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Proteome maps of MAPK/ERK pathway dependent protein expression and modification for the analysis of the biochemical functions of the MAPK/ERK pathway

In our part of the project we analysed cells expressing either conditional forms of Raf or cells with deleted raf (KO cells) using proteomic methods for differentially expressed/modified proteins. Next to conditional Raf we also used serum or TPA. We detected more than 90 Raf regulated phosphoproteins in total. From these we could identify 28 using mass spectrometry. 8 of theses proteins are known to be localised in the nucleus, 4 can shuttle between nucleus and cytoplasm, 15 are found in the nucleus and 1 in the mitochondria. 7 of the proteins are involved in nuclear structure and heterochromatin assembly, 7 in signal transduction, 3 in mRNA transport or stabilisation, 4 have a role in the cytoskeleton, 3 in mRNA transport, 2 are involved in translation the rest have various functions, like e.g. in the proteasome. Using Raf inhibitors U0126/PD98059 we detected several proteins that are phosphorylated after Raf activation independent of the MEK/ERK part of the pathway. The substrates that we identified so far can, after evaluation, be valuable targets for identifying the activation state of the Raf/ERK pathway or for inhibition or augmentation of the pathway in therapeutically interventions.

We also investigated the mitochondrial proteome under Raf/ERK stimulation. Using newly developed methods for mitochondrial isolation, we could detect diversity in mitochondrial populations. The mitochondria are divided into 7 different subpopulations, four of which are apparent in exponentially growing cells, while in cells with acute stimulation of the Raf/ERK pathway three different forms arise, which over the period of some hours change into the formerly observed populations. Their apparent isoelectrical point defines these populations. Therefore, we assume that charges or compositions of proteins integral to the outer membrane, or associated with it, change.

In minor projects we analysed the proteomes of haematopoietic cells. Using different cell lines and blood samples we have identified some three proteins that might be over expressed in cells from acute myeloid leukaemia as opposed to normal CD34+ cells.

Using 2D Gel Difference Electrophoresis (DiGE) we have compared alterations in protein expression and modification in Raf-1 KO fibroblasts and cells reconstituted with Raf-1 or a kinase negative Raf-1 mutant under conditions of serum starvation and stimulation with serum. Most changes in the Raf-1 KO cells were reversed by expression of either wt Raf-1 or kinase negative Raf-1. The main changes observed included changes in proteins that regulate apoptosis and the cytoskeleton. These are consistent with the role of Raf-1 in apoptosis and cell migration that is observed on the cellular and organismal level.

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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University of Manchester Faculty of Life Science
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