Unravelling KRAS Biology: characterization of the KRAS-RAF signalosome and its functional dynamics at the cell membrane

The RAS-RAF-MEK-ERK mitogen-activated kinase signaling (MAPK) pathway plays an essential role not only in driving normal physiological cellular proliferation but also in the dysregulation of the signaling cascades commonly occurring during cancer development. A key event in the complex process of activation of this signaling pathway is the change of RAS localization as a function of its activation state. The most relevant known RAS effectors are RAF kinases. Although the RAS family members are the most studied proteins in the context of cancer, a detailed understanding of RAS-RAF membrane dynamics is still far from being complete. Therefore, the main goal of this work is to investigate the biology and the role of individual isoforms of RAF proteins in the MAPK signaling pathway using new cell models. We generated RAF isoforms constructs containing a CAAX motif, which causes forced localization of proteins to the plasma membrane. In the first part, we focus on the analysis of the effect of forced localization of RAFCAAX proteins in RAS-less MEFs, where we show the rescue of cell proliferation by our constructs in the absence of RAS. In part two, we describe the impact of RAFCAAX mutants in RAF-less MEFs and human BEAS-2B and ATII cells. The data show that depending on the cell context, the signaling outcome is different. Finally, we performed a bioinformatic analysis of data available on online databases to better understand the relationships and differences between RAF isoforms in the tumorigenesis process in patient samples. Overall, our data provide a new perspective on cell membrane complex dynamics, leading to a better understanding of KRAS-RAF signalosome-dependent mechanisms.