Anaphase-promoting Complex Research Articles

Tissue-Specific Control of the Endocycle by the Anaphase Promoting Complex/Cyclosome Inhibitors UVI4 and DEL1.

The endocycle represents a modified mitotic cell cycle that in plants is often coupled to cell enlargement and differentiation. Endocycle onset is controlled by activity of the Anaphase Promoting Complex/Cyclosome (APC/C), a multisubunit E3 ubiquitin ligase targeting cell-cycle factors for destruction. CELL CYCLE SWITCH52 (CCS52) proteins represent rate-limiting activator subunits of the APC/C. In Arabidopsis (Arabidopsis thaliana), mutations in either CCS52A1 or CCS52A2 activators result in a delayed endocycle onset, whereas their overexpression triggers increased DNA ploidy levels. Here, the relative contribution of the APC/CCCS52A1 and APC/CCCS52A2 complexes to different developmental processes was studied through analysis of their negative regulators, being the ULTRAVIOLET-B-INSENSITIVE4 protein and the DP-E2F-Like1 transcriptional repressor, respectively. Our data illustrate cooperative activity of the APC/CCCS52A1 and APC/CCCS52A2 complexes during root and trichome development, but functional interdependency during leaf development. Furthermore, we found APC/CCCS52A1 activity to control CCS52A2 expression. We conclude that interdependency of CCS52A-controlled APC/C activity is controlled in a tissue-specific manner.

Abstract 3456: Centromere gene misregulation is a major source of chromosomal instability in human cancers and predicts patient response to specific therapies

Abstract Chromosomal instability (CIN) is a cancer hallmark that contributes to tumor heterogeneity and other malignant properties. Aberrant centromere function causes CIN through chromosome missegregation in experimental systems. CENP-A is a histone H3 variant found at centromeres. Centromere is regulated epigenetically by replenishing CENP-A chromatin during cell divisions. This process requires a dedicated CENP-A chaperone called HJURP and other factors. However, we are lack of a comprehensive understanding about whether these proteins are involved in clinical cancers, and how centromere misregulation may contribute to cancer development. Here, basing on our previous findings in Drosophila melanogaster, we identified a conserved mutual protection mechanism between CENP-A and HJURP between flies and human cells, where their physical interaction protects each other from being targeted for degradation. Moreover, we showed that HJURP stability is regulated both in vitro and in vivo by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin E3 ligase. We further demonstrated that CENP-A and APC/C compete for the same binding site on HJURP. Using isogenic breast cancer cell culture progression series, we found that many centromere proteins are progressively overexpressed at the protein level during tumorigenesis. These results led us to hypothesize that overexpression of centromere proteins may cause centromere misregulation and CIN, and contribute to human cancers. To test the idea, we developed a CES (Centromere and kinetochore gene Expression Score) signature that quantitates the mRNA levels of 14 key genes required for centromere structure in cancers. High tumor CES values strongly correlate with increased copy number alterations and mutation frequencies across many cancer types, and prognosticate poor patient survival for many cancers. High CES values also signify high levels of genomic instability that sensitize cancer cells to additional genotoxicity. Indeed, the CES signature forecasts patient response to adjuvant chemo- or radiotherapy for lung and breast cancers. In conclusion, we demonstrate regulation of HJURP by APC/C ubiquitin E3 ligase, mutual protection between the key centromere proteins CENP-A and HJURP, and a mode of misregulation of centromere protein genes in human cancers. We show the efficacy of the CES signature as a potential biomarker to identify patients who likely respond to specific treatments, thus spare the non-responding patients from less effective treatments. These findings are expected to help address the over-treatment problem prevalent in cancer treatments. Our approach validates the critical importance of incorporating basic knowledge of chromosome segregation pathways into cancer research and clinical applications. Note: This abstract was not presented at the meeting. Citation Format: Weiguo Zhang, Jian-Hua Mao, Wei Zhu, Anshu K. Jain, Ke Liu, James B. Brown, Gary H. Karpen. Centromere gene misregulation is a major source of chromosomal instability in human cancers and predicts patient response to specific therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3456. doi:10.1158/1538-7445.AM2017-3456

Comparison of Genome-Wide and Gene-Specific DNA Methylation Profiling in First-Trimester Chorionic Villi From Pregnancies Conceived With Infertility Treatments.

