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The cyclin-dependent kinase Cdk1 is best known for its function as master regulator of the cell cycle. It phosphorylates several key proteins to control progression through the different phases of the cell cycle. However, studies conducted several decades ago with mammalian cells revealed that Cdk1 also directly regulates the basal transcription machinery, most notably RNA polymerase II. More recent studies in the budding yeast Saccharomyces cerevisiae have revisited this function of Cdk1 and also revealed that Cdk1 directly controls RNA polymerase III activity. These studies have also provided novel insight into the physiological relevance of this process. For instance, cell cycle-stage-dependent activity of these complexes may be important for meeting the increased demand for various proteins involved in housekeeping, metabolism, and protein synthesis. Recent work also indicates that direct regulation of the RNA polymerase II machinery promotes cell cycle entry. Here, we provide an overview of the regulation of basal transcription by Cdk1, and we hypothesize that the original function of the primordial cell-cycle CDK was to regulate RNAPII and that it later evolved into specialized kinases that govern various aspects of the transcription machinery and the cell cycle.

Purpose: Despite significant benefit for other cancer subtypes, immune checkpoint blockade (ICB) therapy has not yet been shown to significantly improve outcomes for men with castration-resistant prostate cancer (CRPC). Prior data have shown that DNA damage response (DDR) deficiency, via genetic alteration and/or pharmacologic induction using DDR inhibitors (DDRi), may improve ICB response in solid tumors in part due to induction of mitotic catastrophe and innate immune activation. Discerning the underlying mechanisms of this DDRi-ICB interaction in a prostate cancer-specific manner is vital to guide novel clinical trials and provide durable clinical responses for men with CRPC., Experimental Design: We treated prostate cancer cell lines with potent, specific inhibitors of ATR kinase, as well as with PARP inhibitor, olaparib. We performed analyses of cGAS-STING and DDR signaling in treated cells, and treated a syngeneic androgen-indifferent, prostate cancer model with combined ATR inhibition and anti-programmed death ligand 1 (anti-PD-L1), and performed single-cell RNA sequencing analysis in treated tumors., Results: ATR inhibitor (ATRi; BAY1895433) directly repressed ATR-CHK1 signaling, activated CDK1-SPOP axis, leading to destabilization of PD-L1 protein. These effects of ATRi are distinct from those of olaparib, and resulted in a cGAS-STING-initiated, IFN-β-mediated, autocrine, apoptotic response in CRPC. The combination of ATRi with anti-PD-L1 therapy resulted in robust innate immune activation and a synergistic, T-cell-dependent therapeutic response in our syngeneic mouse model., Conclusions: This work provides a molecular mechanistic rationale for combining ATR-targeted agents with immune checkpoint blockade for patients with CRPC. Multiple early-phase clinical trials of this combination are underway., (©2021 The Authors; Published by the American Association for Cancer Research.)

At present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.

Targeting the activities of endoplasmic reticulum (ER)-mitochondrial-dependent metabolic reprogramming is considered one of the most promising strategies for cancer treatment. Here, we present biochemical subcellular fractionation, coimmunoprecipitation, gene manipulation, and pharmacologic evidence that induction of mitochondria-localized phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)-cyclin B1 complexes by apigenin in nasopharyngeal carcinoma (NPC) cells impairs the ER-mitochondrial bioenergetics and redox regulation of calcium (Ca ++ ) homeostasis through suppressing the B cell lymphoma 2 (BCL-2)/BCL-2/B-cell lymphoma-extra large (BCL-x L )-modulated anti-apoptotic and metabolic functions. Using a specific inducer, inhibitor, or short hairpin RNA for acid sphingomyelinase (ASM) demonstrated that enhanced lipid raft-associated ASM activity confers alteration of the lipid composition of lipid raft membranes, which leads to perturbation of protein trafficking, and induces formation of p110α free p85α-unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 complexes in the lipid raft membranes, causing disruption of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated signaling, thus triggering the p-CDK1 (Thr 161))-cyclin B1-mediated BCL-2 (Thr 69/Ser 87)/BCL-x L (Ser 62) phosphorylation and accompanying impairment of ER-mitochondria-regulated bioenergetic, redox, and Ca ++ homeostasis. Inhibition of apigenin-induced reactive oxygen species (ROS) generation by a ROS scavenger N-acetyl-L-cysteine blocked the lipid raft membrane localization and activation of ASM and formation of ceramide-enriched lipid raft membranes, returned PI3K-Akt-GTP-Rac1-modulated CDK1-cyclin B1 activity, and subsequently restored the BCL-2/BCL-x L -regulated ER-mitochondrial bioenergetic activity. Thus, this study reveals a novel molecular mechanism of the pro-apoptotic activity of ASM controlled by oxidative stress to modulate the ER-mitochondrial bioenergetic metabolism, as well as suggests the disruption of CDK1-cyclin B1-mediated BCL-2/BCL-x L oncogenic activity by triggering oxidative stress-ASM-induced PI3K-Akt-GTP-Rac1 inactivation as a therapeutic approach for NPC.

