Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed on the apical membrane of epithelial cells, where it plays a pivotal role in chloride transport and overall tissue homeostasis. CFTR constitutes a unique member of the ATP-binding cassette transporter superfamily, due to its distinctive cytosolic regulatory (R) domain carrying multiple phosphorylation sites that allow the tight regulation of channel activity and gating. Mutations in the CFTR gene cause cystic fibrosis, the most common lethal autosomal genetic disease in the Caucasian population. In recent years, major efforts have led to the development of CFTR modulators, small molecules targeting the underlying genetic defect of CF and ultimately rescuing the function of the mutant channel. Recent evidence has highlighted that this class of drugs could also impact on the phosphorylation of the R domain of the channel by protein kinase A (PKA), a key regulatory mechanism that is altered in various CFTR mutants. Therefore, the aim of this review is to summarize the current knowledge on the regulation of the CFTR by PKA-mediated phosphorylation and to provide insights into the different factors that modulate this essential CFTR modification. Finally, the discussion will focus on the impact of CF mutations on PKA-mediated CFTR regulation, as well as on how small molecule CFTR regulators and PKA interact to rescue dysfunctional channels.

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

Biochemical Journal ◽

10.1042/bj20021428 ◽

2004 ◽

Vol 378 (1) ◽

pp. 151-159 ◽

Cited By ~ 25

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.008427 ◽

2020 ◽

Vol 295 (14) ◽

pp. 4577-4590 ◽

Cited By ~ 1

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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CFTR Chloride Channel Regulation by an Interdomain Interaction

Science ◽

10.1126/science.286.5439.544 ◽

1999 ◽

Vol 286 (5439) ◽

pp. 544-548 ◽

Cited By ~ 93

Author(s):

Anjaparavanda P. Naren ◽

Estelle Cormet-Boyaka ◽

Jian Fu ◽

Matteo Villain ◽

J. Edwin Blalock ◽

...

Keyword(s):

Cystic Fibrosis ◽

Protein Kinase ◽

Protein Kinase A ◽

Chloride Channel ◽

Cystic Fibrosis Gene ◽

Physical Interaction ◽

Amino Terminal ◽

Interdomain Interaction ◽

Kinase A ◽

R Domain

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A–dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH2-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e07-02-0126 ◽

2007 ◽

Vol 18 (9) ◽

pp. 3388-3397 ◽

Cited By ~ 35

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.

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Cystic Fibrosis Transmembrane Conductance Regulator Inverts Protein Kinase A-mediated Regulation of Epithelial Sodium Channel Single Channel Kinetics

Journal of Biological Chemistry ◽

10.1074/jbc.272.22.14037 ◽

1997 ◽

Vol 272 (22) ◽

pp. 14037-14040 ◽

Cited By ~ 136

Author(s):

M. Jackson Stutts ◽

Bernard C. Rossier ◽

Richard C. Boucher

Keyword(s):

Cystic Fibrosis ◽

Protein Kinase ◽

Sodium Channel ◽

Protein Kinase A ◽

Single Channel ◽

Transmembrane Conductance Regulator ◽

Channel Kinetics ◽

Transmembrane Conductance ◽

Cystic Fibrosis Transmembrane ◽

Kinase A

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The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gaz037 ◽

2019 ◽

Vol 25 (10) ◽

pp. 587-600 ◽

Cited By ~ 2

Author(s):

Héctor Zapata-Carmona ◽

Lina Barón ◽

Lidia M Zuñiga ◽

Emilce Silvina Díaz ◽

Milene Kong ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinase A ◽

Current Knowledge ◽

Human Sperm ◽

Adenyl Cyclase ◽

Sperm Capacitation ◽

Time Treatment ◽

Proteasome Subunits ◽

Pka Pathway ◽

Kinase A

Abstract One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P < 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P < 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures.

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Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Biochemical Journal ◽

10.1042/bj20110855 ◽

2011 ◽

Vol 441 (1) ◽

pp. 387-397 ◽

Cited By ~ 18

Author(s):

Xiaofeng Wang ◽

Xin-an Lu ◽

Xiaomin Song ◽

Wei Zhuo ◽

Lin Jia ◽

...

Keyword(s):

Heat Shock ◽

Protein Kinase ◽

Protein Kinase A ◽

Binding Affinity ◽

Heat Shock Protein 90 ◽

Proliferating Cells ◽

Interacting Protein ◽

Fk506 Binding Protein ◽

The Impact ◽

Kinase A

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.

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