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Phosphopeptide Screen Uncovers JNK1 as a Potentiator of Nedd4‐2‐Mediated Epithelial Na+ Channel Inhibition
Author(s) -
Bhalla Vivek,
Oyster Nicholas M,
Wijngaarden Marjolein,
Lee Jeffrey,
Li Hui,
Xia Xiaoyu,
Huang Zhirong,
Chalkley Robert,
Burlingame Alma,
Pearce David,
Hallows Ken
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.611.18
Subject(s) - nedd4 , sgk1 , ubiquitin ligase , phosphorylation , kinase , chemistry , epithelial sodium channel , mapk/erk pathway , microbiology and biotechnology , protein kinase a , ubiquitin , biochemistry , biology , gene , organic chemistry , sodium
The ubiquitin ligase Nedd4‐2 inhibits several ion transport proteins including the epithelial Na + channel (ENaC). To define new regulatory inputs to Nedd4‐2 function, we screened for novel sites of Nedd4‐2 phosphorylation using tandem mass spectrometry. Three of seven identified Nedd4‐2 Ser/Thr phosphorylation sites corresponded to previously identified target sites for serum‐and‐glucocorticoid kinase 1 (SGK1), while four were novel, including Ser‐293, which matched the consensus for a mitogen‐activated protein kinase (MAPK) target sequence. Nedd4‐2 was found to be a target of c‐Jun N‐terminal kinase 1 (JNK1), but not of p38‐MAPK or extracellular‐regulated kinase (ERK1/2). Further rounds of tandem mass spectrometry identified two additional JNK1‐phosphorylated residues within Nedd4‐2: Thr‐408 and Thr‐899. Mutations at all three JNK1 sites markedly inhibited JNK1‐dependent phosphorylation, ENaC‐inhibitory activity by Nedd4‐2, and Nedd4‐2 ubiquitin ligase activity. Together, these data establish JNK1 as a novel potentiator of Nedd4‐2, which may work in conjunction with inhibitory kinases (e.g. SGK1 and PKA) to govern Nedd4‐2 regulation of epithelial ion transport. Supported by the NIH NIDDK.