Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele.
When confronted with a marked increase in external osmolarity, budding yeast (Saccharomyces cerevisiae) cells utilize a conserved mitogen-activated protein kinase (MAPK) signaling cascade (the high-osmolarity glycerol or HOG pathway) to elicit cellular responses necessary to permit continued growth. One input that stimulates the HOG pathway requires the integral membrane protein and putative osmosensor Sho1, which recruits and enables activation of the MAPK kinase kinase Ste11. In mutants that lack the downstream MAPK kinase (pbs2Delta) or the MAPK (hog1Delta) of the HOG pathway, Ste11 activated by hyperosmotic stress is able to inappropriately stimulate the pheromone response pathway. This loss of signaling specificity is known as cross talk. To determine whether it is the Hog1 polypeptide per se or its kinase activity that is necessary to prevent cross talk, we constructed a fully functional analog-sensitive allele of HOG1 to permit acute inhibition of this enzyme without other detectable perturbations of the cell. We found that the catalytic activity of Hog1 is required continuously to prevent cross talk between the HOG pathway and both the pheromone response and invasive growth pathways. Moreover, contrary to previous reports, we found that the kinase activity of Hog1 is necessary for its stress-induced nuclear import. Finally, our results demonstrate a role for active Hog1 in maintaining signaling specificity under conditions of persistently high external osmolarity.
Westfall
Patrick J
PJ
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.
Thorner
Jeremy
J
D000483:Alleles; D002467:Cell Nucleus / Q000378:metabolism; D005786:Gene Expression Regulation; D028884:Karyopherins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009997:Osmotic Pressure; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D028884:Karyopherins; C473023:NMD5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases