The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition

The Aurora B abscission checkpoint delays cytokinesis until resolution of DNA trapped in the cleavage furrow. This process involves PKCε phosphorylation of Aurora B S227. Assessing if this PKCε-Aurora B module provides a more widely exploited genome-protective control for the cell cycle, we show Aurora B phosphorylation at S227 by PKCε also occurs during mitosis. Expression of Aurora B S227A phenocopies inhibition of PKCε in by-passing the delay and resolution at anaphase entry that is associated with non-disjunction and catenation of sister chromatids. Implementation of this anaphase delay is reflected in PKCε activation following cell cycle dependent cleavage by caspase 7; knock-down of caspase 7 phenocopies PKCε loss, in a manner rescued by ectopically expressing/generating a free PKCε catalytic domain. Molecular dynamics indicates that Aurora B S227 phosphorylation induces conformational changes and this manifests in a profound switch in specificity towards S29 TopoIIα phosphorylation, a response necessary for catenation resolution during mitosis.This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001130), the UK Medical Research Council (FC001130) and the Wellcome Trust (FC001130).Peer reviewe

Mitotic catenation is monitored and resolved by a PKCε-regulated pathway.

Exit from mitosis is controlled by silencing of the spindle assembly checkpoint (SAC). It is important that preceding exit, all sister chromatid pairs are correctly bioriented, and that residual catenation is resolved, permitting complete sister chromatid separation in the ensuing anaphase. Here we determine that the metaphase response to catenation in mammalian cells operates through PKCε. The PKCε-controlled pathway regulates exit from the SAC only when mitotic cells are challenged by retained catenation and this delayed exit is characterized by BubR1-high and Mad2-low kinetochores. In addition, we show that this pathway is necessary to facilitate resolution of retained catenanes in mitosis. When delayed by catenation in mitosis, inhibition of PKCε results in premature entry into anaphase with PICH-positive strands and chromosome bridging. These findings demonstrate the importance of PKCε-mediated regulation in protection from loss of chromosome integrity in cells failing to resolve catenation in G2

Studying kinetochore kinases

Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics of their localized activation and deactivation is therefore instrumental for understanding the spatiotemporal control of chromosome segregation

Purification of Reversibly Oxidized Proteins (PROP) Reveals a Redox Switch Controlling p38 MAP Kinase Activity

Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity

Centriole movements in mammalian epithelial cells during cytokinesis

<p>Abstract</p> <p>Background</p> <p>In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules.</p> <p>Results</p> <p>Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent.</p> <p>Conclusions</p> <p>These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.</p

Targeting Sphingosine Kinase 1 in Carcinoma Cells Decreases Proliferation and Survival by Compromising PKC Activity and Cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy

14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells

Amygdala 14-3-3ζ as a Novel Modulator of Escalating Alcohol Intake in Mice

Alcoholism is a devastating brain disorder that affects millions of people worldwide. The development of alcoholism is caused by alcohol-induced maladaptive changes in neural circuits involved in emotions, motivation, and decision-making. Because of its involvement in these processes, the amygdala is thought to be a key neural structure involved in alcohol addiction. However, the molecular mechanisms that govern the development of alcoholism are incompletely understood. We have previously shown that in a limited access choice paradigm, C57BL/6J mice progressively escalate their alcohol intake and display important behavioral characteristic of alcohol addiction, in that they become insensitive to quinine-induced adulteration of alcohol. This study used the limited access choice paradigm to study gene expression changes in the amygdala during the escalation to high alcohol consumption in C57BL/6J mice. Microarray analysis revealed that changes in gene expression occurred predominantly after one week, i.e. during the initial escalation of alcohol intake. One gene that stood out from our analysis was the adapter protein 14-3-3ζ, which was up-regulated during the transition from low to high alcohol intake. Independent qPCR analysis confirmed the up-regulation of amygdala 14-3-3ζ during the escalation of alcohol intake. Subsequently, we found that local knockdown of 14-3-3ζ in the amygdala, using RNA interference, dramatically augmented alcohol intake. In addition, knockdown of amygdala 14-3-3ζ promoted the development of inflexible alcohol drinking, as apparent from insensitivity to quinine adulteration of alcohol. This study identifies amygdala 14-3-3ζ as a novel key modulator that is engaged during escalation of alcohol use

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism