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

• Published in: American Journal of Physiology: Cell Physiology 2009-07, Vol.297 (1), p.C94-C101
• Main Authors: King, J Darwin, Jr, Fitch, Adam C, Lee, Jeffrey K, McCane, Jill E, Mak, Don-On Daniel, Foskett, J. Kevin, Hallows, Kenneth R
• Format: Article
• Language: English
• Subjects: AMP-Activated Protein Kinases - chemistry; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Animals; Biochemistry; Catalytic Domain; Cell Line; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - metabolism; Cystic Fibrosis Transmembrane Conductance Regulator - chemistry; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Enzyme Activation; Enzyme Activators - pharmacology; Humans; Ions; Kinases; Membrane Potentials; Membrane Transporters, Ion Channels, and Pumps; Metabolism; Mutation; Patch-Clamp Techniques; Phosphorylation; Protein Kinase C - metabolism; Protein Structure, Tertiary; Recombinant Proteins - metabolism; Sensors; Serine; Signal Transduction - drug effects; Time Factors; Transfection; Xenopus laevis
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00677.2008

1 Renal-Electrolyte Division, Department of Medicine and 3 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and 2 Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania Submitted 30 December 2008 ; accepted in final form 30 April 2009 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. Cl – channels; patch-clamp; metabolism; Xenopus oocytes; cystic fibrosis; transmembrane conductance regulator Address for re