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Understanding Signaling Specificity in Saccharomyces cerevisiae

  • Author(s): Shock, Teresa Ryan
  • Advisor(s): Madhani, Hiten D
  • et al.
Abstract

All cells must respond correctly to changes in their environment. Paradoxically, many signaling pathways share components, raising the question of how signaling specificity is maintained. In the budding yeast, Saccharomyces cerevisiae, three pathways share components. These are the mating, filamentous growth (FG), and high osmolarity glycerol (HOG) pathways. In this work we both identify a mechanism for cross pathway inhibition between the HOG and FG pathways and further elucidate the molecular events involved in the cross pathway inhibition between the mating and FG pathways.

Specifically, we found that both Ste7 and Kss1, the MAPKK and MAPK of the FG pathway respectively, are phosphorylated during activation of the HOG pathway. However, this phosphorylation does not lead to the expression of FG pathway transcriptional targets because Hog1 prevents the FG transcription factor Tec1 from binding to its target promoters. We determined that Hog1 does not prevent Tec1 from activating transcription by mediating its degradation, as is the case for the mating pathway. Overall, our results demonstrate a novel mechanism whereby HOG signaling interrupts FG pathway signal transduction between the phosphorylation of Kss1 and DNA binding by Tec1.

Next, we further investigated the mechanism for maintaining signaling specificity between the mating and FG pathways. Activation of the mating pathway leads not only to transcription of mating pathway genes, but also to degradation of the FG pathway transcription factor, Tec1. This cross pathway inhibition prevents FG pathway genes from being erroneously transcribed during activation of the mating pathway. Specifically, Fus3, the MAPK of the mating pathway, phosphorylates Tec1, resulting in its recognition by the SCF ubiquitin ligase complex and its subsequent proteolysis. Previous work found that Tec1 is phosphorylated in a sequence, LLpTP, identical to a defined binding site for Cdc4, an F-box protein in the SCF complex. However, continuing work on Cdc4 substrate interactions has shown that Cdc4 binds optimally to dually phosphorylated substrates. Here we find, through detailed mutagenesis and binding studies of the Tec1 phosphodegron, that Tec1 recognition also requires two adjacent phosphorylations (pTPpTA) suggesting that this represents a conserved mechanism of substrate recognition.

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