Assisted reproductive technologies are associated with altered methylation in term placenta. However, it is unclear whether methylation patterns are the result of fertility treatments or intrauterine environment. Thus, we set out to determine whether there are differences in the first-trimester placenta that may be altered by the underlying fertility treatments. Genome-wide DNA methylation analyses from chorionic villus sampling (CVS) from matched singleton pregnancies conceived using in vitro fertilization (IVF), non-IVF fertility treatment (NIFT), or those conceived spontaneously were performed using Illumina Infinium HumanMethylation450 BeadChip from 15 matched CVS samples. Nanofluidic quantitative polymerase chain reaction (qPCR) of differently methylated genes was performed in a confirmatory cohort of 23 IVF conceptions and 24 NIFT conceptions. Global methylation was similar among the IVF, NIFT, and spontaneous conceptions. However, differential methylation from IVF and NIFT pregnancies was present at 34 CpG sites, which was significantly different. Of those, 14 corresponded to known genes, with methylation changes detected at multiple loci in 3 genes, anaphase-promoting complex subunit 2 ( ANAPC2), C-X-C motif chemokine ligand 14 ( CXCL14), and regulating synaptic membrane exocytosis 1 ( RIMS1). Nanofluidic qPCR of differentially methylated genes identified pre T-cell antigen receptor alpha ( PTCRA) to be significantly downregulated in IVF versus NIFT conceptions. Although global methylation patterns are similar, there are differences in methylation of specific genes in IVF compared to NIFT conceptions, leading to altered gene expression. PTCRA was differentially methylated and downregulated in IVF conceptions, warranting further investigation. It remains to be determined whether these changes affect placentation and whether it is due to the more profound underlying infertility requiring IVF, yet these data provide unique insight into the first-trimester placental epigenome.

Fam64a is a novel cell cycle promoter of hypoxic fetal cardiomyocytes in mice

Fetal cardiomyocytes actively proliferate to form the primitive heart in utero in mammals, but they stop dividing shortly after birth. The identification of essential molecules maintaining this active cardiomyocyte proliferation is indispensable for potential adult heart regeneration. A recent study has shown that this proliferation depends on a low fetal oxygen condition before the onset of breathing at birth. We have established an isolation protocol for mouse fetal cardiomyocytes, performed under strict low oxygen conditions to mimic the intrauterine environment, that gives the highest proliferative activities thus far reported. Oxygen exposure during isolation/culture markedly inhibited cell division and repressed cell cycle-promoting genes, and subsequent genome-wide analysis identified Fam64a as a novel regulatory molecule. Fam64a was abundantly expressed in hypoxic fetal cardiomyocyte nuclei, but this expression was drastically repressed by oxygen exposure, and in postnatal cardiomyocytes following the onset of breathing and the resulting elevation of oxygen tension. Fam64a knockdown inhibited and its overexpression enhanced cardiomyocyte proliferation. Expression of a non-degradable Fam64a mutant suggested that optimum Fam64a expression and subsequent degradation by anaphase-promoting complex/cyclosome (APC/C) during the metaphase-to-anaphase transition are required for fetal cardiomyocyte division. We propose that Fam64a is a novel cell cycle promoter of hypoxic fetal cardiomyocytes in mice.

APC/C is essential for hematopoiesis and impaired in aplastic anemia.

Anaphase promoting complex/cyclosome (APC/C) is essential for cell cycle progression. Recently, its non-mitotic functions were also reported but less studied in several tissues including hematopoietic cells. Here, we developed an inducible Anapc2 (a core subunit of APC/C) knockout mice. The animals displayed a fatal bone marrow failure within 7 days after knockout induction. Their hematopoietic stem and progenitor cells (HSPCs) demonstrated a sharp decline and could form little colony. Further, the results of BrdU label-retaining cell assay showed that the dormant HPSCs lost rapidly. Analysis of cell cycle regulators, Skp2, P27, Cdk2, and Cyclin E1, suggested that these quiescent stem cells underwent a shift from quiescence to mitosis followed by apoptosis. We next detected Anapc2-expression in the CD34+ HSPCs of patients with aplastic anemia. CD34+ cells were markedly decreased in the bone marrow and Anapc2-expression in the residual CD34+ cells was undetectable, suggesting that APC/C was deficient and might have a relationship with the pathogenesis of aplastic anemia.

