Identifizierung von K-Ras-Interaktionspartnern in der humanen Pankreaskarzinomzelllinie PANC-1 mittels des tandem affinity purification-Systems

Abstract

Oncogenic K-ras mutations are found in up to 90 % of pancreatic ductal adenocarcinoma. Knowledge about the interaction of K-Ras proteins with effectors and signalling cascades is essential for understanding oncogenic cellular processes and for the development of potential therapies. With this thesis the tandem affinity purification (TAP) method, which was originally developed in yeast to analyze protein-protein interactions, was established in PANC-1 human pancreatic carcinoma cells to identify interacting proteins of oncogenic K-Ras. At first suitable plasmids for the expression of the fusion proteins TAP-EGFP, constitutively active TAP-EGFP-K-Ras(G12V) and dominant negative TAP-EGFP-K-Ras(S17N) in PANC-1 cells were generated. Expression of the TAP fusion proteins was proved by immune blot analysis and fluorescence microscopy. As the localization of Ras proteins in the cell membrane is necessary for their function, subcellular fractionation by ultracentrifugation and fluorescence microscopy were performed to analyze the subcellular localization. In doing so, it was shown that the fusion protein TAP-EGFP-K-Ras(G12V) is localized in the cell membrane. By performing function analysis it was verified that TAP-EGFP-K-Ras(G12V) is still active despite the N-terminal modification through fusion with TAP-EGFP. As a key result of this thesis, it was shown that the constitutively active mutant K-Ras(G12V) but not the dominant negative mutant K-Ras(S17N) interacts with the p85alpha subunit of phosphatidylinositol 3-kinase (PI3-kinase). So by using the TAP method it was demonstrated that the PI3-kinase and its downstream signalling pathway is an effector pathway of oncogenic K-Ras and may potentially be used for the development of new drugs in cancer therapy.