The kinase Raf plays a key role in regulation of proliferation,differentiation and transformation. Although investigated for a long timeand by many researchers the complex regulation of Raf proteins is stillnot solved in all details. We will focus on the scaffold protein CNK1. Onethe one hand we will study the activation mechanism of Raf-1 and thefunction of CNK1 herein. A detailed understanding of Raf signaling isessential for specific intervention in this pathway, especially in respectto the recent findings that point mutations activating Raf proteins arefound in human tumors. On the other hand CNK seems to be an integrator ofdifferent signal pathways. Our preliminary data show that CNK may act as aplatform that binds to three kinases, Src, Raf and the MEK-like kinaseClk1 and to the scaffold protein FHOS. Thereby CNK may connect differentsignal pathway activated by a common stimulus, e.g. insulin. CNK couldallow a cross-talk of signal pathways which may result in synergistic orantagonistic effects.
Yeast two-hybrid screens were performed recently to identify novelinteraction partners for CNK1. Here, these protein-protein interactionswill be verified in mammalian overexpressing systems as well as in tissueor cell extracts. Mutational analysis will be used to localize the regionsessential for binding. The effect of CNK1 and its interaction partners ondifferent signalling pathways will be studied by immunoblotting withphospho-specific antibodies directed against kinases or transcriptionalregulators. To prove the biological relevance of CNK1, siRNA’s targetingCNK1 will be used to down-regulate the expression level of endogenousCNK1. Subsequently the role of CNK1 in signalling can be analysed bybiochemical methods e.g. immunoblotting with phospho-specific antibodiesor by cell biological methods e.g. analysing cell proliferation or cellmorphology.