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

The small GTPase RAS is a signalling hub activating multiple pathways, which may respond differently to a constitutively active RAS mutation. We explored this issue in fission yeast, where RAS-mediated pheromone signalling (PS) activates two downstream pathways: the MAPKSpk1 and Cdc42 pathways. We observed that the yeast RAS mutation ras1.G17V, an equivalent of the mammalian ras.G12V oncogenic mutation, causes prolonged Cdc42 activation, whereas MAPKSpk1 activation was transient and attenuated. To explain this observation, we generated a PS framework by conducting genetic epistasis analysis of PS mutants and biochemical analysis of two Ras1 effectors, Cdc42-GEFScd1 and MAPKKKByr2, each of which triggers activation of the Cdc42 and MAPKSpk1 pathways, respectively. Cdc42-GEFScd1 and MAPKKKByr2 directly interacted with Ras1 in vitro in a competitive manner, and overexpression of the Ras binding domain of either Cdc42-GEFScd1 or MAPKKKByr2 in cells inhibited both downstream pathways, confirming that Ras1 signalling branches into the MAPKSpk1 and Cdc42 pathways. In conjunction with the genetic epistasis analysis, we developed the PS framework-based mathematical model to test which network structures can explain the transient MAPKSpk1 activation profile. Incorporating a negative-feedback circuit acting on pheromone production or sensing enabled the model to quantitatively reproduce MAPKSpk1 dynamics in the wild type and 20 additional PS mutants. The predicted PS negative-feedback was experimentally confirmed by deleting Sxa2, the carboxypeptidase that degrades one of the mating pheromones, which led to hyperactivation of both MAPKSpk1 and Cdc42. Our study provides a holistic understanding of the fission yeast pheromone signalling network, explaining how RAS signalling propagates differently through two downstream pathways. Our PS mathematical model may serve as a valuable reference framework for analysing other RAS signalling systems.