There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immunecompromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and in the face of osmotic stress. These studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The principal mechanism by which yeast cells detect and respond to cell wall stress is through the Cell Wall Integrity (CWI) signaling pathway, which transmits stress signals generated at the cell surface to a GTPase switch that activates a MAP kinase cascade. However, there is an additional pathway that contributes to the structural integrity of the cell wall in response to hypo-osmotic shock. This pathway culminates in the reduction of turgor pressure by release of intracellular glycerol through the Fps1 glycerol channel.
The specific aims of this project are 1) To determine the function of the Mpk1 cell wall stress MAP kinase when bound to the Paf1-RNA polymerase transcription elongation complex (PafC). Recent discoveries have revealed that Mpk1 activates a subset of its transcriptional program through a non-catalytic mechanism that involves its association with the promoters and coding regions of its transcriptional targets. New data suggests that Mpk1 moves from the promoter to the PafC. Experiments are described to dissect the function of Mpk1 within the context of this complex. 2) To establish the nature of the relationship between CWI signaling and DNA damage checkpoint signaling. Mpk1 is activated in reponse to DNA damaging agents, but its cell wall transcriptional program is not activated under these conditions. It is hypothesized that novel serine phosphorylations on Mpk1 provoked by DNA damage checkpoint kinases redirect it from cell wall targets to other functions relevant to the DNA damage response. Experiments are proposed to test this hypothesis and to identify the role of Mpk1 in this response. 3) To establish the mechanism by which a pair of novel regulators of the Fps1 glycerol channel, Rgc1 and Rgc2, activate Fps1 and to identify regulatory phosphorylation sites on these proteins in response to various stress signals. Preliminary data suggest that Rgc1/2 serve as regulatory nodes for multiple protein kinases. Experiments are proposed to identify pathway components both upstream and downstream of Rgc1/2. 4) To determine if the Pkc1 protein kinase of the CWI pathway contributes to the G2/M transition through the RSC chromatin remodeling complex. A recently-discovered physical interaction between Pkc1 and the Rsc1 subunit of the RSC complex suggests a mechanism by which Pkc1 contributes to the G2/M transition. Experiments are proposed to test this possibility.
There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immune-compromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. Proposed studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells.
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