In response to DNA damage, eukaryotic cells activate a complex signaling network to arrest cell proliferation and activate repair mechanisms, or kill the damaged cell by apoptosis. Tumor suppressor
p53 is a major effector of DNA damage signaling pathways. Under normal conditions
p53 is maintained at low levels by the MDM2 protein, an E3 ubiquitin ligase. Recent studies have shown that MDM2 can also neddylate mammalian
p53 and its paralog
p73 to antagonize their transcriptional activity. However, it is not known whether the C. elegans
p53 ortholog, CEP-1, is regulated by neddylation and/or ubiquitin proteolysis pathways. To investigate this we inhibited components of the neddylation pathway by RNAi and quantified DNA damage-induced apoptosis in the germline. Animals depleted of
ned-8 had significantly higher levels of germ cell apoptosis in response to the DNA alkylating agent N-ethyl-N-nitrosourea (ENU) compared with controls. Similarly, inhibiting expression of the NED-8 E1 activating and E2 conjugating enzymes, encoded by the
ula-1/rfl-1 and
ubc-12 genes, also lead to increased levels of ENU-induced germline apoptosis. The increased levels of ENU-induced apoptosis were blocked by the
cep-1(
gk138) deletion allele, indicating that CEP-1 is negatively regulated through the neddylation pathway. The best characterized substrates for Nedd8 conjugation are proteins of the cullin family, which act as scaffolds for the assembly of multi-subunit E3 ubiquitin ligases. We inactivated the six cullin genes by RNAi and found that inhibition of only
cul-1 increased ENU-induced germ cell apoptosis. Since CUL-1 is a component of the Skp1/Cullin1/F-box (SCF) complex, these results suggested that DNA damage-induced germ cell apoptosis is negatively regulated by neddylation through an SCF E3 ligase. We next evaluated 21 Skp1-related (skr), and 520 predicted F-box genes, and found that inhibiting
skr-1 and 18 F-box genes by RNAi caused a significant increase in ENU-induced germ cell apoptosis. These results suggest that neddylation pathway regulates the apoptotic activity of CEP-1 in response to DNA damage through multiple SCF E3 ubiquitin ligases.