Proliferating cell nuclear antigen-dependent rapid recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged sites after UV irradiation in HeLa cells

The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.     

SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

The eukaryotic cell cycle is regulated by multiple ubiquitin-mediated events, such as the timely destruction of cyclins and replication licensing factors. The histone H4 methyltransferase SET8 (Pr-Set7) is required for chromosome compaction in mitosis and for maintenance of genome integrity. In this study, we show that SET8 is targeted for degradation during S phase by the CRL4(CDT2) ubiquitin ligase in a proliferating cell nuclear antigen (PCNA)-dependent manner. SET8 degradation requires a conserved degron responsible for its interaction with PCNA and recruitment to chromatin where ubiquitylation occurs. Efficient degradation of SET8 at the onset of S phase is required for the regulation of chromatin compaction status and cell cycle progression. Moreover, the turnover of SET8 is accelerated after ultraviolet irradiation dependent on the CRL4(CDT2) ubiquitin ligase and PCNA. Removal of SET8 supports the modulation of chromatin structure after DNA damage. These results demonstrate a novel regulatory mechanism, linking for the first time the ubiquitin-proteasome system with rapid degradation of a histone methyltransferase to control cell proliferation.     

A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1

Cul4 E3 ubiquitin ligases contain the cullin 4 scaffold and the triple beta propeller Ddb1 adaptor protein, but few substrate receptors have been identified. Here, we identify 18 Ddb1- and Cul4-associated factors (DCAFs), including 14 containing WD40 repeats. DCAFs interact with multiple surfaces on Ddb1, and the interaction of WD40-containing DCAFs with Ddb1 requires a conserved "WDXR" motif. DCAF2/Cdt2, which is related to S. pombe Cdt2, functions in Xenopus egg extracts and human cells to destroy the replication licensing protein Cdt1 in S phase and after DNA damage. Depletion of human Cdt2 causes rereplication and checkpoint activation. In Xenopus, Cdt2 is recruited to replication forks via Cdt1 and PCNA, where Cdt1 ubiquitylation occurs. These studies uncover diverse substrate receptors for Cul4 and identify Cdt2 as a conserved component of the Cul4-Ddb1 E3 that is essential to destroy Cdt1 and ensure proper cell cycle regulation of DNA replication.     

p21(WAF1) is associated with CDK2 and CDK4 protein during HL-60 cell differentiation by TPA treatment

TPA-treated HL-60 cells are mainly arrested in G1 by p21(WAF1) accumulation. We investigate the downstream changes following such accumulation. Increased p21(WAF1) is associated with CDK2 and CDK4. pRb is dephosphorylated in the presence of p21-CDK2/4 complexes, and the Rb-E2F1 complex increases after TPA treatment, whereas the Rb-HDAC1 complex decreases slightly. Our results suggest that increased p21(WAF1) is associated with CDK2/4, and that these complexes induce pRb dephosphorylation. In turn, hypophosphorylated pRb are mainly complexed with E2F1, but HDAC1 appears not to be a key component in this process.     

An evolutionarily conserved function of proliferating cell nuclear antigen for Cdt1 degradation by the Cul4-Ddb1 ubiquitin ligase in response to DNA damage

The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCF(Skp2), evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage.     

Two different replication factor C proteins, Ctf18 and RFC1, separately control PCNA-CRL4Cdt2-mediated Cdt1 proteolysis during S phase and following UV irradiation

Recent work identified the E3 ubiquitin ligase CRL4(Cdt2) as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4(Cdt2)-dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4(Cdt2) were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4(Cdt2) to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4(Cdt2)-dependent proteolysis of Cdt1 during DNA replication and repair.     

CDK inhibitor p57Kip2 is downregulated by Akt during HER2-mediated tumorigenicity

HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57^Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57^Kip2 regulation, and that Akt is a negative regulator of p57^Kip2. Ectopic expression of Akt can decrease the expression of p57^Kip2, while Akt inhibition leads to p57^Kip2 stabilization. Mechanistic studies show that Akt interacts with p57^Kip2 and causes cytoplasmic localization of p57^Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57^Kip2 were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57^Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.     

Degradation of Cdt1 during S phase is Skp2-independent and is required for efficient progression of mammalian cells through S phase

Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854-30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807-30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.     

Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2

The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) is emerging as an important cell cycle regulator that targets numerous proteins for destruction in S phase and after DNA damage, including Cdt1, p21, and Set8. CRL4(Cdt2) substrates contain a "PIP degron," which consists of a canonical proliferating cell nuclear antigen (PCNA) interaction motif (PIP box) and an adjacent basic amino acid. Substrates use their PIP box to form a binary complex with PCNA on chromatin and the basic residue to recruit CRL4(Cdt2) for substrate ubiquitylation. Using Xenopus egg extracts, we identify an acidic residue in PCNA that is essential to support destruction of all CRL4(Cdt2) substrates. This PCNA residue, which adjoins the basic amino acid of the bound PIP degron, is dispensable for substrate binding to PCNA but essential for CRL4(Cdt2) recruitment to chromatin. Our data show that the interaction of CRL4(Cdt2) with substrates requires molecular determinants not only in the substrate degron but also on PCNA. The results illustrate a potentially general mechanism by which E3 ligases can couple ubiquitylation to the formation of protein-protein interactions.     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double strand break repair, and stress-induced cell-cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27, and show that Cul4A-DDB1^DDB2-Artemis regulates G₁ phase cell cycle progression in mammalian cells.     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double-strand break repair and stress-induced cell cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27 and show that Cul4A-DDB1^DDB2-Artemis regulates G₁-phase cell cycle progression in mammalian cells.Key words: artemis, DDB2, p27, Cul4A-DDB1, ubiquitylation     

p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21^-/-, RPE p21^-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.     

