Scoring of predicted GRK2 phosphorylation sites in Nedd4-2.
BACKGROUND
Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4-2. These ligases bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of this inhibition leads to hypertension. We have previously reported that ENaC channels are maintained in the active state by the G protein coupled receptor kinase, GRK2. The enzyme has been implicated in the development of essential hypertension [R. D. Feldman (2002) Mol. Pharmacol., 61, 707-709]. Additional findings in our lab pointed towards a possible role for GRK2 in the phosphorylation and inactivation of Nedd4-2.


RESULTS
We have predicted GRK2 phosphorylation sites on Nedd4-2 by combining sequence analysis, homology modeling and surface accessibility calculations. A total of 24 potential phosphorylation sites were predicted by sequence analysis. Of these, 16 could be modeled using homology modeling and 6 of these were found to have sufficient surface exposure to be accessible to the GRK2 enzyme responsible for the phosphorylation of Nedd4-2. The method provides an ordered list of the most probable GRK2 phosphorylation sites on Nedd4-2 providing invaluable guidance to future experimental studies aimed at mutating certain Nedd4-2 residues in order to prevent phosphorylation by GRK2. The method developed could be applied in a wide variety of biological applications involving the binding of one molecule to a protein. The relative effectiveness of the technique is determined mainly by the quality of the homology model built for the protein of interest.


BACKGROUND
jarthur@med.usyd.edu.au
Arthur
Jonathan W
JW
Department of Medicine, University of Sydney Sydney, Australia. jarthur@med.usyd.edu.au
Sanchez-Perez
Angeles
A
Cook
David I
DI
D000465:Algorithms; D000595:Amino Acid Sequence; D001185:Artificial Intelligence; D001665:Binding Sites; D003198:Computer Simulation; D056827:Endosomal Sorting Complexes Required for Transport; D054769:G-Protein-Coupled Receptor Kinase 2; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D016415:Sequence Alignment / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D051552:beta-Adrenergic Receptor Kinases / Q000737:chemistry*
D056827:Endosomal Sorting Complexes Required for Transport; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2