Hormone-regulated Na transport in tight epithelia is essential for maintenance of circulatory volume and regulation of the ionic milieu in mammals. SGK1 is an aldosterone-regulated PI3-kinase-dependent serine-threonine kinase that plays an important role in mediating hormonal regulation of Na transport through its effects on trafficking of the epithelial Na channel (ENaC), the rate limiting step in Na+ reabsorption in tight epithelia. SGK1 phosphorylate and inhibits Nedd4-2, a ubiquitin ligase that inhibits ENaC primarily by altering its trafficking and degradation, thus leading to ENaC accumulation in the plasma membrane. We recently found that SGK1-mediated phosphorylation of Nedd4-2 triggers recruitment of a chaperone protein of the 14-3-3 family. These observations and other recent studies suggest the following hypotheses: 1) Interactions with phosphoinositide lipids activate SGK1 and help direct it to the appropriate cellular location to inhibit Nedd4-2 and stimulate ENaC; 2) SGK1 inhibits Nedd4-2 by stimulating its interaction with the chaperone inhibitor 14-3-3sigma, or another 14-3-3 protein; 3) The opposing effects of Nedd4-2 and SGK1 are manifested in changes in ENaC endocytosis and endosomal localization. We propose the following aims: 1) Determine the functional roles and physical basis of SGK1 interactions with phosphoinositides. We will determine which phosphoinositides interact with SGK1 using phosphoinositide lipids fixed to nitrocellulose membranes, and incorporated into liposomes. We will determine the relationship of phosphoinositide binding and function of this novel domain by examining the effect of SGK1 mutants on ENaC-mediated Na+ currents and on in vitro kinase activity. We will determine the role of the SGK1 phosphoinositide interaction domain in targeting SGK1 to specific cellular compartments in response to PI3K activation. 2) Determine the role of 14-3-3 proteins in inhibiting Nedd4-2. We will test the ability of SGK1 to stimulate ENaC-mediated Na+ currents in the presence of Nedd4-2 mutants that have their 14-3-3 interaction motif disrupted. We will determine which 14-3-3 isoforms are expressed in kidney CD and in cultured CCD cells, and interact with Nedd4-2 in cultured CCD cells using isoform specific antibodies in coimmunoprecipitation assays. We will determine the effect on aldosterone-stimulated Na+ transport of eliminating expression of specific 14-3-3 isoforms using siRNA. 3) Determine the effect of SGK1 and Nedd4-2 on ENaC trafficking, and characterize the mechanistic basis of this effect. We will use live cell staining of epitope-tagged transfected ENaC to examine the effects of SGK1 and Nedd4-2 on plasma membrane localization of ENaC. We will determine the role(s) of exocytosis and endocytosis in regulated ENaC trafficking by examining the effects of inhibitors and dominant negative dynamin on cell surface ENaC, and examine the correlation of ENaC currents and apical trafficking by biotinylation of endogenous ENaC in a model CCD cell (mpkCCD). We will establish systems for examining ENaC post-endocytosis. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK056695-05A2
Application #
7033633
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
2000-08-15
Project End
2010-12-31
Budget Start
2006-01-01
Budget End
2006-12-31
Support Year
5
Fiscal Year
2006
Total Cost
$312,626
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Morze, Cornelius von; Allu, Prasanna K R; Chang, Gene Y et al. (2018) Non-invasive detection of divergent metabolic signals in insulin deficiency vs. insulin resistance in vivo. Sci Rep 8:2088
von Morze, Cornelius; Chang, Gene-Yuan; Larson, Peder E Z et al. (2017) Detection of localized changes in the metabolism of hyperpolarized gluconeogenic precursors13C-lactate and13C-pyruvate in kidney and liver. Magn Reson Med 77:1429-1437
Rashmi, Priyanka; Colussi, GianLuca; Ng, Michael et al. (2017) Glucocorticoid-induced leucine zipper protein regulates sodium and potassium balance in the distal nephron. Kidney Int 91:1159-1177
Marco-Rius, Irene; Cao, Peng; von Morze, Cornelius et al. (2017) Multiband spectral-spatial RF excitation for hyperpolarized [2-13C]dihydroxyacetone13C-MR metabolism studies. Magn Reson Med 77:1419-1428
Lang, Florian; Pearce, David (2016) Regulation of the epithelial Na+ channel by the mTORC2/SGK1 pathway. Nephrol Dial Transplant 31:200-5
Gleason, Catherine E; Frindt, Gustavo; Cheng, Chih-Jen et al. (2015) mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity. J Clin Invest 125:117-28
Soundararajan, Rama; Lu, Ming; Pearce, David (2012) Organization of the ENaC-regulatory machinery. Crit Rev Biochem Mol Biol 47:349-59
Soundararajan, Rama; Pearce, David; Ziera, Tim (2012) The role of the ENaC-regulatory complex in aldosterone-mediated sodium transport. Mol Cell Endocrinol 350:242-7
Schmid, Evi; Bhandaru, Madhuri; Nurbaeva, Meerim K et al. (2012) SGK3 regulates Ca(2+) entry and migration of dendritic cells. Cell Physiol Biochem 30:1423-35
Romero, Y; Vuandaba, M; Suarez, P et al. (2012) The Glucocorticoid-induced leucine zipper (GILZ) Is essential for spermatogonial survival and spermatogenesis. Sex Dev 6:169-77

Showing the most recent 10 out of 39 publications