Endosomal trafficking of receptors and associated proteins plays a critical role in signal processing. Until recently, it was thought that trafficking was shut down during cell division. Thus, remarkably, the regulation of trafficking during division remains poorly characterized. Here we delineate the role of mitotic kinases in receptor trafficking during asymmetric division. Targeted perturbations reveal that Cyclin-dependent Kinase 1 (CDK1) and Aurora Kinase promote storage of Fibroblast Growth Factor Receptors (FGFRs) by suppressing endosomal degradation and recycling pathways. As cells progress through metaphase, loss of CDK1 activity permits differential degradation and targeted recycling of stored receptors, leading to asymmetric induction. Mitotic receptor storage, as delineated in this study, may facilitate rapid reestablishment of signaling competence in nascent daughter cells. However, mutations that limit or enhance the release of stored signaling components could alter daughter cell fate or behavior thereby promoting oncogenesis., Competing Interests: The authors have declared that no competing interests exist.

Cell cycle progression and lipid metabolism are well-coordinated processes required for proper cell proliferation. In liver diseases that arise from dysregulated lipid metabolism, proliferation is diminished. To study the outcome of CDK1 loss and blocked hepatocyte proliferation on lipid metabolism and the consequent impact on whole-body physiology, we performed lipidomics, metabolomics, and RNA-seq analyses on a mouse model. We observed reduced triacylglycerides in liver of young mice, caused by oxidative stress that activated FOXO1 to promote expression of Pnpla2 /ATGL. Additionally, we discovered that hepatocytes displayed malfunctioning β-oxidation, reflected by increased acylcarnitines (ACs) and reduced β-hydroxybutyrate. This led to elevated plasma free fatty acids (FFAs), which were transported to the adipose tissue for storage and triggered greater insulin secretion. Upon aging, chronic hyperinsulinemia resulted in insulin resistance and hepatic steatosis through activation of LXR. Here, we demonstrate that loss of hepatocyte proliferation is not only an outcome but also possibly a causative factor for liver pathology., Competing Interests: JO, MC, GZ, JF, HL, SG, HW, AC, CO, MW, WH, HC, PK No competing interests declared, (© 2020, Ow et al.)

The sequential generation of layer-specific cortical neurons requires radial glia cells (RGCs) to precisely balance self-renewal and lineage commitment. While specific cell-cycle phases have been associated with these decisions, the mechanisms linking the cell-cycle machinery to cell-fate commitment remain obscure. Using single-cell RNA-sequencing, we find that the strongest transcriptional signature defining multipotent RGCs is that of G2/M-phase, and particularly CYCLIN-B1/2, while lineage-committed progenitors are enriched in G1/S-phase genes, including CYCLIN-D1. These data also reveal cell-surface markers that allow us to isolate RGCs and lineage-committed progenitors, and functionally confirm the relationship between cell-cycle phase enrichment and cell fate competence. Finally, we use cortical electroporation to demonstrate that CYCLIN-B1/2 cooperate with CDK1 to maintain uncommitted RGCs by activating the NOTCH pathway, and that CYCLIN-D1 promotes differentiation. Thus, this work establishes that cell-cycle phase-specific regulators act in opposition to coordinate the self-renewal and lineage commitment of RGCs via core stem cell regulatory pathways.

