title: Phosphopeptide screen uncovers novel phosphorylation sites of Nedd4-2 that potentiate its inhibition of the epithelial Na+ channel.
abstract: 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.
authors.affiliation: Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hallows
authors.forename: Vivek
authors.initials: V
authors.lastname: Bhalla
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Oyster
authors.forename: Marjolein A
authors.initials: MA
authors.lastname: Wijngaarden
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Lee
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Sindhu
authors.initials: S
authors.lastname: Chandran
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Xia
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Huang
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Chalkley
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: David
authors.initials: D
authors.lastname: Pearce
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry*; D053503:Epithelial Sodium Channels / Q000737:chemistry*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D051379:Mice; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D014981:Xenopus; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D048055:Mitogen-Activated Protein Kinase 8; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; C000618460:nedd4l protein, Xenopus
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