Cyclin B1-Cdk1 Research Articles

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

Prognostic Value and Functional Consequences of Calcium Signalling in Acute Myeloid Leukaemia

Ca 2+ is a ubiquitous second messenger that regulates a large number of cellular functions by acting on various proteins and signaling pathways. Abnormal expression/function of protein involved in Ca 2+ signaling have been associated with oncogenesis. However, its implication in the mechanisms that drive normal hematopoietic cells to leukemic transformation remain largely unknown. In order to identify actors of Ca 2+ signaling of potential prognosis value in acute myeloid leukemia (AML), , we performed a systematic screening on databases and identified expression of STIM2 as negatively correlated with disease free and overall survival while ORAI2 expression presented the opposite pattern. Interestingly, we noticed that ORAI2 and STIM2 expression differed depending on FAB subtypes. Indeed the 50% of patients in the upper quartile of STIM2 expression presented a myelo-monocytic differentiation while monocytic differentiation was associated with a lower ORAI2 expression. On a series of 407 AML patients from the ALFA 07-02 protocol, we observed that higher STIM2 was found in intermediate and high ELN17 while a lower ORAI2 level was seen in ELN17 high-risk patients. We quantified STIM2 and ORAI2 expression in two AML cell lines with monocytic potential, THP1 and OCI-AML3. When cells were driven into monocytic differentiation, we observed at RNA and protein level a decreased ORAI2 expression while STIM2 increased significantly. We then focused our studies on STIM2. To decipher its function in monocytic cell lines, we chose a shRNA-mediated gene knockdown approach. We first measured the capacitive Ca 2+ entry after STIM2 knockdown and found it increased, due to a predominant downregulation of the 2.1 STIM2 isoform, which has a negative effect on Ca 2+ entry, while 2.2 STIM2 isoform was less impacted. We then performed a transcriptomic analysis using the Nanostring technology, and identified a STIM2 signature characterized by dysregulation of genes involved in apoptosis, cell cycle and chromatin modification. Phenotypically, STIM2 knockdown drastically decreased cell proliferation and viability, through activation of the mitochondrial pathway of apoptosis and blockage of cell cycle at the G2/M phase. We observed that STIM2 knockdown led to a genomic stress with DNA double strand breaks as shown by an increased p-ϒH2AX, followed by activation of the p53 pathway, decreased expression of cell cycle regulators such as CDK1-cyclin B1 and the CDC25c phosphatase, and a decreased apoptosis threshold with a low antiapoptotic/proapoptotic protein ratio. Inhibition of p53 using Pifithrin-α reverted apoptosis and cell cycle blockage whereas p-ϒH2AX induction remained high. Moreover, STIM2 knockdown led to p-ϒH2AX activation but neither apoptosis nor cell cycle blockage in the p53-deficient cell line HL60, highlighting the fact that the genomic stress occurred downstream p53 induction in cells after STIM2 knockdown. At last, using in vitro monocytic differentiation of CD34 + cells, we observed a higher STIM2 expression at RNA level in differentiated cells. Acquisition of monocytic markers was faster after STIM2 forced expression, while STIM2 knockdown impaired CD14 expression and led to a p-ϒH2AX, p53 induction, apoptosis and cell cycle blockage in agreement with data observed in leukemic cell lines. In summary, we describe here STIM2, the “forgotten” member of SOCE, as a new actor in the proliferation, survival and differentiation of human normal and malignant monocytic cells. Considering (i) the association of a high STIM2 expression with an adverse prognosis in AML and (ii) the association of STIM2 with AML harboring monocytic/myelomonocytic differentiation, STIM2 may represent an interesting protein to target in these types of leukemia in the future

Protein Kinase C Epsilon Overexpression in Prostate Adenocarcinoma is Associated with Oncogenesis

