Mating in the budding yeast S. cerevisiae provides an excellent paradigm for negative growth control and differentiation in higher eukaryotes. In response to peptide pheromones, dividing haploid cells stop dividing in G1 phase and express specialized genes required for cell-cell contact and fusion. Mating is regulated by a conserved MAP kinase cascade that consists of two MAP kinases, a MEK, a MEKK, and a PAK-type kinase. The cascade is regulated by heterotrimeric and Rho-type G proteins, an SH3 domain protein, and a novel scaffolding protein called Ste5 that spatially organizes the protein kinases. This proposal aims to study two fundamental problems of signal transduction that pertain to growth and differentiation in all eukaryotes--(1) the mechanism by which a MAP kinase cascade is activated and (2) how a MAP kinase cascade performs specialized functions. The grant consists of distinct and interrelated projects, all of which are a continuation of work done during the previous granting period. The PI proposes to: 1. Investigate the different possible functions of Ste5 with respect to protein kinase regulation by isolating and analyzing Ste5 mutants defective in specific functions and interactions with protein kinases and G proteins. Mutant proteins will be characterized biochemically for the ability to bind to pathway components, activate kinases, and direct substrate phosphorylation. 2. Cell biological and genetic approaches will be used to investigate the role of cell cycle- and pheromone-dependent regulation of Ste5 localization to determine whether it is important for sequestering MAPK cascade enzymes to different cellular compartments for specific responses. 3. Mechanisms of activation of the MEKK Ste11 will be analyzed with respect to both Ste11 and Ste5 dimerization and association between the regulatory domain of Ste11 and Ste5 and Ste50. 4. The role of Ste5 in mediating pathway specificity will be investigated by isolating and analyzing mutant forms of Ste5 and Ste11 that activate other MAP kinase cascades.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM046962-06
Application #
2417985
Study Section
Special Emphasis Panel (ZRG5-BM-1 (02))
Project Start
1992-08-01
Project End
2001-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
6
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Wang, Xiaoyan; Sheff, Mark A; Simpson, David M et al. (2011) Ste11p MEKK signals through HOG, mating, calcineurin and PKC pathways to regulate the FKS2 gene. BMC Mol Biol 12:51
Elion, Elaine A (2006) Methods for analyzing MAPK cascades. Methods 40:207-8
Flotho, Annette; Simpson, David M; Qi, Maosong et al. (2004) Localized feedback phosphorylation of Ste5p scaffold by associated MAPK cascade. J Biol Chem 279:47391-401
Andersson, Jessica; Simpson, David M; Qi, Maosong et al. (2004) Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes. EMBO J 23:2564-76
Cherkasova, Vera A; McCully, Ryan; Wang, Yunmei et al. (2003) A novel functional link between MAP kinase cascades and the Ras/cAMP pathway that regulates survival. Curr Biol 13:1220-6
Wang, Yunmei; Elion, Elaine A (2003) Nuclear export and plasma membrane recruitment of the Ste5 scaffold are coordinated with oligomerization and association with signal transduction components. Mol Biol Cell 14:2543-58
Elion, E A (2000) Pheromone response, mating and cell biology. Curr Opin Microbiol 3:573-81
Choi, K Y; Kranz, J E; Mahanty, S K et al. (1999) Characterization of Fus3 localization: active Fus3 localizes in complexes of varying size and specific activity. Mol Biol Cell 10:1553-68
Leza, M A; Elion, E A (1999) POG1, a novel yeast gene, promotes recovery from pheromone arrest via the G1 cyclin CLN2. Genetics 151:531-43
Farley, F W; Satterberg, B; Goldsmith, E J et al. (1999) Relative dependence of different outputs of the Saccharomyces cerevisiae pheromone response pathway on the MAP kinase Fus3p. Genetics 151:1425-44

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