Acute inhibition of Na/H exchanger NHE-3 by cAMP: Role of protein kinase a and NHE-3 phosphoserines 552 and 605

Hui Zhao, Michael R. Wiederkehr, Lingzhi Fan, Roberto L. Collazo, Ladonna A. Crowder, Orson W. Moe

Research output: Contribution to journalArticle

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Abstract

Regulation of the renal Na/H exchanger NHE-3 by protein kinase A (PKA) is a key intermediate step in the hormonal regulation of acid-base and salt balance. We studied the role of NHE-3 phosphorylation in this process in NHE- deficient AP-1 cells transfected with NHE-3 and in OKP cells expressing native NHE-3. A dominant-negative PKA-regulatory subunit completely abolished the effect of cAMP on NHE-3 activity demonstrating a role of PKA in the functional regulation of NHE-3 by cAMP. NHE-3 isolated from cAMP-treated cells showed lower phosphorylation by purified PKA in vitro suggesting that NHE-3 is a PKA substrate in vivo. Although changes in NHE-3 whole protein phosphorylation is difficult to detect in response to cAMP addition, the tryptic phosphopeptide map of in vivo phosphorylated NHE.3 showed a complex pattern of constitutive and cAMP-induced phosphopeptides. To test the causal relationship between phosphorylation and activity, we mutated eight serines in the cytoplasmic domain to glycine or alanine. Single or multiple mutants harboring S552A or S605G showed no PKA activation or reduced regulation by PKA activation. Ser-552 and Ser-605 were phosphorylated in vivo. However, multiple mutations of serines other than Ser-552 or Set-605 also reduced the functional PKA regulation. We conclude that regulation of NHE-3 by PKA in vivo involves complex mechanisms, which include phosphorylation of Ser-552 and Ser-605.

Original languageEnglish (US)
Pages (from-to)3978-3987
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number7
DOIs
StatePublished - Feb 12 1999

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Phosphoserine
Sodium-Hydrogen Antiporter
Cyclic AMP-Dependent Protein Kinases
Protein Kinases
Phosphorylation
Phosphopeptides
Serine
Chemical activation
Cells
Acid-Base Equilibrium
Transcription Factor AP-1
Alanine
Glycine
Salts
Kidney
Mutation
Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Acute inhibition of Na/H exchanger NHE-3 by cAMP : Role of protein kinase a and NHE-3 phosphoserines 552 and 605. / Zhao, Hui; Wiederkehr, Michael R.; Fan, Lingzhi; Collazo, Roberto L.; Crowder, Ladonna A.; Moe, Orson W.

In: Journal of Biological Chemistry, Vol. 274, No. 7, 12.02.1999, p. 3978-3987.

Research output: Contribution to journalArticle

Zhao, Hui ; Wiederkehr, Michael R. ; Fan, Lingzhi ; Collazo, Roberto L. ; Crowder, Ladonna A. ; Moe, Orson W. / Acute inhibition of Na/H exchanger NHE-3 by cAMP : Role of protein kinase a and NHE-3 phosphoserines 552 and 605. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 7. pp. 3978-3987.
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