This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Under conditions of metabolic stress, the expression and activity of many membrane transport proteins including the epithelial sodium channel, ENaC, are inhibited, thereby limiting the dissipation of ionic gradients and preserving the cellular energy required to maintain them. The cellular mechanisms that link membrane transport to energy production and metabolic status are only beginning to be revealed. The authors have previously shown that ENaC is inhibited by AMP-activated protein kinase (AMPK, a ubiquitous serine/theonine kinase that participates as a metabolic sensor and metabolic regulator in many systems). AMPK does not directly phosphorylate ENaC, but may interact with regulators of ENaC-mediated Na+ transport such as the ubiquitin ligase, Nedd4-2. Preliminary experiments indicate that AMPK is unable to inhibit ENaC-mediated Na+ current when using variants of ENaC subunits unable to interact with Nedd4-2. Additionally, AMPK phosphorylates Nedd4-2 in vitro. This line of evidence suggests the following hypothesis: Nedd4-2 may serve as a direct target of AMPK, and AMPK may phosphorylate Nedd4-2 and therefore augment its ability to bind to ENaC or its ubiquitin ligase activity. In order to test this hypothesis, the authors need to establish whether AMPK phosphorylates Nedd4-2 in vivo and at what sites this phosphorylation occurs. We will modulate AMPK activity in cultured cells and purify Nedd4-2 for analysis. We will analyze sites of AMPK phosphorylation of Nedd4-2 by strong cation exchange chromatography fractionation of a tryptic digest of Nedd4-2 followed by non-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001614-27
Application #
7957405
Study Section
Special Emphasis Panel (ZRG1-BCMB-M (40))
Project Start
2009-06-01
Project End
2010-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
27
Fiscal Year
2009
Total Cost
$7,250
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
MacRae, Andrew J; Mayerle, Megan; Hrabeta-Robinson, Eva et al. (2018) Prp8 positioning of U5 snRNA is linked to 5' splice site recognition. RNA 24:769-777
Katsuno, Yoko; Qin, Jian; Oses-Prieto, Juan et al. (2018) Arginine methylation of SMAD7 by PRMT1 in TGF-?-induced epithelial-mesenchymal transition and epithelial stem-cell generation. J Biol Chem 293:13059-13072
Sahoo, Pabitra K; Smith, Deanna S; Perrone-Bizzozero, Nora et al. (2018) Axonal mRNA transport and translation at a glance. J Cell Sci 131:
Tran, Vy M; Wade, Anna; McKinney, Andrew et al. (2017) Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion. Mol Cancer Res 15:1623-1633
Liu, Tzu-Yu; Huang, Hector H; Wheeler, Diamond et al. (2017) Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics. Cell Syst 4:636-644.e9
Bikle, Daniel D (2016) Extraskeletal actions of vitamin D. Ann N Y Acad Sci 1376:29-52
Twiss, Jeffery L; Fainzilber, Mike (2016) Neuroproteomics: How Many Angels can be Identified in an Extract from the Head of a Pin? Mol Cell Proteomics 15:341-3
Cil, Onur; Phuan, Puay-Wah; Lee, Sujin et al. (2016) CFTR activator increases intestinal fluid secretion and normalizes stool output in a mouse model of constipation. Cell Mol Gastroenterol Hepatol 2:317-327
Posch, Christian; Sanlorenzo, Martina; Vujic, Igor et al. (2016) Phosphoproteomic Analyses of NRAS(G12) and NRAS(Q61) Mutant Melanocytes Reveal Increased CK2? Kinase Levels in NRAS(Q61) Mutant Cells. J Invest Dermatol 136:2041-2048
Julien, Olivier; Zhuang, Min; Wiita, Arun P et al. (2016) Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc Natl Acad Sci U S A 113:E2001-10

Showing the most recent 10 out of 630 publications