Characterization of the interactions between Nedd4-2, ENaC, and sgk-1 using surface plasmon resonance.
Previous studies have characterized interactions between the ubiquitin ligase Nedd4-1 and the epithelial Na(+) channel (ENaC). Such interactions control the channel cell surface expression and activity. Recently, evidence has been provided that a related protein, termed Nedd4-2, is likely to be the true physiological regulator of the channel. Unlike Nedd4-1, Nedd4-2 also interacts with the aldosterone-induced channel activating kinase sgk-1. The current study uses surface plasmon resonance to quantify the binding of the four WW domains of Nedd4-2 to synthetic peptides corresponding to the PY motifs of ENaC and sgk-1. The measurements demonstrate that WW3 and WW4 are the only Nedd4-2 domains interacting with both ENaC and sgk-1 and that their binding constants are in the 1-6 microM range.
Asher
Carol
C
Department of Biological Chemistry, The Weizmann Institute of Science, 76100, Rehovot, Israel.
Sinha
Indranil
I
Garty
Haim
H
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D008025:Ligases; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D015222:Sodium Channels; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618461:Nedd4l protein, mouse