Background: PKCε, an isozyme of serine-threonine kinase, has been implicated in epithelial cancer metastasis and progression. This study investigates the impact of the oncogenic PKCε, overexpressed abnormally in human Prostate tumor samples and cell lines, to understand its efficacy. Methods: The microarray dataset, GSE86257, was processed for normalization. The identification of upregulated and downregulated genes was based on FDR >1 and p <0.05 values. Cytoscape analysis and functional enrichment of significant genes were done. The identified genes were validated on the TCGA dataset and survival analysis was performed by Kaplan-Meier analysis. Results: A total of 1524 DEGs were identified with 728 upregulated genes and 818 downregulated genes. The two significant modules with MCODE score:9.0 and Venn analysis provided cyclin-dependent kinase inhibitor protein (CDK1), Cyclin B1 (CCNB1), Phospholipase C Gamma 1 (PLCG1), Cyclin Dependent Kinase 9 (CDK9), Phosphoinositide-3-Kinase Regulatory Subunit 3 (PIK3R3), H4 Clustered Histone 6 (H4C6), Phospholipase C Gamma 2 (PLCG2) as most interacting genes. TCGA data analysis and Prognostic analysis revealed CCNBI, CDK9, and PLCG1 associated with poor prognosis. Conclusion: PKCε regulates genes that are responsible for cancer progression. Therefore, targeting PKCε in Prostate cancer may serve as an important regulatory effect and may improve the prognosis of the disease.

Meiotic Cell Cycle Progression in Mouse Oocytes: Role of Cyclins.

All eukaryotic cells, including oocytes, utilize an engine called cyclin-dependent kinase (Cdk) to drive the cell cycle. Cdks are activated by a co-factor called cyclin, which regulates their activity. The key Cdk-cyclin complex that regulates the oocyte cell cycle is known as Cdk1-cyclin B1. Recent studies have elucidated the roles of other cyclins, such as B2, B3, A2, and O, in oocyte cell cycle regulation. This review aims to discuss the recently discovered roles of various cyclins in mouse oocyte cell cycle regulation in accordance with the sequential progression of the cell cycle. In addition, this review addresses the translation and degradation of cyclins to modulate the activity of Cdks. Overall, the literature indicates that each cyclin performs unique and redundant functions at various stages of the cell cycle, while their expression and degradation are tightly regulated. Taken together, this review provides new insights into the regulatory role and function of cyclins in oocyte cell cycle progression.

Mcrs1 regulates G2/M transition and spindle assembly during mouse oocyte meiosis.

Microspherule protein 1 (Mcrs1) is a component of the nonspecific lethal (NSL) complex and the chromatin remodeling INO80 complex, which participates in transcriptional regulation during mitosis. Here, we investigate the roles of Mcrs1 during female meiosis in mice. We demonstrate that Mcrs1 is a novel regulator of the meiotic G2/M transition and spindle assembly in mouse oocytes. Mcrs1 is present in the nucleus and associates with spindle poles and chromosomes of oocytes during meiosis I. Depletion of Mcrs1 alters HDAC2-mediated H4K16ac, H3K4me2, and H3K9me2 levels in nonsurrounded nucleolus (NSN)-type oocytes, and reduces CDK1 activity and cyclin B1 accumulation, leading to G2/M transition delay. Furthermore, Mcrs1 depletion results in abnormal spindle assembly due to reduced Aurora kinase (Aurka and Aurkc) and Kif2A activities, suggesting that Mcrs1 also plays a transcription-independent role in regulation of metaphase I oocytes. Taken together, our results demonstrate that the transcription factor Mcrs1 has important roles in cell cycle regulation and spindle assembly in mouse oocyte meiosis.

