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Entry into mitosis has been classically attributed to the activation of a cyclin B/Cdk1 amplification loop via a partial pool of this kinase becoming active at the end of G2 phase. However, how this initial pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, depletion of the orthologue of FAM122A in C. elegans prevents entry into mitosis in germline stem cells. Moreover, data from Xenopus egg extracts strongly suggest that FAM122A-dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. Inhibition of this phosphatase allows subsequent phosphorylation of early mitotic substrates by cyclin A/Cdk, resulting in full cyclin B/Cdk1 and Greatwall (Gwl) kinase activation. Subsequent to Greatwall activation, Arpp19/ENSA become phosphorylated and now compete with FAM122A, promoting its dissociation from PP2A-B55 and taking over its phosphatase inhibition role until the end of mitosis., (© 2024. The Author(s).)

During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cells require major physical changes to induce a proper repartition of the DNA. Nuclear envelope breakdown, DNA condensation and spindle formation are promoted at mitotic entry by massive protein phosphorylation and reversed at mitotic exit by the timely and ordered dephosphorylation of mitotic substrates. This phosphorylation results from the balance between the activity of kinases and phosphatases. The role of kinases in the control of mitosis has been largely studied, however, the impact of phosphatases has long been underestimated. Recent data have now established that the regulation of phosphatases is crucial to confer timely and ordered cellular events required for cell division. One major phosphatase involved in this process is the phosphatase holoenzyme PP2A-B55. This review will be focused in the latest structural, biochemical and enzymatic insights provided for PP2A-B55 phosphatase as well as its regulators and mechanisms of action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lacroix, Lorca and Castro.)

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) plays an instrumental role in driving CME initiation. The F-BAR domain-only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remain elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining minimal reconstituted in vitro and cellular systems. Our results indicate that PI(4,5)P 2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like-shaped protein patches. We show that the binding of FCHo2 on cellular membranes promotes PI(4,5)P 2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P 2 -interacting proteins at endocytic sites., Competing Interests: FE, IC, DS, CA, RR, VB, AC, TL, JV, SV, AC, LP No competing interests declared, (© 2022, El Alaoui et al.)

PP2A is a major serine/threonine phosphatase class involved in the regulation of cell signaling through the removal of protein phosphorylation. This class of phosphatases is comprised of different heterotrimeric complexes displaying distinct substrate specificities. The present review will focus on one specific heterocomplex, the phosphatase PP2A-B55. Herein, we will report the direct substrates of this phosphatase identified to date, and its impact on different cell signaling cascades. We will additionally describe its negative regulation by its inhibitors Arpp19 and ENSA and their upstream kinase Greatwall. Finally, we will describe the essential molecular features defining PP2A-B55 substrate specificity that confer the correct temporal pattern of substrate dephosphorylation. The main objective of this review is to provide the reader with a unique source compiling all the knowledge of this particular holoenzyme that has evolved as a key enzyme for cell homeostasis and cancer development., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Arpp19 is a potent PP2A-B55 inhibitor that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by the Greatwall kinase on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as a competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signalling mechanism is elusive. Here, we characterize the molecular determinants conferring high affinity and slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Protein phosphorylation is a post-translational modification essential for the control of the activity of most enzymes in the cell. This protein modification results from a fine-tuned balance between kinases and phosphatases. PP2A is one of the major serine/threonine phosphatases that is involved in the control of a myriad of different signaling cascades. This enzyme, often misregulated in cancer, is considered a tumor suppressor. In this review, we will focus on PP2A-B55, a particular holoenzyme of the family of the PP2A phosphatases whose specific role in cancer development and progression has only recently been highlighted. The discovery of the Greatwall (Gwl)/Arpp19-ENSA cascade, a new pathway specifically controlling PP2A-B55 activity, has been shown to be frequently altered in cancer. Herein, we will review the current knowledge about the mechanisms controlling the formation and the regulation of the activity of this phosphatase and its misregulation in cancer.

Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Greatwall (GWL) is an essential kinase that indirectly controls PP2A-B55, the phosphatase counterbalancing cyclin B/CDK1 activity during mitosis. In Xenopus laevis egg extracts, GWL-mediated phosphorylation of overexpressed ARPP19 and ENSA turns them into potent PP2A-B55 inhibitors. It has been shown that the GWL/ENSA/PP2A-B55 axis contributes to the control of DNA replication, but little is known about the role of ARPP19 in cell division. By using conditional knockout mouse models, we investigated the specific roles of ARPP19 and ENSA in cell division. We found that Arpp19 , but not Ensa , is essential for mouse embryogenesis. Moreover, Arpp19 ablation dramatically decreased mouse embryonic fibroblast (MEF) viability by perturbing the temporal pattern of protein dephosphorylation during mitotic progression, possibly by a drop of PP2A-B55 activity inhibition. We show that these alterations are not prevented by ENSA, which is still expressed in Arpp19 Δ/Δ MEFs, suggesting that ARPP19 is essential for mitotic division. Strikingly, we demonstrate that unlike ARPP19, ENSA is not required for early embryonic development. Arpp19 knockout did not perturb the S phase, unlike Ensa gene ablation. We conclude that, during mouse embryogenesis, the Arpp19 and Ensa paralog genes display specific functions by differentially controlling cell cycle progression., (© 2019 Hached et al.)

Mitosis is controlled by a subtle balance between kinase and phosphatase activities that involve the master mitotic kinase cyclin-B-Cdk1 and its antagonizing protein phosphatase 2A-B55 (PP2A-B55). Importantly, the Greatwall (Gwl; known as Mastl in mammals, Rim15 in budding yeast and Ppk18 in fission yeast) kinase pathway regulates PP2A-B55 activity by phosphorylating two proteins, cAMP-regulated phosphoprotein 19 (Arpp19) and α-endosulfine (ENSA). This phosphorylation turns these proteins into potent inhibitors of PP2A-B55, thereby promoting a correct timing and progression of mitosis. In this Cell Science at a Glance article and the accompanying poster, we discuss how Gwl is regulated in space and time, and how the Gwl-Arpp19-ENSA-PP2A-B55 pathway plays an essential role in the control of M and S phases from yeast to human. We also summarize how Gwl modulates oncogenic properties of cells and how nutrient deprivation influences Gwl activity., (© 2018. Published by The Company of Biologists Ltd.)

Mitosis is induced by the activation of the cyclin B/cdk1 feedback loop that creates a bistable state. The triggering factor promoting active cyclin B/cdk1 switch has been assigned to cyclin B/cdk1 accumulation during G2. However, this complex is rapidly inactivated by Wee1/Myt1-dependent phosphorylation of cdk1 making unlikely a triggering role of this kinase in mitotic commitment. Here we show that cyclin A/cdk1 kinase is the factor triggering mitosis. Cyclin A/cdk1 phosphorylates Bora to promote Aurora A-dependent Plk1 phosphorylation and activation and mitotic entry. We demonstrate that Bora phosphorylation by cyclin A/cdk1 is both necessary and sufficient for mitotic commitment. Finally, we identify a site in Bora whose phosphorylation by cyclin A/cdk1 is required for mitotic entry. We constructed a mathematical model confirming the essential role of this kinase in mitotic commitment. Overall, our results uncover the molecular mechanism by which cyclin A/cdk1 triggers mitotic entry., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Greatwall/Ensa/PP2A-B55 pathway is essential for controlling mitotic substrate phosphorylation and mitotic entry. Here, we investigate the effect of the knockdown of the Gwl substrate, Ensa, in human cells. Unexpectedly, Ensa knockdown promotes a dramatic extension of S phase associated with a lowered density of replication forks. Notably, Ensa depletion results in a decrease of Treslin levels, a pivotal protein for the firing of replication origins. Accordingly, the extended S phase in Ensa-depleted cells is completely rescued by the overexpression of Treslin. Our data herein reveal a new mechanism by which normal cells regulate S-phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.The Greatwall/Ensa/PP2A-B55 pathway controls mitotic substrate phosphorylation and mitotic entry. Here the authors show that cells regulate S phase duration by controlling the ubiquitin-proteasome degradation of Treslin in a Gwl/Ensa-dependent pathway.

In human eggs, aneuploidy increases with age and can result in infertility and genetic diseases. Studies in mouse oocytes suggest that reduced centromere cohesion and spindle assembly checkpoint (SAC) activity could be at the origin of chromosome missegregation. Little is known about these two features in humans. Here, we show that in human eggs, inter-kinetochore distances of bivalent chromosomes strongly increase with age. This results in the formation of univalent chromosomes during metaphase I (MI) and of single chromatids in metaphase II (MII). We also investigated SAC activity by checking the localization of BUB1 and BUBR1. We found that they localize at the kinetochore with a similar temporal timing than in mitotic cells and in a MPS1-dependent manner, suggesting that the SAC signalling pathway is active in human oocytes. Moreover, our data also suggest that this checkpoint is inactivated when centromere cohesion is lost in MI and consequently cannot inhibit premature sister chromatid separation. Finally, we show that the kinetochore localization of BUB1 and BUBR1 decreases with the age of the oocyte donors. This could contribute to oocyte aneuploidy.

