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

L. Daniel HOWELL; Roy BORCHARDT; Jolanta KOLE; Andrew M KAZ; Christoph RANDAK; Jonathan A COHN

doi:10.1042/bj20021428

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

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

L. Daniel HOWELL, Roy BORCHARDT, Jolanta KOLE, Andrew M KAZ, Christoph RANDAK and Jonathan A COHN

The Biochemical journal, Vol.378(1), pp.151-159

02/15/2004

DOI: 10.1042/bj20021428

PMCID: PMC1223935

PMID: 14602047

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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 Km for ATP (from 70 to 250 µM) and the Hill coefficient (from 1 to 1.7) without changing the Vmax. When fusion proteins were photolabelled with 8-azido-[α-32P]ATP, PKA phosphorylation increased the apparent kd 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.

Details

Title: Subtitle: Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain
Creators: L. Daniel HOWELL - Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, U.S.A
Roy BORCHARDT - Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, U.S.A
Jolanta KOLE - Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, U.S.A
Andrew M KAZ - Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, U.S.A
Christoph RANDAK - Kinderklinik im Dr. von Haunerschen Kinderspital, Ludwig-Maximilians-Universität, Munich, Germany
Jonathan A COHN - Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, U.S.A
Resource Type: Journal article
Publication Details: The Biochemical journal, Vol.378(1), pp.151-159
DOI: 10.1042/bj20021428
PMID: 14602047
PMCID: PMC1223935
NLM abbreviation: Biochem J
ISSN: 0264-6021
eISSN: 1470-8728
Language: English
Date published: 02/15/2004
Academic Unit: Pulmonary Medicine; Stead Family Department of Pediatrics
Record Identifier: 9984093343702771

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