In mammals, IMP/Brap2 (BRCA1 associated protein 2) is associated with the Ras signaling pathway and cytoplasmic retention of BRCA-1 and the cell cycle inhibitor
p21 (1,2). The EEED8.16 protein in C.elegans has been identified as a homolog of the Brap2 gene in C.elegans and has been designated
brap-2. It consists of a Brap2 domain, a RING-H2 domain, a ubiquitin-protease-like zinc-finger domain (ZnF-UBP) and leucine heptad repeats. A
brap-2 deletion mutant was obtained from the Caenorhabditis Genetics Center (CGC) which contains a 1540bp deletion that removes the ZnF-UBP and leucine heptad repeats. Here we show that the
brap-2 gene is associated with oxidative stress response in C.elegans. Exposure of the
brap-2 mutant to either paraquat or peroxide resulted in developmental arrest at the L1 stage at significantly lower concentration compared to N2 worms. Nematodes exposed to oxidative stress have been shown to increase its expression of the phase II detoxification enzyme,
gst-4. The
gst-4::gfp reporter construct was expressed in the
brap-2 deletion mutant and showed elevated levels of expression in the intestine compared to expression in the body wall muscles in the
gst-4::gfp strain alone. Since Brap2 has shown to influence
p21 activity, we also focused on the interaction between
brap-2 and the
p21 homologue in C.elegans,
cki-1 as the cause for L1 arrest under oxidative stress conditions. Incorporation of the transcriptional reporter
cki-1::gfp in the
brap-2 mutant showed increase expression in many cells, most notably in the blast cells, upon exposure to paraquat. Co-transfection of
cki-1 and
brap-2 in HEK293T cells showed that
brap-2 modified
cki-1 localization by retaining it in the cytoplasm. We are currently reintroducing the wild-type
brap-2 gene back into the
brap-2 deletion strain to rescue its oxidative stress sensitivity phenotype and determine the stage of the cell cycle which arrest is occurring. References: 1.Asada M., et al. (2004). Molecular and Cellular Biology, 24(18): 8236-8243. 2.Li S, et al. (1998). Journal of Biological Chemistry, 273(11): 6183-6189. 3.An J.H, et al. (2003). Genes and Development. 17(15): 1882-1993.