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.     

Identification of She3 as an SCFGrr1 Substrate in Budding Yeast

The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF) complex. Acting in concert with the substrate-binding F-box protein Grr1, SCF^Grr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth.     

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Plant casein kinases coordinate cell cycle by regulating the stability of a cyclin-dependent kinase inhibitor through promoting interaction with E3 ubiquitin ligases and proteasomal degradation by phosphorylation.     

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis anddegradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainlycontrolled by the ubiquitineproteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26Sproteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsiblefor the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such ascancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promotingrole of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimategoal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore,altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.     

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 ligase SCF(betaTrCP) regulates the degradation of the growth hormone receptor

SCF ubiquitin ligases play a pivotal role in the regulation of cell division and various signal transduction pathways, which in turn are involved in cell growth, survival, and transformation. SCF(TrCP) recognizes the double phosphorylated DSGPhiXS destruction motif in beta-catenin and IkappaB. We show that the same ligase drives endocytosis and degradation of the growth hormone receptor (GHR) in a ligand-independent fashion. The F-box protein beta-TrCP binds directly and specifically with its WD40 domain to a novel recognition motif, previously designated as the ubiquitin-dependent endocytosis motif. Receptor degradation requires an active neddylation system, implicating ubiquitin ligase activity. GHR-TrCP binding, but not GHR ubiquitination, is necessary for endocytosis. TrCP2 silencing is more effective on GHR degradation and endocytosis than TrCP1, although overexpression of either isoform restores TrCP function in silenced cells. Together, these findings provide direct evidence for a key role of the SCF(TrCP) in the endocytosis and degradation of an important factor in growth, immunity, and life span regulation.     

Control models of cell cycle transit, exit, and arrest

Several distinct cycles mediate the events which occur between one cell division and the next. In micro-organisms there are generally two cycles. One governs biomass growth, the other DNA synthesis and cell division. In higher eukaryotes there can be as many as four distinct cycles, with growth, DNA synthesis, cell division, and nuclear division each possessing its own functional sequence of events. These cycles are controlled and coordinated by several different regulatory mechanisms. Restriction points are specific steps in the cycle whose completion is governed by external regulatory agents. One set of restriction points requires nutrients and growth hormones for step completion. Another set serves as receptors for differentiating factors which cause cycle arrest and initiate cellular differentiation. There is currently a debate as to whether restriction point inhibition involves permanent arrest or temporary arrest with a stochastic arrested-state residence time controlled by a transition probability mechanism. Tissue sizing is a process of negative feedback inhibition mediated by intercellular communication via cell surface contact and the extracellular matrix. Sizers commonly operate throughout broad portions of the cycle and appear to cause a slowing of cycle transit velocity rather than arrest. Sizers are probably the major regulatory mechanism for cell growth under conditions of nutrient and growth factor excess. They also generate compensatory proliferation following wounding or cell death. A growing body of evidence suggests that both the transit velocity, with which cells move through their several cycles, and the coordination of the cycles are controlled by intracellular regulatory mechanisms which behave as biological oscillators. These oscillators trigger complex sequences of events such as DNA synthesis and cell division.     

NIPA defines an SCF-type mammalian E3 ligase that regulates mitotic entry

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.     

Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors

Members of the F-box protein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by Skp1, cullin, and one of many Fbps) act as protein-ubiquitin ligases that control the G(1)/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias beta-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25-->q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding beta-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.     

Plant CULLIN-based E3s: phytohormones come first

CULLIN (CUL)-dependent ubiquitin ligases form a class of structurally related multi-subunit enzymes that control the rapid and selective degradation of important regulatory proteins involved in cell cycle progression and development, among others. Several classes of these E3s are also conserved in plants and genetic analyses, using Arabidopsis thaliana, indicate that they play an important function during plant development and responses to the environment. In this review, we will discuss the molecular composition and function of these enzymes in plants with a major emphasis on phytohormone signal transduction pathways.     

SCF(Skp1-Cul1-F-box蛋白)复合物及其在细胞周期中的作用

泛素 -蛋白酶体降解途径在多种蛋白浓度的调节中发挥重要作用。泛素连接酶家族的一个成员SCF复合物 (Skp1- Cul1- F -box蛋白 )通过降解多个细胞周期调节蛋白促使细胞进入增殖周期。本文综述了SCF复合物的组成和结构以及这一复合物对细胞周期调控因子的调节作用。     

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.     

Overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p inhibits ubiquitin ligase activities of their SCF complexes

Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Induction of cell senescence by targeting to Cullin-RING Ligases (CRLs) for effective cancer therapy

Cullin-RING ligases (CRLs) are the biggest family of multiunit ubiquitin E3 ligases, controlling many biological processes by promoting the degradation of a broad spectrum of proteins associated with cell cycle, signal transduction and cell growth. The dysfunction of CRLs causes a lot of diseases including cancer, which meanwhile offers us a promising approach to cancer therapy by targeting to CRLs. Recent studies have demonstrated that genetic or pharmaceutical inactivation of CRLs often leads to cancer cell death by activating multiple cell-killing pathways including senescence, an emerging anticancer mechanism of therapeutic agents. Here, we summarize the induction of cellular senescence and its mechanism of action, triggered by targeting to specific subunits of CRLs via multiple approaches including siRNA silencing, genetic knockout as well as small molecule inhibitor, exhibiting anticancer effect in vitro and in vivo.     

The Flp1/Clp1 phosphatase cooperates with HECT-type Pub1/2 protein-ubiquitin ligases in Schizosaccharomyces pombe

The Schizosaccharomyces pombe Flp1p serine-threonine phosphatase is required for the degradation of the mitotic inducer Cdc25p at the end of mitosis. Cdc25p degradation prevents Cdc2p-tyrosine 15 dephosphorylation and, thus, contributes to the timely inactivation of mitotic CDK-associated kinase activity. Both RING- and HECT-type protein-ubiquitin ligases are involved in Cdc25p destabilization. Flp1p function is required for Cdc25p ubiquitination via anaphase-promoting complex/cyclosome or APC/C (RING-type) and the absence of Pub1p (HECT-type) stabilizes the mitotic inducer. In the present report, we study the functional relationship of Flp1p with Pub1p and Pub2p HECT-type-protein ubiquitin ligases. We show that Flp1p is required for the rapid degradation of Cdc25p while Pub1p is responsible for the long-term destabilization of the mitotic inducer. Accordingly, flp1 and pub1 mutants have a strong genetic interaction, correlating defects in the coordination of mitosis and cytokinesis with the stabilization of hyperactive Cdc25p. However, we also show that Flp1 and Pub2p proteins functionally interact in vivo suggesting that both proteins belong to the same regulatory network in S. pombe cells. Thus Flp1p appears to have an important role in integrating HECT- and RING-type ubiquitin ligases in cell cycle control.     

Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner

Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle.     

Skp1-Cul1-F-box Ubiquitin Ligase (SCFβTrCP)-mediated Destruction of the Ubiquitin-specific Protease USP37 during G2-phase Promotes Mitotic Entry

Ubiquitin-mediated proteolysis is a key regulatory process in cell cycle progression. The Skp1-Cul1-F-box (SCF) and anaphase-promoting complex (APC) ubiquitin ligases target numerous components of the cell cycle machinery for destruction. Throughout the cell cycle, these ligases cooperate to maintain precise levels of key regulatory proteins, and indirectly, each other. Recently, we have identified the deubiquitinase USP37 as a regulator of the cell cycle. USP37 expression is cell cycle-regulated, being expressed in late G₁ and ubiquitinated by APC^Cdh1 in early G₁. Here we report that in addition to destruction at G₁, a major fraction of USP37 is degraded at the G₂/M transition, prior to APC substrates and similar to SCF^βTrCP substrates. Consistent with this hypothesis, USP37 interacts with components of the SCF in a βTrCP-dependent manner. Interaction with βTrCP and subsequent degradation is phosphorylation-dependent and is mediated by the Polo-like kinase (Plk1). USP37 is stabilized in G₂ by depletion of βTrCP as well as chemical or genetic manipulation of Plk1. Similarly, mutation of the phospho-sites abolishes βTrCP binding and renders USP37 resistant to Plk1 activity. Expression of this mutant hinders the G₂/M transition. Our data demonstrate that tight regulation of USP37 levels is required for proper cell cycle progression.     

Cell cycle: the key to plant growth control?

Experimental evidence on the role of the cell cycle in plant growth regulation does not exclusively fit the cellular (division drives growth) or the organismal perspective (division merely accompanies growth). Here we present a broader, integrated concept of plant growth regulatory interactions, which accommodates experimental results gathered to date. This model can serve as a basis for future research, and prompts experimental approaches to encompass both measurements of cell growth and division parameters.