Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis

Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication.     

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     

The COP9 signalosome regulates Skp2 levels and proliferation of human cells

The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCF(Skp2), and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27(Kip1). CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27(Kip1). Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCF(Skp2) complex and therefore contributes to cell cycle regulation of human cells.     

Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.     

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.     

The COP9 signalosome inhibits p27(kip1) degradation and impedes G1-S phase progression via deneddylation of SCF Cul1

The COP9 signalosome (CSN) is a conserved protein complex with homologies to the lid subcomplex of the 26S proteasome. It promotes cleavage of the Nedd8 conjugate (deneddylation) from the cullin component of SCF ubiquitin ligases. We provide evidence that cullin neddylation and deneddylation is highly dynamic, that its equilibrium can be effectively modulated by CSN, and that neddylation allows Cul1 to form larger protein complexes. CSN2 integrates into the CSN complex via its C-terminal region and its N-terminal half region is necessary for direct interaction with Cul1. The polyclonal antibodies against CSN2 but not other CSN subunits cause accumulation of neddylated Cul1/Cul2 in HeLa cell extract, indicating that CSN2 is essential in cullin deneddylation. Further, CSN inhibits ubiquitination and degradation of the cyclin-dependent kinase inhibitor p27(kip1) in vitro. Microinjection of the CSN complex impeded the G1 cells from entering the S phase. Moreover, anti-CSN2 antibodies negate the CSN-dependent p27 stabilization and the G1/S blockage, suggesting that these functions require the deneddylation activity. We conclude that CSN inhibits SCF ubiquitin ligase activity in targeting p27 proteolysis and negatively regulates cell cycle at the G1 phase by promoting deneddylation of Cul1.     

CRL4-DDB1-VPRBP ubiquitin ligase mediates the stress triggered proteolysis of Mcm10

When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of Mcm10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that Mcm10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered Mcm10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates Mcm10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro, proving that Mcm10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets Mcm10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, Mcm10.     

CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation

Previous reports showed that chromatin-associated PCNA couples DNA replication with Cul4-DDB1(Cdt2)-dependent proteolysis of the licensing factor Cdt1. The CDK inhibitor p21, another PCNA-binding protein, is also degraded both in S phase and after UV irradiation. Here we show that p21 is degraded by the same ubiquitin-proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1(Cdt2) were silenced or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co-immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1(Cdt2) complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G(2) phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1(Cdt2). Our findings provide a new aspect of proteolytic control of p21 during the cell cycle.     

Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway.

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the G(0)-G(1) transition of the cell cycle has now been shown to proceed normally in Skp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G(0)-G(1) transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27(Kip1) accumulation during S-G(2) phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of p27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2(-/-) cells, even with a threonine 187 --> alanine mutant of p27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G(0) to G(1).     

F-box/WD-repeat proteins pop1p and Sud1p/Pop2p form complexes that bind and direct the proteolysis of cdc18p

Ubiquitin-dependent proteolysis plays an important role in cell-cycle control [1] [2]. In budding yeast, the protein Skp1p, the cullin-family member Cdc53p, and the F-box/WD-repeat protein Cdc4p form the SCFCdc4p ubiquitin ligase complex, which targets the cyclin-dependent kinase (Cdk) inhibitor Sic1p for proteolysis [3] [4] [5] [6] [7] [8]. Sic1p is recruited to the SCFCdc4p complex by binding to the WD-repeat region of Cdc4p [5] [6], while Skp1p binds to the F-box of Cdc4p [9]. In fission yeast, two distinct Cdc4p-related proteins, Pop1p/Ste16p [10] [11] and the recently identified Sud1p/Pop2p [12], regulate the stability of the replication initiator Cdc18p and the Cdk inhibitor Rum1p. We show here that, despite their structural and functional similarities, the pop1 and pop2 genes fail to complement each other's deletion phenotypes, indicating that they perform non-redundant, but potentially interdependent, functions in proteolysis. Consistent with this hypothesis, Pop1p and Pop2p formed heterooligomeric complexes when overexpressed, and binding of Cdc18p to Pop2p was dependent on Pop1p. The Pop1p-Pop2p interaction was mediated by the amino-terminal domain of Pop2p which, when fused to full-length Pop1p, rescued the phenotype of a Deltapop1Deltapop2 double mutant. Thus, close physical proximity of two distinct F-box/WD-repeat proteins directs proteolysis mediated by the SCFPop ubiquitin ligase complex.     

Regulating the stability and localization of CDK inhibitor p27Kip1 via CSN6-COP1 axis

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27^Kip1 stability, and that COP1 is a negative regulator of p27^Kip1. Ectopic expression of CSN6 can decrease the expression of p27^Kip1, while CSN6 knockdown leads to p27^Kip1 stabilization. Mechanistic studies show that CSN6 interacts with p27^Kip1 and facilitates ubiquitin-mediated degradation of p27^Kip1. CSN6-mediated p27 degradation depends on the nuclear export of p27^Kip1, which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.     

Isolation and characterization of a novel F-box protein Pof10 in fission yeast

The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1. Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats. Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast. Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1. Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm. These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast.     

Multiple functions of Jab1 are required for early embryonic development and growth potential in mice

Jab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways.     

CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6

Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57^Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57^Kip2. CSN6 associates with p57^Kip2, and its overexpression can decrease the steady-state expression of p57^Kip2; accordingly, CSN6 deficiency leads to p57^Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57^Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57^Kip2. Loss of Skp2 compromised CSN6-mediated p57^Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57^Kip2. CSN6’s negative impact on p57^Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57^Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57^Kip2 and result in promoted cancer cell growth.     

CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6

Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57^Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57^Kip2. CSN6 associates with p57^Kip2, and its overexpression can decrease the steady-state expression of p57^Kip2; accordingly, CSN6 deficiency leads to p57^Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57^Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57^Kip2. Loss of Skp2 compromised CSN6-mediated p57^Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57^Kip2. CSN6’s negative impact on p57^Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57^Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57^Kip2 and result in promoted cancer cell growth.     

DDB1 maintains genome integrity through regulation of Cdt1

DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.     

Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export

Phosphorylation of the cyclin-dependent kinase inhibitor p27(Kip1) has been thought to regulate its stability. Ser(10) is the major phosphorylation site of p27(Kip1), and phosphorylation of this residue affects protein stability. Phosphorylation of p27(Kip1) on Ser(10) has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27(Kip1) protein was translocated from the nucleus to the cytoplasm at the G(0)-G(1) transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27(Kip1) at the G(0)-G(1) transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) export, whereas substitution of Ser(10) with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27(Kip1) on Ser(10) is required for its binding to CRM1 and for its subsequent nuclear export.