A Dynamical Model for Activating and Silencing the Mitotic Checkpoint

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism, exclusively sensitive to the states of kinetochores attached to microtubules. During metaphase, the anaphase-promoting complex/cyclosome (APC/C) is inhibited by the SAC but it rapidly switches to its active form following proper attachment of the final spindle. It had been thought that APC/C activity is an all-or-nothing response, but recent findings have demonstrated that it switches steadily. In this study, we develop a detailed mathematical model that considers all 92 human kinetochores and all major proteins involved in SAC activation and silencing. We perform deterministic and spatially-stochastic simulations and find that certain spatial properties do not play significant roles. Furthermore, we show that our model is consistent with in-vitro mutation experiments of crucial proteins as well as the recently-suggested rheostat switch behavior, measured by Securin or CyclinB concentration. Considering an autocatalytic feedback loop leads to an all-or-nothing toggle switch in the underlying core components, while the output signal of the SAC still behaves like a rheostat switch. The results of this study support the hypothesis that the SAC signal varies with increasing number of attached kinetochores, even though it might still contain toggle switches in some of its components.

Downregulation of Talin-1 expression associates with increased proliferation and migration of vascular smooth muscle cells in aortic dissection

BackgroundThis study aimed to assessed whether Talin-1 is involved in the pathogenesis of aortic dissection via regulating vascular smooth muscle cell (VSMC) biological function.MethodsHuman aortic samples were obtained from organ donors who died from nonvascular diseases as normal controls and from patients undergoing surgical repair of thoracic aortic dissection. The expression level and distribution of Talin-1 were detected using westernblot analysis and immunohistochemistry in each sample. We inhibited the expression of Talin-1 via RNA interference in VSMCs. VSMC proliferation was detected by Cell-counting Kit-8 analyses. Scratch test and flow cytometry were used to identify the migration and apoptosis ability. Antibody microarray analysis and qRT-PCR were used to detect some protein and mRNA changes which were induced by Talin-1 downregulation.ResultsTalin-1 was significantly downregulated in the media of aortic dissection samples compared with controls (P < 0.05). Talin-1 knockdown significantly induced VSMC proliferation and migration in vitro. Proteins which involved in cell cycle can be regulated by downregulating Talin-1. Down regulation of Talin-1 can significanly increased the expression of anaphase-promoting complex subunit 2 (APC2) and decreased p19 alternative reading frame (p19ARF), Cullin-3, and beta actin’s expression.ConclusionsTalin-1 induces VSMCs proliferation and migration. It downregulated in aortic dissection, which might play a potential role in the development of aortic dissection.

Precocious centriole disengagement and centrosome fragmentation induced by mitotic delay

The spindle assembly checkpoint (SAC) delays mitotic progression until all sister chromatid pairs achieve bi-orientation, and while the SAC can maintain mitotic arrest for extended periods, moderate delays in mitotic progression have significant effects on the resulting daughter cells. Here we show that when retinal-pigmented epithelial (RPE1) cells experience mitotic delay, there is a time-dependent increase in centrosome fragmentation and centriole disengagement. While most cells with disengaged centrioles maintain spindle bipolarity, clustering of disengaged centrioles requires the kinesin-14, HSET. Centrosome fragmentation and precocious centriole disengagement depend on separase and anaphase-promoting complex/cyclosome (APC/C) activity, which also triggers the acquisition of distal appendage markers on daughter centrioles and the loss of procentriolar markers. Together, these results suggest that moderate delays in mitotic progression trigger the initiation of centriole licensing through centriole disengagement, at which point the ability to maintain spindle bipolarity becomes a function of HSET-mediated spindle pole clustering.

Kinetochores accelerate or delay APC/C activation by directing Cdc20 to opposing fates.

Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. Here, we show that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1, which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates.

