signaling crosstalk...the!pheromone!response!pathway! pheromone mating ste2/3 + g!"# ste20...
TRANSCRIPT
![Page 1: Signaling crosstalk...The!pheromone!response!pathway! pheromone mating Ste2/3 + G!"# Ste20 Ste11/ Ste50 Ste7/ Ste5 Fus3 Ste12 Receptors!and!GJprotein! MAP!kinase!cascade! Transcrip+on!factor!](https://reader031.vdocument.in/reader031/viewer/2022020306/5e341f679c09523a6e39ebbe/html5/thumbnails/1.jpg)
Figure 6.32 The Biology of Cancer (© Garland Science 2007)
Signaling crosstalk
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nutrient limita+on
ma+ng pheromone
Yeast Morphogene+c Transi+ons
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MAP Kinase Cascade
MAP kinase kinase kinase (eg Ste11)
Phosphorylates and thereby ac+vates
Phosphorylates and thereby ac+vates
MAP kinase kinase (eg Ste7)
MAP kinase (eg Fus3)
Which then phosphorylates and regulates downstream targets (eg transcrip+on factors and Cdk regulators) that together achieve the appropriate outcome
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The pheromone response pathway
pheromone
mating
Ste2/3 + G!"#Ste20
Ste11/ Ste50Ste7/ Ste5
Fus3Ste12
Receptors and G-‐protein
MAP kinase cascade
Transcrip+on factor
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Signaling pathways share components
glucose deple*on
filamentous growth
Sho1 Ste20
Ste11/ Ste50 Ste7 Kss1
Ste12/Tec1
pheromone
mating
Ste2/3 + G!"#Ste20
Ste11/ Ste50Ste7/ Ste5
Fus3Ste12
salt/sorbitol
osmolarityresponse
Sho1 Ste20
Ste11/ Ste50Pbs2Hog1
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Lamson et al. (Pryciak) Current Biology 16:618
Scaffold proteins may help solve some of the specificity problem
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Scaffolds also exist in mammalian cells
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Another example of scaffolds and specificity
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X Y
Two-Hybrid System to Detect Protein Interactions
DNA-binding!domain!
Activation!domain!
Reporter gene!Binding site!
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Plasmids for expression of 2-‐hybrid constructs
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Another depic+on ofa 2-‐hybrid experiment
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Ste5 interacts with each member of the MAP Kinase cascade
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2-‐Hybrid tests localize interac+on sites on Ste5
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IP experiments reveal that Ste5 interacts with each member of the MAPK cascade
Ste11 and Fus3
Ste5-‐Ste11 interac+on does not require Ste7
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IP experiments, con+nued
Likewise, Ste7 interacts with Ste5
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Fus3 is ac+vated by alpha-‐factor in a Ste5-‐dependent manner
Fus3 kinase assays
Lanes 1&2: no Ste5
Lanes 3&4: wildtype Ste5
Lanes 5&6: mutant form of Ste5 unable to bind Fus3
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Glycerol gradient centrifuga+on reveals a mul+-‐protein complex
Molecular weight standards
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Quan+ta+on of protein levels across gradient
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Figure 1 Yeast mating and high-osmolarity MAPK pathways require scaffold proteins Ste5 and Pbs2.
S Park et al. Science 2003;299:1061-1064
Published by AAAS
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Engineered protein-‐protein interac+ons can subs+tute for the na+ve Ste5-‐Ste7 and Ste5-‐Ste11 interac+on
PDZ domains are protein-‐protein interac+on domains
* Indicates mutant binding sites on Ste5
Ste5 is tagged with HA epitope; IP with an+-‐HA, probe with an+-‐NOS
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Ar+ficial interac+ons demonstrated by ma+ng test
Ma+ng test: strain with Ste5 construct is leu-‐; test for ma+ng to trp-‐ cells of other ma+ng type. Growth will be observed on minimal medium only if ma+ng has occurred.
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… and by quan+ta+ve ma+ng tests and phosphoryla+on of Fus3
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Can one engineer a new scaffold to direct a different output response?
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The diverter scaffold works as designed Diverter interacts with appropriate kinases
Growth on salt occurs only in presence of alpha-‐factor
Diverter directs phosphoryla+on of Hog1
Diverter directs same change in gene expression as wildtype osmo-‐response
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Figure 4 Mutational analysis of diverter scaffold requirements.
S Park et al. Science 2003;299:1061-1064
Published by AAAS
Growth on high salt in presence of α-‐factor
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Addi+onal regulators of pheromone response
Can a scaffold be engineered to alter kine+cs of response to these regulators?
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Recruited regulators agenuate or s+mulate response
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Recruited regulators change +me course and dose response
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Two MAPK targets for Ste7
What is role of Ste5 in ac+va+on of Fus3
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A new role for Ste5
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Ste5 is required for ac+va+on of Fus3 but not Kss1
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Requirements for ac+va+on of Fus3 by Ste7
Defini+on of a minimal scaffold version of Ste5 Fus3 binding site on Ste5 is not required but docking sites on Ste7 are
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The idea is, Ste7 tethers Fus3 to the Ste5 minimal scaffold
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Ste5 changes the Kcat of Ste7 for Fus3, not the Km
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Ste5 mutants with greater than 100-‐fold reduc+on in phosphoryla+on of Fus3 by Ste7
Mutants fall in two regions on Ste5 surface
The known Ste7 binding site
A region dubbed the coac+vator loop
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Ste5ms mutants affect different aspects of phosphoryla+on of Fus3 by Ste7
Mutant B affects Ste7 binding Mutant C affects k-‐cat
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2 possibili+es for how Ste5 s+mulates phosphoryla+on of Fus3 by Ste7
1. Ste5 makes Ste7 a beger kinase
2. Ste5 make Fus3 a beger substrate
The data support the 2nd possibility; perhaps Ste5 induces a conforma+onal change in Fus3 that makes its ac+va+on loop accessible to Ste7
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Model