Centrioles are essential for the assembly of both centrosomes and cilia. Centriole biogenesis occurs once and only once per cell cycle and is temporally coordinated with cell-cycle progression, ensuring the formation of the right number of centrioles at the right time. The formation of new daughter centrioles is guided by a pre-existing, mother centriole. The proximity between mother and daughter centrioles was proposed to restrict new centriole formation until they separate beyond a critical distance. Paradoxically, mother and daughter centrioles overcome this distance in early mitosis, at a time when triggers for centriole biogenesis Polo-like kinase 4 (PLK4) and its substrate STIL are abundant. Here we show that in mitosis, the mitotic kinase CDK1-CyclinB binds STIL and prevents formation of the PLK4-STIL complex and STIL phosphorylation by PLK4, thus inhibiting untimely onset of centriole biogenesis. After CDK1-CyclinB inactivation upon mitotic exit, PLK4 can bind and phosphorylate STIL in G1, allowing pro-centriole assembly in the subsequent S phase. Our work shows that complementary mechanisms, such as mother-daughter centriole proximity and CDK1-CyclinB interaction with centriolar components, ensure that centriole biogenesis occurs once and only once per cell cycle, raising parallels to the cell-cycle regulation of DNA replication and centromere formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Entry into mitosis is induced by the activation of cyclin-B-Cdk1 and Greatwall (Gwl; also known as MASTL in mammals) kinases. Cyclin-B-Cdk1 phosphorylates mitotic substrates, whereas Gwl activation promotes the phosphorylation of the small proteins Arpp19 and ENSA. Phosphorylated Arpp19 and/or ENSA bind to and inhibit PP2A comprising the B55 subunit (PP2A-B55; B55 is also known as PPP2R2A), the phosphatase responsible for cyclin-B-Cdk1 substrate dephosphorylation, allowing the stable phosphorylation of mitotic proteins. Upon mitotic exit, cyclin-B-Cdk1 and Gwl kinases are inactivated, and mitotic substrates are dephosphorylated. Here, we have identified protein phosphatase-1 (PP1) as the phosphatase involved in the dephosphorylation of the activating site (Ser875) of Gwl. Depletion of PP1 from meioticXenopusegg extracts maintains phosphorylation of Ser875, as well as the full activity of this kinase, resulting in a block of meiotic and mitotic exit. By contrast, preventing the reactivation of PP2A-B55 through the addition of a hyperactive Gwl mutant (GwlK72M) mainly affected Gwl dephosphorylation on Thr194, resulting in partial inactivation of Gwl and in the incomplete exit from mitosis or meiosis. We also show that when PP2A-B55 is fully reactivated by depleting Arpp19, this protein phosphatase is able to dephosphorylate both activating sites, even in the absence of PP1., (© 2016. Published by The Company of Biologists Ltd.)

Entry into mitosis requires the coordinated activation of various protein kinases and phosphatases that together activate sequential signaling pathways allowing entry, progression and exit of mitosis. The limiting step is thought to be the activation of the mitotic Cdk1-cyclin B kinase. However, this model has recently evolved with new data showing that in addition to the Cdk1-cyclin B complex, Greatwall (Gwl) kinase is also required to enter into and maintain mitosis. This new concept proposes that entry into mitosis is now based on the combined activation of both kinases Cdk1-cyclin B and Gwl, the former promoting massive phosphorylation of mitotic substrates and the latter inhibiting PP2A-B55 phosphatase responsible for dephosphorylation of these substrates. Activated Gwl phosphorylates both Arpp19 and ENSA, which associate and inhibit PP2A-B55. This pathway seems relatively well conserved from yeast to humans, although some differences appear based on models or techniques used. While Gwl is activated by phosphorylation, its inactivation requires dephosphorylation of critical residues. Several phosphatases such as PP1, PP2A-B55 and FCP1 are required to control the dephosphorylation and inactivation of Gwl and a properly regulated mitotic exit. Gwl has also been reported to be involved in cancer processes and DNA damage recovery. These new findings support the idea that the Gwl-Arpp19/ENSA-PP2A-B55 pathway is essential to achieve an efficient division of cells and to maintain genomic stability.