On the assembly of the mitotic spindle, bistability and hysteresis

During cell division, the transition from interphase to mitosis is dictated by activation of the cyclin B-cdk1 (Cdk1) complex, master mitotic kinase. During interphase, Cdk1 accumulates in an inactive state (pre-Cdk1). When Cdk1 overcomes a certain threshold of activity upon initial activation of pre-Cdk1, then the stockpiled pre-Cdk1 is rapidly converted into overshooting active Cdk1, and mitosis is established irreversibly in a switch-like fashion. This is granted by positive Cdk1 activation loops and the concomitant inactivation of Cdk1 counteracting phosphatases, empowering Cdk1 activity and favoring the Cdk1-dependent phosphorylations that are required to establish mitosis. These circuitries prevent backtracking and ensure unidirectionality so that interphase and mitosis are considered bistable states. Mitosis also shows hysteresis, meaning that the levels of Cdk1 activity needed to establish mitosis are higher than those required to maintain it; therefore, once in mitosis cells can tolerate moderate drops in Cdk1 activity without exiting mitosis. Whether these features have other functional implications in addition to the general action of preventing backtracking is unknown. Here, we contextualize these concepts in the view of recent evidence indicating that loss of activity of small and compartmentalized amounts of Cdk1 within mitosis is necessary to assemble the mitotic spindle, the structure required to segregate replicated chromosomes. We further propose that, in addition to prevent backtracking, the stability and hysteresis properties of mitosis are also essential to move forward in mitosis by allowing cells to bear small, localized, drops in Cdk1 activity that are necessary to build the mitotic spindle.

The interplay between noncoding RNAs and p21 signaling in gastrointestinal cancer: from tumorigenesis to metastasis.

Non-coding RNAs (ncRNAs) are emerging as important regulators in various pathological conditions, including human cancers. NcRNAs exert potentially crucial effects on cell cycle progression, proliferation, and invasion in cancer cells by targeting various cell cycle-related proteins at transcriptional and post-transcriptional levels. As one of the key cell cycle regulatory proteins, p21 is involved in various processes, including the cellular response to DNA damage, cell growth, invasion, metastasis, apoptosis, and senescence. P21 has been shown to have either a tumor-suppressive or oncogenic effect depending on the cellular localization and posttranslational modifications. P21 exerts a significant regulatory effect on both G1/S and G2/M checkpoints by regulating the function of cyclin-dependent kinase enzymes (CDKs) or interacting with proliferating cell nuclear antigen (PCNA). P21 has an important effect on the cellular response to DNA damage by separating DNA replication enzymes from PCNA and inhibiting DNA synthesis resulting in G1 phase arrest. Furthermore, p21 has been shown to negatively regulate the G2/M checkpoint through the inactivation of cyclin-CDK complexes. In response to any cell damage caused by genotoxic agents, p21 exerts its regulatory effects by nuclear preservation of cyclin B1-CDK1 and preventing their activation. Notably, several ncRNAs, including lncRNAs and miRNAs, have been shown to be involved in tumor initiation and progression through the regulation of the p21 signaling axis. In this review, we discuss the miRNA/lncRNA-dependent mechanisms that regulate p21 and their effects on gastrointestinal tumorigenesis. A better understanding of the regulatory effects of ncRNAs on the p21 signaling may help to discover novel therapeutic targets in gastrointestinal cancer.

Cyclin A and Cks1 promote kinase consensus switching to non-proline-directed CDK1 phosphorylation

Ordered protein phosphorylation by CDKs is a key mechanism for regulating the cell cycle. How temporal order is enforced in mammalian cells remains unclear. Using a fixed cell kinase assay and phosphoproteomics, we show how CDK1 activity and non-catalytic CDK1 subunits contribute to the choice of substrate and site of phosphorylation. Increases in CDK1 activity alter substrate choice, with intermediate- and low-sensitivity CDK1 substrates enriched in DNA replication and mitotic functions, respectively. This activity dependence is shared between Cyclin A- and Cyclin B-CDK1. Cks1 has a proteome-wide role as an enhancer of multisite CDK1 phosphorylation. Contrary to the model of CDK1 as an exclusively proline-directed kinase, we show that Cyclin A and Cks1 enhance non-proline-directed phosphorylation, preferably on sites with a+3 lysine residue. Indeed, 70% of cell-cycle-regulated phosphorylations, where the kinase carrying out this modification has not been identified, are non-proline-directed CDK1 sites.

Cell cycle oscillations driven by two interlinked bistable switches.

