The modulation of tumour suppressor MST2 and proto-oncogene Raf-1 kinases by the scaffold protein CNK1

Urcia, Roby Joseph (2011) The modulation of tumour suppressor MST2 and proto-oncogene Raf-1 kinases by the scaffold protein CNK1. PhD thesis, University of Glasgow.

Abstract

An emerging concept in the regulation of signal transduction specificity is the mediation
of scaffold proteins embedded in the circuitry of signalling pathways. The multidomainbased
architecture of scaffold proteins facilitates the assembly and modulation of protein
complexes to regulate cellular signals to bring about an exacting biological output. The
work presented in this thesis aimed to investigate the mechanisms of the protein scaffold
CNK1 (connector enhancer of Ras 1) in the pro-apoptotic MST2 pathway and the prooncogenic
Raf-1 signalling pathways. Here, by using several molecular, biochemical and
cell biology techniques, I demonstrated that CNK1 regulates the interaction of the protooncogene
Raf-1 and the tumour suppressor MST2 kinase. Perturbations of CNK1 levels
exhibit a biphasic signalling response typical of a scaffold protein. Transient expression
of CNK1 upon growth factor withdrawal results in a concentration-dependent increase
of the Raf-1/MST2 complex, thus preventing apoptosis, but this complex dissociates at
higher expression levels, hence promoting an apoptotic response. Moreover, CNK1 is
involved in the regulation of Fas-induced apoptosis via the MST2/RASSF1A pathway
by influencing the time-scale kinetics of MST2 docking and release from the Raf-
1/CNK1 complex and its eventual activation. SiRNA-silencing of CNK1 destabilizes the
Raf-1 and MST2 interaction, and enhanced MST2/LATS1 interaction that promotes
apoptosis. Thus, CNK1 is required for Raf-1 inhibitory function, but is also necessary
for MST2-mediated apoptosis. Remarkably, CNK1 selects and switches complex
formation of opposing anchored proteins depending on the stimulus. In response to Fas
ligand stimulation, MST2 is released, whereas Raf-1 is retained in complex with CNK1.
Conversely, CNK1 retains MST2 and whilst releasing Raf-1 from the complex following
growth factor treatment. Mapping the multidomain binding sites of CNK1 using peptide
array demonstrates specific interaction sites of client protein complexes. Specific CNK1
point mutants were generated, and found to alter wild-type regulation of client protein
complexes. Thus, the work described in this thesis may reveal a regulatory crosstalk
between the MST2 apoptotic pathway and the Raf-1 proliferative pathway through
CNK1 by coordinating assembly of appropriate pathway components to possibly drive
discrete stimulus-specific responses.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH301 Biology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Pitt, Prof. Andrew
• Date of Award: 2011
• Depositing User: Mrs Marie Cairney
• Unique ID: glathesis:2011-4183
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Apr 2013 14:25
• Last Modified: 13 May 2016 14:50
• URI: https://theses.gla.ac.uk/id/eprint/4183