Phosphopeptide screen uncovers novel phosphorylation sites of Nedd4-2 that potentiate its inhibition of the epithelial Na+ channel.
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.
PubMed ID: 20466724
PMC ID: PMC2898378
Article link: J Biol Chem.
Grant support: K08 DK071648 NIDDK NIH HHS , P30 DK079307 NIDDK NIH HHS , R01 DK056695 NIDDK NIH HHS , R01 DK075048-01A1 NIDDK NIH HHS , R01 DK075048-02 NIDDK NIH HHS , R01 DK075048-03 NIDDK NIH HHS , R01 DK075048-04 NIDDK NIH HHS , R01DK075048 NIDDK NIH HHS , RR015804 NCRR NIH HHS , RR01614 NCRR NIH HHS , P30 DK072506 NIDDK NIH HHS , R01 DK075048 NIDDK NIH HHS
Genes referenced: mapk1 mapk14 mapk8 nedd4 nedd4l sgk1