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

QH301 Biology

Urcia, Roby Joseph ; Pitt, Andrew

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: QH301 Biology;   

Others  :  http://theses.gla.ac.uk/4183/1/2011urciaphd.pdf

来源: University of Glasgow

【 摘 要 】

An emerging concept in the regulation of signal transduction specificity is the mediationof scaffold proteins embedded in the circuitry of signalling pathways. The multidomainbasedarchitecture of scaffold proteins facilitates the assembly and modulation of proteincomplexes to regulate cellular signals to bring about an exacting biological output. Thework presented in this thesis aimed to investigate the mechanisms of the protein scaffoldCNK1 (connector enhancer of Ras 1) in the pro-apoptotic MST2 pathway and the prooncogenicRaf-1 signalling pathways. Here, by using several molecular, biochemical andcell biology techniques, I demonstrated that CNK1 regulates the interaction of the protooncogeneRaf-1 and the tumour suppressor MST2 kinase. Perturbations of CNK1 levelsexhibit a biphasic signalling response typical of a scaffold protein. Transient expressionof CNK1 upon growth factor withdrawal results in a concentration-dependent increaseof the Raf-1/MST2 complex, thus preventing apoptosis, but this complex dissociates athigher expression levels, hence promoting an apoptotic response. Moreover, CNK1 isinvolved in the regulation of Fas-induced apoptosis via the MST2/RASSF1A pathwayby 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 theRaf-1 and MST2 interaction, and enhanced MST2/LATS1 interaction that promotesapoptosis. Thus, CNK1 is required for Raf-1 inhibitory function, but is also necessaryfor MST2-mediated apoptosis. Remarkably, CNK1 selects and switches complexformation of opposing anchored proteins depending on the stimulus. In response to Fasligand 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 followinggrowth factor treatment. Mapping the multidomain binding sites of CNK1 using peptidearray demonstrates specific interaction sites of client protein complexes. Specific CNK1point mutants were generated, and found to alter wild-type regulation of client proteincomplexes. Thus, the work described in this thesis may reveal a regulatory crosstalkbetween the MST2 apoptotic pathway and the Raf-1 proliferative pathway throughCNK1 by coordinating assembly of appropriate pathway components to possibly drivediscrete stimulus-specific responses.