Article

Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2

Discipline of Physiology, School of Medical Science, Faculty of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia.
Journal of Biological Chemistry (Impact Factor: 4.6). 11/2007; 282(41):29866-73. DOI: 10.1074/jbc.M701923200
Source: PubMed

ABSTRACT The epithelial sodium channel (ENaC) plays an important role in transepithelial Na(+) absorption; hence its function is essential for maintaining Na(+) and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1(K179M), decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser(342) and Ser(428), completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na(+) absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.

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However, the transgenic overexpression of SGK-1 in the intestine suppressed the dauer formation constitutive (Daf-c) phenotype of daf-2(e1370) mutants, demonstrating a role for SGK 1 in dauer formation. In addition, using qRT-PCR a downregulation of sgk-1 transcription in a daf 2 mutant was observed, in which DAF-16 is constitutively activated. This repression was significantly reversed in a daf 16; daf-2 double mutant, suggesting a DAF-16-dependent transcription of sgk-1. In the second part of this study, developmental programmed cell death was analyzed in both soma and germline of sgk 1(ok538) mutant. Using 4D time-lapse microscopy, the accelerated kinetics of apoptotic cell engulfment during somatic programmed cell death could be shown in the sgk 1(ok538). Reverse genetic approaches combined with developmental features of the programmed cell death indicate that SGK-1 negatively regulates apoptotic cell engulfment via the CED 5/CED-12/CED-10 pathway, in part independently on CED-2. The loss of sgk-1 only reduced the number of cell corpses in strong loss-of-function mutants of ced-1, ced-6, ced-7, and ced-2 but not of the alternative pathway of ced-5, ced-12, and ced 10. Triple mutant analyses confirmed placement of sgk 1 in the ced-5 pathway. When in addition to ced-7 or ced-1, sgk 1 was mutated together with ced-5, the concomitant mutation of sgk-1 and ced-5 could not reduce the number of persistent cell corpses in ced-7 or ced-1 mutants. In addition, sgk-1 genetically interacted with mig-2 and unc-73, which are described to affect actin cytoskeleton rearrangements and promote apoptotic cell engulfment via the ced 5/ced 12/ced-10 pathway. The analyses of the programmed cell death in the germline showed a similar function of SGK-1 in the removal of apoptotic germ cell corpses. In addition, the migration of the distal tip cells (DTC) during gonad development was analyzed in sgk-1 mutant, because mutations in ced-2, ced-5, ced-12, ced-10, mig-2 and unc-73 display in addition to engulfment deficiency also abnormal migration of the DTC The sgk 1(ok538) mutation also resulted in mild DTC migration defect and enhanced the DTC migration phenotype of ced-12 and mig-2. The DTC migration phenotype of strong loss-of-function allele of ced-10 was significantly suppressed by the sgk-1 mutation. In the third part of this study, in vitro phosphorylation and pull-down analyses provide the first biochemical evidence that CED-5 and CED-12 are substrates of the SGK-1 kinase in C. elegans. Moreover, the phosphorylation of the PXXP domain of CED-5 by SGK-1 did not affect the physical interaction of CED-5 and CED-2. However, CED-5 was no longer phosphorylated by SGK-1 when CED-2 was bound, indicating that the interaction of CED 2 with the PXXP domain of CED-5 prevented the phosphorylation by SGK 1. The interaction of CED-2 with CED-5 did not prevent the interaction of SGK-1 with CED-5 as a CED 2/CED 5/SGK 1 ternary complex was observed. The biochemical analyses confirm the genetic data and corroborate the model of sgk-1 acting within the ced 5/ced 12/ced 10 pathway. Moreover, they demonstrate that SGK-1 acts as a negative modulator of CED 5/CED-12 upstream of CED-10 to control cytoskeleton dynamics during apoptotic cell engulfment and DTC migration in C. elegans. In an attempt to further identify factors involved in the CED 5/CED-12/SGK-1/CED-10 signaling pathway of C. elegans, a yeast two-hybrid split-ubiquitin screen was performed using CED-12 as bait. The screen yielded 11 putative interactors from cellular pathways including metabolism, development, and stress response. Die Isoformen der Serum- und Glukokortikoid-induzierbaren Kinase (SGK) spielen sowohl in der Embryonalentwicklung als auch in der Pathophysiologie einer Reihe von Krankheiten eine bedeutende Rolle, indem sie die Aktivität diverser Ionenkanäle, der Glukoseaufnahme der Zelle, der Zellzyklus-Progression sowie des Zelltodes regulieren. Im Nematoden Caenorhabditis elegans (C. elegans) vermittelt das einzige sgk-1-Genprodukt den DAF 2/insulin-ähnlichen