Characterization of interactions between Nedd4 and beta and gammaENaC using surface plasmon resonance.
Cell surface expression of the epithelial Na(+) channel ENaC is regulated by the ubiquitin ligase Nedd4. Binding of the WW domains of Nedd4 to the PY region in the carboxy tails of beta and gammaENaC, results in channel ubiquitination and degradation. Kinetic analysis of these interactions has been done using surface plasmon resonance. Synthetic peptides corresponding to the PY regions of beta and gammaENaC were immobilized on a sensor chip and "real-time" kinetics of their binding to recombinant WW proteins was determined. Specificity of the interactions was established by competition experiment, as well as by monitoring effects of a point mutation known to impair Nedd4/ENaC binding. These data provides the first determination of association, dissociation and equilibrium constants for the interactions between WW2 and beta or gammaENaC.
Asher
C
C
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
Chigaev
A
A
Garty
H
H
D000818:Animals; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015222:Sodium Channels; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618484:Nedd4 protein, rat; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008025:Ligases