K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK.
We purified His-tagged ROMK1 and carried out in vitro phosphorylation assays with (32)P-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [(32)P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. However, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. Furthermore, phosphopeptide mapping identified two phosphopeptides from the trypsin-digested ROMK1 protein. In contrast, no phosphorylated peptide has been found in the trypsin-digested R1Y337A protein. This suggested that two phosphorylated peptides might contain the same tyrosine residue. Also, addition of c-Src and [(32)P]ATP phosphorylated the synthesized peptide corresponding to amino acid sequence 333-362 of the COOH terminus of ROMK1. We then examined the effect of dietary K intake on the tyrosine-phosphorylated ROMK level. Although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on a K-deficient diet or on a high-K diet, more ROMK channels were phosphorylated by PTK in rats on a K-deficient diet than those on a high-K diet. We conclude that ROMK1 can be phosphorylated by PTK and that tyrosine residue 337 is the key site for the phosphorylation. Also, the tyrosine phosphorylation of ROMK is modulated by dietary K intake. This strongly suggests that PTK is an important member of the aldosterone-independent signal transduction pathway for regulating renal K secretion.
Lin
Dao-Hong
DH
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
Sterling
Hyacinth
H
Lerea
Kenneth M
KM
Welling
Paul
P
Jin
Lianhong
L
Giebisch
Gerhard
G
Wang
Wen-Hui
WH
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D008297:Male; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011188:Potassium / Q000502:physiology*; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D017419:Potassium, Dietary / Q000494:pharmacology; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
C510692:Kcnj1 protein, rat; D010748:Phosphopeptides; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D017419:Potassium, Dietary; D011994:Recombinant Proteins; D014443:Tyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; D011188:Potassium