CFTR activation: Additive effects of stimulatory and inhibitory phosphorylation sites in the R domain

Daniel J. Wilkinson, Theresa V. Strong, Monique K. Mansoura, Deborah L. Wood, Stephen S. Smith, Francis S. Collins, David C. Dawson

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, - 712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (K(A)) for activation by 3-isobutyl-1- methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the K(A) for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume273
Issue number1 17-1
StatePublished - Jul 1997
Externally publishedYes

Fingerprint

Cystic Fibrosis Transmembrane Conductance Regulator
Serine
Phosphorylation
Alanine
1-Methyl-3-isobutylxanthine
Consensus Sequence
Xenopus
Cyclic AMP-Dependent Protein Kinases
Oocytes

Keywords

  • Adenosine 3',5'- cyclic monophosphate-dependent activation
  • Chloride channels
  • Consensus
  • Cystic fibrosis transmembrane conductance regulator
  • Mutagenesis
  • Protein kinaseA
  • Serines
  • Xenopus oocytes

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Cell Biology
  • Physiology
  • Physiology (medical)

Cite this

Wilkinson, D. J., Strong, T. V., Mansoura, M. K., Wood, D. L., Smith, S. S., Collins, F. S., & Dawson, D. C. (1997). CFTR activation: Additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. American Journal of Physiology - Lung Cellular and Molecular Physiology, 273(1 17-1).

CFTR activation : Additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. / Wilkinson, Daniel J.; Strong, Theresa V.; Mansoura, Monique K.; Wood, Deborah L.; Smith, Stephen S.; Collins, Francis S.; Dawson, David C.

In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 273, No. 1 17-1, 07.1997.

Research output: Contribution to journalArticle

Wilkinson, DJ, Strong, TV, Mansoura, MK, Wood, DL, Smith, SS, Collins, FS & Dawson, DC 1997, 'CFTR activation: Additive effects of stimulatory and inhibitory phosphorylation sites in the R domain', American Journal of Physiology - Lung Cellular and Molecular Physiology, vol. 273, no. 1 17-1.
Wilkinson, Daniel J. ; Strong, Theresa V. ; Mansoura, Monique K. ; Wood, Deborah L. ; Smith, Stephen S. ; Collins, Francis S. ; Dawson, David C. / CFTR activation : Additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. In: American Journal of Physiology - Lung Cellular and Molecular Physiology. 1997 ; Vol. 273, No. 1 17-1.
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