Ustilaginoidea virens is an important fungus causing rice false smut, a devastating disease on spikelets of rice. In this study, we identified and characterized two CMGC (CDK/MAPK/GSK3/CLK) kinase genes, UvPmk1 and UvCDC2, in U. virens. Although UvPmk1 and UvCDC2 are, respectively, homologous to Fus3/Kss1 mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs), they all have a conserved serine/threonine protein kinase domain. The qRT-PCR analysis of the relative expression of UvPmk1 and UvCDC2 during the infection of U. virens showed that these two genes were highly expressed during infection. UvPmk1 and UvCDC2 knockout mutants exhibited no significant changes in mycelial vegetative growth but decreases in conidiation. In addition, both UvPmk1 and UvCDC2 knockout mutants showed increases in tolerance to hyperosmotic and cell wall stresses, but they, respectively, exhibited decreases and increases in tolerance to oxidative stress compared with the wild-type strain HWD-2. Pathogenicity and infection assays demonstrated the defective growth of infection hyphae and significant loss of virulence in UvPmk1 and UvCDC2 knockout mutants. Taken together, our results demonstrate that UvPmk1 and UvCDC2 play important roles in the conidiation, stress response, and pathogenicity of U. virens.

Background: Colorectal cancer (CRC) is one of the most common malignancies of the digestive system, which causes severe financial burden worldwide. However, the specific mechanisms involved in CRC are still unclear., Methods: To identify the significant genes and pathways involved in the initiation and progression of CRC, the microarray dataset GSE126092 was downloaded from Gene Expression Omnibus (GEO) database, and then, the data was analyzed to identify differentially expressed genes (DEGs). Subsequently, the Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on these DEGs using the DAVID database, and the protein-protein interaction (PPI) network was constructed using the STRING database and analyzed using the Cytoscape software. Finally, hub genes were screened, and the survival analysis was performed on these hub genes using the Kaplan-Meier curves in the cBioPortal database., Results: In total, 937 DEGs were obtained, including 316 upregulated genes and 621 downregulated genes. GO analysis revealed that the DEGs were mostly enriched in terms of nuclear division, organelle fission, cell division, and cell cycle process. KEGG pathway analysis showed that the DEGs were mostly enriched in cell cycle, oocyte meiosis, cytokine-cytokine receptor interaction, and cGMP-PKG signaling pathway. The PPI network comprised 608 nodes and 3100 edges, and 4 significant modules and 10 hub genes with the highest degree were identified using the Cytoscape software. Finally, survival analysis showed that overexpression of CDK1 and CDC20 in patients with CRC were statistically associated with worse overall survival., Conclusions: This bioinformatics analysis revealed that CDK1 and CDC20 might be candidate targets for diagnosis and treatment of CRC, which provided valuable clues for CRC.

Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of the Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr-187. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was enough for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.

Accurate division of cells into two daughters is a process that is vital to propagation of life. Protein phosphorylation and selective degradation have emerged as two important mechanisms safeguarding the delicate choreography of mitosis. Protein phosphatases catalyze dephosphorylation of thousands of sites on proteins, steering the cells through establishment of the mitotic phase and exit from it. A large E3 ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) becomes active during latter stages of mitosis through G1 and marks hundreds of proteins for destruction. Recent studies have revealed the complex interregulation between these two classes of enzymes. In this review, we highlight the direct and indirect mechanisms by which phosphatases and the APC/C mutually influence each other to ensure accurate spatiotemporal and orderly progression through mitosis, with a particular focus on recent insights and conceptual advances.

Telomeres located at the ends of linear chromosomes play important roles in the maintenance of life. Rap1, a component of the shelterin telomere protein complex, interacts with multiple proteins to perform various functions; further, formation of shelterin requires Rap1 binding to other components such as Taz1 and Poz1, and telomere tethering to the nuclear envelope (NE) involves interactions between Rap1 and Bqt4, a nuclear membrane protein. Although Rap1 is a hub for telomere protein complexes, the regulatory mechanisms of its interactions with partner proteins are not fully understood. Here, we show that Rap1 is phosphorylated by casein kinase 2 (CK2) at multiple sites, which promotes interactions with Bqt4 and Poz1. Among the multiple CK2-mediated phosphorylation sites of Rap1, phosphorylation at Ser496 was found to be crucial for both Rap1-Bqt4 and Rap1-Poz1 interactions. These mechanisms mediate proper telomere tethering to the NE and the formation of the silenced chromatin structure at chromosome ends., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SE translocation (SET), an inhibitor of protein phosphatase 2A (PP2A), plays important roles in mitosis and possesses oncogenic activity in several types of cancer. However, little is known regarding its regulation. Here we reveal a novel phosphorylation site of SET isoform 1, and we have determined its biological significance in tumorigenesis. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates SET isoform 1 in vitro and in vivo at serine 7 during antitubulin drug-induced mitotic arrest and normal mitosis. SET deletion resulted in massive multipolar spindles, chromosome misalignment and missegregation, and centrosome amplification during mitosis. Moreover, mitotic phosphorylation of SET isoform 1 is required for cell migration, invasion, and anchorage-independent growth in vitro and tumorigenesis in xenograft animal models. We further documented that SET phosphorylation affects Akt activity. Collectively, our findings suggest that SET isoform 1 promotes oncogenesis in a mitotic phosphorylation-dependent manner.