Polymerase eta and p53 jointly regulate cell survival,apoptosis and Mre11 recombination during S phase checkpoint arrest after UV irradiation

Xeroderma pigmentosum variant (XPV) cells lack the damage-specific polymerase eta and undergo a protracted arrest at the S phase checkpoint(s) following UV damage. The S phase checkpoints encompass several qualitatively different processes, and stimulate downstream events that are dependent on the functional state of p53. Primary fibroblasts with wild-type p53 arrest in S, and require a functional polymerase eta (pol eta) to carry out bypass replication, but do not recruit recombination factors for recovery. XPV cells with non-functional p53, as a result of transformation by SV40 or HPV16 (E6/E7), recruit the hMre11/hRad50/Nbs1 complex to arrested replication forks, coincident with PCNA, whereas normal transformed cells preferentially use the pol eta bypass replication pathway. The formation of hMre11 foci implies that arrested replication forks rapidly undergo a collapse involving double strand breakage and rejoining. Apoptosis occurs after UV only in cells transformed by SV40, and not in normal or XPV fibroblasts or HPV16 (E6/E7) transformed cells. Conversely, ultimate cell survival in XPV cells was much less in HPV16 (E6/E7) transformed cells than in SV40 transformed cells, indicating that apoptosis was not a reliable predictor of cell survival. Inhibition of p53 transactivation by pifithrin-alpha or inhibition of protein synthesis by cycloheximide did not induce hMre11 foci or apoptosis in UV damaged fibroblasts. Inhibition of kinase activity with wortmannin did not increase killing by UV, unlike the large increase seen with caffeine. Since HPV16 (E6/E7) transformed XPV cells were highly UV sensitive and not further sensitized by caffeine, it appears likely that caffeine sensitization proceeds through a p53 pathway. The S phase checkpoints are therefore, a complex set of different checkpoints that are coordinated by p53 with the capacity to differentially modulate cell survival, apoptosis, bypass replication and hMre11 recombination.     

Inhibition of colon cancer cell proliferation by the dietary compound conjugated linoleic acid is mediated by the CDK inhibitor p21CIP1/WAF1

Our previous studies indicated that dietary conjugated linoleic acid (CLA) inhibits colon tumor cell proliferation in vitro and in vivo. To identify mechanisms by which CLA regulates growth arrest, the HT-29 human colon carcinoma cell line was treated with various physiological concentrations of CLA and analyzed by flow cytometry. We detected a dose-dependent increase in the percentage of cells arrested in G1 after CLA treatment that was accompanied by induction of the cyclin dependent kinase (CDK) inhibitor p21CIP1/WAF. CLA addition also led to increased p21 expression in HCT116 and SW480 cells, indicating that p21 induction is a general consequence of CLA treatment in colon cancer cells. Since both HT-29 and SW480 cells have mutant p53, our data indicate that p53 is not essential for induction of p21. In addition to an increase in p21 levels, HT-29 cell growth arrest was also accompanied by moderate decreases in Cyclin A, D1, E, and proliferating cell nuclear antigen (PCNA) levels. Following CLA treatment, p21 associated with and inhibited CDK4 and CDK2, and this correlated with reduced phosphorylation of retinoblastoma proteins. Increased association of p21 with PCNA was also detected. Dietary CLA inhibits cell cycle progression by inducing p21, which negatively regulates the growth promoting activities of CDK/cyclins and PCNA. These studies indicate that physiological concentrations of CLA inhibit growth of colon cancer cells with either wild-type or mutant p53, and may have therapeutic benefits in vivo.     

Selective ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G ubiquitin-conjugating enzymes via the CRL4Cdt2 ubiquitin ligase complex

CRL4(Cdt2) is a cullin-based E3 ubiquitin ligase that promotes the ubiquitin-dependent proteolysis of various substrates implicated in the control of cell cycle and various DNA metabolic processes such as DNA replication and repair. Substrates for CRL4(Cdt2) E3 ubiquitin ligase include the replication licensing factor Cdt1 and the cyclin-dependent kinase (Cdk) inhibitor p21. Inhibition of this E3 ligase leads to serious abnormalities of the cell cycle and cell death. The ubiquitin-conjugating enzyme (UBC) involved in this important pathway, however, remains unknown. By a proteomic analysis of Cdt2-associated proteins and an RNA interference-based screening approach, we show that CRL4(Cdt2) utilizes two different UBCs to target different substrates. UBCH8, a member of the UBE2E family of UBCs, ubiquitylates and promotes the degradation of p21, both during the normal cell cycle and in UV-irradiated cells. Importantly, depletion of UBCH8 by small interfering RNA (siRNA) increases p21 protein level, delays entry into S phase of the cell cycle, and suppresses the DNA damage response after UV irradiation. On the other hand, members of the UBE2G family of UBCs (UBE2G1 and UBE2G2) cooperate with CRL4(Cdt2) to polyubiquitylate and degrade Cdt1 postradiation, an activity that is critical for preventing origin licensing in DNA-damaged cells. Finally, we show that UBCH8, but not UBE2G1 or UBE2G2, is required for CRL4(Cdt2)-mediated ubiquitylation and degradation of the histone H4 lysine 20 monomethyltransferase Set8, a previously identified CRL4(Cdt2) substrate, as well as for CRL4(Cdt2)-dependent monoubiquitylation of PCNA in unstressed cells. These findings identify the UBCs required for the activity of CRL4(Cdt2) on multiple substrates and demonstrate that different UBCs are involved in the selective ubiquitylation of different substrates by the same E3 complex.