The PP2A phosphatase is often inactivated in cancer and is considered as a tumour suppressor. A new pathway controlling PP2A activity in mitosis has been recently described. This pathway includes the Greatwall (GWL) kinase and its substrates endosulfines. At mitotic entry, GWL is activated and phosphorylates endosulfines that then bind and inhibit PP2A. We analysed whether GWL overexpression could participate in cancer development. We show that GWL overexpression promotes cell transformation and increases invasive capacities of cells through hyperphosphorylation of the oncogenic kinase AKT. Interestingly, AKT hyperphosphorylation induced by GWL is independent of endosulfines. Rather, GWL induces GSK3 kinase dephosphorylation in its inhibitory sites and subsequent SCF-dependent degradation of the PHLPP phosphatase responsible for AKT dephosphorylation. In line with its oncogenic activity, we find that GWL is often overexpressed in human colorectal tumoral tissues. Thus, GWL is a human oncoprotein that promotes the hyperactivation of AKT via the degradation of its phosphatase, PHLPP, in human malignancies.

Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (∼ 10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G 2 phase onwards. Addition of low doses of RO3306 in G 2 phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G 2 phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G 2 phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.

New data have recently established that protein phosphorylation during mitosis is the result of a controlled balance between kinase and phosphatase activities and that, as for mitotic kinases, phosphatases are also regulated during cell division. This regulation is at least in part induced by the activation of the Greatwall (Gwl) kinase at mitotic entry. Activated Gwl phosphorylates its substrates cAMP-regulated phospho protein 19 (Arpp19) and α-endosulfine (ENSA), promoting their binding to and the inhibition of PP2A. Interestingly, besides the role of the Gwl-Arpp19/ENSA in the control of mitotic division, new data in yeast support the involvement of this pathway in mRNA stabilization during G(0) program initiation, although in this case the phosphatase PP2A appears not to be implicated. Finally, Gwl activity has been shown to be required for DNA checkpoint recovery. These new findings support the view that Gwl, Arpp19 and ENSA could function as the core of a new signalization pathway that, by targeting different final substrates, could participate in a variety of physiological functions.

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A(Cdc55) phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2A(Cdc55) in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2A(Cdc55) activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2A(Cdc55), timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2A(Cdc55), which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms., Competing Interests: The authors have declared that no competing interests exist.

Mitotic division is induced by protein phosphorylation. For a long time the supported hypothesis was that mitotic entry and exit were the exclusive result of cyclin B-Cdk1 kinase activation and inactivation, whereas the phosphatase activity required to dephosphorylate mitotic substrates was thought to be constant during mitosis. Recent data demonstrate that phosphatase activity must also be tightly regulated to promote correct cell division. Here we describe the new pathway involved in phosphatase regulation and the questions that this discovery raises concerning the classic view of cell cycle regulation.

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.

Here we investigate the mechanisms regulating Greatwall (Gwl), a serine/threonine kinase essential for promoting the correct timing of mitosis. We identify Gwl as a unique AGC kinase that, unlike most AGC members, appears to be devoid of a hydrophobic motif despite the presence of a functional hydrophobic pocket. Our results suggest that Gwl activation could be mediated by the binding of its hydrophobic pocket to the hydrophobic motif of another AGC kinase. Our molecular modeling and mutagenic analysis also indicate that Gwl displays a conserved tail/linker site whose phosphorylation mediates kinase activation by promoting the interaction of this phosphorylated residue with two lysines at the N terminus. This interaction could stabilize the αC-helix and maintain kinase activity. Finally, the different phosphorylation sites on Gwl are identified, and the role of each one in the regulation of Gwl kinase activity is determined. Our data suggest that only the phosphorylation of the tail/linker site, located outside the putative T loop, appears to be essential for Gwl activation. In summary, our results identify Gwl as a member of the AGC family of kinases that appears to be regulated by unique mechanisms and that differs from the other members of this family.

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.

Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phosphatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.

Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.

The spindle assembly checkpoint (SAC) prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The MAP kinase (MAPK) is an important player in this pathway, however its exact role is not fully understood. One major target of MAPK is the p90 ribosomal protein S6 kinase (RSKs) family. In this study, we analyse whether Rsk2 could participate in the activation of the SAC. Our data indicate that this protein is localized at the kinetochores under checkpoint conditions. Moreover, it is essential for the SAC activity in Xenopus egg extracts as its depletion prevents metaphase arrest as well as the kinetochore localization of the other SAC components. We also show that this kinase might also participate in the maintenance of the SAC in mammalian cells as Rsk2 knockdown in these cells prevents the kinetochore localization of Mad1, Mad2 and CENP-E under checkpoint conditions.

