Dr Kayoko Tanaka E: firstname.lastname@example.org
Dr Andrey Revyakin E: email@example.com
Dr Cyril Dominguez E: firstname.lastname@example.org
The RAS family of small GTPases act as signalling hubs regulating cell proliferation and differentiation. The physiological importance of RAS signalling is evident as 20% of all human cancers harbour mutations in RAS genes (COSMIC). However, there is no anti-Ras inhibitor is available except the one which specifically targets G12C oncogenic mutation through the thiol group of the G12C cysteine. As G12C mutation contributes to about 10% of human Kras oncogenic mutations, it is vital to develop effective inhibitors against other oncogenic Ras variants. Towards this goal, we need to understand the mode of action of oncogenic RAS molecules.
It is generally assumed that oncogenic RAS molecules over-activate all the downstream effectors. Well-characterised RAS downstream pathways include MAPK and Akt pathways, both of which are hyper-activated upon over-expression of oncogenic RAS mutants. However, accumulating evidence shows that the physiological phenotype of genomic RAS mutation is different from the outcome caused by over-expression of oncogenic RAS (Perez-Mancera and Tuveson, 2006). In Kras.G12D mouse model, where the oncogenic G12D mutation causes preneoplastic epithelial hyperplasias to the mice, over-activation of ERK (MAPK) nor Akt was not observed. Instead, a morphological change was noted in the mouse embryonic fibroblasts generated from the Kras.G12D mouse (Tuveson et al., 2004). In a HEK293 tissue culture system, chromosomally integrated oncogenic RAS mutation increases cellular invasiveness, which is dependent on a small GTPase RalB, but not dependent on ERK nor Akt (Zago et al., 2018). Using a simple model organism, fission yeast, we also showed that genomic RAS oncogenic mutation causes prolonged activation of a yeast small GTPase, leading to highly deformed cells, whereas MAPK hyper-activation does not occur (Kelsall, Vertesy et al., bioRxiv, 2019). These findings prompted us to hypothesise that oncogenic RAS signalling causes biased over-activation of small GTPases, such as RalB in humans, that regulate cell morphology and motility, but not ERK nor Akt pathways.
It is through direct protein-protein interaction the way RAS activates down-stream pathways; active RAS directly interacts with the “effector” molecules to cause structural changes to them, which then trigger activation of corresponding downstream pathways. Representative effectors include BRAF that primes ERK pathway activation and PI3K that leads to Akt activation. For small GTPase pathways, GDP-GTP exchange factors (GEFs) of the small GTPases act as a RAS effector and then activate the small GTPases such as RalB. Domains required for RAS interaction have been identified in various RAS effectors and termed Ras Bind Domains (RBDs) or Ras Associating domains (RAs). Primary structures of RBDs or RAs vary, and hence, identification of potential RBDs/RAs through genome database analysis was challenging. Meanwhile, structural analyses of existing effector molecules revealed that RBDs and RAs share a common structural feature; they all have ubiquitin-like folding structures consisting of ββαββαβ.
Although the essentiality of RAS-effector interaction in the oncogenic RAS signalling is well-recognised, the dynamic mode of RAS-effector interaction has been elusive.
-Does RAS simultaneously interact with multiple effectors?
-Does RAS jump between different effectors?
-Does interaction with one of the effectors influence the next interaction?
These questions have not been addressed previously because RAS-effector interactions were assessed in biochemical assays that monitor bulk molecular behaviours or by X-ray crystallography where molecular dynamics cannot be studied.
In the PhD project, we will address these questions by single-molecule analysis using optical microscopy, which is one of the most powerful techniques to measure molecular dynamics. It allows us to obtain kon and koff rate of interacting molecules through live observation and to record multiple effector interactions along a time-course of ms resolution. This part of the project will involve biochemical purification of recombinant proteins, operation of total internal reflection fluorescence (TIRF) microscopy and data analysis of single molecules. The findings will be further validated using a combination of structural biology (NMR, X-ray crystallography) and cell biology (generation of human cell lines with Kras G12 oncogenic mutant variants). Successful delivery of the project will bring a novel concept of RAS signalling and help design inhibitors targeting RAS signalling.
The project involves gene editing of human tissue culture cells, cell biology using live-cell imaging, structural biology and single-molecule analyses. Thus, by definition, it will be interdisciplinary.
Kelsall, Vertesy et al., (2019) Constitutively active RAS in S.pombe causes persistent Cdc42 signalling but only transient MAPK activation, bioRxiv, doi: https://doi.org/10.1101/380220
Grimm et al., (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy, Nature Methods, 12, 244-50, doi: https://doi:10.1038/nmeth.3256
Lee et al., (2013) Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions, Nature Protocol, 8, 2045–2060, doi: https://doi:10.1038/nprot.2013.116