Structure, function and mechanism of the anaphase promoting complex (APC/C)

The complex molecular events responsible for coordinating chromosome replication and segregation with cell division and growth are collectively known as the cell cycle. Progression through the cell cycle is orchestrated by the interplay between controlled protein synthesis and degradation and protein phosphorylation. Protein degradation is primarily regulated through the ubiquitin proteasome system, mediated by two related E3 protein ubiquitin ligases, the Skp1 cullin F-box (SCF) and the anaphase promoting complex (also known as the cyclosome) (APC/C). The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that regulates progression through the mitotic phase of the cell cycle and controls entry into S phase by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D-box and KEN-box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. The aim of this article is to discuss the APC/C from a structural and mechanistic perspective. Although an atomic structure of the APC/C is still lacking, a combination of genetic, biochemical, electron microscopy studies of intact APC/C and crystallographic analysis of individual subunits, together with analogies to evolutionarily related E3 ligases of the RING family, has provided deep insights into the molecular mechanisms of catalysis and substrate recognition, and structural organisation of the APC/C.     

ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity

We have identified two highly conserved RING finger proteins, ROC1 and ROC2, that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolished their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins proteins potentially constitute a wide variety of ubiquitin ligases.     

Activation of UBC5 ubiquitin-conjugating enzyme by the RING finger of ROC1 and assembly of active ubiquitin ligases by all cullins

Protein ubiquitination plays an important role in regulating the abundance and conformation of a broad range of eukaryotic proteins. This process involves a cascade of enzymes including ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). E1 and E2 represent two families of structurally related proteins and are relatively well characterized. In contrast, the nature and mechanism of E3, proposed to contain activities in catalyzing isopeptide bond formation (ubiquitin ligation) and substrate targeting, remains inadequately understood. Two major families of E3 ubiquitin ligases, the HECT (for homologous to E6-AP C terminus) family and the RING family, have been identified that utilize distinct mechanisms in promoting isopeptide bond formation. Here, we showed that purified RING finger domain of ROC1, an essential subunit of SKP1-cullin/CDC53-F box protein ubiquitin ligases, was sufficient to activate UBCH5c to synthesize polyubiquitin chains. The sequence flanking the RING finger in ROC1 did not contribute to UBCH5c activation, but was required for binding with CUL1. We demonstrated that all cullins, through their binding with ROC proteins, constituted active ubiquitin ligases, suggesting the existence in vivo of a large number of cullin-RING ubiquitin ligases. These results are consistent with the notion that the RING finger domains allosterically activate E2. We suggest that RING-E2, rather than cullin-RING, constitutes the catalytic core of the ubiquitin ligase and that one major function of the cullin subunit is to assemble the RING-E2 catalytic core and substrates together.     

Proteolysis and the cell cycle: with this RING I do thee destroy

The ubiquitin system drives the cell division cycle by the timely destruction of numerous regulatory proteins. Remarkably, the two main activities that catalyze substrate ubiquitination in the cell cycle, the Skp1-Cdc53/cullin-F-box protein (SCF) complexes and the anaphase-promoting complex/cyclosome (APC/C), define a new superfamily of E3 ubiquitin ligases, all based on related cullin and RING-H2 finger protein subunits. The circuits that interconnect the SCF, APC/C and cyclin-dependent kinase activities form a master oscillator that coordinates the replication and segregation of the genome.     

The ubiquitin proteasome system — Implications for cell cycle control and the targeted treatment of cancer

Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin–proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (anaphase promoting complex or cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/C^Cdc20) and establishes a stable G1 phase (APC/C^Cdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Degradation of Postsynaptic Scaffold GKAP and Regulation of Dendritic Spine Morphology by the TRIM3 Ubiquitin Ligase in Rat Hippocampal Neurons

Changes in neuronal activity modify the structure of dendritic spines and alter the function and protein composition of synapses. Regulated degradation of postsynaptic density (PSD) proteins by the ubiquitin-proteasome system is believed to play an important role in activity-dependent synaptic remodeling. Stimulating neuronal activity in vitro and in vivo induces the ubiquitination and degradation of GKAP/SAPAP and Shank, major scaffold proteins of the PSD. However, the specific ubiquitin ligases that regulate postsynaptic protein composition have not been identified. Here we identify the RING finger-containing protein TRIM3 as a specific E3 ubiquitin ligase for the PSD scaffold GKAP/SAPAP1. Present in PSD fractions from rat brain, TRIM3 stimulates ubiquitination and proteasome-dependent degradation of GKAP, and induces the loss of GKAP and associated scaffold Shank1 from postsynaptic sites. Suppression of endogenous TRIM3 by RNA interference (RNAi) results in increased accumulation of GKAP and Shank1 at synapses, as well as enlargement of dendritic spine heads. RNAi of TRIM3 also prevented the loss of GKAP induced by synaptic activity. Thus, TRIM3 is a novel E3 ligase that mediates activity-dependent turnover of PSD scaffold proteins and is a negative regulator of dendritic spine morphology.     

