scholarly journals Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

2010 ◽  
Vol 191 (2) ◽  
pp. 313-329 ◽  
Author(s):  
Liviu Malureanu ◽  
Karthik B. Jeganathan ◽  
Fang Jin ◽  
Darren J. Baker ◽  
Janine H. van Ree ◽  
...  
Keyword(s):  
De Novo ◽  
Cyclin B1 ◽  
Anaphase Promoting Complex ◽  
De Novo Synthesis ◽  
Cytoplasmic Polyadenylation ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Element Binding Protein ◽  
Sister Chromosome ◽  
Chromosome Separation

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element–binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

scholarly journals The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

2014 ◽  
Vol 25 (5) ◽  
pp. 594-605 ◽  
Author(s):  
Sushama Sivakumar ◽  
John R. Daum ◽  
Aaron R. Tipton ◽  
Susannah Rankin ◽  
Gary J. Gorbsky
Keyword(s):  
Cyclin B1 ◽  
Mitotic Exit ◽  
Anaphase Promoting Complex ◽  
Metaphase Arrest ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Chromosome Alignment ◽  
Partial Degradation ◽  
Interfering Rna ◽  
Transient Alignment

The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.

scholarly journals SCAP/SREBP pathway is required for the full steroidogenic response to cyclic AMP

2016 ◽  
Vol 113 (38) ◽  
pp. E5685-E5693 ◽  
Author(s):  
Masami Shimizu-Albergine ◽  
Brian Van Yserloo ◽  
Martin G. Golkowski ◽  
Shao-En Ong ◽  
Joseph A. Beavo ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Leydig Cells ◽  
De Novo ◽  
Regulatory Element ◽  
De Novo Synthesis ◽  
Intracellular Lipid ◽  
Element Binding Protein ◽  
Short Palindromic Repeat ◽  
Sterol Regulatory Element

Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP–SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP/SREBP activation and subsequent regulation of steroidogenesis.

scholarly journals A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zhejian Ji ◽  
Haishan Gao ◽  
Luying Jia ◽  
Bing Li ◽  
Hongtao Yu
Keyword(s):  
Spindle Checkpoint ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Checkpoint Activation ◽  
Checkpoint Signaling ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Phosphorylation Cascade ◽  
Mitotic Checkpoint Complex

The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.

Over-expression of Aurora-A targets cytoplasmic polyadenylation element binding protein and promotes mRNA polyadenylation of Cdk1 and cyclin B1

2005 ◽  
Vol 10 (7) ◽  
pp. 627-638 ◽  
Author(s):  
Takashi Sasayama ◽  
Tomotoshi Marumoto ◽  
Naoko Kunitoku ◽  
Dongwei Zhang ◽  
Norihiko Tamaki ◽  
...  
Keyword(s):  
Binding Protein ◽  
Cyclin B1 ◽  
Aurora A ◽  
Cytoplasmic Polyadenylation ◽  
Over Expression ◽  
Element Binding Protein ◽  
Mrna Polyadenylation

scholarly journals Sequestration of CDH1 by MAD2L2 prevents premature APC/C activation prior to anaphase onset

2013 ◽  
Vol 203 (1) ◽  
pp. 87-100 ◽  
Author(s):  
Tamar Listovsky ◽  
Julian E. Sale
Keyword(s):  
Cyclin B1 ◽  
Human Cells ◽  
Anaphase Promoting Complex ◽  
Bistable Switch ◽  
Anaphase Onset ◽  
Subsequent Degradation ◽  
Sequential Activation

The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.

scholarly journals 253BOVINE OOCYTE CYCLIN B1 MRNA UNDERGOES CYTOPLASMIC POLYADENYLATION BEFORE THE BEGINNING OF IN VITRO MATURATION

