Cooperation of HECT-domain Ubiquitin Ligase hHYD and DNA Topoisomerase II-binding Protein for DNA Damage Response

Abstract

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast twohybrid screen showed that DNA topoisomerase II␤-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with ␥-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.The ubiquitin-proteosome proteolytic system is involved in a variety of fundamental cell regulations including gene expression, stress response, DNA repair, and cell cycles (1-6). Ubiquitination includes a cascade of three classes of enzymes, the ubiquitin-activating enzyme E1, 1 the ubiquitin-conjugating enzyme Ubc or E2, and the ubiquitin ligase E3. After activation of a ubiquitin by E1, E2 and E3 cooperate to catalyze the formation of a multiubiquitin chain on a protein substrate. The E3 enzymes or the protein complexes with a ligase activity are believed to target the substrate for selective ubiquitination and a subsequent turnover by a large protease complex, the 26 S proteosome. There are two distinct groups of human ubiquitin ligases, the HECT domain E3 enzymes, including E6-associated protein (E6AP) and NEDD4, and the RING finger E3 enzymes, including SCF (Skp1-cullin-F box), VBC (VHL-Elongin B-Elongin C), APC (anaphase-promoting complex) and other single RING finger proteins such as c-Cbl and MDM2 (7). The HECT sequence of ϳ200 amino acids is highly conserved in the C-terminal catalytic domain of family members from yeast to mammals. The HECT-domain protein forms a thiol ester bond with a ubiquitin at the active cysteine residue and transfers the ubiquitin directly to the substrate (8 -10), whereas RING finger proteins form complexes containing an E2 enzyme, which facilitate ubiquitination of the substrate. However, the overall mechanisms by which these ubiquitin ligases recognize the target proteins remain to be elucidated.In the present study, we focused on characterizing the human HECT-domain protein, a counterpart of the regulator of cell proliferation and the putative tumor suppressor hyperplastic discs in Drosophil...

The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro

“…For example, expression of ERCC1 and XPA, components of the nucleotide excision repair pathways, are associated with platinum resistance in ovarian cancer (Damia et al, 1998;Selvakumaran et al, 2003); methylation and silencing of the mismatch repair gene MLH1 in some ovarian cancers also result in loss of cisplatin sensitivity (Strathdee et al, 1999); and disruption of the FANC/ BRCA pathway by methylation of BRCA1 and FANCF alters sensitivity to cisplatin in ovarian cancer (Taniguchi et al, 2003). As one function of EDD is in the cellular response to DNA damage (Henderson et al, 2002(Henderson et al, , 2006Honda et al, 2002;Munoz et al, 2007), we postulated that the ability of EDD to predict disease recurrence in serous ovarian cancer might similarly be related to the emergence of chemoresistance. The survival curves for those patients who initially responded to treatment were consistent with this hypothesis: all of the patients had a similar survival rate until approximately 12 months (disease recurrence) before diverging into two groups based on EDD expression with clearly different outcomes, suggestive of the emergence of chemoresistant cancer cells.…”

“…To date, there is no evidence that these proteins are ubiquitinylation substrates of EDD. Others have identified topoisomerase IIb-binding protein, required for cell survival after DNA damage (Yamane et al, 2002) and associated with an increased risk of familial breast and ovarian cancer (Karppinen et al, 2006), as a ubiquitinylation target of EDD (Honda et al, 2002).…”

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer

“…This property suggests that the EDD gene might play a role in human cancer. Our interest in this possibility was strengthened by recent evidence that EDD interacts with one or more proteins in the DNA damage response (Henderson et al, 2002;Honda et al, 2002). As an initial approach to evaluate EDD's involvement in cancer, we used microsatellite allelotyping to examine a narrow region of chromosome 8q that encompasses the locus for EDD (8q22.3) and identified this as a significant and specific area of allelic imbalance in ovarian cancer, hepatocellular carcinoma, squamous cell carcinoma of the tongue, breast cancer and metastatic melanoma.…”

“…This possibility is reinforced by an implied role for EDD in DNA damage signalling. A recent study found that topoisomerase IIb binding protein (TopBP1) is ubiquitinylated and targeted for proteosomal degradation by EDD (Honda et al, 2002). TopBP1 colocalizes with BRCA1 at stalled replication forks, evidence that TopBP1 functions in the DNA damage response (Makiniemi et al, 2001).…”

A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis

“…The mammalian orthologue E3 identified by Differential Display (EDD) was first discovered through the use of differential display to identify progestin-regulated genes in a breast cancer cell line (23). Evidence suggesting an involvement of EDD1 in human cancer includes the finding from several studies demonstrating that the degree by which EDD1 interacts with CIB, TOPBP1, and CHK2 is regulated by the DNA damage response (22,24,25). Moreover, EDD1 expression has been shown to functionally correlate with the responsiveness of patients with ovarian cancer to DNA-damaging cytotoxic therapies (26).…”