Characterization of RapGAP1 from Dictyostelium discoideum

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.