In vitro phosphorylation of COOH termini of the epithelial Na(+) channel and its effects on channel activity in Xenopus oocytes.
Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na(+) channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the gamma-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on beta(S620) was phosphorylated as well. The possible role of gammaT630 and betaS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na(+) and had a high basal Na(+) current. Thus mutating gammaT630 or betaS620 may limit the maximal channel activity achieved by a combination of sgk and low Na(+).
Chigaev
A
A
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Lu
G
G
Shi
H
H
Asher
C
C
Xu
R
R
Latter
H
H
Seger
R
R
Garty
H
H
Reuveny
E
E
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D003106:Colon / Q000166:cytology; D003600:Cytosol / Q000201:enzymology; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D015870:Gene Expression / Q000502:physiology; D005982:Glutathione Transferase / Q000235:genetics; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D008025:Ligases / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D009865:Oocytes / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D000099184:Serum-Glucocorticoid Regulated Kinases
D002135:Calcium-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005982:Glutathione Transferase; D017874:Immediate-Early Proteins; D008025:Ligases; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D010761:Phosphorus Radioisotopes; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D012964:Sodium; D015222:Sodium Channels; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases