About the Project
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.
UK/EU applicants only.
Applicants are required to hold/or expect to obtain a UK Bachelor Degree 2:1 or better in a relevant subject.
The University of Leicester English language requirements apply where applicable: https://le.ac.uk/study/research-degrees/entry-reqs/eng-lang-reqs/ielts-65
How to apply:
To apply for the PhD please refer to the guidelines and use the application link at https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp
Please also submit your MIBTP notification form at https://warwick.ac.uk/fac/cross_fac/mibtp/pgstudy/phd_opportunities/application/
Project / Funding Enquiries: [Email Address Removed]
Application enquiries to [Email Address Removed]
UK/EU fees and stipend at UKRI rates. For 2020 this will be £15,285 pa
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