Functional Characterisation of Raf-kinases and proteomics
Raf-protein kinases are central regulatory elements of cellular signal transduction pathways regulating proliferation, differentiation, senescence and apoptosis. Additionally Raf-1 is instrumental for the tumorigenic transformation of cells by oncogenic ras. Raf-1 is well recognised for heading the Raf/MEK/MAP kinase cascade but further unknown signalling pathways emerging from Raf-1 are predicted from several lines of evidence. Different cellular localisations and various activating mechanisms for Raf in specific cellular situations suggest that it utilises specific pathways and substrates for each of its functions. Elucidation of these pathways is central in order to understand how the activation of Raf-1 in different situations can lead to a multitude of biological responses. One major aim is to identify and correlate the different signal transduction pathways emerging from Raf-1 with its biological functions. To identify target proteins we use proteome analysis, whereby all proteins and their different modifications expressed within the cells are displayed by two-dimensional electrophoresis and analysed by mass spectrometry. To reveal causal relationships in the highly dynamic proteome we use conditional forms of Raf-1, that are activated upon hormone addition (Fig. 1).
Using these kinases in combination with metabolic labelling of cells we can analyse phosphorylation changes accumulating within minutes. One of the targets of Raf kinases identified using proteome analysis is stathmin. It is a phosphorylation-dependent regulator of micro tubular dynamics in vivo, suggesting a model to explain the morphological changes observed during cellular transformation, following the activation of Raf. Other targets we identified so far include transcription factors and ribo-nucleoproteins.