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

• Published in: The Journal of biological chemistry Vol. 285; no. 28; pp. 21671 - 21678
• Main Authors: Hallows, Kenneth R., Bhalla, Vivek, Oyster, Nicholas M., Wijngaarden, Marjolein A., Lee, Jeffrey K., Li, Hui, Chandran, Sindhu, Xia, Xiaoyu, Huang, Zhirong, Chalkley, Robert J., Burlingame, Alma L., Pearce, David
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.07.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; E3 Ubiquitin Ligase; Endosomal Sorting Complexes Required for Transport - chemistry; Epithelial Sodium Channels - chemistry; Humans; Immediate-Early Proteins - metabolism; JNK; Mass Spectrometry (MS); Mice; Mitogen-Activated Protein Kinase 8 - metabolism; Molecular Sequence Data; Nedd4 Ubiquitin Protein Ligases; Nedd4-2; Phosphorylation; Protein Phosphorylation; Protein Serine-Threonine Kinases - metabolism; Recombinant Proteins - chemistry; SGK1; Signal Transduction; Sodium Channels; Tandem Mass Spectrometry - methods; Ubiquitin - chemistry; Ubiquitin-Protein Ligases - chemistry; Xenopus; Xenopus Proteins; E3 Ubiquitin Ligase; JNK; Sodium Channels; Mass Spectrometry (MS); Protein Phosphorylation; Nedd4-2; SGK1
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M109.084731

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.