ABCMdb

PMID: 19419994

King JD Jr, Fitch AC, Lee JK, McCane JE, Mak DO, Foskett JK, Hallows KR
AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR.
Am J Physiol Cell Physiol. 2009 Jul;297(1):C94-101. Epub 2009 May 6., [PubMed]

Sentences

No. Mutations Sentence Comment

78 ABCC7 p.Ser768Ala One day after harvesting, oocytes were injected with either wild-type CFTR or CFTR-S768A cRNA (1 ng). Login to comment 79 ABCC7 p.Ser768Ala One day after harvesting, oocytes were injected with either wild-type CFTR or CFTR-S768A cRNA (1 ng). Login to comment 139 ABCC7 p.Ser768Ala AMPK-dependent phosphorylation of CFTR-S768A was substantially reduced (by ϳ70%) compared with that of wild-type CFTR, to levels similar to that of the CFTR-10SA mutant (Fig. 4, B and C). Login to comment 140 ABCC7 p.Ser768Ala AMPK-dependent phosphorylation of CFTR-S768A was substantially reduced (by ϳ70%) compared with that of wild-type CFTR, to levels similar to that of the CFTR-10SA mutant (Fig. 4, B and C). Login to comment 143 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp It was reported previously that CFTR-S768A exhibits a very high open probability, whereas the phospho-mimic Ser-to-Asp S768D CFTR mutant has a very low open probability relative to wild-type CFTR following PKA stimulation (8, 27, 28). Login to comment 144 ABCC7 p.Ser768Ala ABCC7 p.Ser768Asp It was reported previously that CFTR-S768A exhibits a very high open probability, whereas the phospho-mimic Ser-to-Asp S768D CFTR mutant has a very low open probability relative to wild-type CFTR following PKA stimulation (8, 27, 28). Login to comment 146 ABCC7 p.Ser768Ala The relevance of Ser768 for AMPK modulation of CFTR activity was further investigated in TEV studies of Xenopus oocytes expressing either wild-type CFTR or CFTR-S768A. Login to comment 147 ABCC7 p.Ser768Ala The relevance of Ser768 for AMPK modulation of CFTR activity was further investigated in TEV studies of Xenopus oocytes expressing either wild-type CFTR or CFTR-S768A. Login to comment 150 ABCC7 p.Ser768Ala Peak currents following stimulation with PKA agonists were significantly greater in oocytes expressing CFTR-S768A than in those expressing CFTR-WT, consistent with previous results (8). Login to comment 151 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala Peak currents following stimulation with PKA agonists were significantly greater in oocytes expressing CFTR-S768A than in those expressing CFTR-WT, consistent with previous results (8). Login to comment 152 ABCC7 p.Ser768Ala Of note, ZMP was without effect on the peak conductance of oocytes expressing CFTR-S768A, suggesting that this mutant is resistant to Fig. 4. Login to comment 156 ABCC7 p.Ser768Ala The WT and S768A images shown are from a different membrane than the rest of the mutants shown. Login to comment 157 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala The WT and S768A images shown are from a different membrane than the rest of the mutants shown. Login to comment 158 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala C: in vitro phosphorylation, corrected for CFTR protein expression and normalized to that of CFTR-WT (*P Ͻ 0.01, unpaired t-tests compared with the S768A, 10SA, and 10SA-A737S mutants; n ϭ 4-6 separate experiments). Login to comment 159 ABCC7 p.Ser768Ala There were no significant differences in phosphorylation intensity among the S768A, 10SA, and 10SA-A737S mutants (P Ͼ 0.10, unpaired t-tests). Login to comment 162 ABCC7 p.Ser768Ala In addition, baseline, unstimulated CFTR conductances were enhanced by approximately fivefold in oocytes expressing CFTR-S768A (Fig. 5C), suggesting that CFTR-WT conductance is tonically inhibited in oocytes by Ser768 phosphorylation. Login to comment 163 ABCC7 p.Ser768Ala In addition, baseline, unstimulated CFTR conductances were enhanced by approximately fivefold in oocytes expressing CFTR-S768A (Fig. 5C), suggesting that CFTR-WT conductance is tonically inhibited in oocytes by Ser768 phosphorylation. Login to comment 173 ABCC7 p.Ser768Ala A: representative whole cell conductance recordings from control [potassium gluconate (KG)]-injected oocytes expressing either CFTR-WT or CFTR-S768A. Login to comment 174 ABCC7 p.Ser768Ala A: representative whole cell conductance recordings from control [potassium gluconate (KG)]-injected oocytes expressing either CFTR-WT or CFTR-S768A. Login to comment 175 ABCC7 p.Ser768Ala B: mean (Ϯ SE) peak conductances normalized to the mean control (KG)-injected, CFTR-WT peak conductance for that experimental day and batch in CFTR-WT versus CFTR-S768A-expressing oocytes injected with either control (KG) or AMPK activator [5-aminoimidazole-4-carboxamide ribonucleoside monophosphate (ZMP)]. Login to comment 176 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala B: mean (Ϯ SE) peak conductances normalized to the mean control (KG)-injected, CFTR-WT peak conductance for that experimental day and batch in CFTR-WT versus CFTR-S768A-expressing oocytes injected with either control (KG) or AMPK activator [5-aminoimidazole-4-carboxamide ribonucleoside monophosphate (ZMP)]. Login to comment 177 ABCC7 p.Ser768Ala A significant decrease in relative peak conductance was observed for ZMP-injected, CFTR-WT-expressing oocytes (*P Ͻ 0.01) but not for ZMP-injected CFTR-S768A-expressing oocytes. Login to comment 178 ABCC7 p.Ser768Ala CFTR-S768A-expressing oocytes had a dramatically elevated prestimulation starting conductance relative to that of CFTR-WT-expressing oocytes. Login to comment 179 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala CFTR-S768A-expressing oocytes had a dramatically elevated prestimulation starting conductance relative to that of CFTRWT-expressing oocytes. Login to comment 180 ABCC7 p.Ser768Ala There was also a ϳ40-50% decrease in starting conductance of CFTR-WT-expressing oocytes injected with ZMP versus KG (#P Ͻ 0.05), whereas there was no difference in starting conductance between the two conditions for CFTR-S768A-expressing oocytes. Login to comment 181 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala Whole cell CFTR conductance before and after addition of PKA agonists with or without AMPK activation in Xenopus oocytes Starting Conductance, ␮S Peak Conductance, ␮S CFTR-WT ϩ KG 12.4Ϯ1.4 67.0Ϯ9.3 CFTR-WT ϩ ZMP 8.5Ϯ0.9 30.5Ϯ3.8 CFTR-S768A ϩ KG 146.9Ϯ13.0 256.6Ϯ15.6 CFTR-S768A ϩ ZMP 128.5Ϯ9.0 238.7Ϯ13.7 Values are means Ϯ SE of whole cell two-electrode voltage clamp conductances measured before and at the peak after infusion of 1 ␮M forskolin and 100 ␮M IBMX. Login to comment 182 ABCC7 p.Ser768Ala Whole cell CFTR conductance before and after addition of PKA agonists with or without AMPK activation in Xenopus oocytes Starting Conductance, ␮S Peak Conductance, ␮S CFTR-WT ϩ KG 12.4Ϯ1.4 67.0Ϯ9.3 CFTR-WT ϩ ZMP 8.5Ϯ0.9 30.5Ϯ3.8 CFTR-S768A ϩ KG 146.9Ϯ13.0 256.6Ϯ15.6 CFTR-S768A ϩ ZMP 128.5Ϯ9.0 238.7Ϯ13.7 Values are means Ϯ SE of whole cell two-electrode voltage clamp conductances measured before and at the peak after infusion of 1 ␮M forskolin and 100 ␮M IBMX. Login to comment 189 ABCC7 p.Ser768Ala Consistent with previous observations (8), we found that mutation of this site to Ala (S768A) greatly enhanced baseline, unstimulated conductance of CFTR expressed in Xenopus oocytes, and it reduced the relative activation of CFTR by PKA agonists (Fig. 5). Login to comment 190 ABCC7 p.Ser768Ala ABCC7 p.Ser768Ala Consistent with previous observations (8), we found that mutation of this site to Ala (S768A) greatly enhanced baseline, unstimulated conductance of CFTR expressed in Xenopus oocytes, and it reduced the relative activation of CFTR by PKA agonists (Fig. 5). Login to comment 191 ABCC7 p.Ser768Ala The apparent constitutive activation of CFTR-S768A is reminiscent of that observed for CFTR-WT in cells with AMPK activity inhibited by overexpression of AMPK-DN (Fig. 1). Login to comment 192 ABCC7 p.Ser768Ala The importance of AMPK phosphorylation at Ser768 is further demonstrated by the observation that whereas PKA stimulation of CFTR-WT conductance was inhibited by the AMPK activator ZMP, PKA stimulation of CFTR-S768A was insensitive to ZMP. Login to comment 193 ABCC7 p.Ser768Ala The importance of AMPK phosphorylation at Ser768 is further demonstrated by the observation that whereas PKA stimulation of CFTR-WT conductance was inhibited by the AMPK activator ZMP, PKA stimulation of CFTR-S768A was insensitive to ZMP. Login to comment 197 ABCC7 p.Ser768Ala Indeed, a residual AMPK-mediated phosphorylation of 25-30% was observed in CFTR-S768A (Fig. 4), suggesting that other AMPK phosphorylation sites in CFTR may exist. Login to comment 198 ABCC7 p.Ser768Ala Indeed, a residual AMPK-mediated phosphorylation of 25-30% was observed in CFTR-S768A (Fig. 4), suggesting that other AMPK phosphorylation sites in CFTR may exist. Login to comment