2004 ◽  
Vol 16 (2) ◽  
pp. 246 ◽  
Author(s):  
K. Tremblay ◽  
C. Vigneault ◽  
G. Bujold ◽  
M.-A. Sirard
Keyword(s):  
In Vitro Maturation ◽  
De Novo ◽  
Mrna Level ◽  
Tail Length ◽  
Cyclin B1 ◽  
Cytoplasmic Polyadenylation ◽  
Cis Acting ◽  
Significant Difference ◽  
Bovine Oocytes

Maternal oocyte Cyclin B1 mRNA is known to be stored in the cytoplasm with a short poly(A) tail and be translationally dormant at GV stage. During maturation, Cyclin B1 poly(A) tail is elongated by a process called cytoplasmic polyadenylation and driven by A/U-rich cis-acting elements in its 3′ untranslated region (UTR) known as cytoplasmic polyadenylation elements (CPEs). The objective of this study was to elucidate whether GV-stage bovine oocytes possess a stockpile of Cyclin B1 mRNA stored with a short a poly(A) tail that is elongated during maturation by CPE regulation. The mRNA poly(A) tail length was measured by Rapid Amplification of cDNA Ends Polyadenylation test (Race-PAT) on oocytes (n=100) at the GV stage and 3, 5, 8, 10, 15, 20, and 25h of in vitro maturation. The mRNA poly(A) tail length was also measured in triplicate (n=20) on cold oocytes in GV (all manipulations on ice), warm oocytes in GV (ovaries transported in warm saline and manipulations on ice) and warm+2h 30min oocytes in GV (oocytes left for an additional 2h and 30min at room temperature). To assess for variation in mRNA quantity, Cyclin B1 mRNA level was quantified by real-time PCR (Lightcycler, Roche, Indianapolis, IN, USA) in cold, warm or warm+2h 30min GV oocytes (n=20). The data were treated as factorial design, using treatment and type of RT as factors, and analysed by ANOVA (SAS Inst., Cary, NC, USA). Differences between means were checked using Tukey’s test. Oocyte Cyclin B1 transcript show two different 3′ UTRs. These transcripts had the same ORF but different 3′ UTR lengths because of an alternative nuclear polyadenylation element AAUAAA (NPE). The longest form (Cyclin B1L) that possessed a putative CPE (UUUUAAUAAA) fused to the last NPE was studied. In warm GV oocytes, Cyclin B1L had a long poly(A) tail of 100 adenosine residues, and this length did not change during in vitro maturation. Interestingly, we found that Cyclin B1L showed an expected short poly(A) tail when the ovaries and the oocytes were transported and manipulated on ice. We showed that Cyclin B1L mRNA is cytoplasmically polyadenylated (addition of 75 adenosine residues) between the time of collection and the end of manipulation. This lengthening is most probably sufficient to promote translation. There was no significant difference between the Cyclin B1 mRNA quantity of cold oocytes or warm oocytes when the oligo used for the reverse transcription was either dt or decamers. Therefore, we believe that the increase in poly(A) tail length is not the result of Cyclin B1L mRNA degradation in cold oocytes or de novo transcription in warm oocytes. We report for the first time that Cyclin B1L cytoplasmic polyadenylation is carried out well before the beginning of in vitro maturation in bovine oocytes when ovaries are transported from the slaughterhouse in warm saline. Studying the real early mechanisms leading to resumption of meiosis in bovine oocytes is complicated by Cyclin B1 polyadenylation occurring prior to in vitro maturation. (Supported by NSERC.)