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in S. cerevisiae . This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division., Competing Interests: The authors have declared that no competing interests exist.

Oogenesis in the urochordate, Oikopleura dioica, occurs in a large coenocyst in which vitellogenesis precedes oocyte selection in order to adapt oocyte production to nutrient conditions. The animal has expanded Cyclin-Dependant Kinase 1 (CDK1) and Cyclin B paralog complements, with several expressed during oogenesis. Here, we addressed functional redundancy and specialization of CDK1 and cyclin B paralogs during oogenesis and early embryogenesis through spatiotemporal analyses and knockdown assays. CDK1a translocated from organizing centres (OCs) to selected meiotic nuclei at the beginning of the P4 phase of oogenesis, and its knockdown impaired vitellogenesis, nurse nuclear dumping, and entry of nurse nuclei into apoptosis. CDK1d-Cyclin Ba translocated from OCs to selected meiotic nuclei in P4, drove meiosis resumption and promoted nuclear envelope breakdown (NEBD). CDK1d-Cyclin Ba was also involved in histone H3S28 phosphorylation on centromeres and meiotic spindle assembly through regulating Aurora B localization to centromeres during prometaphase I. In other studied species, Cyclin B3 commonly promotes anaphase entry, but we found O. dioica Cyclin B3a to be non-essential for anaphase entry during oogenic meiosis. Instead, Cyclin B3a contributed to meiotic spindle assembly though its loss could be compensated by Cyclin Ba.

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial ( Escherichia coli ) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan ( Saccharomyces cerevisiae ) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: The epidermal growth factor (EGF) receptor (EGFR) pathway is one of the most dysregulated and extensively investigated signaling pathways in human cancers and plays important roles in the regulation of nuclear functions through both cytoplasmic and nuclear EGFR pathways. However, the current understanding of the nuclear phosphorylation responses to activated EGFR pathways remains limited. In the present study, phosphoproteomics analysis revealed the increased phosphorylation of 90 nuclear proteins, primarily involved in RNA processing, pre-mRNA splicing and cell cycle regulation, upon EGF stimulation in MDA-MB-468 cells. Cellular splicing assays of the β-globin (HBB) minigene confirmed that EGF induced constitutive pre-mRNA splicing. Further analysis of phosphoproteomics data identified multiple CDK1/2 substrates in pre-mRNA splicing-related proteins, and both CDK1/2 inhibitors and CDK1/2 knockdowns reduced EGF-regulated pre-mRNA splicing. In conclusion, the results of the present study provide evidence that CDK1/2 participate in the regulation of constitutive pre-mRNA splicing by EGF stimulation in MDA-MB-468 cells., Significance: In this study, we successfully carried out a survey of nuclear phosphorylation changes in response to EGF stimulation. The results from the functional category analysis and pre-mRNA splicing assay strongly indicated that EGFR activation increased constitutive pre-mRNA splicing in MDA-MB-468 cells, revealing additional role of EGFR on regulation of mRNA maturation beyond alternative pre-mRNA splicing reported by previous studies. Furthermore, we found that CDK1/2 participated in constitutive pre-mRNA splicing regulation by EGF in MDA-MB-468 cells. Our study provides new knowledge for understanding the regulation of constitutive pre-mRNA splicing by EGF stimulation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2'-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Meiotic progression requires the translation of maternal mRNAs in a strict temporal order. In isolated animal oocytes, translation of maternal mRNAs containing a cytoplasmic polyadenylation element (CPE), such as cyclin B, is activated by in vitro stimulation of meiotic resumption which induces phosphorylation of CPEB (CPE-binding protein) and elongation of their polyadenosine (poly(A)) tails; whether or not this model can be applied in vivo to oocytes arrested at metaphase of meiosis I in ovaries is unknown. In this study, we found that active CDK1 (cyclin-dependent kinase 1) phosphorylated CPEB in ovarian oocytes arrested at metphase I in the starfish body cavity, but phosphorylation of CPEB was not sufficient for elongation of cyclin B poly(A) tails. Immediately after spawning, however, mRNA was polyadenylated, suggesting that an increase in intracellular pH (pHi ) upon spawning triggers the elongation of poly(A) tails. Using a cell-free system made from maturing oocytes at metaphase I, we demonstrated that polyadenylation was indeed suppressed at pH below 7.0. These results suggest that a pH-sensitive process, functioning after CPEB phosphorylation, is blocked under physiologically low pHi (<7.0) in metaphase-I-arrested oocytes. The increase in pHi (>7.0) that occurs after spawning triggers polyadenylation of cyclin B mRNA and progression into meiosis II., (© 2015 Wiley Periodicals, Inc.)

Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity., (© 2015. Published by The Company of Biologists Ltd.)

The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho-SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing., (© 2015 Tavernier et al.)

In Schizosaccharomyces pombe, late mitotic events are coordinated with cytokinesis by the septation initiation network (SIN), an essential spindle pole body (SPB)-associated kinase cascade, which controls the formation, maintenance, and constriction of the cytokinetic ring. It is not fully understood how SIN initiation is temporally regulated, but it depends on the activation of the GTPase Spg1, which is inhibited during interphase by the essential bipartite GTPase-activating protein Byr4-Cdc16. Cells are particularly sensitive to the modulation of Byr4, which undergoes cell cycle-dependent phosphorylation presumed to regulate its function. Polo-like kinase, which promotes SIN activation, is partially responsible for Byr4 phosphorylation. Here we show that Byr4 is also controlled by cyclin-dependent kinase (Cdk1)-mediated phosphorylation. A Cdk1 nonphosphorylatable Byr4 phosphomutant displays severe cell division defects, including the formation of elongated, multinucleate cells, failure to maintain the cytokinetic ring, and compromised SPB association of the SIN kinase Cdc7. Our analyses show that Cdk1-mediated phosphoregulation of Byr4 facilitates complete removal of Byr4 from metaphase SPBs in concert with Plo1, revealing an unexpected role for Cdk1 in promoting cytokinesis through activation of the SIN pathway., (© 2014 Rachfall et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Errors in chromosome segregation are safeguarded by the spindle assembly checkpoint. Yet the very defects that trigger this checkpoint are inescapable consequences of sister chromatid segregation in anaphase. Three new studies provide clues to how cells cope with this problem., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Activation of anaphase-promoting complex/cyclosome (APC/C(Cdc20)) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/C(Cdc20) degrades cyclin B and securin, which activates separase [1]. The latter then cleaves cohesin holding sister chromatids together [2]. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1 [3-5]. To test this, we analyzed mouse oocytes and embryos expressing nondegradable cyclin B together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/C(Cdc20) activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/C(Cdc20) inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/C(Cdc20) substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores [1]. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/C(Cdc20)) [2]. Upon satisfaction of both pathways, the APC/C(Cdc20) elicits the degradation of securin and cyclin B [3]. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood [4]. Here we show that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/C(Cdc20) is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. We propose that the simultaneous destruction of securin and cyclin B elicited by the APC/C(Cdc20) couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cdc2 kinase is a catalytic subunit of the maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocytes. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized. In this study, a complete cDNA sequence of Cdc2 kinase was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. The Cdc2 cDNA (1,769 bp) encodes for a 299 amino acid protein with a calculated molecular weight of 34.7 kDa. Quantitative real-time PCR demonstrated that Cdc2 mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. Immunohistochemistry analysis revealed that the Cdc2 protein relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that Cdc2 kinase may play an important role in the gametogenesis and gonad development in C. quadricarinatus., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Human SERPINB5, commonly known as maspin, has diverse functions as a tumor suppressor. In this study, we discovered that maspin has a novel role in cell cycle control, and common variants were discovered to be associated with gastric cancer. The genotypes of 836 unrelated Korean participants (including 430 with gastric cancer) were examined for 12 tag single-nucleotide polymorphisms (SNPs) and imputed for 178 SNPs in the maspin gene. Susceptibility to diffuse-type gastric cancer was strongly and significantly associated with several SNPs including rs3744941 (C>T) in the promoter (TT versus CC+CT, odds ratio = 0.56 [0.37-0.83], P = 0.0038) and rs8089104 (C>T) in intron 1 (TT+CT versus CC, odds ratio = 1.7 [1.2-2.5], P = 0.0021). No SNPs were associated with susceptibility to intestinal-type gastric cancer. A haplotype of three highly correlated promoter SNPs associated with higher cancer risk showed 40% of the activity of a non-risk-associated haplotype promoter in the diffuse-type gastric cancer cell line MKN45. Maspin downregulation achieved either by a short hairpin RNA targeting maspin or overexpression of the E2F1-DP1 complex in MKN45 cells dramatically accelerated cell cycle progression and caused an increase of active CDC25C levels and a decrease of inactive CDK1 levels. In contrast, maspin upregulation had the opposite effect, substantially retarding cell proliferation. Therefore, our results suggest that a maspin promoter haplotype that reduces maspin gene expression accelerates cell cycle progression and, consequently, is associated with increased susceptibility to diffuse-type gastric cancer. Furthermore, a novel maspin-related pathway is demonstrated to underlie gastric carcinogenesis.

Cell division requires careful orchestration of three major events: entry into mitosis, chromosomal segregation, and cytokinesis. Signaling within and between the molecules that control these events allows for their coordination via checkpoints, a specific class of signaling pathways that ensure the dependency of cell-cycle events on the successful completion of preceding events. Multiple positive- and negative-feedback loops ensure that a cell is fully committed to division and that the events occur in the proper order. Unlike other signaling pathways, which integrate external inputs to decide whether to execute a given process, signaling at cell division is largely dedicated to completing a decision made in G1 phase-to initiate and complete a round of mitotic cell division. Instead of deciding if the events of cell division will take place, these signaling pathways entrain these events to the activation of the cell-cycle kinase cyclin-dependent kinase 1 (CDK1) and provide the opportunity for checkpoint proteins to arrest cell division if things go wrong.

The epidermis is a self-renewing tissue, the homeostasis of which is dependent upon the tight balance between proliferation and differentiation based on appropriate regulation of the cell cycle. The cell cycle regulation is dependent on the interactions among a number of cell cycle regulatory molecules, including the pituitary tumor-transforming gene 1 (PTTG1), also known as securin, a regulator of sister chromatid separation and transition from metaphase to anaphase. This study was conducted to clarify the less-known functions of PTTG1 in the epidermis by the use of keratinocytes cultured under two-dimensional (2D) or three-dimensional (3D) conditions. Forced overexpression of PTTG1 caused upregulation of cyclin B1, cyclin-dependent kinase 1 (CDK1), and c-Myc, resulting in enhanced proliferation and suppression of early differentiation without apparent alterations in terminal differentiation, and the exogenous PTTG1 was downregulated in association with cell cycle exit. In contrast, depletion of PTTG1 caused their downregulation and constrained proliferation with retention of differentiation capacity. These findings suggested that PTTG1 could alter the proliferation status by modulating the expression levels of the other cell cycle regulatory proteins, and excess PTTG1 primarily affects early differentiation of keratinocytes under the stability regulation associated with cell cycle exit.

The duration of S phase in early embryos is often short, and then increases as development proceeds because of the appearance of late-replicating regions of the genome. In the April 1, 2012, issue of Genes & Development, Farrell and colleagues (pp. 714-725) demonstrate that the down-regulation of cyclin-dependent kinase 1 (Cdk1) activity triggers the onset of late-replicating DNA and an increase in S-phase length in Drosophila embryos, revealing an unexpected role for Cdk1 in replication control during development.

