mTOR signalling hyperphosphorylation of 4E-BP1 and translational control during myogenic differentiation

During muscle cell regeneration or following injury, critical information stored in the gene sequences of the genetic material (DNA) has to be decoded by the cell to produce a wide variety of essential proteins of the right type and in the right amount to allow for this process. The general transfer of information from DNA to protein is carried out by the messenger RNA (mRNA), which is a copy of the DNA sequence. This mRNA has to be decoded into protein by a complex, highly regulated machine termed a ribosome, in a process known as translation. To work efficiently, accurately, and to allow the ribosome to function in the best interests of the cell, this machinery requires helper proteins (translation initiation factors; eIF) that interact with each other, and also make sure that the mRNA and the ribosome come together into a highly regulated, large initiation complex to make the proteins required. So how does the cell control this? The interaction of the initiation factors themselves is a major site for regulation in mammalian cells. One protein, 4E-binding protein 1 (4E-BP1) prevents the interaction of eIF4E with the scaffold protein, eIF4G, and stops the recruitment of mRNA to the ribosome and halts protein synthesis. When protein synthesis is needed, the cell signals to 4E-BP1 to release the eIF4E/mRNA from the 4E-BP1/eIF4E/mRNA complex to let it work. The cell does this by marking the 4E-BP1 with phosphate groups in a process known as phosphorylation. This modification promotes its release from eIF4E/mRNA which can subsequently bind to eIF4G and form the multi-protein initiation complex required to make the correct types and amounts of protein needed for muscle cell regeneration. In the work described here we want to investigate the signals required to bring about the phosphorylation of 4E-BP1 and identify the tagged sites which are important for controlling this process during muscle cell regeneration. We also want to understand more about what other cellular components are required to assemble the stored forms of mRNA in the 4E-BP1/eIF4E/mRNA complex.

The regulation of translation initiation is a key point in modulating gene expression during myogenic differentiation, rapidly altering the temporal and spatial expression of specific mRNAs, without requiring new transcription. Central to this is the recruitment of mRNA to the ribosome during translation initiation, mediated by eIF4E and eIF4G. eIF4E interacts directly with the mRNA cap structure and forms mutually-exclusive complexes with either inhibitory regulatory proteins (4E-BP1) or with the eIF4G scaffold proteins. The resulting eIF4G/eIF4E/mRNA complex and additional protein factors, facilitates ribosome binding and initiation at the correct start site. The association of eIF4E with its negative regulator, 4E-BP1, is modulated by phosphorylation of 4E-BP1 controlled via mTORC1 signalling. Current models suggest that hypo-phosphorylated 4E-BP1 binds eIF4E/mRNA thereby recruiting specific mRNAs into translationally inactive storage granules, structures which are also often associated with miRs. Following mitogenic stimulation, 4E-BP1 is hyper-phosphorylated, dissociates from eIF4E and allows interaction of released eIF4E/mRNA with eIF4G, promoting translation initiation on stored mRNAs. We have found that whilst inhibition of mTORC1 during differentiation delays increased translation rates, paradoxically phosphorylation of 4E-BP1 is increased. The hyper-phosphorylated form(s) of 4E-BP1 that remains could not be ascribed to increased phosphorylation at known important regulatory sites. Using C2C12 myoblasts and employing siRNAs and biochemical approaches, we will investigate the role for 4E-BP1 and its hyper-phosphorylation in the post-transcriptional regulation of gene expression of selected mRNAs during differentiation by: (a) identifying the sites of phosphorylation, and characterising the kinases/phosphatase activities involved; (b) identifying interacting proteins and miRNAs associated with selected inactive 4E-BP1/eIF4E/mRNA complexes

The main beneficiaries of the proposed work will be research colleagues and those in the Pharma industry with an interest in cell signalling during differentiation, growth and wound healing. In addition to presentation of this 'basic, blue-sky research' at international meetings, the main impact will be on: New Collaborations; Inhibitors of mTORC1 signalling are currently used as immunosuppressants for transplant patients, in surgical stents, and are in Phase II/III clinical trials for cancer patients with solid tumours. Based on a previous agreement using the Mnk1 inhibitor, CGP57380, we secured a supply of RAD001 about 2 years ago to investigate its effects on muscle cell growth and differentiation. Our preliminary data associated with this application for funding used both of these drugs. In future, we will build upon this collaboration to secure access to the next generation of signalling inhibitors which are currently entering clinical trials. The benefits for the Pharma industry is that their products get tested in different cell types and are engaged in answering scientific questions which they might not have the facilities or expertise for, or may have not considered. This work will also help foster new investigations into how these signalling pathways impinge on cell growth in general. This has promoted a new BBSRC-funded collaboration with Dr. Sarah Newbury, research faculty at the Medical School at Sussex (BSMS), to look at the role of phosphorylation events in wound healing using Drosophila as a model system. We have already secured pilot funding with Dr.Tim Chevassut, a BSMS clinician, to investigate the differential sensitivity of leukaemic stem cells to our signalling inhibitors. With an MRC Senior Clinical Research Fellow at BSMS, Dr. Anthony Chalmers, we have started collaborating to investigate whether RAD001 can sensitise human glioma tumour cells to radiotherapy. Skills, training and knowledge economy; The PDRA will have to master a number of timely and intensive techniques and approaches, optimising them accordingly. This will provide a fertile environment for training and development of new skills. The on-going collaboration with Dr. Bushell (Nottingham) on analysis of miRNAs recovered in association with repressed mRNA will expand our way of thinking about translational regulation and how mRNAs are triaged by the cells. New networking groups; South Coast RNA Group. As part of an efficient vehicle for efficient dissemination of our work, with Dr. Sarah Newbury and funded by Sussex IP, we have started a group of like-minded workers who will meet every quarter at various locations on the south coast. The format will be seminars and discussions to promote interactions and collaborations; the first meeting will be in November 2009 at Sussex. Sussex Cancer Group, is a group of researchers and clinicians at Sussex working in themes of cancer, growth control, patient care and clinical trials, brought together by Dr. Chalmers. This group meets 4 times per year and is funded by Sussex IP and Industrial sponsors. This networking opportunity has led to a number of fruitful discussions and the collaborations outlined in above. Schools and public engagement; As part of our current lab remit to publicise our work, I have talked at a local girl's 6th form college and we will have 6th form students carrying out 'hands-on' projects in the lab again over the summer. Both my PhD student and myself have interacted with 8-14yr olds at the Brighton Science Fair in the form of 'hands-on' playdough model sculpturing of cells, their structures and morphology. I have also participated in discussions about the use of animal research in science at a public forum in Brighton and regularly attend local Café Scientifique presentations to talk about what we do in the lab.