Photo of Simon Morley

Simon Morley
Professor of Signal Transduction (Biochemistry)
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T: +44 (0)1273 872794


Research

Current research interests
Background

The research carried out in my lab over the past 20 years, funded by a Senior Research Fellowship from The Wellcome Trust and research grants from the BBSRC, has been directed at the identification of the intracellular signalling mechanisms involved in the regulation of mRNA recruitment to the ribosome during different phases of the cell cycle. In particular, we have chosen to determine the role of individual initiation factors and their post-translational modification in the activation of protein synthesis following the induction of cell growth, and in the maintenance of translation rates during cell death and differentiation. More recently we have become interested in the role for localised translation in controlling cell polarity and migration. My laboratory has made a wide range of important contributions to understanding the function of translation factors in mammalian cells. These relate to a number of different components of the translational machinery, including several initiation factors (eIF4E, eIF4G, PABP), a family of protein kinases with initiation factors as substrates (e.g. Mnk1), and the regulation of initiation factor localisation in mammalian cells under different growth conditions. In summary, our main achievements have been:

(a) eIF4E phosphorylation  We have made a number of important discoveries in this area:

(i) we demonstrated that the mTOR signalling pathway is not required for the acute phosphorylation of eIF4E in Xenopus oocytes or primary mammalian cells;

(ii) we challenged the dogma that eIF4E was rate-limiting for translation in mammalian cells;

(iii) we showed that the phosphorylation of eIF4E was increased in response to stress and physiological activation of the T-cell receptor complex, mediated by ERK/p38 MAP kinases;

(iv) we conclusively showed that the phosphorylation of eIF4E was not a pre-requisite for the activity of this protein in vitro or in vivo and that the phosphorylation of eIF4E was not required for de novo protein synthesis following recovery of cell from salt shock. In fact, this reflected a novel cross-talk between the MEK1/2 and mTOR signaling pathways in mammalian cells.

(b) eIF4F complex assembly and translational control Using a combination of approaches to isolate eIF4E and its associated proteins, we have conclusively shown that :

(i) serum stimulated the association of eIF4F and PABP in Xenopus kidney cells;

(ii) using regenerating mouse liver, mTOR signaling in required for the assembly of eIF4F complex and cyclin D1 protein expression under physiological conditions;

(iv) the proteasome inhibitor, MG132, promoted a re-programming of translation and generation of a novel eIF4F complex containing hsp25;

(v) the activation of Pak2 results in the phosphorylation of eIF4G and decreased levels of eIF4F;

(vi) multiple forms of eIF4GI exists in human cells with different abilities to maintain protein synthesis rates but isoforms lacking individual domains can still drive protein synthesis in vitro.

(c) Proteolytic cleavage of eIF4G during viral infection and cell death Over a number of years, our Wellcome Trust/BBSRC funded research has demonstrated that:

(i) translation of uncapped mRNA could be stimulated by pre-cleaved eIF4G to the untreated reticulocyte lysate, with the C-terminus of eIF4G sufficient to support cap-independent translation;

(ii) binding of eIF4E to eIF4G altered the structure of the latter allowing cleavage by L protease;

(iii) eIF4G was cleaved during apoptosis and to map these novel cleavage sites;

(iv) we used caspase-deficient cell lines to show the role for caspase-8 in eIF4G cleavage;

(v) caspases target the PABP/eIF4B interaction to decrease translation during apoptosis.

(d) Localisation of initiation factors in mammalian cells In work largely funded by the BBRSC, we used antisera and Alexa-fluor coupled antibodies tools to conclusively demonstrate:

(i) a population of eIF4G resides in the nucleus where it is actively associated with the splicing machinery, providing a functional link between the nuclear and cytoplasmic cap-binding proteins;

(ii) PABP co-localises with paxillin at the dense ER and the leading edges of migrating cells;

(iii) eIF4GI possesses a functional nuclear localization signal in its N-terminus responsible for the nuclear accumulation of an apoptotic fragment of eIF4G;

(iv) MG132 promotes the re-localisation of initiation factors in myoblasts in culture;

(v) Mnk1 actively shuttles into the nucleus where it might target a nuclear pool of eIF4E;

(vi) the selective compartmentalisation and localisation of eIF4F in normally growing fibroblasts;

(vii) used a novel pumomycylation assay to show localised protein synthesis in spreading cells and co-localisation of initiation factor and specific mRNAs at the leading edge of the cell.

