AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR

Abstract

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.

Activating AMP-activated protein kinase (AMPK) slows renal cystogenesis

“…AMPK phosphorylates and directly inhibits CFTR and indirectly antagonizes mTOR through phosphorylation of tuberous sclerosis protein 2 (TSC2) and Raptor (10)(11)(12)(13). Both of these actions are consistent with the role of AMPK as a regulator that decreases energy-consuming processes such as transport, secretion, and growth when cellular ATP levels are low (14).…”

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

“…This view is supported by evidence indicating that removal of portions of the R region encompassing residues 760 -783 or 817 -838 produces channels that open without activation by phosphorylation (Baldursson et al 2001;Xie et al 2002). Activation of the channel requires PKA phosphorylation of multiple serines (there are .10 sites), most of which are in the R region (Picciotto et al 1992;Chappe et al 2004), although there are also protein kinase C (PKC) (Picciotto et al 1992;Chappe et al 2004) and inhibitory adenosine monophosphate-stimulated kinase (AMPK) sites (King et al 2009;Kongsuphol et al 2009). Critically, there is no requirement for phosphorylation at any one specific site (Cheng et al 1991;).…”

Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

“…51,[93][94][95][96] In particular, AMPK inhibits the Cl -channel CFTR (cystic transmembran transport regulator), which is expressed in the apical cell membrane of Cl --secreting epithelial cells. 51,[93][94][95][96] AMPK activity slows the inactivation of the voltage-gated cardiac Na + channel Nav1.5 and shifts the voltage activation curve toward hyperpolarized values, an effect which could prolong the action potential. 97 Activation of AMPK and subsequent AMPK-sensitive regulation of this channel may contribute to the arrhythmia following cardiac ischemia.…”