ABCMdb

PMID: 19095655

Kongsuphol P, Cassidy D, Hieke B, Treharne KJ, Schreiber R, Mehta A, Kunzelmann K
Mechanistic insight into control of CFTR by AMPK.
J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18., 2009-02-27 [PubMed]

Sentences

No. Mutations Sentence Comment

43 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Leu1430Ala ABCC7 p.Leu1430Ala ABCC7 p.Leu1431Ala ABCC7 p.Leu1431Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala ABCC7 p.Ser737Asp ABCC7 p.Ser737Asp ABCC7 p.Ser573Ala ABCC7 p.Ser573Ala ABCC7 p.Ser768Asp ABCC7 p.Ser768Asp ABCC7 p.Glu1474* ABCC7 p.Glu1474* ABCC7 p.Ser1248Ala ABCC7 p.Ser1248Ala EXPERIMENTAL PROCEDURES cRNAs for CFTR and Double Electrode Voltage Clamp-Oocytes were injected with cRNA (10 ng, 47 nl of double-distilled water) encoding wtCFTR, L1430A/L1431A, S573A, S1248A, F508del-CFTR, G551D-CFTR, S768A, S737A, S768D, S737D, E1474X, and AMPK␣1. Login to comment 70 ABCC7 p.Ser768Ala The S768A mutant of CFTR abrogated almost entirely by phosphorylation of AMPK. Login to comment 83 ABCC7 p.Leu1430Ala ABCC7 p.Leu1431Ala To exclude a nonspecific effect of compound C, we expressed a CFTR mutant (L1430A/ L1431A), which has been proposed to eliminate binding of AMPK␣1 to a C-terminal region of CFTR (5). Login to comment 85 ABCC7 p.Gly551Asp Also two common CFTR mutants, F508del-CFTR and G551D-CFTR, could not be further activated by inhibition of AMPK with compound C (Fig. 1F). Login to comment 86 ABCC7 p.Leu1430Ala ABCC7 p.Leu1431Ala This enhanced activity of L1430A/L1431A-CFTR was similar to that seen with wild-type CFTR first exposed to compound C and then activated by PKA. Login to comment 93 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Fig. 2B demonstrates that AMPK indeed phosphorylates the R domain in vitro and this AMPK phosphorylation is largely reduced in R domain mutants S737A and S768A (compare lanes 2 and 3 in Fig. 2B, lower panel). Login to comment 94 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala The data also suggest that serine 768 has a much greater reductive impact because S768A almost abolished all the phosphorylation, whereas some were preserved with S737A. Login to comment 95 ABCC7 p.Ser768Ala Crucially, the double mutant labeling was similar to S768A alone. Login to comment 96 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala We quantified data (supplemental Fig. S2) from three independent experiments and found that the S737A mutation leads to a small 23 Ϯ 8% reduction in counts, whereas S768A leads to a major 81 Ϯ 5% (mean Ϯ range) reduction similar to the double mutant (87% Ϯ 4%) when compared with the wild type (100%). Login to comment 103 ABCC7 p.Leu1430Ala ABCC7 p.Leu1431Ala D, summary of whole cell conductances generated by wtCFTR and L1430A/L1431A-CFTR and effects of activators and inhibitors of AMPK. Login to comment 104 ABCC7 p.Leu1430Ala ABCC7 p.Leu1431Ala E, whole cell currents activated by IBMX and forskolin in wtCFTR and L1430A/L1431A-CFTR expressing oocytes. Login to comment 105 ABCC7 p.Gly551Asp F, comparison of the effects of compound C (80 ␮M) on whole conductances generated by wtCFTR, F508del-CFTR, and G551D-CFTR. Login to comment 125 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala It is clear that S768A (lower left panel) has a more profound inhibitory effect on R domain phosphorylation compared with S737A (upper right) given that all these experiments were run simultaneously with similar concentrations of R domain protein and kinase, and each imaged for identical lengths of time. Login to comment 126 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Broadly, prolonged incubation that previously created two major spots found with AMPK alone was now supplemented by multiple small spots that almost disappeared after S768A mutation, but much less so after S737A mutation (Fig. 2D, compare lower two panels with upper). Login to comment 128 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala When expressed in oocytes, CFTR bearing the R domain mutants S737A and S768A as well as the double mutant S737A/ S768A (Fig. 3A) produced dramatically enhanced conductances (S768A Ͼ S737A; Fig. 3B) upon stimulation with forskolin (2 ␮M) and IBMX (1 mM). Login to comment 129 ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp In contrast, the S768D mutation, mimicking phosphorylation at Ser768 , produced a whole cell conductance that was significantly smaller than even wtCFTR (note that conductance not lowered with S737D; see also Fig. 3C for phenformin sensitivity of these mutants). Login to comment 130 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala The large Cl-conductances generated by S737A/S768A were inhibited by 5 ␮M of the specific chloride channel blocker CFTRinh-172 (Fig. 3D), or alternatively, the PKA inhibitor KT5720 and Cl- replacement by gluconate (not shown). Login to comment 131 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Once again the differential roles of these two serines were observed because the conductance observed with S737A was almost 100 ␮S lower than that found with S768A. Login to comment 135 