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Proteome map of Raf-1 signalling complexes for the analysis of the role of protein interactions in regulation of the functions of the MAPK/ERK pathway

In this part of the project we isolated and characterised Raf-1 signalling complexes and identified their components by mass spectrometry. We used three strategies to identify functionally important proteins:

- Comparison of complexes formed by Raf-1 with complexes formed by mutant Raf-1 proteins that induce interesting biological phenotypes. For instance, the Raf-1 S259A point mutant efficiently stimulates the ERK pathway as well as proliferation, but fails to induce transformation in fibroblasts and differentiation in PC12 cells. The use of this mutant led to the identification of PP5 a protein phosphatase that selectively removes an activating phosphorylation in Raf-1 after mitogen stimulation.

- Comparison of complexes formed by Raf-1 under different conditions of cellular stimulation, e.g. serum starvation versus mitogen stimulation. This approach led to the identification of MST2 which selectively associates with Raf-1 in serum starved cells and induces apoptosis in response to stress signals. Raf-1 inhibits MST2 activation.

- Comparison of complexes formed by Raf-1 in different subcellular compartments. Density gradient fractionation of Raf-1 complexes allowed the separation of at least 5 different Raf-1 complexes. The proteomic characterisation of these complexes led to the identification of synaptojanin2 a protein that is involved in receptor endocytosis and also has a splice variant with mitochondrial localisation. Thus, all three strategies were applied successfully and can serve as a design template for the analysis of signalling pathways by proteomics.

The main value of the result obtained is of scientific nature, and resides in the knowledge acquired on the function of the proteins of the MAPK cascade and its organisation in terms of protein complexes and subcellular compartmentalisation. The descriptions of the result have been/will be published in a series of publication from the partner labs, and thus disseminated to the scientific community. The result is being/will be used by the partners themselves, to carry out specific scientific projects, and by other scientists without the consortium who are interested in exploiting our models to ask a specific question in their system.

We have already received such requests, and have complied with all of them. Biomedical/pharma companies value the expertise and the knowledge we are accumulating on these eminently druggable kinase module. In particular, the results has already revealed alternative targets of the kinases in the pathway; and it has predictive value in the context of the possible side effects of therapeutic approaches aimed at interfering with a given component of the pathway. Both issues are eminently relevant when evaluating the suitability of a given protein as a therapeutic target.

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Reported by

Beatson Institute for Cancer Research
Garscube Estate, Switchback Road
G61 1BD Glasgow
United Kingdom