During embryonic cell cycles, B-cyclin-CDKs function as the core component of an autonomous oscillator. Current models for the cell-cycle oscillator in nonembryonic cells are slightly more complex, incorporating multiple G1, S phase, and mitotic cyclin-CDK complexes. However, periodic events persist in yeast cells lacking all S phase and mitotic B-cyclin genes, challenging the assertion that cyclin-CDK complexes are essential for oscillations. These and other results led to the proposal that a network of sequentially activated transcription factors functions as an underlying cell-cycle oscillator. Here we examine the individual contributions of a transcription factor network and cyclin-CDKs to the maintenance of cell-cycle oscillations. Our findings suggest that while cyclin-CDKs are not required for oscillations, they do contribute to oscillation robustness. A model emerges in which cyclin expression (thereby, CDK activity) is entrained to an autonomous transcriptional oscillator. CDKs then modulate oscillator function and serve as effectors of the oscillator., (Copyright © 2012 Elsevier Inc. All rights reserved.)

During mitosis, equal segregation of chromosomes depends on proper kinetochore-microtubule attachments. Merotelic kinetochore orientation, in which a single kinetochore binds microtubules from both spindle poles [1], is a major cause of chromosome instability [2], which is commonly observed in solid tumors [3, 4]. Using the fission yeast Schizosaccharomyces pombe, we show that a proper force balance between kinesin motors on interpolar spindle microtubules is critical for correcting merotelic attachments. Inhibition of the plus-end-directed spindle elongation motors kinesin-5 (Cut7) and kinesin-6 (Klp9) reduces spindle length, tension at kinetochores, and the frequency of merotelic attachments. In contrast, merotely is increased by deletion of the minus-end-directed kinesin-14 (Klp2) or overexpression of Klp9. Also, Cdk1 regulates spindle elongation forces to promote merotelic correction by phosphorylating and inhibiting Klp9. The role of spindle elongation motors in merotelic correction is conserved, because partial inhibition of the human kinesin-5 homolog Eg5 using the drug monastrol reduces spindle length and lagging chromosome frequency in both normal (RPE-1) and tumor (CaCo-2) cells. These findings reveal unexpected links between spindle forces and correction of merotelic attachments and show that pharmacological manipulation of spindle elongation forces might be used to reduce chromosome instability in cancer cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore-microtubule attachment. Removal of the checkpoint-activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi-oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC-induced arrest. We show that Cdk1 promotes kinetochore bi-orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC-induced mitotic arrest by regulating bi-orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti-mitotic drugs.

