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Jacob, Jibi (2006): Characterization of RapGAP1 from Dictyostelium discoideum. Dissertation, LMU München: Faculty of Biology
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Abstract

Rap1 is a ubiquitous Ras-like guanine-nucleotide-binding protein that is involved in a variety of signal-transduction processes especially during cytoskeletal rearrangements. Rap1 is regulated by guanine-nucleotide- exchange factors (GEFs) and GTPase-activating proteins (GAPs) which increase the slow intrinsic GTPase activity by many orders of magnitude and allow tight regulation of signaling. In this study a new Dictyostelium RapGAP1 gene was cloned and characterized. RapGAP1 was discovered by screening the sequences of the D. discoideum database. The Dictyostelium RapGAP1 gene encodes a protein with 1212 amino acids protein which shows at the C-terminal region 53% sequence similarity to human RapGAP. RapGAP1 mRNA was present during all stages of D. discoideum development with a strong upregulation at 9 hours of development. Furthermore, to investigate the role of RapGAP1 in cellular processes RapGAP1 null cells where generated by inserting a gene replacement construct into the endogenous gene. RapGAP1 minus mutants did not show any significant phenotypic abnormalities except that there was a slight delay in development. This delay by about three hours was confirmed by testing the expression of developmentally regulated genes like csA, a cell adhesion protein, and MUD1, a prespore-specific cell surface antigen. However, the mutant was able to complete normal developmental and to form fruiting bodies containing mature spores. Studies on cell motility showed that RapGAP1 null cells moved faster than AX2 wild type cells. This finding suggests that RapGAP1 belongs to a signal transduction chain which ultimately leads to changes in cytoskeletal dynamics.