Stress-activated MAPK signaling pathways respond to a wide variety of stress conditions, including DNA damage, oxidative stress, heat shock, endoplasmic reticulum stress, hyper- and hypo-osmotic stress, shear stress, and a growing number of chemical toxins. Despite their organization with sensors at the cell surface connected to signaling components that typically culminate in the control of transcription factors and other proteins that impact cell physiology, there is growing evidence that many stressors activate these pathways through intracellular inputs rather than by signals that emanate from the cell surface. We have developed evidence in baker's yeast that a multitude of stress signals stimulate two stress-activated MAPK pathways, the Cell Wall Integrity (CWI) pathway and the High Osmolarity Glycerol (HOG) pathway, through intracellular inputs at various points along these pathways. This proposal is focused on identifying and characterizing the stress-specific components that feed into the MAPK cascades with a special focus on DNA damage-induced activation of the CWI pathway.
Aim 1 extends our recent discovery that protein kinase C (Pkc1), which is the top protein kinase of the CWI MAPK cascade is a target of DNA damage checkpoint signaling. Pkc1 plays important roles in the response to and survival of DNA damage. Thus, we propose to take a combined phospho-proteomic, biochemical, and molecular genetic approach to understanding the impact of checkpoint signaling on the regulation of Pkc1 and the role of this pathway in the maintenance of genomic stability.
Aim 2 seeks to identity stress-specific components involved in the intracellular activation of the CWI pathway MAPK cascade. These will focus on DNA damage, which activates the CWI MAPK (Mpk1) without the need for regulation of its upstream pathway components.
This aim will also examine cell wall stress inputs, which surprisingly enter the pathway at a point within the MAPK cascade. We will use a combination of biochemical and mass spectrometric approaches, as well as genetic screens.
Aim 3 is to identify and characterize intracellular stress inputs to the HOG MAPK cascade. The toxic metalloid arsenite and citric acid both activate the MAPK (Hog1) through an input that does not require regulation of upstream pathway components. Curcumin, a promising colon cancer and Alzheimer's therapeutic, signals to Hog1 through an input within the MAPK cascade. Completion of these aims will provide novel insights into the mechanisms by which stress signals activate MAPK pathways and will delineate a novel branch of the DNA damage response pathway.

Public Health Relevance

This project is focused on the identification of novel intracellular inputs to stress-activated signaling pathways. These inputs are critical to elicit th normal cellular responses to a wide variety of chemical and physical stress conditions, including DNA damage, and responses to environmental toxins. Such cellular responses help to maintain genomic stability and are important in the treatment and prevention of cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM048533-22
Application #
9028359
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Reddy, Michael K
Project Start
1992-09-30
Project End
2019-12-31
Budget Start
2016-01-19
Budget End
2016-12-31
Support Year
22
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
Lee, Jongmin; Levin, David E (2018) Intracellular mechanism by which arsenite activates the yeast stress MAPK Hog1. Mol Biol Cell 29:1904-1915
Liu, Li; Levin, David E (2018) Intracellular mechanism by which genotoxic stress activates yeast SAPK Mpk1. Mol Biol Cell 29:2898-2909
Lee, Jongmin; Liu, Li; Levin, David E (2018) Stressing out or stressing in: intracellular pathways for SAPK activation. Curr Genet :
Lee, Jongmin; Levin, David E (2015) Rgc2 Regulator of Glycerol Channel Fps1 Functions as a Homo- and Heterodimer with Rgc1. Eukaryot Cell 14:719-25
Lee, Jongmin; Reiter, Wolfgang; Dohnal, Ilse et al. (2013) MAPK Hog1 closes the S. cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators. Genes Dev 27:2590-601
Beese-Sims, Sara E; Pan, Shih-Jung; Lee, Jongmin et al. (2012) Mutants in the Candida glabrata glycerol channels are sensitized to cell wall stress. Eukaryot Cell 11:1512-9
Beese-Sims, Sara E; Lee, Jongmin; Levin, David E (2011) Yeast Fps1 glycerol facilitator functions as a homotetramer. Yeast 28:815-9
Kim, Ki-Young; Levin, David E (2011) Mpk1 MAPK association with the Paf1 complex blocks Sen1-mediated premature transcription termination. Cell 144:745-56
Levin, David E (2011) Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics 189:1145-75
Kim, Ki-Young; Truman, Andrew W; Caesar, Stefanie et al. (2010) Yeast Mpk1 cell wall integrity mitogen-activated protein kinase regulates nucleocytoplasmic shuttling of the Swi6 transcriptional regulator. Mol Biol Cell 21:1609-19

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