Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK

Mol Biol Cell. 2015 Sep 15;26(18):3359-71. doi: 10.1091/mbc.E15-01-0037. Epub 2015 Jul 15.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Dual-Specificity Phosphatases / metabolism
  • MAP Kinase Signaling System*
  • Mitogen-Activated Protein Kinases / metabolism*
  • Models, Biological
  • Pheromones / metabolism
  • Phosphorylation
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • Pheromones
  • Saccharomyces cerevisiae Proteins
  • FUS3 protein, S cerevisiae
  • Mitogen-Activated Protein Kinases
  • Dual-Specificity Phosphatases