scholarly journals The Formation of the cAMP/Protein Kinase A-dependent Annexin 2–S100A10 Complex with Cystic Fibrosis Conductance Regulator Protein (CFTR) Regulates CFTR Channel Function

2007 ◽  
Vol 18 (9) ◽  
pp. 3388-3397 ◽  
Author(s):  
Lee A. Borthwick ◽  
Jean Mcgaw ◽  
Gregory Conner ◽  
Christopher J. Taylor ◽  
Volker Gerke ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Complex Analysis ◽  
Short Circuit ◽  
Disulfonic Acid ◽  
Intestinal Biopsy ◽  
Regulator Protein ◽  
Annexin 2 ◽  
Kinase A

Cystic fibrosis results from mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/protein kinase A (PKA) and ATP-regulated Cl− channel. CFTR is increasingly recognized as a component of multiprotein complexes and although several inhibitory proteins to CFTR have been identified, protein complexes that stimulate CFTR function remain less well characterized. We report that annexin 2 (anx 2)–S100A10 forms a functional cAMP/PKA/calcineurin (CaN)-dependent complex with CFTR. Cell stimulation with forskolin/3-isobutyl-1-methylxanthine significantly increases the amount of anx 2–S100A10 that reciprocally coimmunoprecipitates with cell surface CFTR and calyculin A. Preinhibition with PKA or CaN inhibitors attenuates the interaction. Furthermore, we find that the acetylated peptide (STVHEILCKLSLEG, Ac1-14), but not the nonacetylated equivalent N1-14, corresponding to the S100A10 binding site on anx 2, disrupts the anx 2–S100A10/CFTR complex. Analysis of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and CFTRinh172-sensitive currents, taken as indication of the outwardly rectifying Cl− channels (ORCC) and CFTR-mediated currents, respectively, showed that Ac1-14, but not N1-14, inhibits both the cAMP/PKA-dependent ORCC and CFTR activities. CaN inhibitors (cypermethrin, cyclosporin A) discriminated between ORCC/CFTR by inhibiting the CFTRinh172-, but not the DIDS-sensitive currents, by >70%. Furthermore, peptide Ac1-14 inhibited acetylcholine-induced short-circuit current measured across a sheet of intact intestinal biopsy. Our data suggests that the anx 2–S100A10/CFTR complex is important for CFTR function across epithelia.

scholarly journals Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

2009 ◽  
Vol 135 (1) ◽  
pp. 43-58 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Owen M. Woodward ◽  
Damian B. van Rossum ◽  
Nicholas C. Zachos ◽  
Linxi Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Chinese Hamster Ovary Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Chinese Hamster ◽  
T84 Cells ◽  
Cl Secretion ◽  
Intestinal Chloride Secretion ◽  
Kinase A

Intestinal Cl− secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl− secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl− secretion. FSK-stimulated Cl− secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl− secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl− secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl− conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl−>Br−>I− permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl− secretion, which is carried by a novel, previously undescribed Cl− channel.

Rectal epithelial expression of protein kinase A phosphorylation of cystic fibrosis transmembrane conductance regulator

1994 ◽  
Vol 106 (4) ◽  
pp. 890-898 ◽  
Author(s):  
Mrinalini C. Rao ◽  
Grace B. Bissonnette ◽  
Teresa Mahaffey ◽  
William B. Guggino ◽  
Jay L. Goldstein
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A

scholarly journals β3-Adrenoceptor Control the Cystic Fibrosis Transmembrane Conductance Regulator through a cAMP/Protein Kinase A-independent Pathway

1999 ◽  
Vol 274 (10) ◽  
pp. 6107-6113 ◽  
Author(s):  
Véronique Leblais ◽  
Sophie Demolombe ◽  
Geneviève Vallette ◽  
Dominique Langin ◽  
Isabelle Baró ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A

The protein kinase A-regulated cardiac CI− channel resembles the cystic fibrosis transmembrane conductance regulator

Nature ◽  
1992 ◽  
Vol 360 (6399) ◽  
pp. 81-84 ◽  
Author(s):  
Georg Nagel ◽  
Tzyh-Chang Hwang ◽  
Kent L. Nastiuk ◽  
Angus C. Nairn ◽  
David C. Gadsbyt
Keyword(s):  
Cystic Fibrosis ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane ◽  
Kinase A

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.

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