The RAS superfamily of small GTPases consists of approximately 50 protein members divided into several subfamilies. Although the Ras subfamily has been the most extensively studied for its role in malignant transformation, there is an increasing interest in characterizing the Rho GTPases due to the role that these proteins play in the control of cell homeostasis. The Rho GTPases are biological molecular """"""""switches"""""""" that control multiple cellular processes such as growth, differentiation, and transformation by cycling between an inactive (GDP-bound) and an active (GTP- bound) state. The Rho guanine nucleotide exchange factors (GEFs) regulate the Rho GTPases. The Rho GTPase cascade, however, is far from being completely resolved and the contributions of particular Rho GEFs or Rho GTPases to human diseases remain undetermined. We have recently identified a novel gene whose sequence predicts it to be a Rho GEF. This gene called Leukemia Associated Rho GEF, or LARG was found fused with a transcription factor in acute leukemia. We hypothesize that LARG has oncogenic activity that is mediated by its putative Rho GEF activity and, in turn, by its ability to activate Rho GTPase pathways. We propose to test this hypothesis by characterizing the functions of LARG and assessing its relevance to leukemogenesis. We have identified three specific aims to achieve these goals. In the first aim we will characterize in vitro LARG as a GEF for Rho GTPases. In the second specific aim we will determine the role of LARG within the Rho GTPase cascade in vivo by generating transgenic Drosophila lines, and we will characterize e x p ression patterns of the LARG murine homolog (Larg) during mouse embryogenesis by in situ hybridization. In the third specific aim, we will assess the relevance of LARG to the pathogenesis of acute leukemia by analyzing the leukemogenic potential of the LARG fusion gene and other LARG mutants in vitro and in vivo by retroviral transfection of murine stem cells. Ultimately, we believe that this K08 proposal will provide important insights into the regulation of cell functions by this novel Rho GEF, and will provide a firm foundation for future independent investigative basic science work by the principal investigator (P.I.).
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