translocation.

Myocardium undergoing remodeling in vivo exhibits insulin resistance that has been attributed to a shift from the insulin-sensitive glucose transporter GLUT4 to the fetal, less insulin-sensitive, isoform GLUT1. To elucidate the role of altered GLUT4 expression in myocardial insulin resistance, glucose uptake and the expression of the glucose transporter isoforms GLUT4 and GLUT1 were measured in adult rat cardiomyocytes (ARC). ARC in culture spontaneously undergo dedifferentiation, hypertrophy-like spreading, and return to a fetal-like gene expression pattern. Insulin stimulation of 2-deoxy-D-glucose uptake was completely abolished on day 2 and 3 of culture and recovered thereafter. Although GLUT4 protein level was reduced, the time-course of unresponsiveness to insulin did not correlate with altered expression of GLUT1 and GLUT4. However, translocation of GLUT4 to the sarcolemma in response to insulin was completely abolished during transient insulin resistance. Insulin-mediated phosphorylation of Akt was not reduced, indicating that activation of phosphatidylinositol 3-kinase (PI3K) was preserved. On the other hand, total and phosphorylated Cbl was reduced during insulin resistance, suggesting that activation of Cbl/CAP is essential for insulin-mediated GLUT4 translocation, in addition to activation of PI3K. Pharmacological inhibition of contraction in insulin-sensitive ARC reduced insulin sensitivity and lowered phosphorylated Cbl. The results suggest that transient insulin resistance in ARC is related to impairment of GLUT4 translocation. A defect in the PI3K-independent insulin signaling pathway involving Cbl seems to contribute to reduced insulin responsiveness and may be related to contractile arrest.

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape.

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Differential phosphoproteomics of fragmenting yeast vacuoles

Many organelles exist in an equilibrium of fragmentation into smaller units and fusion into larger structures, which is coordinated with cell division, the increase in cell mass, and envi¬ronmental conditions. In yeast cells, organelle homeostasis can be studied using the yeast vacuole (lysosome) as a model system. Yeast vacuoles are the main compartment for degrada¬tion of cellular proteins and storage of nutrients, ions and metabolites. Fission and fusion of vacuoles can be induced by hyper- and hypotonic shock in vivo, respectively, and have also been reconstituted in vitro using isolated vacuoles. The conserved serine/threonine kinase TOR (target of rapamycin) is a central nutrient sensor and regulates cell growth and metabolism. In yeast, there are two TOR proteins, Torlp and Tor2p, which are part of larger protein complexes, TORCI and TORC2. Only TORCI is rapamycin-sensitive. Disregulation of TOR signaling is linked to a multitude of diseases in humans, e.g. cancer, neurodegenerative diseases and metabolic syndrome. It has been shown that TORCI localizes to the vacuole membrane, and recent findings of our laboratory demonstrated that TORCI positively regulates vacuole fragmentation. This suggests that the fragmentation machinery should contain target proteins phosphorylated by TORCI. I explored the rapamycin-and fission-dependent vacuolar phosphoproteome during frag¬mentation, using a label-free mass-spectrometry approach. I identified many vacuolar factors whose phosphorylation was downregulated in a TORCI- and fission-dependent manner. Among them were known protein complexes that are functionally linked to fission or fusion, like the HOPS, VTC and FAB1 complexes. Hence, TORCI-dependent phosphorylations might positively regulate vacuole fission. Several candidates were chosen for detailed microscopic analysis of in vivo vacuole frag-mentation, using deletion mutants. I was able to identify novel factors not previously linked to fission phenotypes, e.g. the SEA complex, Pib2, and several vacuolar amino acid transporters. Transport of neutral and basic amino acids across the membrane seems to control vacuole fission, possibly via TORCI. I analyzed vacuolar fluxes of amino acids in wildtype yeast cells and found evidence for a selective vacuolar export of basic amino acids upon hyperosmotic stress. This leads me to propose a model where vacuolar export of amino acids is necessary to reshape the organelle under salt stress. - Le nombre et la taille de certaines organelles peut être déterminé par un équilibre entre la fragmentation qui produit des unités plus petites et la fusion qui génère des structures plus larges. Cet équilibre est coordonné avec la division cellulaire, l'augmentation de la masse cellulaire, et les conditions environnementales. Dans des cellules de levure, l'homéostasie des organelles peut être étudié à l'aide d'un système modèle, la vacuole de levure (lysosome). Les vacuoles constituent le principal compartiment de la dégradation des protéines et de stockage des nutriments, des ions et des métabolites. La fragmentation et la fusion des vacuoles peuvent être respectivement induites par un traitement hyper- ou hypo-tonique dans les cellules vivantes. Ces processus ont également été reconstitués in vitro en utilisant des vacuoles isolées. La sérine/thréonine kinase conservée TOR (target of rapamycin/cible de la rapamycine) est un senseur de nutriments majeur qui régule la croissance cellulaire et le métabolisme. Chez la levure, il existe deux protéines TOR, Torlp et Tor2p, qui sont les constituants de plus grands complexes de protéines, TORCI et TORC2. TORCI est spécifiquement inhibé par la rapamycine. Une dysrégulation de la signalisation de TOR est liée à une multitude de maladies chez l'homme comme le cancer, les maladies neurodégénératives et le syndrome métabolique. Il a été montré que TORCI se localise à la membrane vacuolaire et les découvertes récentes de notre laboratoire ont montré que TORCI régule positivement la fragmentation de la vacuole. Ceci suggère que le mécanisme de fragmentation doit être contrôlé par la phosphorylation de certaines protéines cibles de TORCI. J'ai exploré le phosphoprotéome vacuolaire lors de la fragmentation, en présence ou absence de rapamycine et dans des conditions provoquant la fragmentation des organelles. La méthode choisie pour réaliser la première partie de ce projet a été la spectrométrie de masse différentielle sans marquage. J'ai ainsi identifié plusieurs facteurs vacuolaires dont la phosphorylation est régulée d'une manière dépendante de TORCI et de la fragmentation. Parmi ces facteurs, des complexes protéiques connus qui sont fonctionnellement liées à fragmentation ou la fusion, comme les complexes HOPS, VTC et FAB1 ont été mis en évidence. Par conséquent, la phosphorylation dépendante de TORCI peut réguler positivement la fragmentation des vacuoles. Plusieurs candidats ont été choisis pour une analyse microscopique détaillée de la fragmentation vacuolaire in vivo en utilisant des mutants de délétion. J'ai été en mesure d'identifier de nouveaux facteurs qui n'avaient pas été encore associés à des phénotypes de fragmentation tels que les complexes SEA, Pib2p, ainsi que plusieurs transporteurs vacuolaires d'acides aminés. Le transport des acides aminés à travers la membrane semble contrôler la fragmentation de la vacuole. Puisque ces transporteurs sont phosphorylés par TORCI, ces résultats semblent confirmer la