Regular transitions between interphase and mitosis during the cell cycle are driven by changes in the activity of the enzymatic protein complex cyclin B with cyclin-dependent kinase 1 (Cdk1). At the most basic level, this cell cycle oscillator is driven by negative feedback: active cyclin B-Cdk1 activates the anaphase-promoting complex/cyclosome, which triggers the degradation of cyclin B. Such cell cycle oscillations occur fast and periodically in the early embryos of the frog Xenopus laevis, where several positive-feedback loops leading to bistable switches in parts of the regulatory network have been experimentally identified. Here, we build cell cycle oscillator models to show how single and multiple bistable switches in parts of the underlying regulatory network change the properties of the oscillations and how they can confer robustness to the oscillator. We present a detailed bifurcation analysis of these models.

Design, synthesis, and discovery of Eudistomin Y derivatives as lysosome-targeted antiproliferation agents

Eudistomin Y is a novel class of β-carbolines of marine origin with potential antiproliferation activity against MDA-MB-231 cells (triple-negative breast carcinoma). However, the subcellular target or the detailed mechanism against cancer cell proliferation has not yet been identified. In this study, based on its special structure, a novel series of Eudistomin Y fluorescent derivatives were designed and synthesized by enhancing the electron-donor effect of N-9 to endow it with fluorescent properties through N-alkylation. The structure–activity relationships against the proliferation of cancer cells were also analyzed. A quarter of Eudistomin Y derivatives showed much higher potency against cancer cell proliferation than the original Eudistomin Y1. Fluorescent derivative H1k with robust antiproliferative activity could arrest MDA-MB-231 cells in the G2–M phase. The subcellular localization studies of the probes, including H1k, and Eudistomin Y1 were performed in MDA-MB-231 cells, and the co-localization and competitive inhibition assays revealed their lysosome-specific localization. Moreover, H1k could dose-dependently increase the autophagy signal and downregulate the expression of cyclin-dependent kinase (CDK1) and cyclin B1 which principally regulated the G2–M transition. Furthermore, the specific autophagy inhibitor 3-methyladenine significantly inhibited the H1k-triggered antiproliferation of cancer cells and the downregulation of CDK1 and cyclin B1. Overall, the lysosome is identified as the subcellular target of Eudistomin Y for the first time, and derivative H1k showed robust antiproliferative activity against MDA-MB-231 cells by decreasing Cyclin B1–CDK1 complex via a lysosome-dependent pathway.

Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.

Nuclear-cytoplasmic compartmentalization of cyclin B1-Cdk1 promotes robust timing of mitotic events

The cyclin-dependent kinase (Cdk1) oscillator is widely characterized in homogenized cytosolic extracts, leaving unclear the impact of nucleocytoplasmic compartmentalization. Here, by developing a Förster resonance energy transfer (FRET) biosensor, we track Cdk1 spatiotemporal dynamics in reconstituted cells with or without side by side and find compartmentalization significantly modulates clock properties previously found in bulk studies. Although nucleus-absent cells display highly tunable frequency, the nucleus-present cells maintain constant frequency against cyclin B1 variations. Despite high expression variability, cyclin degraded within the same duration, enabling a robust mitotic phase. Moreover, Cdk1 and cyclin B1 cycle rigorously out-of-phase, ensuring wide phase-plane orbits, essential for oscillation robustness. Although Cdk1 in homogeneous extracts is well known for delayed switch-like activation, we find active cyclin B1-Cdk1 accumulates in nuclei, without delay, until the nuclear envelope breakdown (NEB) when another abrupt activation triggers anaphase. Cdk1 biphasic activation and spatial compartmentalization may together coordinate the accurate ordering of different downstream events.

