K depletion increases protein tyrosine kinase-mediated phosphorylation of ROMK

We purified Histagged ROMK1 and carried out in vitro phosphorylation assays with P-32-radiolabeled ATP to determine whether ROMK1 protein is a substrate for PTK. Addition of active c-Src and [P-32]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.