Cyclin A and Nek2A: APC/C–Cdc20 substrates invisible to the mitotic spindle checkpoint

2010 ◽  
Vol 38 (1) ◽  
pp. 72-77 ◽  
Author(s):  
Wouter van Zon ◽  
Rob M.F. Wolthuis
Keyword(s):  
Nuclear Envelope ◽  
Spindle Checkpoint ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Sister Chromatids ◽  
Spindle Microtubules

Active cyclin B1–Cdk1 (cyclin-dependent kinase 1) keeps cells in mitosis, allowing time for spindle microtubules to capture the chromosomes and for incorrect chromosome-spindle attachments to be repaired. Meanwhile, securin, an inhibitor of separase, secures cohesion between sister chromatids, preventing anaphase onset. The spindle checkpoint is a signalling pathway emerging from improperly attached chromosomes that inhibits Cdc20, the mitotic activator of the APC/C (anaphase-promoting complex/cyclosome) ubiquitin ligase. Blocking Cdc20 stabilizes cyclin B1 and securin to delay mitotic exit and anaphase until all chromosomes reach bipolar spindle attachments. Cells entering mitosis in the absence of a functional spindle checkpoint degrade cyclin B1 and securin right after nuclear-envelope breakdown, in prometaphase. Interestingly, two APC/C substrates, cyclin A and Nek2A, are normally degraded at nuclear-envelope breakdown, even when the spindle checkpoint is active. This indicates that the APC/C is activated early in mitosis, whereas cyclin B1 and securin are protected as long as the spindle checkpoint inhibits Cdc20. Remarkably, destruction of cyclin A and Nek2A also depends on Cdc20. The paradox of Cdc20 being both active and inhibited in prometaphase could be explained if cyclin A and Nek2A are either exceptionally efficient Cdc20 substrates, or if they are equipped with ‘stealth’ mechanisms to effectively escape detection by the spindle checkpoint. In the present paper, we discuss recently emerging models for spindle-checkpoint-independent APC/C–Cdc20 activity, which might even have implications for cancer therapy.

scholarly journals Cyclin B1 Expression Regulated by Cytoplasmic Polyadenylation Element Binding Protein in Astrocytes

2011 ◽  
Vol 31 (34) ◽  
pp. 12118-12128 ◽  
Author(s):  
K. C. Kim ◽  
W. J. Oh ◽  
K. H. Ko ◽  
C. Y. Shin ◽  
D. G. Wells
Keyword(s):  
Binding Protein ◽  
Cyclin B1 ◽  
Cytoplasmic Polyadenylation ◽  
Element Binding Protein

Functional amyloid: widespread in Nature, diverse in purpose

2014 ◽  
Vol 56 ◽  
pp. 207-219 ◽  
Author(s):  
Chi L.L. Pham ◽  
Ann H. Kwan ◽  
Margaret Sunde
Keyword(s):  
Spider Silk ◽  
Epigenetic Inheritance ◽  
Fibrillar Structure ◽  
Cytoplasmic Polyadenylation ◽  
Functional Amyloid ◽  
Interacting Protein ◽  
Diverse Range ◽  
Element Binding Protein ◽  
Functional Amyloids ◽  
Micro Organisms

Amyloids are insoluble fibrillar protein deposits with an underlying cross-β structure initially discovered in the context of human diseases. However, it is now clear that the same fibrillar structure is used by many organisms, from bacteria to humans, in order to achieve a diverse range of biological functions. These functions include structure and protection (e.g. curli and chorion proteins, and insect and spider silk proteins), aiding interface transitions and cell–cell recognition (e.g. chaplins, rodlins and hydrophobins), protein control and storage (e.g. Microcin E492, modulins and PMEL), and epigenetic inheritance and memory [e.g. Sup35, Ure2p, HET-s and CPEB (cytoplasmic polyadenylation element-binding protein)]. As more examples of functional amyloid come to light, the list of roles associated with functional amyloids has continued to expand. More recently, amyloids have also been implicated in signal transduction [e.g. RIP1/RIP3 (receptor-interacting protein)] and perhaps in host defence [e.g. aDrs (anionic dermaseptin) peptide]. The present chapter discusses in detail functional amyloids that are used in Nature by micro-organisms, non-mammalian animals and mammals, including the biological roles that they play, their molecular composition and how they assemble, as well as the coping strategies that organisms have evolved to avoid the potential toxicity of functional amyloid.

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