L2DTL/CDT2 Interacts with the CUL4/DDB1 Complex and PCNA and Regulates CDT1 Proteolysis in Response to DNA Damage

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate cell cycle, DNA replication, and DNA damage response. To determine the composition of CUL4 ubiquitin E3 ligase complex, we used anti-CUL4 antibody affinity chromatography to isolate the proteins that associated with human CUL4 complexes and identified them by mass-spectrometry. A novel and conserved WD40 domain-containing protein, the human homologue of Drosophila lethal(2) denticleless protein (L2DTL), was found to associate with CUL4 and DDB1. L2DTL also interacts with replication licensing protein CDT1 in vivo. Loss of L2DTL in Drosophila S2 and human cells suppressed proteolysis of CDT1 in response to DNA damage. We further isolated the human L2DTL complexes by anti-L2DTL immuno-affinity chromatography from HeLa cells and found it associates with DDB1, components of the COP9-signalosome complex (CSN), and PCNA. We found that PCNA interacts with CDT1 and loss of PCNA suppressed CDT1 proteolysis after DNA damage. Our data also revealed that in vivo, inactivation of L2DTL causes the dissociation of DDB1 from the CUL4 complex. Our studies suggest that L2DTL and PCNA interact with CUL4/DDB1 complexes and are involved in CDT1 degradation after DNA damage.     

L2DTL/CDT2 and PCNA Interact with p53 and Regulate p53 Polyubiquitination and Protein Stability through MDM2 and CUL4A/DDB1 Complexes

The CUL4-ROC1 E3 ligase complex regulates genome stability, replication, and cell cycle progression. A novel WD40 domain-containing protein, L2DTL, and PCNA were identified as proteins associated with CUL4/DDB1 complexes. Inactivation of CUL4A, L2DTL, PCNA, DDB1, or ROC1 induced p53 stabilization and growth arrest. L2DTL, PCNA, and DDB1/CUL4A complexes were found to physically interact with p53 tumor suppressor and its regulator MDM2/HDM2. The isolated CUL4A complexes display potent and robust polyubiquitination activity towards p53 and this activity is dependent on L2DTL, PCNA, DDB1, ROC1, and MDM2/HDM2. We also found that the interaction between p53 and CUL4 complex is regulated by DNA damage. Our data further showed that MDM2/HDM2 is rapidly proteolyzed in response to UV irradiation and this process is regulated by CUL4/DDB1 and PCNA. Our studies demonstrate that PCNA, L2DTL, and the DDB1-CUL4A complex play critical and differential roles in regulating the protein stability of p53 and MDM2/HDM2 in unstressed and stressed cells.     

ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites.     

FBXO44-Mediated Degradation of RGS2 Protein Uniquely Depends on a Cullin 4B/DDB1 Complex

The ubiquitin-proteasome system for protein degradation plays a major role in regulating cell function and many signaling proteins are tightly controlled by this mechanism. Among these, Regulator of G Protein Signaling 2 (RGS2) is a target for rapid proteasomal degradation, however, the specific enzymes involved are not known. Using a genomic siRNA screening approach, we identified a novel E3 ligase complex containing cullin 4B (CUL4B), DNA damage binding protein 1 (DDB1) and F-box protein 44 (FBXO44) that mediates RGS2 protein degradation. While the more typical F-box partners CUL1 and Skp1 can bind FBXO44, that E3 ligase complex does not bind RGS2 and is not involved in RGS2 degradation. These observations define an unexpected DDB1/CUL4B-containing FBXO44 E3 ligase complex. Pharmacological targeting of this mechanism provides a novel therapeutic approach to hypertension, anxiety, and other diseases associated with RGS2 dysregulation.     

Cullin-4A·DNA damage-binding protein 1 E3 ligase complex targets tumor suppressor RASSF1A for degradation during mitosis

Tumor suppressor RASSF1A (RAS association domain family 1, isoform A) is known to play an important role in regulation of mitosis; however, little is known about how RASSF1A is regulated during the mitotic phase of the cell cycle. In the present study, we have identified Cullin-4A (CUL4A) as a novel E3 ligase for RASSF1A. Our results demonstrate that DNA damage-binding protein 1 (DDB1) functions as a substrate adaptor that directly interacts with RASSF1A and bridges RASSF1A to the CUL4A E3 ligase complex. Depletion of DDB1 also diminishes intracellular interactions between RASSF1A and CUL4A. Our results also show that RASSF1A interacts with DDB1 via a region containing amino acids 165-200, and deletion of this region abolishes RASSF1A and DDB1 interactions. We have found that CUL4A depletion results in increased levels of RASSF1A protein due to increased half-life; whereas overexpression of CUL4A and DDB1 markedly enhances RASSF1A protein ubiquitination resulting in reduced RASSF1A levels. We further show that CUL4A-mediated RASSF1A degradation occurs during mitosis, and depletion of CUL4A markedly reverses mitotic-phase-stimulated RASSF1A degradation. We also note that overexpression of CUL4A antagonizes the ability of RASSF1A to induce M-phase cell cycle arrest. Thus, our present study demonstrates that the CUL4A·DDB1 E3 complex is important for regulation of RASSF1A during mitosis, and it may contribute to inactivation of RASSF1A and promoting cell cycle progression.     

Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.     

Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.     

DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.     

Cullin 4B protein ubiquitin ligase targets peroxiredoxin III for degradation

Cullin 4B (CUL4B) is a scaffold protein that assembles cullin-RING ubiquitin ligase (E3) complexes. Recent studies have revealed that germ-line mutations in CUL4B can cause mental retardation, short stature, and many other abnormalities in humans. Identifying specific CUL4B substrates will help to better understand the physiological functions of CUL4B. Here, we report the identification of peroxiredoxin III (PrxIII) as a novel substrate of the CUL4B ubiquitin ligase complex. Two-dimensional gel electrophoresis coupled with mass spectrometry showed that PrxIII was among the proteins up-regulated in cells after RNAi-mediated CUL4B depletion. The impaired degradation of PrxIII observed in CUL4B knockdown cells was confirmed by Western blot. We further demonstrated that DDB1 and ROC1 in the DDB1-CUL4B-ROC1 complex are also indispensable for the proteolysis of PrxIII. In addition, the degradation of PrxIII is independent of CUL4A, a cullin family member closely related to CUL4B. In vitro and in vivo ubiquitination assays revealed that CUL4B promoted the polyubiquitination of PrxIII. Furthermore, we observed a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells, which was associated with increased resistance to hypoxia and H(2)O(2)-induced apoptosis. These findings are discussed with regard to the known function of PrxIII as a ROS scavenger and the high endogenous ROS levels required for neural stem cell proliferation. Together, our study has identified a specific target substrate of CUL4B ubiquitin ligase that may have significant implications for the pathogenesis observed in patients with mutations in CUL4B.     

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.     

ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.     

PUVA-induced apoptosis involves mitochondrial dysfunction caused by the opening of the permeability transition pore

Damaged DNA-binding activity comprises two major protein components, DDB1 and DDB2, which are implicated in the repair of ultraviolet (UV) radiation-induced DNA damage. The possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. The abundance of DDB2 in UV-resistant HeLa cell lines was increased compared with that in the parental UV-sensitive cells. Stable transfection of the resistant cells with DDB2 antisense cDNA resulted in marked depletion of DDB2 protein and restored cellular sensitivity to UV-induced apoptosis. Whereas the extent of UV-induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase-3 were reduced in the UV-resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. In contrast, overexpressing DDB2 in DDB2-depleted cells with recombinant adenovirus, which carries ddb2 cDNA, markedly inhibited the extent of UV-induced activation of DNA fragmentation factor, and caspase-3. Interestingly, a mutated form of DDB2, which is defective in interacting with DDB1 and binding to UV-damaged DNA, also markedly inhibited the activation of apoptosis executioners. These results indicate that DDB2 is a modulator of UV-induced apoptosis, and that UV resistance can be overcome by inhibition of DDB2. The findings also suggest that modulation of UV-induced apoptosis by DDB2 may be independent of DNA repair.     

Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells

Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin-proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells.     

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.