Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain.
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.
Howell
L Daniel
LD
Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, USA.
Borchardt
Roy
R
Kole
Jolanta
J
Kaz
Andrew M
AM
Randak
Christoph
C
Cohn
Jonathan A
JA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D001386:Azides / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005982:Glutathione Transferase / Q000235:genetics; D006868:Hydrolysis; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001386:Azides; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases