The osmotic response in the yeast is mediated in part by the HOG1 MAP kinase cascade, a signal transduction pathway whose activity is regulated by members of the two-component signal transduction family. Sln1p is a plasma-membrane localized two-component sensor-kinase that autophosphorylates on a conserved histidine upon stimulation. The Sln1p phosphoryl group is relayed to aspartyl and histidyl groups on sln1p and Ypd1p respectively and finally to aspartyl groups on the two response regulators, Ssk1p and Skn7p. The phosphorylation state of Ssk1p regulates HOG1 pathway activity, while Skn7p is a bifunctional transcription factorwhose phosphorylation state dictates whether osmotic response or oxidative response genes will be activated. Our isolation and characterization of a set of three s1nl* mutants that cause a constitutive kinase phenotype and activation of the Skn7p response regulator have focused our attention on various aspects of the SLN1-YPD1-SKN7 phosphorelay. In this proposal we discuss the molecular basis of Sln1 kinase activation, the details of signal propagation and the molecular basisfor regulation of Skn7p function by phosphorylation.
The specific aims of this grant are: (1) to investigate the role of a cytoplasmic coiled-coil region in regulating Sln1 kinase activity using activating sln1* mutations and SLN1 chimeras as tools to dissect the process; (2) to investigate how thesln1p signal is propagated to the nucleus by careful analysis ofSkn7p and Ypd1p localization and by defining the cis-and trans-acting requirements for their localization; and (3) to investigate the molecular details of SLN1-SKN7 dependent transcriptional events by analysis of DNA and protein interaction with constitutive and non-phosphorylatable forms of the Skn7 protein. The power of genetics approaches has made the yeast S. cerevisiae an important model for the study of stress response. Regulation of the yeast osmotic stress pathway by a two-component type histidine kinase introduces added health implications since, given the restricted presence of two-component systems in lower eukaryotes and plants, the histidine kinase are an excellent potential anti-fungal drug target.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM056719-05
Application #
6437148
Study Section
Special Emphasis Panel (ZRG1-MBC-2 (02))
Program Officer
Anderson, James J
Project Start
1998-01-01
Project End
2005-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
5
Fiscal Year
2002
Total Cost
$302,820
Indirect Cost
Name
University of Iowa
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Fassler, Jan S; West, Ann H (2013) Histidine phosphotransfer proteins in fungal two-component signal transduction pathways. Eukaryot Cell 12:1052-60
Fassler, Jan S; West, Ann H (2011) Fungal Skn7 stress responses and their relationship to virulence. Eukaryot Cell 10:156-67
Mulford, K E; Fassler, J S (2011) Association of the Skn7 and Yap1 transcription factors in the Saccharomyces cerevisiae oxidative stress response. Eukaryot Cell 10:761-9
Fassler, Jan S; West, Ann H (2010) Genetic and biochemical analysis of the SLN1 pathway in Saccharomyces cerevisiae. Methods Enzymol 471:291-317
He, Xin-Jian; Mulford, KariAn E; Fassler, Jan S (2009) Oxidative stress function of the Saccharomyces cerevisiae Skn7 receiver domain. Eukaryot Cell 8:768-78
Shankarnarayan, Sandhya; Narang, Sandhya S; Malone, Cheryl L et al. (2008) Modulation of yeast Sln1 kinase activity by the CCW12 cell wall protein. J Biol Chem 283:1962-73
He, Xin-Jian; Fassler, Jan S (2005) Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae. Mol Microbiol 58:1454-67
Lu, Jade Mei-Yeh; Deschenes, Robert J; Fassler, Jan S (2004) Role for the Ran binding protein, Mog1p, in Saccharomyces cerevisiae SLN1-SKN7 signal transduction. Eukaryot Cell 3:1544-56
Lu, Jade Mei-Yeh; Deschenes, Robert J; Fassler, Jan S (2003) Saccharomyces cerevisiae histidine phosphotransferase Ypd1p shuttles between the nucleus and cytoplasm for SLN1-dependent phosphorylation of Ssk1p and Skn7p. Eukaryot Cell 2:1304-14
Tao, Wei; Malone, Cheryl L; Ault, Addison D et al. (2002) A cytoplasmic coiled-coil domain is required for histidine kinase activity of the yeast osmosensor, SLN1. Mol Microbiol 43:459-73

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