 Aims of the current work

The aims of my current work are to investigate the signalling pathways regulating mRNA utilisation in eukaryotic cells during proliferation and differentiation. My previous studies have clearly shown the co-ordinate phosphorylation of components of the translational machinery has a central role in the regulation of protein synthesis, and may mediate the selection of specific mRNA species for translation. One of these components, which is the main focus of my work, is the initiation factor complex, eIF4F. Increased phosphorylation of two components of eIF4F (eIF4E and eIF4G) is an early response to stimulation of translation by a wide variety of agents (serum, hormones, mitogens). We are currently investigating the assembly of this complex during different phases of the cell cycle, the localised regulation of protein synthesis and the control of eIF4F complex assembly during myogenic differentiation. Although the regulation of protein synthesis is fundamental to cell growth and/or survival, relatively little is actually known about the role of phosphorylation of translation initiation factors in modulating this process. In particular, there is, as yet no direct evidence that phosphorylation of eIF4E or eIF4G at physiological sites has any effect on translation rates or cell growth in vivo.
In light of my previous findings, we are currently:
(a) investigating the signalling pathways utilised to modulate eIF4F phosphorylation and rates of protein synthesis in vivo, with particular emphasis on the role of the eIF4E kinase, Mnk1, in translational control and cell mobility;
(b) determining how the phosphorylation status of eIF4E and other RNA binding proteins influences the selection of specific mRNAs for translation at distinct intracellular locations;
(c) investigating the role of intracellular localisation of initiation factors in translational control;
(d) investigating the role for initiation factor phosphorylation in controlling cellular differentiation.
(e) initiating new links to pioneer studies into the role for translational control in leukaemic stem cell proliferation (Dr. T. Chevassut), loss of growth control in glioma cells (Dr. A Chalmers, Glasgow), during wound healing (Dr. S. Newbury), as well as the role for SUMOylation of initiation factors in controlling protein synthesis (Dr. F. Watts). 
We have been addressing these related questions by parallel studies using a range of human cells in culture. We believe that these studies will substantially increase our understanding of the significance of the control of protein synthesis in the regulation of cell growth or death.

Current project (BBSRC)

    Protein synthesis is carried out in three linked stages (initiation, elongation and termination), with binding of mRNA to the ribosome a major site of translational regulation. This step is facilitated by the assembly of initiation factors (eIFs) into a multi-protein complex, eIF4F (eIF4E, eIF4A, eIF4G), associated with eIF3 and the ribosome. eIF4F formation is strictly controlled by both regulatory proteins (such as 4E-BP1 and CYFIP) and their intracellular localisation, regulating cell migration, growth and survival.

    Localised protein synthesis coupled to mRNA targeting spatially restricts the synthesis of specific proteins required for cell spreading and migration; how the cell regulates this is unclear. With MRC5 fibroblasts, we have used confocal microscopy and IF to show an enrichment of initiation factors, actively translating ribosomes, specific mRNAs and novel regulatory mRNA binding proteins (CYFIP, FMRP) in lamellipodia at the leading edges of spreading cells. Furthermore, FISH, IP and TIRF microscopy show that mRNAs encoding structural and regulatory proteins of the WAVE complex are in the same mRNP complexes as FMRP/CYFIP at the leading edge. As protein synthesis is a requirement for cell spreading, we suggest that the regulation of localised translation has a pivotal role in controlling cell spreading and migration. However, outside of neuronal cells, little is known about how FMRP/CYFIP/DDX3X regulate localised translation during cell spreading. Loss of CYFIP, a negative regulator of eIF4E, is associated with increased mobility on tumour cells, whilst either loss of over-expression can affect mobility, depending upon the cell type. To address this lack of knowledge, we will use fibroblasts and: (a) determine if there is a regulatory role for phosphorylation and/or arginine methylation of FMRP during cell spreading; (b) analyse whether CYFIP/FMRP/eIF4E or eIF4E//DDX3X complexes are regulated at the leading edge of cells; (c) investigate structural elements in target mRNAs required for recruiting such mRNA to regulatory binding proteins in lamellopodia.