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala B, phosphoryl- ationoftheRdomainwithPKAandAMPK.AMPKphosphorylationwaslargely reduced or abolished in S737A and S768A, respectively (lower panel). Login to comment 143 ABCC7 p.Ser768Ala The S768A mutant abrogates almost all the phosphorylation. Login to comment 145 ABCC7 p.Ser768Ala ABCC7 p.Ser573Ala Regulation of CFTR by AMPK 5648 respectively) with the S573A but not with the S768A (compare second and third panels of Fig. 3C with the corresponding wild type result in the first panel of the figure). Login to comment 146 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala Overall the data suggest that S737A, S768A, and the double mutant S737A/ S768A were no longer sensitive to stimulation or inhibition of AMPK indicating that phosphorylation at both serines is required (Fig. 3C, middle panels). Login to comment 147 ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp In contrast the residual CFTR conductances generated by S768D (but not S737D) were not only further inhibited by phenformin, but neither phospho-mimic mutant could be augmented by the AMPK inhibitor compound C. Login to comment 151 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala In contrast to wtCFTR the CFTR mutants S737A (not shown), S768A, and S737A/S768A produced a high Cl- con- ductance under basal conditions, i.e. in the absence of IBMX and forskolin (Fig. 4, A and B). Login to comment 155 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala A, whole cell currents activated by IBMX (1 mM) and forskolin (2 ␮M) in wtCFTR and S737A/ S768A-CFTRexpressingoocytes.B,summaryofwholecellconductancesgen- erated by wtCFTR and different CFTR mutants. Login to comment 156 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala C, summary of CFTR whole cell conductances generated by wtCFTR and different CFTR mutants, and effects of phenformin and compound C. D, summary of the whole cell conductance activated by S737A/S768A-CFTR and inhibition by the CFTR blocker CFTRinh-172. Login to comment 161 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala S737A/S768A-CFTR generates a baseline conductance. Login to comment 162 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala A, effect of extracellular Cl- replacement by gluconate (glcn) on whole cell currents generated by wtCFTR, S768A-CFTR, and S737A/S768A-CFTR in the absence of IBMX and forskolin. Login to comment 164 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala C, summary of the whole cell conductances generated by wtCFTR and S737A/S768A-CFTRintheabsenceofstimulationwithIBMXandforskolinand effectsofphenformin,compoundC,andCFTRinh-172.DandE,baselinewhole cell conductances generated by wild type CFTR (D) and S737A/S768A-CFTR (E) and effects of compound C and the PKA inhibitor KT520. Login to comment 170 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Moreover, the PKA inhibitor KT5720 (50 ␮M) inhibited this enhanced baseline CFTR conductance generated by S737A/ S768A irrespective of the presence of compound C (Fig. 4E). Login to comment 172 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala We interpret this finding to suggest that the sensitivity of S737A/ S768A-CFTR toward PKA inhibition is retained, which implies that PKA is now active after the loss of AMPK sensitivity. Login to comment 178 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala The initial recovery from PKA stimulation was equal in S737A/ S768A (1.1 Ϯ 0.3 ␮S/min) and wtCFTR (1.1 Ϯ 0.1 ␮S/min) (Fig. 5C), but was enhanced in ooctyes coexpressing kinase-dead AMPK␣1-K45R (2.8 Ϯ 0.4 ␮S/min), whereas overexpression of wtAMPK␣1beta1␥1 literally eliminated CFTR currents (Fig. 5B). Login to comment 180 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Although AMPK largely antagonizes activation of CFTR, the mutated serines S737A and S768A do not seem to influence the recovery time from forskolin stimulation. Login to comment 184 ABCC7 p.Glu1474* We eliminated the PDZ binding domain of CFTR (E1474X-CFTR) and gradually increased stimulation of the oocytes as shown in Fig. 5D. Login to comment 187 ABCC7 p.Glu1474* In marked contrast 3 mM 8Br- -cAMP alone was unable to promote full activation of E1474X-CFTR but instead required costimulation by 8Br- -cAMP, forskolin, and IBMX, and compound C further augmented whole cell conductance (Fig. 5D) suggesting AMPK sensitivity was retained by this mutant. Login to comment 210 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala C, inactivation of conductances generated by wtCFTR and S737A/S768A-CFTR. Login to comment 225 ABCC7 p.Ser737Ala ABCC7 p.Ser768Ala Even maximal stimulation of wtCFTR with a mixture of 8-Br- - cAMP, IBMX, and forskolin does not produce the same high level of conductance as S737A-CFTR or S768A-CFTR. Login to comment 252 ABCC7 p.Gly551Asp Our failure to reverse the gating defects in two common disease causing mutants (G551D and F508delCFTR) suggests that their mechanism of dysfunction is not due to an overactive AMPK but further work will have to establish whether inhibition of AMPK might be a therapeutic option in other mutations. Login to comment