Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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Quantitative Profiling of Dual Phosphorylation of Fus3 MAP Kinase in Saccharomyces cerevisiae

  • Hur, Jae-Young (Department of Biological Sciences, Seoul National University) ;
  • Kang, Gum-Yong (Department of Molecular Biotechnology, Konkuk University) ;
  • Choi, Min-Yeon (Department of Biological Sciences, Seoul National University) ;
  • Jung, Jin Woo (Department of Molecular Biotechnology, Konkuk University) ;
  • Kim, Kwang-Pyo (Department of Molecular Biotechnology, Konkuk University) ;
  • Park, Sang-Hyun (Department of Biological Sciences, Seoul National University)
  • Received : 2008.04.01
  • Accepted : 2008.04.07
  • Published : 2008.07.31
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Abstract

Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracelluar stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved $T{\times}Y$ motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the $T{\times}Y$ motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic $T{\times}Y$ motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.

Keywords

Acknowledgement

Supported by : Korea Research Foundation, Korea Science and Engineering Foundation, Ministry of Health and Welfare

References

  1. Alessi, D.R., Saito, Y., Campbell, D.G., Cohen, P., Sithanandam, G., Rapp, U., Ashworth, A., Marshall, C.J., and Cowley, S. (1994). Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 13, 1610-1619
  2. Andersson, J., Simpson, D.M., Qi, M., Wang, Y., and Elion, E.A. (2004). Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes. EMBO J. 23, 2564-2576 https://doi.org/10.1038/sj.emboj.7600250
  3. Asara, J.M., Christofk, H.R., Freimark, L.M., and Cantley, L.C. (2008). A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen. Proteomics 8, 994-999 https://doi.org/10.1002/pmic.200700426
  4. Ballif, B.A., Roux, P.P., Gerber, S.A., MacKeigan, J.P., Blenis, J., and Gygi, S.P. (2005). Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. Proc. Natl. Acad. Sci. USA102, 667-672
  5. Bhattacharyya, R.P., Remenyi, A., Good, M.C., Bashor, C.J., Falick, A.M., and Lim, W.A. (2006). The Ste5 scaffold allosterically modulates signaling output of the yeast mating pathway. Science 311, 822-826 https://doi.org/10.1126/science.1120941
  6. Brill, J.A., Elion, E.A., and Fink, G.R. (1994). A role for autophosphorylation revealed by activated alleles of FUS3, the yeast MAP kinase homolog. Mol. Biol. Cell. 5, 297-312 https://doi.org/10.1091/mbc.5.3.297
  7. Canagarajah, B.J., Khokhlatchev, A., Cobb, M.H., and Goldsmith, E.J. (1997). Activation mechanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859-869 https://doi.org/10.1016/S0092-8674(00)80351-7
  8. Cobb, M.H., and Goldsmith, E.J. (1995). How MAP kinases are regulated. J. Biol. Chem. 270, 14843-14846 https://doi.org/10.1074/jbc.270.25.14843
  9. Elion, E.A. (2000). Pheromone response, mating and cell biology. Curr. Opin. Microbiol. 3, 573-581 https://doi.org/10.1016/S1369-5274(00)00143-0
  10. Errede, B., Gartner, A., Zhou, Z., Nasmyth, K., and Ammerer, G. (1993). MAP kinase-related FUS3 from cerevisiae is activated by STE7 in vitro. Nature 361, 261-264
  11. Ferrell, J.E., Jr. (1996). Tripping the switch fantastic: how a protein kinase cascade can convert graded inputs into switch-like outputs. Trends Biochem. Sci. 21, 460-466 https://doi.org/10.1016/S0968-0004(96)20026-X
  12. Ferrell, J.E., Jr., and Bhatt, R.R. (1997). Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase. J. Biol. Chem. 272, 19008-19016 https://doi.org/10.1074/jbc.272.30.19008
  13. Gartner, A., Nasmyth, K., and Ammerer, G. (1992). Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1. Genes Dev. 6, 1280-1292 https://doi.org/10.1101/gad.6.7.1280
  14. Gerber, S.A., Rush, J., Stemman, O., Kirschner, M.W., and Gygi, S.P. (2003). Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proc. Natl. Acad. Sci. USA 100, 6940-6945
  15. Gustin, M.C., Albertyn, J., Alexander, M., and Davenport, K. (1998). MAP kinase pathways in the yeast Saccharomyces cerevisiae . Microbiol. Mol. Biol. Rev. 62, 1264-1300
  16. Herskowitz, I. (1995). MAP kinase pathways in yeast: for mating and more. Cell 80, 187-197 https://doi.org/10.1016/0092-8674(95)90402-6
  17. Lee, S.J., Lee, J.R., Kim, Y.H., Park, Y.S., Park, S.I., Park, H.S., and Kim, K.P. (2007). Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom 21, 2797-2804 https://doi.org/10.1002/rcm.3145
  18. Morrison, D.K., and Davis, R.J. (2003). Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu Rev. Cell Dev. Biol. 19, 91-118 https://doi.org/10.1146/annurev.cellbio.19.111401.091942
  19. Park, S.H., Zarrinpar, A., and Lim, W.A. (2003). Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science 229, 1061-1064
  20. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B.E., Karandikar, M., Berman, K., and Cobb, M.H. (2001). Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr. Rev. 299, 153-183
  21. Steen, H., Jebanathirajah, J.A., Springer, M., and Kirschner, M.W. (2005). Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. Proc. Natl. Acad. Sci. USA 102, 3948-3953
  22. Swain, P.S., and Siggia, E.D. (2002). The role of proofreading in signal transduction specificity. Biophys. J. 82, 2928-2933 https://doi.org/10.1016/S0006-3495(02)75633-6
  23. Warmka, J., Hanneman, J., Lee, J., Amin, D., and Ota, I. (2001). Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1. Mol. Cell. Biol. 21, 51-60 https://doi.org/10.1128/MCB.21.1.51-60.2001
  24. Wolf-Yadlin, A., Hautaniemi, S., Lauffenburger, D.A., and White, F.M. (2007). Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks. Proc. Natl. Acad. Sci. USA 104, 5860-5865
  25. Young, C., Mapes, J., Hanneman, J., Al-Zarban, S., and Ota, I. (2002). Role of Ptc2 type 2C Ser/Thr phosphatase in yeast highosmolarity glycerol pathway inactivation. Eukaryot. Cell 1, 1032- 1040 https://doi.org/10.1128/EC.1.6.1032-1040.2002
  26. Zarrinpar, A., Park, S.H., and Lim, W.A. (2003). Optimization of specificity in a cellular protein interaction network by negative selection. Nature 426, 676-680 https://doi.org/10.1038/nature02178
  27. Zhan, X.L., Deschenes, R.J., and Guan, K.L. (1997). Differential regulation of FUS3 MAP kinase by tyrosine-specific phosphatases PTP2/PTP3 and dual-specificity phosphatase MSG5 in Saccharomyces cerevisiae. Genes Dev. 11, 1690-1702 https://doi.org/10.1101/gad.11.13.1690
  28. Zheng, C.F., and Guan, K.L. (1994). Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues. EMBO J. 13, 1123-1131