Abstract
Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosmotic stress [the High Osmolarity Glycerol (HOG) pathway]. Our method requires stimulating cells with square waves of oscillatory hyperosmotic input (1 M sorbitol) over a range of frequencies and measuring the activity of the HOG pathway in response to this oscillatory input. The input frequency at which the pathway’s steady-state activity drops precipitously because the stimulus is changing too rapidly for the pathway to respond faithfully is defined as the pathway bandwidth. In this chapter, we provide details of the techniques required to measure pathway bandwidth in the HOG pathway. These methods are generally useful and can be applied to signaling pathways in S. cerevisiae and other organisms whenever a rapid reporter of pathway activity is available.
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References
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, Endocrine reviews 22, 153–183.
Raman, M., Chen, W., and Cobb, M. H. (2007) Differential regulation and properties of MAPKs, Oncogene 26, 3100–3112.
Robinson, M. J., and Cobb, M. H. (1997) Mitogen-activated protein kinase pathways, Current opinion in cell biology 9, 180–186.
Banuett, F. (1998) Signalling in the yeasts: an informational cascade with links to the filamentous fungi, Microbiol Mol Biol Rev 62, 249–274.
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.
Klipp, E., Nordlander, B., Kruger, R., Gennemark, P., and Hohmann, S. (2005) Integrative model of the response of yeast to osmotic shock, Nature biotechnology 23, 975–982.
Janiak-Spens, F., Cook, P. F., and West, A. H. (2005) Kinetic analysis of YPD1-dependent phosphotransfer reactions in the yeast osmoregulatory phosphorelay system, Biochemistry 44, 377–386.
Hersen, P., McClean, M. N., Mahadevan, L., and Ramanathan, S. (2008) Signal processing by the HOG MAP kinase pathway, Proceedings of the National Academy of Sciences of the United States of America 105, 7165–7170.
Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., Weissman, J. S., and O’Shea, E. K. (2003) Global analysis of protein localization in budding yeast, Nature 425, 686–691.
Sikorski, R. S., and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae, Genetics 122, 19–27.
Burke, D. D., D and T. Stearns. (2000) Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, in Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual 2000 ed., Cold Spring Harbor Laboratory Woodbury, NY.
Beebe, D. J., Mensing, G. A., and Walker, G. M. (2002) Physics and applications of microfluidics in biology, Annual review of biomedical engineering 4, 261–286.
Weigl, B. H., Bardell, R. L., and Cabrera, C. R. (2003) Lab-on-a-chip for drug development, Advanced drug delivery reviews 55, 349–377.
Agarwal, B. a. G., IJ. (1968) Protein carbohydrate interaction vii: Physical and chemical studies of concanavalin a, the hemagglutinin of the jack bean., Arch Biochem Biophys 124,11.
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McClean, M.N., Hersen, P., Ramanathan, S. (2011). Measuring In Vivo Signaling Kinetics in a Mitogen-Activated Kinase Pathway Using Dynamic Input Stimulation. In: Becskei, A. (eds) Yeast Genetic Networks. Methods in Molecular Biology, vol 734. Humana Press. https://doi.org/10.1007/978-1-61779-086-7_6
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DOI: https://doi.org/10.1007/978-1-61779-086-7_6
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