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.
Department of Medicine, Duke University and VA Medical Centers, Durham, NC 27710-3378, USA.
L Daniel
LD
Howell
Roy
R
Borchardt
Jolanta
J
Kole
Andrew M
AM
Kaz
Christoph
C
Randak
Jonathan A
JA
Cohn
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