The APC11 RING-H2 finger mediates E2-dependent ubiquitination

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.     

Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC's RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC(Cdc20) and APC(Cdh1) complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.     

一个新的与泛素化有关的蛋白家族——TRIM家族

TRIM家族是一个结构保守、进化快速的蛋白家族,它参与了细胞凋亡、周期调控、细胞对病毒的应答等重要的生命过程。结构上的保守预示着TRIM家族可能是以一种共同的机制参与各种生命过程的。最近的一些研究显示TRIM家族可能是一类新的RING指泛素连接酶。     

Herpes simplex virus type 1 immediate-early protein ICP0 and is isolated RING finger domain act as ubiquitin E3 ligases in vitro.

Proteasome-dependent degradation of ubiquitinated proteins plays a key role in many important cellular processes. Ubiquitination requires the E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and frequently a substrate-specific ubiquitin protein ligase (E3). One class of E3 ubiquitin ligases has been shown to contain a common zinc-binding RING finger motif. We have previously shown that herpes simplex virus type 1 ICP0, itself a RING finger protein, induces the proteasome-dependent degradation of several cellular proteins and induces the accumulation of colocalizing conjugated ubiquitin in vivo. We now report that both full-length ICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains in vitro in the presence of E1 and the E2 enzymes UbcH5a and UbcH6. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in ICP0 activity in other assays. We conclude that ICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and to target specific cellular proteins for destruction by the 26S proteasome.     

The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets key cell cycle regulatory proteins for degradation. Blockade of APC activity causes mitotic arrest. Recent evidence suggests that the APC may have roles outside the cell cycle. Several studies indicate that ubiquitin plays an important role in regulating synaptic strength. We previously showed that ubiquitin is directly conjugated to GLR-1, a C. elegans non-NMDA (N-methyl-D-aspartate) class glutamate receptor (GluR), resulting in its removal from synapses. By contrast, endocytosis of rodent AMPA GluRs is apparently regulated by ubiquitination of associated scaffolding proteins. Relatively little is known about the E3 ligases that mediate these effects. We examined the effects of perturbing APC function on postmitotic neurons in the nematode C. elegans. Temperature-sensitive mutations in APC subunits increased the abundance of GLR-1 in the ventral nerve cord. Mutations that block clathrin-mediated endocytosis blocked the effects of the APC mutations, suggesting that the APC regulates some aspect of GLR-1 recycling. Overexpression of ubiquitin decreased the density of GLR-1-containing synapses, and APC mutations blunted this effect. APC mutants had locomotion defects consistent with increased synaptic strength. This study defines a novel function for the APC in postmitotic neurons.     

Cloning and identification of a novel RNF6 transcriptional splice variant Spg2 in human development

RING finger proteins are zinc finger proteins containing the RING motifs. They act mainly as E3 ubiq-uitin ligases, bind the ubiquitin E2 conjugating enzyme and promote degradation of targeted proteins, Many novel genes have been isolated and differentially expressed in human adult and embryo testis by a testis cDNA-array differential display technique. A novel RING finger cDNA is highly expressed in adult testis and at low level in fetal testis. It was named Spg2. It contains a 2055 nucleotide ORF, en-codes a 685-amino-acid RNF6 protein, and has a RING finger in its C terminal. NCBI Blast shows that the gene is located on chromosome 13 and contains five exons. A multiple tissue expression profile also indicates that it is highly expressed in human testis, so we speculate that it may be associated with human spermatogenesis by virtue of the action of its RING domain.     

The plant homeodomain fingers of fission yeast Msc1 exhibit E3 ubiquitin ligase activity

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage. A screen for suppressors of a fission yeast chk1 mutant defective in the checkpoint pathway identified a novel Schizosaccharomyces pombe protein, Msc1. Msc1 contains 3 plant homeodomain (PHD) finger motifs, characteristically defined by a C4HC3 consensus similar to RING finger domains. PHD finger domains in viral proteins and in the cellular protein kinase MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1) have been implicated as ubiquitin E3 protein ligases that affect protein stability. The close structural relationship of PHD fingers to RING fingers suggests that other PHD domain-containing proteins might share this activity. We show that each of the three PHD fingers of Msc1 can act as ubiquitin E3 ligases, reporting for the first time that PHD fingers from a nuclear protein exhibit E3 ubiquitin ligase activity. The function of the PHD fingers of Msc1 is needed to rescue the DNA damage sensitivity of a chk1Delta strain. Msc1 co-precipitates Rhp6, the S. pombe homologue of the human ubiquitin-conjugating enzyme Ubc2. Strikingly, deletion of msc1 confers complete suppression of the slow growth phenotype, UV and hydroxyurea sensitivities of an rhp6 deletion strain and restores deficient histone H3 methylation observed in the rhp6Delta mutant. We speculate that the target of the E3 ubiquitin ligase activity of Msc1 is likely to be a chromatin-associated protein.     

