AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR
AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR
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DOI：
10.1152/ajpcell.00677.2008
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发表时间：
2009-07-01
影响因子：
5.5
通讯作者：
Hallows, Kenneth R.
中科院分区：
文献类型：
--
作者：
King, J. Darwin, Jr.;Fitch, Adam C.;Hallows, Kenneth R.
King JD Jr，Fitch AC，Lee JK，McCane JE，Mak DD，Foskett JK，Hallows KR. AMP激活的蛋白激酶磷酸化的R结构域抑制PKA刺激CFTR。美国生理学杂志细胞生理学297：C94-C101，2009年。首次发表于2009年5月6日; doi：10.1152/ajpcell.00677.2008。代谢传感器AMP活化蛋白激酶（AMPK）已成为细胞代谢状态和离子转运活性之间的重要联系。我们先前发现AMPK在体外结合并磷酸化CFTR，并抑制Calu-3支气管浆液腺上皮细胞中CFTR通道门控的PKA依赖性刺激。为了进一步表征AMPK依赖性调节CFTR的机制，在表达组成型活性或显性阴性AMPK突变体（AMPK-CA或AMPK-DN）的Calu-3细胞中用PKA活化进行全细胞膜片钳测量。表达AMPK-DN的细胞中的基线CFTR电导显著大于对照，表明这些细胞中的紧张性AMPK活性在基础条件下抑制CFTR。尽管表达AMPK-CA的细胞中的基线CFTR电导与对照相当，但在表达AMPK-CA的细胞中CFTR的PKA刺激被完全阻断，表明AMPK活化使CFTR在体内对PKA活化具有抗性。在人胚肾-293细胞中使用四环素诱导的AMPK-DN表达的CFTR磷酸化研究证明了体内CFTR的AMPK依赖性磷酸化。然而，AMPK活性调节对响应于PKA或PKC激动剂的分级剂量的CFTR体内磷酸化没有影响。因此，AMPK依赖性CFTR磷酸化使得通道抵抗PKA和PKC的激活，而不阻止这些激酶的磷酸化。我们发现，Ser 768，CFTR R结构域残基被认为是抑制PKA位点，是AMPK磷酸化在体外的优势位点。在该位点的Ser-Ala突变增强了基线CFTR活性，并使CFTR对AMPK的抑制具有抗性，表明AMPK在Ser 768的磷酸化是CFTR抑制所必需的。总之，我们的研究结果表明，AMPK依赖性磷酸化CFTR抑制CFTR激活PKA，从而调整PKA的反应CFTR代谢和其他压力的细胞。
King JD Jr, Fitch AC, Lee JK, McCane JE, Mak DD, Foskett JK, Hallows KR. AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR. Am J Physiol Cell Physiol 297: C94-C101, 2009. First published May 6, 2009; doi:10.1152/ajpcell.00677.2008.-The metabolic sensor AMP-activated protein kinase (AMPK) has emerged as an important link between cellular metabolic status and ion transport activity. We previously found that AMPK binds to and phosphorylates CFTR in vitro and inhibits PKA-dependent stimulation of CFTR channel gating in Calu-3 bronchial serous gland epithelial cells. To further characterize the mechanism of AMPK-dependent regulation of CFTR, whole cell patch-clamp measurements were performed with PKA activation in Calu-3 cells expressing either constitutively active or dominant-negative AMPK mutants (AMPK-CA or AMPK-DN). Baseline CFTR conductance in cells expressing AMPK-DN was substantially greater than controls, suggesting that tonic AMPK activity in these cells inhibits CFTR under basal conditions. Although baseline CFTR conductance in cells expressing AMPK-CA was comparable to that of controls, PKA stimulation of CFTR was completely blocked in AMPK-CA-expressing cells, suggesting that AMPK activation renders CFTR resistant to PKA activation in vivo. Phosphorylation studies of CFTR in human embryonic kidney-293 cells using tetracycline-inducible expression of AMPK-DN demonstrated AMPK-dependent phosphorylation of CFTR in vivo. However, AMPK activity modulation had no effect on CFTR in vivo phosphorylation in response to graded doses of PKA or PKC agonists. Thus, AMPK-dependent CFTR phosphorylation renders the channel resistant to activation by PKA and PKC without preventing phosphorylation by these kinases. We found that Ser768, a CFTR R domain residue considered to be an inhibitory PKA site, is the dominant site of AMPK phosphorylation in vitro. Ser-to-Ala mutation at this site enhanced baseline CFTR activity and rendered CFTR resistant to inhibition by AMPK, suggesting that AMPK phosphorylation at Ser768 is required for its inhibition of CFTR. In summary, our findings indicate that AMPK-dependent phosphorylation of CFTR inhibits CFTR activation by PKA, thereby tuning the PKA-responsiveness of CFTR to metabolic and other stresses in the cell.