CDK4 protein is degraded by anaphase-promoting complex/cyclosome in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells

CDK4 regulates G1/S phase transition in the mammalian cell cycle by phosphorylating retinoblastoma family proteins. However, the mechanism underlying the regulation of CDK4 activity is not fully understood. Here, we show that CDK4 protein is degraded by anaphase-promoting complex/cyclosome (APC/C) during metaphase-anaphase transition in HeLa cells, whereas its main regulator, cyclin D1, remains intact but is sequestered in cytoplasm. CDK4 protein reaccumulates in the following G1 phase and shuttles between the nucleus and the cytoplasm to facilitate the nuclear import of cyclin D1. Without CDK4, cyclin D1 cannot enter the nucleus. Point mutations that disrupt CDK4 and cyclin D1 interaction impair the nuclear import of cyclin D1 and the activity of CDK4. RNAi knockdown of CDK4 also induces cytoplasmic retention of cyclin D1 and G0/G1 phase arrest of the cells. Collectively, our data demonstrate that CDK4 protein is degraded in late mitosis and reaccumulates in the following G1 phase to facilitate the nuclear import of cyclin D1 for activation of CKD4 to initiate a new cell cycle in HeLa cells.

Maternal age-dependent APC/C-mediated decrease in securin causes premature sister chromatid separation in meiosis II

Sister chromatid attachment during meiosis II (MII) is maintained by securin-mediated inhibition of separase. In maternal ageing, oocytes show increased inter-sister kinetochore distance and premature sister chromatid separation (PSCS), suggesting aberrant separase activity. Here, we find that MII oocytes from aged mice have less securin than oocytes from young mice and that this reduction is mediated by increased destruction by the anaphase promoting complex/cyclosome (APC/C) during meiosis I (MI) exit. Inhibition of the spindle assembly checkpoint (SAC) kinase, Mps1, during MI exit in young oocytes replicates this phenotype. Further, over-expression of securin or Mps1 protects against the age-related increase in inter-sister kinetochore distance and PSCS. These findings show that maternal ageing compromises the oocyte SAC–APC/C axis leading to a decrease in securin that ultimately causes sister chromatid cohesion loss. Manipulating this axis and/or increasing securin may provide novel therapeutic approaches to alleviating the risk of oocyte aneuploidy in maternal ageing.

New Functions of APC/C Ubiquitin Ligase in the Nervous System and Its Role in Alzheimer's Disease.

The E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) regulates important processes in cells, such as the cell cycle, by targeting a set of substrates for degradation. In the last decade, APC/C has been related to several major functions in the nervous system, including axon guidance, synaptic plasticity, neurogenesis, and neuronal survival. Interestingly, some of the identified APC/C substrates have been related to neurodegenerative diseases. There is an accumulation of some degradation targets of APC/C in Alzheimer’s disease (AD) brains, which suggests a dysregulation of the protein complex in the disorder. Moreover, recently evidence has been provided for an inactivation of APC/C in AD. It has been shown that oligomers of the AD-related peptide, Aβ, induce degradation of the APC/C activator subunit cdh1, in vitro in neurons in culture and in vivo in the mouse hippocampus. Furthermore, in the AD mouse model APP/PS1, lower cdh1 levels were observed in pyramidal neurons in CA1 when compared to age-matched wildtype mice. In this review, we provide a complete list of APC/C substrates that are involved in the nervous system and we discuss their functions. We also summarize recent studies that show neurobiological effects in cdh1 knockout mouse models. Finally, we discuss the role of APC/C in the pathophysiology of AD.

Brain metabolism in health, aging, andneurodegeneration.

Brain cells normally respond adaptively to bioenergetic challenges resulting from ongoing activity in neuronal circuits, and from environmental energetic stressors such as food deprivation and physical exertion. At the cellular level, such adaptive responses include the "strengthening" of existing synapses, the formation of new synapses, and the production of new neurons from stem cells. At the molecular level, bioenergetic challenges result in the activation of transcription factors that induce the expression of proteins that bolster the resistance of neurons to the kinds of metabolic, oxidative, excitotoxic, and proteotoxic stresses involved in the pathogenesis of brain disorders including stroke, and Alzheimer's and Parkinson's diseases. Emerging findings suggest that lifestyles that include intermittent bioenergetic challenges, most notably exercise and dietary energy restriction, can increase the likelihood that the brain will function optimally and in the absence of disease throughout life. Here, we provide an overview of cellular and molecular mechanisms that regulate brain energy metabolism, how such mechanisms are altered during aging and in neurodegenerative disorders, and the potential applications to brain health and disease of interventions that engage pathways involved in neuronal adaptations to metabolic stress.

