Interactions of beta and gamma ENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation.
Phosphorylation of the epithelial Na(+) channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the beta and gamma subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of beta and gammaENaC, have been identified by an in vitro assay. One of these phosphorylates betaThr-613 and gammaThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of gammaThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating betaThr-613 and gammaThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions beta613 or gamma623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on betaThr-613 and gammaThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of betaThr-613 and gammaThr-623 down-regulates the channel by facilitating its interaction with Nedd4.
Shi
Haikun
H
Department of Biological Chemistry, The Weizmann Institute of Science, P. O. Box 26, Rehovot 76100, Israel.
Asher
Carol
C
Chigaev
Alexander
A
Yung
Yuval
Y
Reuveny
Eitan
E
Seger
Rony
R
Garty
Haim
H
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques; D015153:Blotting, Western; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005982:Glutathione Transferase / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D029867:Xenopus Proteins
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D007328:Insulin; D010455:Peptides; D020033:Protein Isoforms; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D029867:Xenopus Proteins; D013912:Threonine; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618484:Nedd4 protein, rat; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D020928:Mitogen-Activated Protein Kinases; D008025:Ligases