Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain

  • Howell L
  • Borchardt R
  • Kole J
 et al. 
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Abstract

Gating of the CFTR Cl- channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1-R-GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1-R-GST by increasing the apparent K(m) for ATP (from 70 to 250 microM) and the Hill coefficient (from 1 to 1.7) without changing the V(max). When fusion proteins were photolabelled with 8-azido-[alpha-32P]ATP, PKA phosphorylation increased the apparent k(d) for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1-R-GST but not of R-GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2-MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1-R-GST. Thus when the R domain in NBD1-R-GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.

Author-supplied keywords

  • *Adenosine Triphosphate/aa [Analogs & Derivatives]
  • *Adenosine Triphosphate/me [Metabolism]
  • *Cyclic AMP-Dependent Protein Kinases/me [Metaboli
  • *Cystic Fibrosis Transmembrane Conductance Regulat
  • Azides/me [Metabolism]
  • Cystic Fibrosis Transmembrane Conductance Regulato
  • Dimerization
  • Glutathione Transferase/ge [Genetics]
  • Hydrolysis
  • Phosphorylation
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins/me [Metabolism]
  • Support, Non-U.S. Gov't
  • Support, U.S. Gov't, Non-P.H.S.
  • Support, U.S. Gov't, P.H.S.

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  • PMID: 14602047

Authors

  • L D Howell

  • R Borchardt

  • J Kole

  • A M Kaz

  • C Randak

  • J A Cohn

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