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CFTR Regulation by Phosphorylation

  • Rodrigo AlzamoraEmail author
  • J Darwin KingJr
  • Kenneth R. Hallows
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 741)

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is the gene product mutated in cystic fibrosis, a common lethal genetic disease characterized by abnormal electrolyte transport across epithelia. CFTR functions as an ATP-gated, phosphorylation-regulated Cl channel that mediates agonist-stimulated apical membrane epithelial Cl and bicarbonate secretion and also regulates a variety of other transport proteins and cellular processes. CFTR belongs to the ATP-binding cassette (ABC) transporter superfamily. Its presumed architecture consists of two transmembrane domain regions that form the channel pore, two nucleotide-binding domains that bind and hydrolyze ATP, and a unique regulatory (R) domain that contains numerous protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites. Other kinases have also been shown more recently to phosphorylate and regulate CFTR activity. This chapter describes strategies and methods for studying the phosphorylation of CFTR both in vitro and whole-cell systems.

Key words

Kinases PKA PKC AMPK mass spectrometry immunoprecipitation immunoblotting autoradiography phosphoproteins 

Notes

Acknowledgments

The authors wish to thank Dr. William Reenstra for the past sharing of his laboratory protocols for CFTR in vitro and in vivo phosphorylation. This work was supported by an American Heart Association Postdoctoral Fellowship (AHA 0825540D) to R.A., by the National Institutes of Health (T32 HL007563 to J.D.K. and R01 DK075048 to K.R.H.) and by the Cystic Fibrosis Foundation (HALLOW06P0 to K.R.H.)

References

  1. 1.
    Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073.PubMedCrossRefGoogle Scholar
  2. 2.
    Rommens, J. M., Iannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., et al. (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245, 1059–1065.PubMedCrossRefGoogle Scholar
  3. 3.
    Carson, M. R., Travis, S. M., and Welsh, M. J. (1995) The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity. J. Biol. Chem. 270, 1711–1717.PubMedCrossRefGoogle Scholar
  4. 4.
    Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J., and Smith, A. E. (1991) Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66, 1027–1036.PubMedCrossRefGoogle Scholar
  5. 5.
    Chappe, V., Hinkson, D. A., Zhu, T., Chang, X. B., Riordan, J. R., and Hanrahan, J. W. (2003) Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA. J. Physiol. 548, 39–52.PubMedCrossRefGoogle Scholar
  6. 6.
    Jia, Y., Mathews, C. J., and Hanrahan, J. W. (1997) Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272, 4978–4984.PubMedCrossRefGoogle Scholar
  7. 7.
    Hallows, K. R., Raghuram, V., Kemp, B. E., Witters, L. A., and Foskett, J. K. (2000) Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J. Clin. Invest. 105, 1711–1721.PubMedCrossRefGoogle Scholar
  8. 8.
    Hallows, K. R., McCane, J. E., Kemp, B. E., Witters, L. A., and Foskett, J. K. (2003) Regulation of channel gating by AMP-activated protein kinase modulates cystic fibrosis transmembrane conductance regulator activity in lung submucosal cells. J. Biol. Chem. 278, 998–1004.PubMedCrossRefGoogle Scholar
  9. 9.
    Hallows, K. R., Kobinger, G. P., Wilson, J. M., Witters, L. A., and Foskett, J. K. (2003) Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells. Am. J. Physiol. Cell Physiol. 284, C1297–C1308.PubMedGoogle Scholar
  10. 10.
    King, J. D., Jr., Fitch, A. C., Lee, J. K., McCane, J. E., Mak, D. O., Foskett, J. K., et al. (2009) AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR. Am. J. Physiol. Cell Physiol. 297, C94–C101.PubMedCrossRefGoogle Scholar
  11. 11.
    Kongsuphol, P., Cassidy, D., Hieke, B., Treharne, K. J., Schreiber, R., Mehta, A., et al. (2009) Mechanistic insight into control of CFTR by AMPK. J. Biol. Chem. 284, 5645–5653.PubMedCrossRefGoogle Scholar
  12. 12.
    Fischer, H., and Machen, T. E. (1996) The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel. Biophys. J. 71, 3073–3082.PubMedCrossRefGoogle Scholar
  13. 13.
    Treharne, K. J., Xu, Z., Chen, J. H., Best, O. G., Cassidy, D. M., Gruenert, D. C., et al. (2009) Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR. Cell Physiol. Biochem. 24, 347–360.PubMedCrossRefGoogle Scholar
  14. 14.
    Blaydes, J. P., Vojtesek, B., Bloomberg, G. B., and Hupp, T. R. (2000) The development and use of phospho-specific antibodies to study protein phosphorylation. Methods Mol. Biol. 99, 177–189.PubMedGoogle Scholar
  15. 15.
    Hegedus, T., Aleksandrov, A., Mengos, A., Cui, L., Jensen, T. J., and Riordan, J. R. (2009) Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A. Biochim. Biophys. Acta 1788, 1341–1349.PubMedCrossRefGoogle Scholar
  16. 16.
    Zhou, H., Watts, J. D., and Aebersold, R. (2001) A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378.PubMedCrossRefGoogle Scholar
  17. 17.
    Carr, S. A., Huddleston, M. J., and Annan, R. S. (1996) Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry. Anal. Biochem. 239, 180–192.PubMedCrossRefGoogle Scholar
  18. 18.
    Bloom, N., Sicheritz-Ponten, T., Gupta, R., Gammeltoft, S., and Brunak, S. (2004) Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Rodrigo Alzamora
    • 1
    Email author
  • J Darwin KingJr
    • 1
  • Kenneth R. Hallows
    • 1
  1. 1.Renal-Electrolyte DivisionSchool of Medicine, University of PittsburghPittsburghUSA

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