蛋白质泛素化系统

泛素化是单个或多个泛素在泛素激活酶,泛素结合酶及泛素蛋白质连接酶的作用下共价修饰底物蛋白质的过程,近年来的研究发现,许多含环指的蛋白质本身是蛋白质泛素连接酶,或是多亚基连接酶中的重要成分。由于细胞内可表达200以上的环指蛋白,并且多亚基连接酶可利用同一环指蛋白但不同的底物识别蛋白。这些研究极大地丰富了对泛素化系统酶的认识,也使进一步调节和干预连接酶与底物的相互作用成为可能,新近的研究还发现,泛素化不仅可导致蛋白质的降解,还可直接影响蛋白质的活性和细胞内定位,是调节细胞内蛋白质功能和水平的主要机制之一。     

Identification of novel human Cdt1-binding proteins by a proteomics approach: proteolytic regulation by APC/CCdh1

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIalpha, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/C(Cdh1), SNF2H, topoisomerase I and IIalpha, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/C(Cdh1) indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCF(Skp2) and cullin4-based ubiquitin ligases, APC/C(Cdh1) is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.     

The F box protein AFR is a positive regulator of phytochrome A-mediated light signaling

Light is an important environmental cue to plants, and much of their physiology is influenced by light. The light signals that drive these responses are perceived by photoreceptors including the red/far-red responsive phytochromes (phyA-E). In addition to direct effects, light also exerts its influence by modifying the rhythms generated by the circadian clock. In Arabidopsis thaliana, the molecular makeup of the interface between the central clock and its input/output pathways is not fully defined, but a major point of control is likely to be protein turnover mediated by the ubiquitin/26S proteasome system. To identify additional constituents of this interface, stable double-stranded RNA interference (RNAi) was used to reduce mRNA levels of rhythmically expressed candidate genes encoding putative components of E3 ubiquitin ligases (i.e., F box and RING finger proteins), followed by screening of the transgenic plants for circadian and light signaling defects. RNAi lines with diminished expression of the novel gene ATTENUATED FAR-RED RESPONSE (AFR) display phenotypes consistent with impaired phyA-mediated light signaling. Furthermore, AFR is a true SCF E3 ubiquitin ligase component. SCF(AFR) is expected to mediate the turnover of a repressor of phyA signaling, possibly to prepare the plant to receive light signals at dawn.     

Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated by two different ubiquitin E3 ligases

In animals and fungi, a group of proteins called the cyclin-dependent kinase inhibitors play a key role in cell cycle regulation. However, comparatively little is known about the role of these proteins in plant cell cycle regulation. To gain insight into the mechanisms by which the plant cell cycle is regulated, we studied the cyclin-dependent kinase inhibitor KRP1 in Arabidopsis. KRP1 interacts with the CDKA;1/CYCD2;1 complex in planta and functions in the G1–S transition of the cell cycle. Furthermore, we show that KRP1 is a likely target of the ubiquitin/proteasome pathway. Two different ubiquitin protein ligases, SCF^SKP2 and the RING protein RKP, contribute to its degradation. These results suggest that SCF^SKP2b and RPK play an important role in the cell cycle through regulating KRP1 protein turnover.     

Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.     

FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest

During cell proliferation, protein degradation is strictly regulated by the cell cycle and involves two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F-box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF ligases are constitutively active and generally target only proteins after they have been selected for degradation, usually by phosphorylation. In contrast, APC/C complexes are themselves activated by phosphorylation and their substrates contain a targeting signal known as degron, a consensus amino acid sequence such as a D-Box. SCF complexes degrade proteins during the G1 phase. However, as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. APC-2, a protein crucial to cell division, initiates anaphase by triggering the degradation of multiple proteins. This study explores an unexpected interaction between APC-2 and SCF^FBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Mutagenesis-based experiments show that this interaction requires a D-Box found within the FBG1 F-box domain. Unexpectedly, we demonstrate that co-expression with FBG1 increases total APC2 levels. However, free APC2 is decreased, inhibiting cell proliferation. Finally, FACS analysis of cell populations expressing different forms of FBG1 demonstrate that this ubiquitin ligase induces S-phase arrest, illustrating the functional consequences of the interaction described. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent from its function as ubiquitin ligase, providing the basis for future studies of FBG1 in aging and cancer.