Core component of multiple cullin-RING-based E3 ubiquitin-protein ligase complexes which mediate the ubiquitination and subsequent proteasomal degradation of target proteins. The functional specificity of the E3 ubiquitin-protein ligase complex depends on the variable substrate recognition subunit. CUL4B may act within the complex as a scaffold protein, contributing to catalysis through positioning of the substrate and the ubiquitin-conjugating enzyme. Plays a role as part of the E3 ubiquitin-protein ligase complex in polyubiquitination of CDT1, histone H2A, histone H3 and histone H4 in response to radiation-induced DNA damage. Targeted to UV damaged chromatin by DDB2 and may be important for DNA repair and DNA replication. Required for ubiquitination of cyclin E, and consequently, normal G1 cell cycle progression. Regulates the mammalian target-of-rapamycin (mTOR) pathway involved in control of cell growth, size and metabolism. Specific CUL4B regulation of the mTORC1-mediated pathway is dependent upon 26S proteasome function and requires interaction between CUL4B and MLST8.
By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)-based ubiquitin ligase, DDB1-CUL4A(DDB2). We previously showed that DDB1-CUL4A(DDB2) ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A(DDB2) and DDB1-CUL4B(DDB2). To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B(DDB2) E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B(DDB2) is more efficient than DDB1-CUL4A(DDB2) in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B(DDB2) E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER.
Posttranslational histone modifications play important roles in transcription and other chromatin-based processes. Compared to acetylation, methylation, and phosphorylation, very little is known about the function of histone ubiquitylation. Here, we report the purification and functional characterization of a histone H3 and H4 ubiquitin ligase complex, CUL4-DDB-ROC1. We demonstrate that CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation occurs both in vitro and in vivo. Importantly, CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation is regulated by UV irradiation. Reduction of histone H3 and H4 ubiquitylation by knockdown of CUL4A impairs recruitment of the repair protein XPC to the damaged foci and inhibits the repair process. Biochemical studies indicate that CUL4-DDB-ROC1-mediated histone ubiquitylation weakens the interaction between histones and DNA and facilitates the recruitment of repair proteins to damaged DNA. Thus, our studies uncover CUL4-DDB-ROC1 as a histone ubiquitin ligase and demonstrate that histone H3 and H4 ubiquitylation participates in the cellular response to DNA damage.
Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.
The mammalian target-of-rapamycin (mTOR) signaling pathway serves as a major regulator of cell growth, cell size and metabolism. In vivo, mTOR exists in two complexes, both of which contain the catalytic subunit mTOR, the invariable subunit mLST8, and a complex specific subunit Raptor or Rictor, forming either the rapamycin-sensitive mTORC1 or rapamycin-insensitive mTORC2, respectively. The exact functions of Raptor or Rictor in these complexes are still unclear. Here we demonstrate that mTORC1-mediated signaling events require the function of the 26S proteasome. Inhibition of the 26S proteasome by MG132 leads to the rapid inhibition of phosphorylation of the mTORC1 substrates S6 kinase and 4E-BP1. We have further discovered that the WD40 repeat proteins Raptor and mLST8 bind the CUL4-DDB1 ubiquitin E3 ligase. Loss of CUL4B or DDB1 specifically blocks the phosphorylation of S6 kinase at threonine 389 and 4E-BP1 at serine 65 and threonines 37 and 46, while loss of CUL4B enhances the phosphorylation of AKT at serine 473. These phosphorylation effects are identical to those resulting from the inactivation of Raptor. Our data suggest that the CUL4-DDB1 ubiquitin ligase interacts with Raptor and regulates the mTORC1- mediated signaling pathway through ubiquitin-dependent proteolysis.
CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin alpha1, alpha3, and alpha5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore, the nuclear localization of CUL4B mediated by NLS is critical for its normal function in cell proliferation.
The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, we analyzed the effect of inactivation of CUL4 in both Drosophila and human cells. We found that loss of CUL4 in Drosophila cells causes G(1) cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo with CUL4 abolishes the G(1) cell cycle arrest. In human cells, inactivation of CUL4A induces CDK inhibitor p27(Kip1) stabilization and G(1) cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved. In addition, we found that the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. We provide evidence that human CUL4B, a paralogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. Our studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G(1) cell cycle progression in both Drosophila and human cells.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
The cullin-containing ubiquitin-protein isopeptide ligases (E3s) play an important role in regulating the abundance of key proteins involved in cellular processes such as cell cycle and cytokine signaling. They have multisubunit modular structures in which substrate recognition and the catalysis of ubiquitination are carried out by distinct polypeptides. In a search for proteins involved in regulation of cullin-containing E3 ubiquitin ligases we immunopurified CUL4B-containing complex from HeLa cells and identified TIP120A as an associated protein by mass spectrometry. Immunoprecipitation of cullins revealed that all cullins tested specifically interacted with TIP120A. Reciprocal immunoaffinity purification of TIP120A confirmed the stable interaction of TIP120A with cullin family proteins. TIP120A formed a complex with CUL1 and Rbx1, but interfered with the binding of Skp1 and F-box proteins to CUL1. TIP120A greatly reduced the ubiquitination of phosphorylated IkappaBalpha by SCF(beta-TrCP) ubiquitin ligase. These results suggest that TIP120A functions as a negative regulator of SCF E3 ubiquitin ligases and may modulate other cullin ligases in a similar fashion.
Evidence
2:
Inferred from Physical InteractionDFLAT
Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.
The progression of biochemical and morphological phases and events that occur in a cell during successive cell replication or nuclear replication events. Canonically, the cell cycle comprises the replication and segregation of genetic material followed by the division of the cell, but in endocycles or syncytial cells nuclear replication or nuclear division may not be followed by cell division.
The gene cul-1 (formerly lin-19) is a negative regulator of the cell cycle in C. elegans. Null mutations cause hyperplasia of all tissues. cul-1 is required for developmentally programmed transitions from the G1 phase of the cell cycle to the GO phase or the apoptotic pathway. Moreover, the mutant phenotype suggests that G1-to-S phase progression is accelerated, overriding mechanisms for mitotic arrest and producing abnormally small cells. Significantly, diverse aspects of cell fate and differentiation are unaffected in cul-1 mutants. cul-1 represents a conserved family of genes, designated cullins, with at least five members in nematodes, six in humans, and three in budding yeast.
The process of restoring DNA after damage. Genomes are subject to damage by chemical and physical agents in the environment (e.g. UV and ionizing radiations, chemical mutagens, fungal and bacterial toxins, etc.) and by free radicals or alkylating agents endogenously generated in metabolism. DNA is also damaged because of errors during its replication. A variety of different DNA repair pathways have been reported that include direct reversal, base excision repair, nucleotide excision repair, photoreactivation, bypass, double-strand break repair pathway, and mismatch repair pathway.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways resulting in the breakdown of a protein by the destruction of the native, active configuration, with or without the hydrolysis of peptide bonds.
The chemical reactions and pathways resulting in the breakdown of a protein or peptide by hydrolysis of its peptide bonds, initiated by the covalent attachment of a ubiquitin group, or multiple ubiquitin groups, to the protein.
Protein involved in the complex series of events by which the cell duplicates its contents and divides into two. The eukaryotic cell cycle can be divided in four phases termed G1 (first gap period), S (synthesis, phase during which the DNA is replicated), G2 (second gap period) and M (mitosis). The prokaryotic cell cycle typically involves a period of growth followed by DNA replication, partition of chromosomes, formation of septum and division into two similar or identical daughter cells.
Protein induced by DNA damage or protein involved in the response to DNA damage. Drug- or radiation-induced injuries in DNA introduce deviations from its normal double-helical conformation. These changes include structural distortions which interfere with replication and transcription, as well as point mutations which disrupt base pairs and exert damaging effects on future generations through changes in DNA sequence. Response to DNA damage results in either repair or tolerance.
Protein involved in the repair of DNA, the various biochemical processes by which damaged DNA can be restored. DNA repair embraces, for instance, not only the direct reversal of some types of damage (such as the enzymatic photoreactivation of thymine dimers), but also multiple distinct mechanisms for excising damaged base; termed nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR); or mechanisms for repairing double-strand breaks.
Protein involved in ubiquitin-like modifier processing, activation, conjugation or deconjugation such as Ubl-activating enzymes (E1s), Ubl-conjugating enzymes (E2s), Ubl-protein ligases (E3s), some thiol proteases (Ubiquitin carboxyl-terminal hydrolases (UCH), Ubiquitin- specific processing proteases (UBP) and ubiquitin-like proteases) and the ubiquitin-like modifier proteins. Besides signaling proteolysis, ubiquitination for example can be a signal for trafficking, kinase activation and other nonproteolytic fates.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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