Coal tar pitch extract could induce chromosomal instability of human bronchial epithelial cells mediated by spindle checkpoint-related proteins.

Coal tar pitch (CTP) is a byproduct of coal tar distillation. The workers working with coal tar or in aluminum smelters, potrooms and carbon plants have the opportunities of exposing to coal tar pitch volatiles. Coal tar pitches from which polycyclic aromatic hydrocarbons (PAHs) originate have been shown to exhibit lung carcinogenicity in humans. Chromosomal instability (CIN) is a mechanism in carcinogenesis, however, whether CIN is involved in coal tar pitch-induced lung cancer remains elusive. In this present study, human bronchial epithelial cells (BEAS-2B) were first exposed to coal tar pitch extracts (CTPE) to induce a malignant transformation model. Then, the occurrence of severe chromosomal changes detected using G band, R band and multiplex fluorescence in situ hybridization (M-FISH) staining were examined. It was shown that more clones of transformed BEAS-2B cells at passage 30 following stimulation with CTPE were formed in the soft agar compared with the vehicle control. Moreover, the expression of the spindle checkpoint-related proteins, mitotic arrest defective 2 (Mad2), budding uninhibited in benzimidazole 1 (Bub1), and anaphase-promoting complex (APC), indicators of abnormal chromosomes and carcinogenesis, reduced in CTPE-treated BEAS-2B cells at Passage 30 compared with the vehicle control using real-time PCR and immunohistochemistry. In summary, exposure of BEAS-2B cells to CTPE may induce chromosomal instability through spindle checkpoint-related proteins.

APC/CCdh1-Rock2 pathway controls dendritic integrity and memory

Disruption of neuronal morphology contributes to the pathology of neurodegenerative disorders such as Alzheimer's disease (AD). However, the underlying molecular mechanisms are unknown. Here, we show that postnatal deletion of Cdh1, a cofactor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase in neurons [Cdh1 conditional knockout (cKO)], disrupts dendrite arborization and causes dendritic spine and synapse loss in the cortex and hippocampus, concomitant with memory impairment and neurodegeneration, in adult mice. We found that the dendrite destabilizer Rho protein kinase 2 (Rock2), which accumulates in the brain of AD patients, is an APC/CCdh1 substrate in vivo and that Rock2 protein and activity increased in the cortex and hippocampus of Cdh1 cKO mice. In these animals, inhibition of Rock activity, using the clinically approved drug fasudil, prevented dendritic network disorganization, memory loss, and neurodegeneration. Thus, APC/CCdh1-mediated degradation of Rock2 maintains the dendritic network, memory formation, and neuronal survival, suggesting that pharmacological inhibition of aberrantly accumulated Rock2 may be a suitable therapeutic strategy against neurodegeneration.

FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia

AbstractFZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G1 phase, and activation of the G2-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage–induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

Anaphase-Promoting Complex Adaptor FZR1/CDH1 Blocks BRAF Signaling Both by Targeting BRAF for Proteolytic Degradation and by Disrupting BRAF Dimerization.

<b/> Blocking dimerization and stimulating protein degradation are two mechanisms known to inhibit BRAF activity. The study reported by Wan and colleagues identifies BRAF as a substrate of the APC/CFZR1-ubiqutin-proteasome system. The interaction between FZR1 and BRAF also induces a conformational change that disrupts BRAF dimerization. These findings identify a dynamic interplay between FZR1 and BRAF with strong implications for cell-fate determination and the tumor suppressor role of FZR1. Cancer Discov; 7(4); 356-8. ©2017 AACRSee related article by Wan et al., p. 424.

