title: Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK.
abstract: Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5(ND)) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or "synthetic," supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling.
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: Michal J
authors.initials: MJ
authors.lastname: Nagiec
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: McCarter
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Kelley
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: Gauri
authors.initials: G
authors.lastname: Dixit
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Elston
authors.affiliation: Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 hdohlman@med.unc.edu telston@amath.unc.edu.
authors.forename: Henrik G
authors.initials: HG
authors.lastname: Dohlman
mesh_terms: D054637:Dual-Specificity Phosphatases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D010675:Pheromones / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D010675:Pheromones; D029701:Saccharomyces cerevisiae Proteins; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases; D054637:Dual-Specificity Phosphatases