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B-Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B-Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B-Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B-Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A-dependent dephosphorylation of cyclin B-Cdc2 substrates is increased in GW-depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B-Cdc2 but also by the regulation of PP2A by GW.

Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified beta-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.

The anaphase-promoting complex (APC) early mitotic inhibitor 1 (Emi1) is required to induce S- and M-phase entries by stimulating the accumulation of cyclin A and cyclin B through APC(Cdh1/cdc20) inhibition. In this report, we show that Emi1 proteolysis can be induced by cyclin A/cdk (cdk for cyclin-dependent kinase). Paradoxically, Emi1 is stable during G2 phase, when cyclin A/cdk, Plx1 and SCF(betatrcp) (SCF for Skp1-Cul1-Fbox protein)--which play a role in its degradation--are active. Here, we identify Pin1 as a new regulator of Emi1 that induces Emi1 stabilization by preventing its association with SCF(betatrcp). We show that Pin1 binds to Emi1 and prevents its association with betatrcp in an isomerization-dependent pathway. We also show that Emi1-Pin1 binding is present in vivo in XL2 cells during G2 phase and that this association protects Emi1 from being degraded during this phase of the cell cycle. We propose that S- and M-phase entries are mediated by the accumulation of cyclin A and cyclin B through a Pin1-dependent stabilization of Emi1 during G2.

The transcription factors MyoD and Myf5 present distinct patterns of expression during cell cycle progression and development. In contrast to the mitosis-specific disappearance of Myf5, which requires a D-box-like motif overlapping the basic domain, here we describe a stable and inactive mitotic form of MyoD phosphorylated on its serine 5 and serine 200 residues by cyclin B-cdc2. In mitosis, these modifications are required for releasing MyoD from condensed chromosomes and inhibiting its DNA-binding and transcriptional activation ability. Then, nuclear MyoD regains instability in the beginning of G1 phase due to rapid dephosphorylation events. Moreover, a non-phosphorylable MyoD S5A/S200A is not excluded from condensed chromatin and alters mitotic progression with apparent abnormalities. Thus, the drop of MyoD below a threshold level and its displacement from the mitotic chromatin could present another window in the cell cycle for resetting the myogenic transcriptional program and to maintain the myogenic determination of the proliferating cells.

Xenopus RINGO/Speedy (XRINGO) is a potent inducer of oocyte meiotic maturation that can directly activate Cdk1 and Cdk2. Here, we show that endogenous XRINGO protein accumulates transiently during meiosis I entry and then is downregulated. This tight regulation of XRINGO expression is the consequence of two interconnected mechanisms: processing and degradation. XRINGO processing involves recognition of at least three distinct phosphorylated recognition motifs by the SCF(betaTrCP) ubiquitin ligase, followed by proteasome-mediated limited degradation, resulting in an amino-terminal XRINGO fragment. XRINGO processing is directly stimulated by several kinases, including protein kinase A and glycogen synthase kinase-3beta, and may contribute to the maintenance of G2 arrest. On the other hand, XRINGO degradation after meiosis I is mediated by the ubiquitin ligase Siah-2, which probably requires phosphorylation of XRINGO on Ser 243 and may be important for the omission of S phase at the meiosis-I-meiosis-II transition in Xenopus oocytes.

Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation. Xenopus egg extracts present many advantages for the study of the cell cycle, including the availability of a large quantity of material synchronized at a particular phase of the cell cycle. In this chapter, we describe various protocols used in Xenopus egg extracts to study the ubiquitination and degradation of different cell cycle regulators. We first provide the method used to obtain interphase- and metaphase II-arrested egg extracts. Subsequently, we describe the protocol employed in these extracts to test the putative ubiquitination and degradation of a protein. Moreover, we describe a detailed practical procedure to test the role of different regulators in the ubiquitin-dependent degradation pathway of a specific protein. To that, we show how to eliminate some of these regulators from the extracts by immunodepletion and how to activate ectopically their function by the translation of their messenger ribonucleic acid. Finally, the Notes provide a series of practical details that explain the different problems that can occur and the possible solutions used to overcome them.

