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Obtaining molecular insights of effector competition of constitutively active oncogenic RAS signalling

Primary Supervisor: Dr Kayoko Tanaka, Department of Molecular and Cell Biology

Secondary supervisor: Dr Andrey Revyakin

PhD project title: Obtaining molecular insights of effector competition of constitutively active oncogenic RAS signalling

University of Registration: University of Leicester

Project outline:

The RAS family of small GTPases act as signalling hubs regulating cell proliferation and differentiation. Importantly, about 20% of all human cancers harbour mutations in RAS genes (COSMIC). It is generally assumed that, when oncogenically mutated, RAS molecules over-activate all the downstream effectors. We recently showed, however, that, a constitutive RAS signal input results in over-activation of one of the downstream pathways, but not all (Kelsall, Vertesy et al., bioRxiv, 2018). In this work, we exploited a relatively simple yet physiological setting of fission yeast mating process, where Ras1, the unique fission yeast RAS homologue, activates two pathways, MAPKSpk1 and Cdc42 (a prototype small G-protein in yeast). We demonstrated that a constitutively active Ras1 mutant causes a prolonged activation of Cdc42, whilst MAPK Spk1 activation is only transient. The result indicates that the activated RAS may develop a preference towards specific downstream pathways or some pathways may be more rigorously downregulated than others.

Compared to the well-characterised process of RAS-mediated MAPK activation, molecular mechanism of RAS-mediated activation of small G-proteins (small-Gs) is largely unknown. We hypothesised that in yeast, Ras1 induces Cdc42 activation by directly interacting with Scd1, a GDP-GTP exchanging factor (GEF) for Cdc42; in other words, by interacting with Ras1, Scd1 is activated and then activates Cdc42. We confirmed the direct interaction between Ras1 and Scd1 for the first time by analysing bacterially expressed recombinant Ras1 and Scd1 complex using NMR and pull-down assays (Tariq et al., manuscript in preparation).

Having established the importance and molecular mechanism of RAS-mediated small-G activation pathway, we now wish to address the next question; How does RAS manage to activate multiple downstream targets in a coordinated manner? Does RAS simultaneously interacts with multiple targets? Or does RAS jump between different targets?

In the PhD project, we will address the question by combining structural biology, cell biology and single molecule analysis. One of the unique features of the single molecule analysis is that it allows us to obtain Kon and Koff rate of interacting molecules through live observation of these molecules. This allows us to study how multiple RAS effectors are competing each other for the active RAS at a molecular level.

We will exploit both fission yeast and human tissue culture systems, where chromosomal endogenous RAS gene loci bear oncogenic constitutively active mutations. Studying two divergent systems allows us to establish core common features of RAS signalling. We firstly focus on the simple yeast model, which helps us to recognise essential key properties of RAS signalling. The outcome will be applied to the human tissue culture system to examine whether such properties are conserved or not.

It is well-established that RAS directly interacts with MAP kinase kinase kinase (MAPKKK) to prime activation of MAPK pathway. We confirmed that, in vitro, RAS binding domains (RBDs) of Scd1 and Byr2 (yeast MAPKKK) compete for Ras1 (Kelsall, Vertesy et al., bioRxiv, 2018). Strikingly, RBD- Scd1 and RBD-Byr2 compete for Ras1 binding in vivo as well, because overexpression of either RBD-Scd1 or RBD-Byr2 in yeast cells interfered with activation of both pathways.

In the PhD project, we will further develop this observation. For in vivo studies, co-localisation of Ras1, RBD-Scd1 and RBD-Byr2 will be examined through live cell imaging through super-resolution microscopy, split-GFP assays and fluorescence resonance energy transfer (FRET). For in vitro studies, single molecule analyses as well as competition NMR analysis will be conducted. In parallel, we will conduct human counterpart experiments using human KRAS and its representative effectors, BRAF, Rgl2 and RALGDS. Collectively, successful delivery of the project will bring a novel conserved concept of RAS signalling.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology & Systems Biology

      Techniques that will be undertaken during the project:

      Gene editing (both yeast and human tissue culture cells), live cell imaging analyses including super-resolution microscopy, split-GFP assays and fluorescence resonance energy transfer (FRET) using a super-resolution confocal microscope, recombinant protein production, NMR, and single molecule analyses using a TIRF microscope.

      Contact: Dr Kayoko Tanaka, University of Leicester