Tumour cell suppression by spiroleucettadine through dual regulation of cell cycle and apoptosis

Background: Spiroleucettadine is an alkaloid originally derived from the yellow Leucetta sea sponge. Spiroleucettadine has previously been shown to inhibit the growth of various cancer cell lines and has a high anti-proliferative activity against a non-small cell lung cancer cell line (H522). The mediators of these anti-proliferative effects have not been determined. Therefore, in this study we measured changes in cell death and cell proliferation and their immediate protein mediators in response to spiroleucettadine, toward the aim of ultimately determining target(s) for spiroleucettadine in cancer cells and a more precise description of its mechanism of action. Methods: We used flow cytometry to investigate changes in the cell cycle apoptosis and Western blot to investigate associated protein changes following exposure of H522 cells to varying concentrations of spiroleucettadine. Results: We found evidence for cell cycle arrest at G2/M and associated increases in cyclin B1 expression and CDK1 phosphorylation, as well as an increase in apoptosis alongside marked increase in Bim expression, consistent with activation of the intrinsic apoptotic pathway Conclusions: Any targets for spiroleucettadine that may be proposed are constrained to those with mechanisms of actions that lead to G2/M arrest, induction of the intrinsic apoptotic pathway, and changes in the expression of associated proteins.

Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation.

As cells prepare to divide, they must ensure that enough space is available to assemble the mitotic machinery without perturbing tissue homeostasis. To do so, cells undergo a series of biochemical reactions regulated by cyclin B1-CDK1 that trigger cytoskeletal reorganization and ensure the coordination of cytoplasmic and nuclear events. Along with the biochemical events that control mitotic entry, mechanical forces have recently emerged as important players in cell-cycle regulation. However, the exact link between mechanical forces and the biochemical pathways that control mitotic progression remains unknown. Here, we identify a tension-dependent signal on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and mitotic entry. This signal relies on actomyosin contractility, which unfolds the nucleus during the G2-M transition, activating the stretch-sensitive cPLA2 on the nuclear envelope and regulating the nuclear translocation of cyclin B1. Our data demonstrate how nuclear tension during the G2-M transition contributes to timely and efficient mitotic spindle assembly and prevents chromosomal instability.

Small changes in phospho-occupancy at the kinetochore-microtubule interface drive mitotic fidelity.

Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.

CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

The disassembly of integrin-containing focal adhesions (FAs) at mitotic entry is essential for cell rounding, mitotic retraction fibre formation, bipolar spindle positioning and chromosome segregation. The mechanism that drives FA disassembly at mitotic entry is unknown. Here, we show that the CDK1–cyclin B1 complex phosphorylates the integrin activator kindlin, which results in the recruitment of the cullin 9–FBXL10 ubiquitin ligase complex that mediates kindlin ubiquitination and degradation. This molecular pathway is essential for FA disassembly and cell rounding, as phospho-inhibitory mutations of the CDK1 motif prevent kindlin degradation, FA disassembly and mitotic cell rounding. Conversely, phospho-mimetic mutations promote kindlin degradation in interphase, accelerate mitotic cell rounding and impair mitotic retraction fibre formation. Despite the opposing effects on kindlin stability, both types of mutations cause severe mitotic spindle defects, apoptosis and aneuploidy. Thus, the exquisite regulation of kindlin levels at mitotic entry is essential for cells to progress accurately through mitosis.

Regulation of the cell cycle under anoxia stress in tail muscle and hepatopancreas of the freshwater crayfish, Orconectes virilis

Regulation of the cell cycle is an understudied response to oxygen deprivation among crustaceans. The virile crayfish, Orconectes virilis, is a freshwater crustacean that when challenged by environmental oxygen limitation undergoes metabolic rate depression (to ~30% of normal levels) and switches to anaerobic metabolism to generate energy. To understand how crayfish regulate the cell cycle in response to anoxia, key proteins involved in cell cycle control were analyzed in muscle and hepatopancreas. At the G1/S barrier, an overall upregulation of positive regulators of cell cycle progression was indicated by the responses of G1 cyclins (cyclin D and cyclin E) and Cyclin dependent kinases (CDK4, CDK6 and CDK2) under anoxia. Although the levels of Cyclin kinase inhibitors (CKIs) at this juncture were also upregulated (P15/16 and P21 (T145) in muscle and P16 (S152) in hepatopancreas), levels of a major regulator of this phase and driver to S-phase, E2F1, were significantly higher in both tissues in conjunction with deactivation of its inhibitor, Retinoblastoma (Rb) protein. At the G2/M barrier, expression profiles of the G2 cyclin B suggested cell cycle progression despite overall trend of higher activities of checkpoint kinases, (Chk1 (S317) and Chk2 (S19)), that also negatively regulate the cyclin B-CDK1 complex via CdC25C (cell division cycle 25) whose levels remained unchanged. Overall, the present study suggests continued cell cycle progression, albeit with potential deceleration, as indicated by checkpoint kinases and kinase inhibitor profiles that might play a role in protecting tissues from apoptotic damage under chronic anoxic stress.