Background: The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s) of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells. To this aim, we undertook the biochemical analysis of the mechanisms governing Myf5 mitotic degradation, using heterologous systems., Results: We show here that mitotic degradation of Myf5 is conserved in non-myogenic cells, and is thus strictly under the control of the cell cycle apparatus. Using Xenopus egg extracts as an in vitro system to dissect the main steps of Myf5 mitotic proteolysis, we show that (1) Myf5 stability is regulated by a complex interplay of phosphorylation/dephosphorylation, probably involving various kinases and phosphatases, (2) Myf5 is ubiquitylated in mitotic extracts, and this is a prerequisite to its degradation by the proteasome and (3) at least in the Xenopus system, the E3 responsible for its mitotic degradation is not the APC/C (the major E3 during mitosis)., Conclusion: Altogether, our data strongly suggest that the mitotic degradation of Myf5 by the ubiquitin-proteasome system is precisely controlled by multiple phosphorylation of the protein, and that the APC/C is not involved in this process.

The success of cell division relies on the activation of its master regulator Cdc2-cyclin B, and many other kinases controlling cellular organization, such as Aurora-A. Most of these kinase activities are regulated by phosphorylation. Despite numerous studies showing that okadaic acid-sensitive phosphatases regulate both Cdc2 and Aurora-A activation, their identity has not yet been established in Xenopus oocytes and the importance of their regulation has not been evaluated. Using an oocyte cell-free system, we demonstrate that PP2A depletion is sufficient to lead to Cdc2 activation, whereas Aurora-A activation depends on Cdc2 activity. The activity level of PP1 does not affect Cdc2 kinase activation promoted by PP2A removal. PP1 inhibition is also not sufficient to lead to Aurora-A activation in the absence of active Cdc2. We therefore conclude that in Xenopus oocytes, PP2A is the key phosphatase that negatively regulates Cdc2 activation. Once this negative regulator is removed, endogenous kinases are able to turn on the activator Cdc2 system without any additional stimulation. In contrast, Aurora-A activation is indirectly controlled by Cdc2 activity independently of either PP2A or PP1. This strongly suggests that in Xenopus oocytes, Aurora-A activation is mainly controlled by the specific stimulation of kinases under the control of Cdc2 and not by downregulation of phosphatase.

Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation by the 26S proteasome. Two different ubiquitin ligases play an important role in the cell cycle: the SCF (Skp1/Cullin/F-box) and the anaphase-promoting complex (APC). In this review, we describe the present knowledge about the APC. We pay particular attention to the latest results concerning APC structure, APC regulation and substrate recognition, and we discuss the implication of these findings in the understanding the APC function.

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.

The molecular events triggered during progesterone-induced oocyte maturation in Xenopus are not well understood. One of the first events is the activation of the MAPK cascade and the maturation-promoting factor (MPF). The latter triggers meiosis I resumption and meiosis II progression until the metaphase II arrest. The release of the metaphase II is mediated by the anaphase-promoting complex (APC)-dependent degradation of cyclin B. This degradation activity requires the APC activator Cdc20 that activates ubiquitination reactions by recruiting substrates to the APC. However, recent reports in different organisms involve other APC regulators during different phases of the meiotic cycle. Therefore, we investigated the role of another APC regulator, XCdh1 during the G2/M transition in meiosis I in the Xenopus oocyte. Here, we report that XCdh1 protein is expressed in oocytes. Besides, injection of specific XCdh1 antisense inhibits progesterone-induced G2/M transition that can be rescued by adding back the purified human Cdh1 protein. On the other hand, ectopic expression of low levels of XCdh1 protein has a positive effect on the G2/M transition by facilitating this process. Moreover, the sole injection of XCdh1 mRNA triggers Mos protein synthesis and biphosphorylation of MAPK in absence of progesterone. Altogether, these data show that XCdh1 has a positive role during the G2/M transition in the oocyte. According to our results, its role could be independent of the APC.

The papillomavirus E1 replicative helicase is essential for replication and maintenance of extrachromosomal viral genomes in infected cells. We previously found that the bovine papillomavirus E1 protein is a substrate of the ubiquitin-dependent proteolytic pathway. Here we show that E1 is targeted for degradation by the anaphase-promoting complex (APC). Inhibition of APC activity by the specific inhibitor Emi1 or point mutations in the D-box and KEN-box motifs of E1 stabilize the protein and increase viral DNA replication in both a cell-free system and in living cells. These findings involve APC as the ubiquitin ligase that controls E1 levels to maintain a constant low copy number of the viral genome during latent infection.