Phosphorylation by cAMP-Dependent Protein Kinase Causes a Conformational Change in the R Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Biochemistry ◽  
1994 ◽  
Vol 33 (13) ◽  
pp. 4072-4079 ◽  
Author(s):  
Ann M. Dulhanty ◽  
John R. Riordan
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Conformational Change ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Cystic Fibrosis Transmembrane ◽  
R Domain

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

2004 ◽  
Vol 378 (1) ◽  
pp. 151-159 ◽  
Author(s):  
L. Daniel HOWELL ◽  
Roy BORCHARDT ◽  
Jolanta KOLE ◽  
Andrew M. KAZ ◽  
Christoph RANDAK ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Protein Kinase A ◽  
Atp Hydrolysis ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A ◽  
R 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 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.

scholarly journals Protein kinase A phosphorylation potentiates cystic fibrosis transmembrane conductance regulator gating by relieving autoinhibition on the stimulatory C terminus of the regulatory domain

2020 ◽  
Vol 295 (14) ◽  
pp. 4577-4590 ◽  
Author(s):  
Jeng-Haur Chen
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Channel Gating ◽  
Burst Duration ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A ◽  
R Domain

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel activated by protein kinase A (PKA) phosphorylation on the regulatory (R) domain. Phosphorylation at several R domain residues stimulates ATP-dependent channel openings and closings, termed channel gating. To explore the protein segment responsible for channel potentiation and PKA-dependent activation, deletion mutations were constructed by removing one to three protein segments of the R domain including residues 708–759 (ΔR708–759), R760–783, and R784–835, each of which contains one or two PKA phosphorylation sites. Deletion of R708–759 or R760–783 had little effect on CFTR gating, whereas all mutations lacking R784–835 reduced CFTR activity by decreasing the mean burst duration and increasing the interburst interval (IBI). The data suggest that R784–835 plays a major role in stimulating CFTR gating. For ATP-associated regulation, ΔR784–835 had minor impact on gating potentiation by 2′dATP, CaATP, and pyrophosphate. Interestingly, introducing a phosphorylated peptide matching R809–835 shortened the IBI of ΔR708–835-CFTR. Consistently, ΔR815–835, but not ΔR784–814, enhanced IBI, whereas both reduced mean burst duration. These data suggest that the entirety of R784–835 is required for stabilizing the open state of CFTR; however, R815–835, through interactions with the channel, is dominant for enhancing the opening rate. Of note, PKA markedly decreased the IBI of ΔR708–783-CFTR. Conversely, the IBI of ΔR708–814–CFTR was short and PKA-independent. These data reveal that for stimulating CFTR gating, PKA phosphorylation may relieve R784–814–mediated autoinhibition that prevents IBI shortening by R815–835. This mechanism may elucidate how the R domain potentiates channel gating and may unveil CFTR stimulation by other protein kinases.

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