Mcl-1 is one of the major anti-apoptotic members of the Bcl-2 family of apoptotic regulatory proteins. In this study we investigated the role of Mcl-1 in mitotic arrest-induced apoptosis. Vinblastine treatment of KB-3 cells initially resulted in a phosphatase-sensitive mobility shift in Mcl-1 and then subsequent loss of Mcl-1 protein expression which was prevented by MG132, suggesting that phosphorylation triggered proteosome-mediated degradation. Mcl-1 phosphorylation/degradation was a specific response to microtubule inhibition and did not occur in response to lethal concentrations of DNA damaging agents. Vinblastine treatment caused degradation of Mcl-1 in cells in which apoptosis was blocked by Bcl-xL overexpression, indicating that Mcl-1 degradation was not a consequence of apoptosis. A partial reversible phosphorylation of Mcl-1 was observed in synchronized cells traversing mitosis, whereas more extensive phosphorylation and subsequent degradation of Mcl-1 was observed if synchronized cells were treated with vinblastine. Mcl-1 phosphorylation closely paralleled cyclin B expression, and specific cyclin-dependent kinase (Cdk) inhibitors blocked vinblastine-induced Mcl-1 phosphorylation, its subsequent degradation, and improved cell viability after mitotic arrest. Co-immunoprecipitation studies indicated that Mcl-1 was complexed with Bak, but not Bax or Noxa, in untreated cells, and that Bak became activated in concert with loss of Mcl-1 expression. These results suggest that Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis via Mcl-1 phosphorylation, promoting its degradation and subsequently releasing Bak from sequestration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Emerging evidence shows that the translocation of apoptosis related factors on cellular organelles, such as mitochondria, endoplasmic reticulum, Golgi apparatus and nucleus, has a crucial role in the apoptotic process. We characterized the effect of paclitaxel (Sigma®) on Golgi involved apoptosis in human hormone refractory prostate cancer., Materials and Methods: FACScan™ flow cytometric analysis was used to determine cell cycle distribution and the subG1 (apoptosis) population. Protein expression and localization were detected by Western blot, confocal microscopic examination and the sucrose gradient separation technique., Results: Paclitaxel induced Golgi apparatus disassembly and interaction between Golgi complexes and mitochondria. Discontinuous sucrose gradient fractionation was used to determine and collect Golgi containing fractions. Data revealed that paclitaxel induced an increase of Cdk1 activity and DR5 expression on the Golgi complex that was associated with increased cleavage of caspase-8, a DR5 downstream factor, and caspase-3 into catalytically active fragments. Data were validated by confocal immunofluorescence microscopy. Golgi associated effects were inhibited by the Cdk1 inhibitor roscovitine (Sigma), suggesting a critical role for Golgi-Cdk1. Also, paclitaxel caused an increase of nuclear but not of Golgi associated PKC-δ activity. The selective PKC-δ inhibitor rottlerin (Sigma) completely inhibited the increase of Golgi-Cdk1 activity, suggesting that nuclear PKC-δ served as an upstream regulator of Golgi-Cdk1., Conclusions: Data suggest that paclitaxel induces nuclear translocation and activation of PKC-δ, which in turn causes Golgi-Cdk1 activation, leading to Golgi associated DR5 up-regulation, and caspase-8 and 3 activation. Golgi mediated signaling cascades facilitate mitochondria involved apoptotic pathways and at least partly explain the anticancer activity of paclitaxel action., (Copyright © 2011 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Protracted mitotic arrest leads to cell death; however, the molecular signals that link these distinct processes remain poorly understood. Here we report that the pro-apoptotic BH3-only family member Bim undergoes phosphorylation in K562 cells following treatment with the microtubule targeting agents Taxol and Nocodazole. The phosphorylation of two Bim isoforms, BimEL and BimL, at the mitochondria correlates with mitotic arrest and precedes cell death induced by Taxol. It was also found that Bim undergoes transient phosphorylation during normal mitosis in K562 cells. In addition, siRNA silencing of Bim reduces sensitivity to Taxol-induced cell death. The transition of K562 cells from mitosis to G1 results in the loss of BimEL and BimL phosphorylation and correlates with the degradation of cyclin B1. The Cdk1 inhibitors, RO-3306 and Purvalanol A, block Bim phosphorylation in mitotically arrested cells. Importantly, it was found that cyclin B1 co-immunoprecipitates with endogenous Bim in mitotic extracts. Furthermore, active recombinant Cdk1/cyclin B1 phosphorylates BimEL and BimL in vitro and Serine 44 on BimL has been identified as a Cdk1 phosphorylation site. Collectively, these results suggest that Cdk1/cyclin B1-dependent hyper-phosphorylation of Bim during prolonged mitotic arrest is an important cell death signal.

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This short review updates the maturation-inducing hormonal signaling in starfish oocytes. In this system, the activation of cyclin B-Cdc2 kinase (Cdk1) that leads to meiotic resumption does not require new protein synthesis. The key intracellular mediator after hormonal stimulation by 1-methyladenine is the protein kinase Akt/PKB, which in turn directly downregulates Myt1 and upregulates Cdc25 toward the activation of cyclin B-Cdc2. Mitotic kinases including Aurora, Plk1 and Greatwall are activated downstream of cyclin B-Cdc2. The starfish oocyte thus provides a simple model system for the study of meiotic resumption., (Copyright © 2011 Wiley Periodicals, Inc.)

DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5' strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11-Rad50-Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase-topoisomerase complex Sgs1-Top3-Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways.

Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.

The mitosis-to-interphase transition involves dramatic cellular reorganization from a state that supports chromosome segregation to a state that complies with all functions of an interphase cell. This process, termed mitotic exit, depends on the removal of mitotic phosphorylations from a broad range of substrates. Mitotic exit regulation involves inactivation of mitotic kinases and activation of counteracting protein phosphatases. The key mitotic exit phosphatase in budding yeast, Cdc14, is now well understood. By contrast, in animal cells, it is now emerging that mitotic exit relies on distinct regulatory networks, including the protein phosphatases PP1 and PP2A.

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

Cells that are deficient in homologous recombination, such as those that lack functional breast cancer-associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclin-dependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA-wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function and consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers.