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.     

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.     

Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or near the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4^DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4^DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4^DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation.Key words: Merlin, NF2, E3 ubiquitin ligase, CRL4, DCAF1, FERM domain protein     

Essential role of E3 ubiquitin ligase activity in Cbl-b-regulated T cell functions

E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or autoimmunity.     

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.     

Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or close to the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4^DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4^DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4^DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation.     

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1^β-TrCP and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase δ, is critical for inhibiting fork progression. CRL4^Cdt2 is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4^Cdt2 and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression.     

The Cullin-RING E3 ubiquitin ligase CRL4-DCAF1 complex dimerizes via a short helical region in DCAF1

The cullin4A-RING E3 ubiquitin ligase (CRL4) is a multisubunit protein complex, comprising cullin4A (CUL4), RING H2 finger protein (RBX1), and DNA damage-binding protein 1 (DDB1). Proteins that recruit specific targets to CRL4 for ubiquitination (ubiquitylation) bind the DDB1 adaptor protein via WD40 domains. Such CRL4 substrate recognition modules are DDB1- and CUL4-associated factors (DCAFs). Here we show that, for DCAF1, oligomerization of the protein and the CRL4 complex occurs via a short helical region (residues 845-873) N-terminal to DACF1's own WD40 domain. This sequence was previously designated as a LIS1 homology (LisH) motif. The oligomerization helix contains a stretch of four Leu residues, which appear to be essential for α-helical structure and oligomerization. In vitro reconstituted CRL4-DCAF1 complexes (CRL4(DCAF1)) form symmetric dimers as visualized by electron microscopy (EM), and dimeric CRL4(DCAF1) is a better E3 ligase for in vitro ubiquitination of the UNG2 substrate compared to a monomeric complex.     

UHRF2, another E3 ubiquitin ligase for p53

UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.     

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.     

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.     

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 δ isoform (PP1cδ) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans. These findings suggest a novel mechanism whereby the ability of MP to modulate myosin light chain might be regulated by the degradation of its targeting subunit MYPT1 through the SIAH2-ubiquitin-proteasomal pathway. In this manner, the turnover of MYPT1 would serve to limit the duration and/or magnitude of MP activity required to achieve a desired physiological effect.     

Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase

The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.     

Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase

Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCFCdc4 ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 A X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.     

The CUL7 E3 ubiquitin ligase targets insulin receptor substrate 1 for ubiquitin-dependent degradation

Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.