A proteomic analysis of Raf-1 signalling pathways

von Kriegsheim, Alexander F. (2005) A proteomic analysis of Raf-1 signalling pathways. PhD thesis, University of Glasgow.

Abstract

Raf-1 is an integral part of the Ras/Raf/MEK/ERK signalling pathway that relays mitogenic and survival signals to the nucleus, leading to suppression of apoptosis, growth and proliferation. The ability of Raf-1 to fulfil these diverse functions is likely to require its ability to form multiple complexes which are regulated by phosphorylation and sub-cellular localisation. The aim of the work described in this thesis was to develop proteomic approaches to study dynamic changes in Raf-1 signalling complexes in vivo. Recent studies have indicated that Raf-1 not only plays a crucial role in relaying growth signals through the MAPK pathway but it also has a kinase independent function in protecting cells from apoptosis. To characterise this anti-apoptotic role of Raf-1, DIGE (different gel electrophoresis) was used to determine changes in protein expression resulting from the reconstitution of Raf-1-/- fibroblasts with kinase-dead or wild-type Raf-1. Numerous pro- and anti-apoptotic proteins were differentially expressed between these three cell lines. Furthermore levels of several potential Raf-1 binding proteins and proteins involved in actin dynamics and motility were altered. The changes in protein expression identified by proteomic analysis are consistent with the hypersensitivity of Raf-l-/- fibroblasts to apoptotic stimuli and the alterations in their actin cytoskeleton and motility. In addition to examining global protein changes by DIGE, we developed a gel-less, mass spectrometry based two-dimensional liquid chromatography approach to analyse protein samples of medium complexity and successfully adapted it to identify Raf-1 protein complexes. We identified over 100 potential Raf-1 interacting partners, some of which required the presence of S259 site to bind to Raf-1 whereas others interacted only when cells were induced with mitogens or apoptotic insults. Two of these interactions (protein phosphatase 5 and SIP1) were verified by co-immunoprecipitations and functional analysis was performed on one of these proteins. Furthermore we developed a highly sensitive and robust method for phosphorylation site mapping and demonstrated its potential by identifying phosphorylation sites of proteins present in Raf-1 complexes. Raf-1 activation is a complex process in which phosphorylation plays a key role. While several kinases have been shown to phosphorylate Raf-1, only one phosphatase, PP2A, has been identified to regulate Raf-1 activity in vivo by dephosphorylating serine 259 upon activation with mitogens. We identified protein phosphatase 5 (PP5) as a potential Raf-1 interacting protein. This interaction was validated by co-immunoprecipitation of endogenous Raf-1 with PP5 in COS-1 cells. PP5 dissociated from Raf-1 upon stimulation with EGF and specifically dephosphorylated a crucial activation site, S338, in-vivo and in- vitro. Furthermore we provide evidence that cross talk between the MAPK pathway and the Gal2 pathway is regulated by PP5. The results presented here show that we have developed robust proteomic methods enabling us to analyse protein complexes as well as changes in protein expression and phosphorylation with high sensitivity and accuracy.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Prof. Walter Humes.
• Keywords: Biochemistry.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Supervisor, not known
• Date of Award: 2005
• Depositing User: Enlighten Team
• Unique ID: glathesis:2005-72539
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 12 Aug 2021 14:00
• URI: https://theses.gla.ac.uk/id/eprint/72539