Molecular dissection of Ras protein signalling in Schizosaccharomyces pombe

Fission yeast mating pheromone triggers the RAS-MAPK signalling pathway essential for meiotic differentiation. It also induces a dramatic morphological change of the cells leading to mating, a cell fusion event between cells of opposite mating types. The system is ideal to dissect the mechanisms by which the RAS-MAPK signal activation is regulated and highlight basic regulatory concepts of RAS protein signalling. In this work we evaluated the role of Ras1 in coordinating both MAPK activation due to pheromone signalling and activation of the Cdc42 pathway responsible for actin reorganisation. We established a condition to induce highly synchronous mating of fission yeast cells and we also established an assay system to monitor the MAPK phosphorylation status with an anti-phospho ERK monoclonal antibody. In addition, the changes in cellular morphology during the time course of meiotic differentiation were monitored. These tools allowed us to carry out quantitative measurements of the MAPK phosphorylation status in cells harbouring various mutations in the signalling components. We confirmed that a constitutively active mutation of the MEK induces constitutive phosphorylation of the MAPK. Strikingly however, with a canonical oncogenic Ras mutation (ras1.val17), although the MAPK activation occurs acutely, it is rapidly down-regulated therefore identifying the role of an unidentified modulator of the pathway at the level of the MEKK or MEK. We have also identified that unlike Ras1 and its GEF, Ste6, the G-protein, Gpa1 and the adaptor protein Ste4 are essential for MAPK phosphorylation although Ras1 appears to be the main activator of MAPK phosphorylation exclusively through the MAPK cascade. We therefore conclude that Ras1 is activating both the MAPK branch - which leads to expression of meiotic genes - and the Cdc42 pathway for polarised growth in response to pheromone. Successful MAPK activation is required for a morphological response even in the presence of oncogenic Ras1 indicating that MAPK activation is likely to be essential for site selection for polarised growth and that preventing MAPK activation can successfully prevent the oncogenic Ras1 phenotype. Importantly, the oncogenic phenotype of ras1.val17 can be rescued by the overexpression of the Ras1GAP, Gap1, therefore highlighting the importance of Ras proteins as potential therapeutic targets.