Different Functionality of Cdc20 Binding Sites within the Mitotic Checkpoint Complex

The mitotic checkpoint is a cellular safeguard that prevents chromosome missegregation in eukaryotic cells [1, 2]. Suboptimal functioning may foster chromosome missegregation in cancer cells [3]. Checkpoint signaling produces the "mitotic checkpoint complex" (MCC), which prevents anaphase by targeting Cdc20, the activator of the anaphase-promoting complex/cyclosome (APC/C). Recent biochemical and structural studies revealed that the human MCC binds two Cdc20 molecules, one (Cdc20M) through well-characterized, cooperative binding to Mad2 and Mad3/BubR1 (forming the "core MCC") and the other one (Cdc20A) through additional binding sequences in Mad3/BubR1 [4-6]. Here, we dissect the different functionality of these sites invivo. We show in fission yeast that, at low Cdc20 concentrations, Cdc20M binding is sufficient for checkpoint activity and Cdc20A binding becomes dispensable. Cdc20A binding is mediated by the conserved Mad3 ABBA-KEN2-ABBA motif [7, 8], which we find additionally required for binding of the MCC to the APC/C and for MCC disassembly. Strikingly, deletion of the APC/C subunit Apc15 mimics mutations in this motif, revealing a shared function. This function of Apc15 may be masked in human cells by independent mediators of MCC-APC/C binding. Our data provide important invivo support for the recent structure-based models and functionally dissect three elements of Cdc20 inhibition: (1) sequestration of Cdc20 in the core MCC, sufficient at low Cdc20 concentrations; (2) inhibition of a second Cdc20 through the Mad3 C terminus, independent of Mad2 binding to this Cdc20 molecule; and (3) occupancy of the APC/C with full MCC, where Mad3 and Apc15 are involved.

Identification of a Ribose-Phosphate Pyrophosphokinase that Can Interact In Vivo with the Anaphase Promoting Complex/Cyclosome.

5-Phospho-d-ribosyl-1-diphosphate (PRPP) synthase (PRS) catalyzes the biosynthesis of PRPP, which is an important compound of metabolism in most organisms. However, no PRS genes have been cloned, let alone studied for their biological function in rubber tree. In this study, we identify a novel protein (PRS4) that interacts in vivo with rubber tree anaphase promoting complex/cyclosome (APC/C) subunit 10 (HbAPC10) by yeast two-hybrid assays. PRS4 has been cloned from rubber tree and named as HbPRS4. Blastp search in the genome of Arabidopsis thaliana showed that HbPRS4 shared the highest similarity with AtPRS4, with 80.71% identity. qRT-PCR was used to determine the expression of HbPRS4 in different tissues and under various treatments. HbPRS4 was preferentially expressed in the bark. Moreover, the expression level of HbPRS4 was significantly induced by the proteasome inhibitor MG132 treatment, suggesting it might be regulated by the ubiquitin/26S proteasome pathway. The amount of HbPRS4 transcript was obviously decreased after mechanical wounding and abscisic acid (ABA) treatments, while a slight increase was observed at 24 h after ABA treatment. HbPRS4 transcript in the latex was significantly upregulated by ethephon (ET) and methyl jasmonate (MeJA) treatments. These results suggested that HbPRS4 may be a specific substrate of HbAPC10 indirectly regulating natural rubber biosynthesis in rubber tree.

Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest

SummaryDuring mitosis, cells must segregate the replicated copies of their genome to their daughter cells with extremely high fidelity. Segregation errors lead to an abnormal chromosome number (aneuploidy), which typically results in disease or cell death [1]. Chromosome segregation and anaphase onset are initiated through the action of the multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C [2]). The APC/C is inhibited by the spindle checkpoint in the presence of kinetochore attachment defects [3, 4]. Here we demonstrate that two non-essential APC/C subunits (Apc14 and Apc15) regulate association of spindle checkpoint proteins, in the form of the mitotic checkpoint complex (MCC), with the APC/C. apc14Δ mutants display increased MCC association with the APC/C and are unable to silence the checkpoint efficiently. Conversely, apc15Δ mutants display reduced association between the MCC and APC/C, are defective in poly-ubiquitination of Cdc20, and are checkpoint defective. In vitro reconstitution studies have shown that human MCC-APC/C can contain two molecules of Cdc20 [5, 6, 7]. Using a yeast strain expressing two Cdc20 genes with different epitope tags, we show by co-immunoprecipitation that this is true in vivo. MCC binding to the second molecule of Cdc20 is mediated via the C-terminal KEN box in Mad3. Somewhat surprisingly, complexes containing both molecules of Cdc20 accumulate in apc15Δ cells, and the implications of this observation are discussed.