Vesicular transport of d-serine in astrocytes

The concept of tripartite synapse suggests that astrocytes make up a functional synapse with pre- and postsynaptic neuronal elements to modulate synaptic transmission through the regulated release of neuromodulators called gliotransmitters. Release of gliotransmitters such as glutamate or D-serine has been shown to depend on Ca21-dependent exocytosis. However, the origin (cytosolic versus vesicular) of the released gliotransmitter is still a matter of debate. The existence of Ca21-regulated exocytosis in astrocytes has been questioned mostly because the nature of secretory organelles which are loaded with gliotransmitters is unknown. Here we show the existence of a population of vesicles that uptakes and stores glutamate and D-serine in astrocytes which are present in situ. Immunoisolated glial organelles expressing synaptobrevin 2 (Sb2) display morphological and biochemical features very similar to synaptic vesicles. We demonstrate that these organelles not only contain and uptake glutamate but also display a glia-specific transport activity for D-serine. Furthermore, we report that the uptake of D-serine is energized by a H1-ATPase present on the immunoisolated vesicles and that cytosolic chloride ions modulate the uptake of D-serine. Finally, we show that serine racemase (SR), the synthesizing enzyme for D-serine, is anchored to the membrane of glial organelles allowing a local and efficient concentration of the gliotransmitter to be transported. We conclude that vesicles in astrocytes do exist with the goal to store and release D-serine, glutamate and most likely other neuromodulators.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Bcl10 controls TCR- and FcgammaR-induced actin polymerization.

Bcl10 plays an essential role in the adaptive immune response, because Bcl10-deficient lymphocytes show impaired Ag receptor-induced NF-kappaB activation and cytokine production. Bcl10 is a phosphoprotein, but the physiological relevance of this posttranslational modification remains poorly defined. In this study, we report that Bcl10 is rapidly phosphorylated upon activation of human T cells by PMA/ionomycin- or anti-CD3 treatment, and identify Ser(138) as a key residue necessary for Bcl10 phosphorylation. We also show that a phosphorylation-deficient Ser(138)/Ala mutant specifically inhibits TCR-induced actin polymerization yet does not affect NF-kappaB activation. Moreover, silencing of Bcl10, but not of caspase recruitment domain-containing MAGUK protein-1 (Carma1) induces a clear defect in TCR-induced F-actin formation, cell spreading, and conjugate formation. Remarkably, Bcl10 silencing also impairs FcgammaR-induced actin polymerization and phagocytosis in human monocytes. These results point to a key role of Bcl10 in F-actin-dependent immune responses of T cells and monocytes/macrophages.