Signalweg. In dieser Arbeit wurde das Allel der Deletionsmutante sgk 1(ok538) im Modellorganismus C. elegans charakterisiert. Die Mutante zeigt sowohl eine reduzierte pharyngeale Pumprate als auch eine reduzierte Fruchtbarkeit ähnlich den in der Nahrungsaufnahme reduzierten Mutanten; sie wies aber eine wildtyp-ähnliche Lebensspanne auf. Außerdem zeigte sie eine erhöhte Empfindlichkeit gegenüber oxidativem und hitzebedingtem Stress, wohingegen bei limitierenden Umweltbedingungen eine normale Entwicklung zur Dauerlarve erfolgte. Dennoch supprimierte die transgene Überexpression von SGK-1 im Darm den Phänotyp der konstitutiven Ausbildung der Dauerlarve (Dauer formation constitutive, Daf-c) der daf 2(e1370)-Mutante, was auf eine Rolle von SGK 1 in der Entwicklung der Dauerlarve hinweist. Mit Hilfe der qRT-PCR-Methode war zusätzlich eine Herunterregulierung der sgk-1-Transkription in der daf-2-Mutante, in welcher der DAF-16 Transkriptionsfaktor konstitutiv aktiv ist, zu beobachten. Diese Repression wurde in einer daf 16; daf-2-Doppelmutante signifikant unterdrückt, womit eine DAF-16-abhängige Transkription von sgk-1 belegt wurde. Im zweiten Teil dieser Arbeit wurde der entwicklungsbedingte programmierte Zelltod im Soma wie auch in der Keimbahn der sgk-1-Mutante untersucht. Mittels 4D-Langzeit- Mikroskopie konnte eine beschleunigte Kinetik der Phagozytose während des programmierten Zelltodes im Soma von sgk-1(ok538) nachgewiesen werden. Reverse genetische Untersuchungen des programmierten Zelltodes deuten darauf hin, dass SGK-1 die Phagozytose der apoptotischen Zellen über den CED 5/CED 12/CED-10-Signalweg negativ reguliert, wobei eine partielle Unabhängigkeit von CED-2 besteht. Der Verlust von sgk 1 führte zu eine Reduktion der persistierenden apoptotischen Zellkörper in starken Funktionsverlustmutanten von ced 1, ced-6, ced-7 und ced-2, nicht aber in ced-5, ced-12 und ced 10, welche den alternativen Signalweg definieren. Die Analyse von Dreifachmutanten bestätigte die Platzierung von sgk-1 im ced-5-Signalweg. Wenn zusätzlich zu ced-7 oder ced-1 sgk 1 zusammen mit ced-5 mutiert waren, konnte die Anzahl der persistierenden apoptotischen Zellkörper von ced-7- oder ced-1-Mutanten nicht mehr reduziert werden. Weiterhin interagierte sgk-1 genetisch mit mig-2 und unc-73, von denen bekannt ist, dass sie die Reorganisation des Aktinzytoskeletts beeinflussen und die Phagozytose der apoptotischen Zellen über den ced 5/ced 12/ced-10-Signalweg fördern. Die Untersuchungen des programmierten Zelltodes in der Keimbahn zeigten eine ähnliche Funktion von SGK-1 in der Beseitigung der apoptotischen Zellkörper. Weil die Mutationen in ced-2, ced-5, ced-12, ced-10, mig-2 und unc-73 neben den Störungen der Phagozytose der apoptotischen Zellen auch Defekte der Migration der distalen Endzellen (distal tip cells, DTC) aufweisen, wurde auch die Migration der DTC während der Entwicklung der Gonade in der sgk-1-Mutante untersucht. Die Mutation von sgk 1(ok538) zeigt einen milden Defekt der Migration der DTC und steigert den Migrationsphänotyp der DTC der ced 12- und mig-2-Mutanten. Der Migrationsphänotyp der DTC in der starken Funktionsverlustmutation von ced-10 wurde durch die sgk-1-Mutation signifikant supprimiert. Im dritten Teil dieser Arbeit liefern in vitro Phosphorylierung- und Protein-Protein-Interaktionsstudien (pull-down analyses) die ersten biochemischen Beweise dafür, dass CED 5 und CED-12 Substrate der SGK-1-Kinase in C. elegans sind. Weiterhin störte die Phosphorylierung der CED-5-PXXP-Domäne von SGK-1 nicht die physische Interaktion von CED-5 und CED-2. Jedoch konnte CED-5 nicht mehr von SGK-1 phosphoryliert werden, wenn CED-2 zuvor gebunden hatte. Dies zeigt, dass die physische Bindung von CED-2 an die PXXP-Domäne von CED-5 die Phosphorylierung von CED-5 durch SGK-1 verhindert wird. Die Bindung von CED-2 an CED-5 verhinderte jedoch nicht die Interaktion von SGK-1 mit CED-5; ursächlich dafür war die Bildung eines ternären CED 2/CED 5/SGK 1-Komplexes zu beobachten. Die biochemischen Analysen bestätigen die genetischen Daten und stützen das Modell der Funktion von sgk-1 innerhalb des ced 5/ced 12/ced 10-Signalwegs. Darüber hinaus demonstrieren sie, dass SGK-1 als ein negativer Regulator von CED-5/CED-12 oberhalb von CED-10 funktioniert und dadurch die Dynamik des Aktinzytoskeletts während der Phagozytose der apoptotischen Zellen und der Migration der DTC in C. elegans reguliert. Um weitere Faktoren des CED 5/CED-12/SGK-1/CED-10-Signalwegs in C. elegans zu identifizieren, wurden ein Protein-Protein-Interaktionsscreen mit einem Split-Ubiquitin-Hefe-Zwei-Hybrid-System und CED-12 als Bait-Protein (Köderprotein) durchgeführt. Dabei wurden elf putative Wechselwirkungspartner identifiziert. Diese Interaktoren besitzen Funktionen in den Bereichen Metabolismus, Entwicklung und Stressreaktion.