The osmoregulation system in Saccharomyces cerevisiae offers a unique opportunity in which to explore key features of signal transduction pathways that are prevalent in both prokaryotic and eukaryotic cells. The initial steps of this signaling pathway involve multiple histidine-to-aspartate phosphoryl transfer events involving three proteins, SLN1, YPD1, and SSK1, that are related to bacterial """"""""two-component"""""""" signal transducers. This phosphorelay system, in turn, regulates a downstream mitogen- activated protein (MAP) kinase cascade. The research proposed here focuses on understanding structure/function relationships that dictate molecular interactions and hence phosphoryl transfer. X-ray crystallographic studies coupled with mutagenesis experiments of the histidine-containing phosphorelay protein YPD1 will yield insight regarding surface(s) of YPD1 that interact with the homologous domains associated with SLN1 and SSK1. The response regulator protein, SSK1, represents a novel regulator of a MAP kinase cascade. Thus, by initiating structural studies of SSK1, it is anticipated that we will uncover unique and important information regarding the role of phosphorylation in regulating SSK1 function and the molecular surface(s) of SSK1 that are available for interaction with the downstream proteins of the MAP kinase cascade. This study will have broad based implications relevant to the many two-component signal transduction systems in bacteria and MAP kinase-dependent signaling pathways in higher eukaryotes. Moreover, because related MAP kinase cascades function in pathways that control cell growth and differentiation and since histidine-to-aspartate phosphoryl transfer is essential for the tumor-suppressive activity of human Nm23 protein, the research proposed here will likely provide insights into the etiology of cancer. Overall, the fundamental objective of this research is to better understand protein phosphorylation as a universal form of cellular regulation.

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National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Oklahoma Norman
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Fassler, Jan S; West, Ann H (2011) Fungal Skn7 stress responses and their relationship to virulence. Eukaryot Cell 10:156-67
Fassler, Jan S; West, Ann H (2010) Genetic and biochemical analysis of the SLN1 pathway in Saccharomyces cerevisiae. Methods Enzymol 471:291-317
Kaserer, Alla O; Andi, Babak; Cook, Paul F et al. (2010) Kinetic studies of the yeast His-Asp phosphorelay signaling pathway. Methods Enzymol 471:59-75
Kaserer, Alla O; Andi, Babak; Cook, Paul F et al. (2009) Effects of osmolytes on the SLN1-YPD1-SSK1 phosphorelay system from Saccharomyces cerevisiae. Biochemistry 48:8044-50
Zhao, Xiaodong; Copeland, Daniel M; Soares, Alexei S et al. (2008) Crystal structure of a complex between the phosphorelay protein YPD1 and the response regulator domain of SLN1 bound to a phosphoryl analog. J Mol Biol 375:1141-51
Tan, Hui; Janiak-Spens, Fabiola; West, Ann H (2007) Functional characterization of the phosphorelay protein Mpr1p from Schizosaccharomyces pombe. FEMS Yeast Res 7:912-21
Weber, Harald; Pesavento, Christina; Possling, Alexandra et al. (2006) Cyclic-di-GMP-mediated signalling within the sigma network of Escherichia coli. Mol Microbiol 62:1014-34
Janiak-Spens, Fabiola; Cook, Paul F; West, Ann H (2005) Kinetic analysis of YPD1-dependent phosphotransfer reactions in the yeast osmoregulatory phosphorelay system. Biochemistry 44:377-86
Chooback, Lilian; West, Ann H (2003) Co-crystallization of the yeast phosphorelay protein YPD1 with the SLN1 response-regulator domain and preliminary X-ray diffraction analysis. Acta Crystallogr D Biol Crystallogr 59:927-9
Porter, Stace W; Xu, Qingping; West, Ann H (2003) Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p. Eukaryot Cell 2:27-33

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