Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7

Mol Cell Biol. 1996 Jul;16(7):3637-50. doi: 10.1128/MCB.16.7.3637.

Abstract

Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in the absence of any additional yeast proteins. These complexes were specific because neither Hog1 nor Mpk1 (two other yeast MAPKs), nor mammalian Erk2, was able to associate detectably with Ste7. Neither the kinase catalytic core of Ste7 nor the phosphoacceptor regions of Ste7 and Kss1 were necessary for complex formation. Ste7-Kss1 (and Ste7-Fus3) complexes were present in yeast cell extracts and were undiminished in extracts prepared from a ste5delta-ste11delta double mutant strain. In Ste7-Kss1 (or Ste7-Fus3) complexes isolated from naive or pheromone-treated cells, Ste7 phosphorylated Kss1 (or Fus3), and Kss1 (or Fus3) phosphorylated Ste7, in a pheromone-stimulated manner; dissociation of the high-affinity complex was shown to be required for either phosphorylation event. Deletions of Ste7 in the region required for its stable association with Kss1 and Fus3 in vitro significantly decreased (but did not eliminate) signaling in vivo. These findings suggest that the high-affinity and active site-independent binding observed in vitro facilitates signal transduction in vivo and suggest further that MEK-MAPK interactions may utilize a double-selection mechanism to ensure fidelity in signal transmission and to insulate one signaling pathway from another.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cloning, Molecular
  • Escherichia coli
  • Fungal Proteins / biosynthesis
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism*
  • Mitogen-Activated Protein Kinase Kinases
  • Mitogen-Activated Protein Kinases*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Phenotype
  • Pheromones / physiology*
  • Protein Binding
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins*
  • Transcription, Genetic

Substances

  • Fungal Proteins
  • Oligodeoxyribonucleotides
  • Pheromones
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Protein Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • FUS3 protein, S cerevisiae
  • KSS1 protein, S cerevisiae
  • Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinase Kinases