Phosphopeptide Screen Uncovers Novel Phosphorylation Sites of Nedd4-2 That Potentiate Its Inhibition of the Epithelial Na+ Channel

Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2010; 285(28):21671-8. DOI: 10.1074/jbc.M109.084731
Source: PubMed


The E3 ubiquitin ligase Nedd4-2 regulates several ion transport proteins, including the epithelial Na+ channel (ENaC). Nedd4-2 decreases apical membrane expression and activity of ENaC. Although it is subject to tight hormonal
control, the mechanistic basis of Nedd4-2 regulation remains poorly understood. To characterize regulatory inputs to Nedd4-2
function, we screened for novel sites of Nedd4-2 phosphorylation using tandem mass spectrometry. Three of seven identified
Xenopus Nedd4-2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for SGK1, whereas four were novel,
including Ser-293, which matched the consensus for a MAPK target sequence. Further in vitro and in vivo phosphorylation experiments revealed that Nedd4-2 serves as a target of JNK1, but not of p38 MAPK or ERK1/2. Additional rounds
of tandem mass spectrometry identified two other phosphorylated residues within Nedd4-2, including Thr-899, which is present
within the catalytic domain. Nedd4-2 with mutations at these sites had markedly inhibited JNK1-dependent phosphorylation,
virtually no ENaC inhibitory activity, and significantly reduced ubiquitin ligase activity. These data identify phosphorylatable
residues that activate Nedd4-2 and may work together with residues targeted by inhibitory kinases (e.g. SGK1 and protein kinase A) to govern Nedd4-2 regulation of epithelial ion transport.

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Available from: Vivek Bhalla, Nov 30, 2015
    • "Rsp5 is the only yeast member of the Nedd4 family of ubiquitin ligases (E3), which have a common C2-WW-HECT modular architecture with C2 calcium and a lipid-binding domain, three WW protein-binding domains and a catalytic HECT domain. The cysteine residue in the M a n u s c r i p t 4 HECT domains (Gallagher et al., 2006) phosphorylation of T899 in the HECT domain of Nedd4-2 increases the activity by unknown mechanism (Hallows et al., 2010). Several kinases are involved in phosphorylation of Nedd4 family members in vivo, including PKA (Snyder et al., 2004). "
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