Cyclin dependent kinase 1-dependent activation of APC/C ubiquitin ligase

Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex/cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by p9/Cks2 (CDK regulatory subunit)-CDK1 controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognised subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target and our findings may facilitate the development of specific inhibitors

Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli

The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the {beta} clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25{degrees}C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25{degrees}C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42{degrees}C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25{degrees}C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25{degrees}C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway

Targeting of Fzr/Cdh1 for timely activation of the APC/C at the centrosome during mitotic exit.

A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/C(Fzr) substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity.Cancer Research UK (Career Development Fellowship), Biotechnology and Biological Sciences Research Council (project grant), Medical Research Council (project grant), Japan Society for the Promotion of Science (Postdoctoral Fellowship for Research Abroad), European Commission (Marie Skłodowska-Curie actions individual fellowship)This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1260

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop

The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin-dependent kinase 1 (Cdk1) promotes Cdc20 co-activator loading in mitosis to form active APC/C-Cdc20. However, detailed phospho-regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo-like kinase (Plx1) and PP2A-B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1-loop500) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1-loop500 in a phosphorylation-dependent manner and promotes the formation of APC/C-Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A-B56 is recruited to the Apc1-loop500 and differentially promotes dissociation of Plx1 and PP2A-B56 through dephosphorylation of Plx1-binding sites. Stable Plx1 binding, which prevents PP2A-B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1-loop500 is controlled by distant Apc3-loop phosphorylation. Our study suggests that phosphorylation-dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle

PP2A-B56 binds to Apc1 and promotes Cdc20 association with the APC/C ubiquitin ligase in mitosis

Cell cycle progression and genome stability are regulated by a ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Cyclin-dependent kinase 1 (Cdk1) has long been implicated in APC/ C activation; however, the molecular mechanisms of governing this process in vivo are largely unknown. Recently, a Cdk1-dependent phosphorylation relay within Apc3-Apc1 subunits has been shown to alleviate Apc1-mediated auto-inhibition by which a mitotic APC/ C co-activator Cdc20 binds to and activates the APC/C. However, the underlying mechanism for dephosphorylation of Cdc20 and APC/C remains elusive. Here, we show that a disordered loop domain of Apc1 (Apc1-loop500) directly binds the B56 regulatory subunit of protein phosphatase 2A (PP2A) and stimulates Cdc20 loading to the APC/C. Using the APC/C reconstitution system in Xenopus egg extracts, we demonstrate that mutations in Apc1- loop500 that abolish B56 binding decrease Cdc20 loading and APC/ C-dependent ubiquitylation. Conversely, a non-phosphorylatable mutant Cdc20 can efficiently bind the APC/C even when PP2A-B56 binding is impeded. Furthermore, PP2A-B56 preferentially dephosphorylates Cdc20 over the Apc1 inhibitory domain. These results indicate that Apc1-loop500 plays a role in dephosphorylating Cdc20, promoting APC/C-Cdc20 complex formation in mitosi

Effects of Recrystallization on Tensile Anisotropic Properties for IN738LC Fabricated by Laser Powder Bed Fusion

This study demonstrates the effects of recrystallization on tensile properties and the anisotropy of IN738LC, a typical γ’ precipitation-strengthened alloy, at both room and high temperatures via the laser powder bed fusion process. The nonrecrystallized columnar microstructure, subjected to standard IN738LC heat treatment up to 1120◦ C, and the almost fully recrystallized microstructure, heat-treated at 1204◦ C, were compared. The tensile properties strongly depend on whether recrystallization was completed as well as the tensile direction. This can be explained by microstructure characterization, featuring the Taylor factor in the tensile direction, average grain size estimated by ellipse approximation, and the relationship between the grain shape and tensile direction. The shape of the recrystallized grains and the distribution of coarse MC carbides inside the recrystallized grains were determined by the microstructure in an as-built state. In high-temperature tensile tests conducted in the horizontal direction, the separation of the columnar grains caused a brittle fracture. In contrast, dimples were observed at the fracture surface after recrystallization, indicating scope for further improvement in ductility.Hibino S., Fujimitsu K., Azuma M., et al. Effects of Recrystallization on Tensile Anisotropic Properties for IN738LC Fabricated by Laser Powder Bed Fusion. Crystals, 12, 6, 842. https://doi.org/10.3390/cryst12060842

CDK1-PP2A-B55 interplay ensures cell cycle oscillation via Apc1-loop300

Cell cycle control relies on a delicate balance of phosphorylation with CDK1 and phosphatases like PP1 and PP2A-B55. Yet, identifying the primary substrate responsible for cell cycle oscillations remains a challenge. We uncover the pivotal role of phospho-regulation in the anaphase-promoting complex/cyclosome (APC/C), particularly through the Apc1-loop300 domain (Apc1-300L), orchestrated by CDK1 and PP2A-B55. Premature activation of PP2A-B55 during mitosis, induced by Greatwall kinase depletion, leads to Apc1-300L dephosphorylation, stalling APC/C activity and delaying Cyclin B degradation. This effect can be counteracted using the B55-specific inhibitor pEnsa or by removing Apc1-300L. We also show Cdc20's dynamic APC/C interaction across cell cycle stages, but dephosphorylation of Apc1-300L specifically inhibits further Cdc20 recruitment. Our study underscores APC/C's central role in cell cycle oscillation, identifying it as a primary substrate regulated by the CDK-PP2A partnership