IFN alpha activates dormant haematopoietic stem cells in vivo

Maintenance of the blood system is dependent on dormant haematopoietic stem cells (HSCs) with long-term self-renewal capacity. After injury these cells are induced to proliferate to quickly reestablish homeostasis(1). The signalling molecules promoting the exit of HSCs out of the dormant stage remain largely unknown. Here we show that in response to treatment of mice with interferon-alpha (IFN alpha), HSCs efficiently exit G(0) and enter an active cell cycle. HSCs respond to IFN alpha treatment by the increased phosphorylation of STAT1 and PKB/Akt (also known as AKT1), the expression of IFN alpha target genes, and the upregulation of stem cell antigen-1 (Sca-1, also known as LY6A). HSCs lacking the IFN alpha/beta receptor (IFNAR)(2), STAT1 (ref. 3) or Sca-1 (ref. 4) are insensitive to IFN alpha stimulation, demonstrating that STAT1 and Sca-1 mediate IFN alpha-induced HSC proliferation. Although dormant HSCs are resistant to the anti-proliferative chemotherapeutic agent 5-fluoro-uracil(1,5), HSCs pre-treated (primed) with IFN alpha and thus induced to proliferate are efficiently eliminated by 5-fluoro-uracil exposure in vivo. Conversely, HSCs chronically activated by IFN alpha are functionally compromised and are rapidly out-competed by non-activatable Ifnar(-/-) cells in competitive repopulation assays. Whereas chronic activation of the IFN alpha pathway in HSCs impairs their function, acute IFN alpha treatment promotes the proliferation of dormant HSCs in vivo. These data may help to clarify the so far unexplained clinical effects of IFN alpha on leukaemic cells(6,7), and raise the possibility for new applications of type I interferons to target cancer stem cells(8).

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease.

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.

Effect of different G protein-coupled receptor kinases on phosphorylation and desensitization of the alpha1B-adrenergic receptor.

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative betaARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 resulted in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor was coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated.

A concerted kinase interplay identifies PPARgamma as a molecular target of ghrelin signaling in macrophages.

The peroxisome proliferator-activator receptor PPARgamma plays an essential role in vascular biology, modulating macrophage function and atherosclerosis progression. Recently, we have described the beneficial effect of combined activation of the ghrelin/GHS-R1a receptor and the scavenger receptor CD36 to induce macrophage cholesterol release through transcriptional activation of PPARgamma. Although the interplay between CD36 and PPARgamma in atherogenesis is well recognized, the contribution of the ghrelin receptor to regulate PPARgamma remains unknown. Here, we demonstrate that ghrelin triggers PPARgamma activation through a concerted signaling cascade involving Erk1/2 and Akt kinases, resulting in enhanced expression of downstream effectors LXRalpha and ABC sterol transporters in human macrophages. These effects were associated with enhanced PPARgamma phosphorylation independently of the inhibitory conserved serine-84. Src tyrosine kinase Fyn was identified as being recruited to GHS-R1a in response to ghrelin, but failure of activated Fyn to enhance PPARgamma Ser-84 specific phosphorylation relied on the concomitant recruitment of docking protein Dok-1, which prevented optimal activation of the Erk1/2 pathway. Also, substitution of Ser-84 preserved the ghrelin-induced PPARgamma activity and responsiveness to Src inhibition, supporting a mechanism independent of Ser-84 in PPARgamma response to ghrelin. Consistent with this, we found that ghrelin promoted the PI3-K/Akt pathway in a Galphaq-dependent manner, resulting in Akt recruitment to PPARgamma, enhanced PPARgamma phosphorylation and activation independently of Ser-84, and increased expression of LXRalpha and ABCA1/G1. Collectively, these results illustrate a complex interplay involving Fyn/Dok-1/Erk and Galphaq/PI3-K/Akt pathways to transduce in a concerted manner responsiveness of PPARgamma to ghrelin in macrophages.

Expression of genes encoding cytotoxic cell-associated serine proteases in thymocytes.

A family of homologous serine esterases designated granzyme A-H and the pore-forming protein perforin are present in cytoplasmic granules of mature peripheral cytolytic T lymphocytes and natural killer cells. In vivo, the majority of cytotoxic T cells containing these granule-associated proteins are of the CD4-CD8+ phenotype. It is generally assumed that these cells are derived from immature CD4-CD8- thymocytes. However, the precise intrathymic differentiation steps leading to functionally mature cytotoxic T cells are unclear. Thus we decided to analyze the expression of genes in the thymus which are preferentially expressed in mature cytotoxic cells, i.e. granzyme A, granzyme B, and perforin. In situ hybridization on tissue sections revealed the expression of genes coding for granzyme A and granzyme B in the thymus. No evidence was found, however, for thymocytes expressing the perforin gene. Granzyme A and granzyme B mRNA positive cells in the thymus are almost exclusively CD4-CD8- thymocytes, particularly of the CD3- IL2R- phenotype.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase.

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. L-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of L-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Pom1 regulates the assembly of Cdr2-Mid1 cortical nodes for robust spatial control of cytokinesis.

Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.