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Identification of Novel Target Proteins of Cyclic GMP Signaling Pathways Using Chemical Proteomics

  • Kim, Eui-Kyung (Department of Life Science, Division of Molecular and Life Science, Pohang University of Science & Technology) ;
  • Park, Ji-Man (Department of Life Science, Division of Molecular and Life Science, Pohang University of Science & Technology)
  • Received : 2002.11.18
  • Accepted : 2003.01.10
  • Published : 2003.05.31

Abstract

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the co-precipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.

Keywords

References

  1. Bergseid, M., Baytan, A. R., Wiley, J. P., Ankener, W. M., Stolowitz, M. L., Hughes, K. A. and Chesnut, J. D. (2000) Small molecule-based chemical affinity system for the purification of proteins. Biotechniques 29, 1126-1133.
  2. Chan, T. F., Carvalho, J., Riles, L. and Zheng, X. F. (2000) A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR). Proc. Natl. Acad. Sci. USA 97, 13227-13232. https://doi.org/10.1073/pnas.240444197
  3. Choi, B. -M., Pae, H. -O., Jang, S. I., Kim, Y.-M. and Chung. H. -T. (2002) Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator. J. Biochem. Mol. Biol. 35. 116-126. https://doi.org/10.5483/BMBRep.2002.35.1.116
  4. Clark, M. S. (1999) Comparative genomics: the key to understanding the Human Genome Project. Bioessays 21, 121-130. https://doi.org/10.1002/(SICI)1521-1878(199902)21:2<121::AID-BIES6>3.0.CO;2-O
  5. Dills, W. L., Beavo, J. A., Bechtel, P. J., Myers, K. R., Sakai, L. J. and Krebs, E. G. (1976) Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives. Biochemistry 15, 3724-3730. https://doi.org/10.1021/bi00662a013
  6. Essayan. D. M. (2001) Cyclic nucleotide phosphodiesterases. J. Allergy Clin. lmmunol. 108, 671-680. https://doi.org/10.1067/mai.2001.119555
  7. Francis. S. H. and Corbin. J. D. (1999) Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit. Rev. Clin. Lab. Sci. 36, 275-328. https://doi.org/10.1080/10408369991239213
  8. Hofmann, F., Ammendola. A. and Schlossmann. J. (2000) Rising behind NO: cGMP-dependent protein kinases. J. Cell Sci. 113. 1671-1676.
  9. Hung, D. T., Jamison. T. F. and Schreiber, S. L. (1996) Understanding and controlling the cell cycle with natural products. Chem. Biol. 3. 623-639. https://doi.org/10.1016/S1074-5521(96)90129-5
  10. Jensen. O. N., Vorm, O. and Mann, M. (1996) Sequence patterns produced by incomplete enzymatic digestion or one-step Edman degradation of peptide mixtures as probes for protein database searches. Electrophoresis 17, 938-944. https://doi.org/10.1002/elps.1150170516
  11. Kim. K -M., Kim. P. K. M., Kwon. Y. -G., Bai. S. -K., Nanl, W. -D. and Kim, Y. -M. (2002) Regulation of apoptosis by nitrosative stress. J. Biochem. Mol. Biol. 35, 127-133. https://doi.org/10.5483/BMBRep.2002.35.1.127
  12. Mortz, E., Vorm. O., Mann. M. and Roepstorff, P. (1994) Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search. Biol. Mass Spectrom 23, 249-261. https://doi.org/10.1002/bms.1200230503
  13. Rotella. D. P. (2002) Phosphodiesterase 5 inhibitors: current status and potential applications. Nat. Rev. Drug Discov. 1, 674-682. https://doi.org/10.1038/nrd893
  14. Schlossmann, J., Ammendola, A., Ashman, K., Zong. X., Huber, A., Neubauer, G., Wang. G. X., Allescher, H. D., Korth. M., Wilm, M., Hofmann. F. and Ruth. P. (2000) Regulation of intracellular calcium by a signaling complex of IRAG, IP3 receptor and cGMP kinase Ibeta. Nature 404, 197-201. https://doi.org/10.1038/35004606
  15. Schwede. F., Maronde, E., Genieser, H. and Jastorff, B. (2000) Cyclic nucleotide analogs as biochemical tools and prospective drugs. Pharmacol. Ther. 87, 199-226. https://doi.org/10.1016/S0163-7258(00)00051-6
  16. Shevchenko. A., Wilm, M., Vorm, O. and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850-858. https://doi.org/10.1021/ac950914h
  17. Shirai, T., Tanaka. K., Terada, Y., Sawada. T., Shirai. R., Hashimoto. Y., Nagata. S., Iwamatsu. A., Okawa, K., Li, S., Hattori, S., Mano, H. and Fukui, Y. (1998) Specific detection of phosphatidylinositol 3.4.5-uisphosphate binding proteins by the PIP3 analogue beads: an application for rapid purification of the PIP3 binding proteins. Biochim. Biophys. Acta 1402, 292-302. https://doi.org/10.1016/S0167-4889(98)00014-7
  18. You. A. J., Jackman. R. J., Whitesides. G. M. and Schreiber, S. L. (1997) A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells. Chem. Biol. 4, 969-975. https://doi.org/10.1016/S1074-5521(97)90305-7

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