Compartmentalized control of Cdk1 drives mitotic spindle assembly.

During cell division, dramatic microtubular rearrangements driven by cyclin B-cdk1 (Cdk1) kinase activity mark the onset of mitosis leading to dismantling of the interphase microtubular cytoskeleton and assembly of the mitotic spindle. During interphase, Cdk1 accumulates in an inactive state, phosphorylated at inhibitory sites by Wee1/Myt1 kinases. At mitosis onset, Cdc25 phosphatase dephosphorylates and activates Cdk1. Once activated, Cdk1 clears cytoplasmic microtubules by inhibiting microtubule-stabilizing and growth-promoting microtubule-associated proteins (MAPs). Nevertheless, some of these MAPs are required for spindle microtubule growth and spindle assembly, creating quite a conundrum. We show here that a Cdk1 fraction bound to spindle structures escapes Cdc25 action and remains inhibited by phosphorylation (i-Cdk1) in mitotic human cells. Loss or restoration of i-Cdk1 inhibits or promotes spindle assembly, respectively. Furthermore, polymerizing spindle microtubules foster i-Cdk1 aggregating with Wee1 and excluding Cdc25. Our data reveal that spindle assembly relies on compartmentalized control of Cdk1 activity.

The Aurora B gradient sustains kinetochore stability in anaphase.

SummaryKinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.

CDK1, CCNB1 and NDC80 are associated with prognosis and progression of hepatitis B virus-associated hepatocellular carcinoma: a bioinformatic analysis

To identify the key genes involved in the transformation of hepatitis B virus (HBV) into hepatocellular carcinoma (HCC) and explore the underlying molecular mechanisms. We analyzed the mRNA microarray data of 119 HBV-related HCC tissues and 252 HBV-related non-tumor tissues in GSE55092, GSE84044 and GSE121248 from the GEO database, and the "sva" R package was used to remove the batch effects. Integration analysis was performed to identify the differentially expressed genes (DEGs) in HBV-related liver cancer and liver tissues with HBV infection. The significant DEGs were functionally annotated using GO and KEGG analyses, and the most important modules and hub genes were explored with STRING analysis. Kaplan-Meier and Oncomine databases were used to verify the HCC gene expression data in the TCGA database to explore the correlations of the hub genes with the occurrence, progression and prognosis of HCC. We also examined the expressions of the hub genes in 17 pairs of surgical specimens of HCC and adjacent tissues using RT-qPCR. We identified a total of 121 DEGs and 3 genetic markers in HCC (P < 0.01). These DEGs included cyclin1 (CDK1), cyclin B1 (CCNB1), and nuclear division cycle 80 (NDC80), which participated in cell cycle, pyrimidine metabolism and DNA replication and were highly correlated (P < 0.05). Analysis of the UALCAN database confirmed high expressions of these 3 genes in HCC tissues, which were correlated with a low survival rate of the patients, as shown by Kaplan-Meier analysis of the prognostic data from the UALCAN database. CDK1, CCNB1 and NDC80 were all correlated with the clinical grading of HCC (P < 0.05). The results of RT-qPCR on the surgical specimens verified significantly higher expressions of CDK1, CCNB1 and NDC80 mRNA in HCC tissues than in the adjacent tissues. CDK1, CCNB1 and NDC80 genes can be used as prognostic markers of HBV-related HCC and may serve as potential targets in preclinical studies and clinical treatment of HCC.