Functional analysis of RAF mutations

The family of RAF proto-oncogenes, consisting of A-RAF, B-RAF, and C-RAF, is the entry point to the mitogen activated protein kinase (MAPK) signalling cascade, a pathway which mainly mediates mitogenic and anti- apoptotic signals from the cell membrane to intracellular effector proteins. Mutations have been described in B- RAF and C-RAF, and their association with the development of human malignancies has been firmly established now. Most of the currently known mutants result in a constitutive activation of the MAPK cascade which in turn has been thought to relay the oncogenic potential of RAF mutants. However, recent observations of activating B- RAF germ-line mutations in cardio-facio-cutaneous syndrome (CFC), a hereditary disorder without predisposition to malignant disorders, as well as the detection of a non-activating oncogenic germ-line mutation in C-RAF by our own group, refute this simplistic hypothesis. Instead, here we hypothesize that the transforming ability of mutant RAF is modulated, suppressed or enabled by hitherto unknown co-factors. These co-factors may be inhibitors, downstream transducers or proteins that mediate spillover crosstalk with other pathways. Thus, we suppose that particular RAF mutations induce different protein complexes, and consequently relay their oncogenic and non-oncogenic effects, respectively, via different downstream pathways. Especially in the case of C- RAF, such MAPK independent downstream effectors, that mainly relay anti-apoptotic properties, have already been shown to exist. In addition, preliminary results of proteomic screens of endogenous B-RAF signalling complexes in PC12 cells in response to different extracellular stimuli demonstrated remarkable differences between the resulting B-RAF signalling complexes. The aim of this project is to identify signalling complexes that are specifically induced by oncogenic and non- oncogenic RAF mutations, respectively. For this purpose we will initially identify and compare protein complexes formed by a representative set of oncogenic and non-oncogenic RAF mutations using quantitative proteomics. Consequently we will use these different protein complexes to perform a further pathway mapping and to identify the downstream pathways specifically used by the oncogenic and non-oncogenic mutants, respectively. A further aim of the project is to delineate the role of RAF homo- and heterodimerisation during physiologic and pathologic cellular signalling. These RAF heterodimers, whose distinct biologic function has been recently proven by the host laboratory, can be present under physiologic conditions, but can also be induced by various oncogenic and eventually non-oncogenic RAF mutations. Again, protein complexes formed before and after RAF homo- and heterodimerisation will be compared by quantitative proteomics, and subsequently, interacting proteins will be used to perform a further pathway mapping and to identify the downstream pathways specific for RAF homo- and heterodimers, respectively.