NHE3 phosphorylation at serines 552 and 605 does not directly affect NHE3 activity.
Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by (22)Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na(+)/H(+) exchange activity.
Kocinsky
Hetal S
HS
Dept. of Pediatrics, Yale University, New Haven, CT 06520-8064, USA. hetal.kocinsky@yale.edu
Dynia
Diane W
DW
Wang
Tong
T
Aronson
Peter S
PS
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D012979:Sodium Radioisotopes; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013268:Stimulation, Chemical
D010281:Parathyroid Hormone; D010726:Phosphodiesterase Inhibitors; C000613387:Slc9a3 protein, rat; D012979:Sodium Radioisotopes; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D005576:Colforsin; D012694:Serine; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D015056:1-Methyl-3-isobutylxanthine