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Articles in PresS, published online ahead of print June 11, 2002
Am J Physiol Renal Physiol, 10.1152/ajprenal.00160.2002
Submitted on April 26, 2002
Accepted on May 23, 2002
1 Department of Pharmacology, New York Medical College, Valhalla, NY, USA; Department of Microbiology, Harbin Medical University, Harbin, China
2 Department of Pharmacology, New York Medical College, Valhalla, NY, USA
3 Department of Anatomy and Cell Biology, New York Medical College, Valhalla, NY, USA
4 Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, USA
5 Department of Microbiology, Harbin Medical University, Harbin, China
6 Department of C & M Physiology, Yale University School of Medicine, New Haven, CT, USA
* To whom correspondence should be addressed. E-mail: wenhui_wang{at}nymc.edu.
We have previously demonstrated that stimulating protein tyrosine kinase (PTK) suppressed whereas stimulating protein tyrosine phosphatase increased the activity of ROMK1. To determine whether ROMK1 protein is a substrate for PTK, we purified His-tagged ROMK1 fusion protein from E.coli and carried out in vitro phosphorylation assays with radio labeled [32P]ATP. Addition of active c-Src and [32P]ATP to the purified ROMK1 protein resulted in the phosphorylation of the ROMK1 protein. In contrast, c-Src did not phosphorylate R1Y337A in which tyrosine residue 337 was mutated to alanine. The notion that tyrosine residue 337 is critical for the phosphorylation of ROMK1 by cSrc was further confirmed by phosphopeptide mapping in which two phosphopeptides were identified 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. Moreover, addition of c-Src and [32P]ATP also phosphorylated the synthesized peptide corresponding to amino acid sequence 333-362 of the C-terminus of ROMK1. We then examined whether dietary K intake affects tyrosine phosphorylation of ROMK channels. Western blot demonstrated that the tyrosine-phosphorylated ROMK channels, which were harvested by immunoprecipitation with 4G10 (antibody that reacts with the tyrosine- phosphorylated proteins), were significantly higher in the renal cortex and outer medulla in rats on a K-deficient (KD) diet than those on a high K (HK) diet. Moreover, although the ROMK channels pulled down by immunoprecipitation with ROMK antibody were the same from rats on KD or HK diet, Western blot with 4G10 showed that more ROMK channels were phosphorylated by PTK in rats on a KD diet than those on a HK 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.
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