A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1

Abstract

The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans.

A walk-through of the yeast mating pheromone response pathway

“…In the yeast mating pathway, putative D-sites are also been found in Gpa1 Gα [102], the Ptp3 phosphatase [154], and the Dig1 and Dig2 transcriptional regulators [79]. Hence, D-sites appear to be portable, modular motifs that mediate the interaction of MAPKs with multiple binding partners, contributing to both signal transmission and specificity.…”

Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates

“…Examples of GST-pull down assays involving MAPKs that were quantified using the above technique have been described [24,[28][29][30]32]. In these studies, GST-MAPKs were used to pull down in vitro-translated MEKs or substrates, or GST-fusions to portions of MEKs, substrates or scaffold proteins were used to pull down in vitro-translated MAPKs.…”

“…Structural and mutagenesis studies indicate that the basic submotif of the D-site contacts two closely spaced acidic patches on the MAPK surface-the 'CD/sevenmaker' region and the 'ED region' [24,[54][55][56][57][58]. The hydrophobic submotif of the D-site binds to a 'hydrophobic docking groove' located close the CD/ED regions [54,56,57,59].…”

“…In this paper, we summarize methods we have used successfully and repeatedly to detect MAPK activation in cells [21][22][23][24], estimate the cellular concentrations of MAPKs and other proteins of interest [25]; detect and quantify the stable physical association of MAPKs with their substrates and regulators [24][25][26][27][28][29][30][31][32], and delineate the MAPK-binding regions or domains of MAPK-interacting proteins [24][25][26][29][30][31][32].…”

“…The docking site sequences, fused to GST or anchored to a membrane, are often sufficient to bind to MAPKs [24,29,30].…”