Constitutively active RAS prolongs Cdc42 signalling, while MAPK signalling is attenuated during fission yeast mating

Area of Science:

• Cellular signalling
• Molecular biology
• Systems biology

Background:

• RAS GTPases are key signalling hubs controlling diverse cellular processes.
• RAS-mediated signalling can activate multiple downstream pathways, potentially with differential responses to mutations.
• Fission yeast pheromone signalling (PS) involves RAS signalling, activating both the MAPKSpk1 and Cdc42 pathways.

Purpose of the Study:

• To investigate how a constitutively active RAS mutation (ras1.G17V) differentially affects downstream MAPKSpk1 and Cdc42 pathway activation in fission yeast.
• To elucidate the network structure governing RAS-mediated pheromone signalling, particularly the transient activation of MAPKSpk1.
• To develop and validate a mathematical model of the fission yeast pheromone signalling framework.

Main Methods:

• Genetic epistasis analysis of pheromone signalling mutants.
• Biochemical analysis of Ras1 effectors (Cdc42-GEFScd1 and MAPKKKByr2).
• Development of a mathematical model based on the pheromone signalling framework.
• Experimental validation of model predictions, including gene deletions (e.g., Sxa2).

Main Results:

• The ras1.G17V mutation caused prolonged Cdc42 activation but transient MAPKSpk1 activation.
• Ras1 signalling branches into MAPKSpk1 and Cdc42 pathways, with competitive interaction between effectors.
• A negative-feedback circuit regulating pheromone production/sensing was identified as essential for explaining MAPKSpk1 dynamics.
• Experimental deletion of Sxa2 confirmed the predicted negative-feedback, leading to hyperactivation of both pathways.

Conclusions:

• RAS signalling propagates distinctly through the MAPKSpk1 and Cdc42 pathways in fission yeast.
• A negative-feedback mechanism is critical for controlling the transient activation of the MAPKSpk1 pathway.
• The developed mathematical model provides a framework for understanding RAS signalling networks and can be adapted for other systems.