Intracellular Stress Inputs to SAPK Signaling Pathways

Summary Stress-activated protein kinase (SAPK) 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. We are interested in two general questions. First, how do diverse stressors activate a small number of evolutionarily conserved SAPKs? We have developed evidence in Saccharomyces cerevisiae that a multitude of stress signals stimulate the two yeast SAPK pathways – the Cell Wall Integrity (CWI) pathway and the High Osmolarity Glycerol (HOG) pathway – through intracellular inputs at various points along these pathways, rather than signaling from the cell surface. Second, how does the cell mobilize coherent, stress-specific outputs from an activated SAPK? The canonical stress pathways respond to cell wall stress (CWI pathway) and high external osmolarity (HOG pathway), but many stresses require the activated SAPKs to drive unrelated responses. We use the two SAPK pathways of yeast as models for understanding the complexity of stress inputs to SAPKs, as well as stress-specific outputs from activated SAPKs. This proposal is focused on identifying and characterizing the stress-specific components and pathways that feed into the two yeast SAPK cascades. Aim 1 is to understand the role of SAPK Hog1 in the intracellular activation of SAPK Mpk1 by heat shock. We have found that heat shock signals both through the canonical CWI SAPK pathway and, independently, through a Hog1-dependent input to either Mpk1 itself or its immediately upstream kinase, Mkk1. We propose a combined, biochemical, mass spectrometric approach, together with a genetic screen to identify and characterize the pathway between Hog1 and Mpk1. Aim 2 is to identify and characterize intracellular inputs to the HOG pathway cascade by curcumin, a promising cancer and Alzheimer’s therapeutic. We have identified two separate curcumin-induced inputs to the Sln1 branch of the HOG pathway, one at the MEK kinase Ssk2 and the other at Ypd1, a component of the HOG pathway phospho-relay. We propose a combination of biochemical and mass spectrometric approaches and a genetic screen to identify novel pathway components to these two inputs. Aim 3 is to identify and characterize the intracellular input to the HOG pathway by acetic acid stress. We have identified an input to the HOG pathway from acetic acid that activates Hog1 either at the level of its MEK Pbs2, or at Hog1 itself. We have also found that acetic acid stress is unusual in that it induces a stable interaction between Hog1 and its MEK Pbs2. Our favored hypothesis is that this stress activates Hog1 through recruitment of a scaffold protein. We propose a combination of biochemical and mass spectrometric approaches to understand the basis for Hog1 activation by acetic acid, as well as the stress-specific Hog1 output elicited by this stress. Completion of these aims will provide novel insights into the mechanisms by which these stress signals activate SAPKs.