Premium
Ubiquitin‐conjugating enzyme UbcD4 as an essential component of Drosophila immune deficiency pathway (LB192)
Author(s) -
Park Eun Sil,
Kim YoungJoon,
Yoo Yung Joon
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb192
Subject(s) - rna interference , gene knockdown , ubiquitin , biology , schneider 2 cells , microbiology and biotechnology , ubiquitin conjugating enzyme , green fluorescent protein , reporter gene , transgene , gene , ubiquitin ligase , genetics , gene expression , rna
Ubiquitination has been emerged as a key regulatory mechanism in the NF‐κB pathway, in which the different types of ubiquitin chains are involved in the discrete steps. Given that the ubiquitin conjugating enzymes (E2s) are key enzymes to determine the chain topologies during ubiquitination, we tried to systematically screen out the E2s required for the IMD pathway in Drosophila , which is very similar to TNF‐α mediated NF‐κB pathway in mammals, via RNAi‐mediated knockdown of individual E2 using GAL4/UAS system. For this purpose, we first generated a transgenic fly containing a reporter dipt‐GPF gene and a fat body driver hml‐Gal4 gene that expressed the reporter protein GFP upon challenge of Gram‐negative bacteria, Ecc15 . When dipt‐GFP, hml‐Gal4 transgenic flies were crossed to each of 35 UAS‐E2‐RNAi IR lines obtained from VDRC and the offspring female adult flies were infected with Ecc15 , and the expressed GFP in each IR offspring was compared by Western immunoblot, it was found that the GFP level was significantly reduced in IR lines targeting UbcD4 in addition to effete and bendless. RNAi‐mediated down‐regulation of a mammalian UbcD4 homolog, Ube2K/E2‐25K, significantly attenuated the TNF‐α mediated NF‐κB activation pathway. Taken together, UbcD4 and Ube2K/E2‐25K play a conserved role as a positive regulator in Drosophila IMD pathway and mammalian TNF‐α mediated NF‐κB pathway.