A positive feedback loop involving Haspin and Aurora B promotes CPC accumulation at centromeres in mitosis

Haspin phosphorylates histone H3 at Thr3 (H3T3ph) during mitosis [1, 2], providing a chromatin binding site for the chromosomal passenger complex (CPC) at centromeres to regulate chromosome segregation [3-5]. H3T3ph becomes increasingly focused at inner centromeres during prometaphase [1, 2], but little is known about how its level or location and the consequent chromosomal localization of the CPC are regulated. In addition, CPC binding to shugoshin proteins contributes to centromeric Aurora B localization [5, 6]. Recruitment of the shugoshins to centromeres requires the phosphorylation of histone H2A at Thr120 (H2AT120ph) by the kinetochore kinase Bub1 [7], but the molecular basis for the collaboration of this pathway with H3T3ph has been unclear. Here, we show that Aurora B phosphorylates Haspin to promote generation of H3T3ph and that Aurora B kinase activity is required for normal chromosomal localization of the CPC, indicating an intimate linkage between Aurora B and Haspin functions in mitosis. We propose that Aurora B activity triggers a CPC-Haspin-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin. We also provide evidence that the Bub1-shugoshin-CPC pathway supplies a signal that boosts the CPC-Haspin-H3T3ph feedback loop specifically at centromeres to produce the well-known accumulation of the CPC in these regions.     

OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases

OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2A^T120 and H3^T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays.     

Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr³ of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg² and Lys⁴ adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys⁴ of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser¹³⁷ of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser¹³⁷ resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser¹³⁷ might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.Eukaryotic protein kinases (ePK)¹ constitute a large family of enzymes that coordinate virtually any cellular processes by the phosphorylation of their target proteins at specific sites (1, 2). Active kinases often modulate the activity of other enzymes, including other kinases, thus amplifying and extending an initial signal that affect sometimes thousands of proteins (3). This creates a highly complex network of feedback and forward loops where multiple kinases can mutually influence each other''s activity. Kinases adopt three molecular strategies to select and specifically phosphorylate their substrates in the crowded environment of a cell (2). First, tight control of cellular kinase localization assures that only proteins present in the close proximity of the kinase can be phosphorylated; second, the kinase specific activity can be regulated via post-translational modifications or the recruitment of cofactor molecules; and third, the recognition of specific consensus motifs on substrates ensures that phosphorylation only occurs at the intended site or sites (2).The Haspin kinase is a member of the ePK family that structurally diverges from most ePKs (1, 4). The Haspin kinase domain displays structural features that have never been observed in other ePK family members (5, 6). Specifically, the possibility of activation loop phosphorylation, a frequent regulatory mechanisms to control kinase activity, is absent in Haspin (5). Haspin is characterized by an active conformation that is stabilized by a hydrophobic lock of the helix αC inducing a stable S conformation of the structurally unique activation segment. These specific structural features also create a structurally diverse substrate binding site comprising a highly electronegative cleft for the histone H3 basic tails (5). Interestingly, the recognition of H3 has been shown to be modulated by methylation at H3 residue Lys⁴, thus coupling Haspin activity with epigenetic mechanisms of chromatin regulation (5). Histone H3 that is phosphorylated at Thr³ is so far the only well-characterized Haspin substrate (7). H3Thr³ phosphorylation (H3Thr^3ph) is required for the localization of Aurora B at the centromere (8–10). Inactivation of Haspin catalytic activity by ATP mimetic inhibitors induces Aurora B centromeric delocalization, leading to a loss of phosphorylation in chromatin associated Aurora B substrates (11, 12). To date, apart from this well-characterized centromeric function of Haspin activity, the broader cellular functions of the kinase and the phosphorylation events that control these remain essentially unknown.In this study, we used an integrated biochemical, proteomic, pharmacologic, and structural biology approach to study the Haspin kinase, its substrates and the cellular consequences of its activity. Specifically, we determined a new mode of kinase substrate binding and identified a Haspin kinase substrate recognition motif. We identified 3964 phosphorylation sites in chromatin-associated proteins, quantified their response to Haspin inhibition, and verified the mitotic phosphorylation of MacroH2A Ser¹³⁷ (13) as directly dependent by Haspin activity. Altogether, our data suggest that Haspin regulates the phosphorylation of proteins involved in mechanisms that control gene expression, including the modifications of histones, and provide evidence for novel molecular effects of Haspin activity on mitotic chromatin.     

HP1α targets the chromosomal passenger complex for activation at heterochromatin before mitotic entry

The chromosomal passenger complex (CPC) is directed to centromeres during mitosis via binding to H3T3ph and Sgo1. Whether and how heterochromatin protein 1α (HP1α) influences CPC localisation and function during mitotic entry is less clear. Here, we alter HP1α dynamics by fusing it to a CENP‐B DNA‐binding domain. Tethered HP1 strongly recruits the CPC, destabilising kinetochore–microtubule interactions and activating the spindle assembly checkpoint. During mitotic exit, the tethered HP1 traps active CPC at centromeres. These HP1‐CPC clusters remain catalytically active throughout the subsequent cell cycle. We also detect interactions between endogenous HP1 and the CPC during G₂. HP1α and HP1γ cooperate to recruit the CPC to active foci in a CDK1‐independent process. Live cell tracking with Fab fragments reveals that H3S10ph appears well before H3T3 is phosphorylated by Haspin kinase. Our results suggest that HP1 may concentrate and activate the CPC at centromeric heterochromatin in G₂ before Aurora B‐mediated phosphorylation of H3S10 releases HP1 from chromatin and allows pathways dependent on H3T3ph and Sgo1 to redirect the CPC to mitotic centromeres.     

Heterochromatin and cohesion protection at human centromeres: the final say of a long controversy?

Mammalian centromeres are embedded within heterochromatin, a specialized chromatin assembled onto repetitive DNA that forms the primary constriction of chromosomes. In early mitosis, the bulk of cohesin dissociates from chromosomes, but a small fraction is spared at the centromere providing the ultimate linker between sister chromatid pairs, essential for their proper attachment to the mitotic spindle. Whether heterochromatin plays a role in the protection of centromere cohesion has long been controversial. In this issue of EMBO Reports, Yi et al show that heterochromatin protein 1 (HP1) isoforms α and γ act redundantly to protect mitotic centromere cohesion through the recruitment of the cohesion protector Haspin 1 .     

Cohesion and centromere activity are required for phosphorylation of histone H3 in maize

Haspin‐mediated phosphorylation of histone H3 at threonine 3 (H3T3ph) promotes proper deposition of Aurora B at the inner centromere to ensure faithful chromosome segregation in metazoans. However, the function of H3T3ph remains relatively unexplored in plants. Here, we show that in maize (Zea mays L.) mitotic cells, H3T3ph is concentrated at pericentromeric and centromeric regions. Additional weak H3T3ph signals occur between cohered sister chromatids at prometaphase. Immunostaining on dicentric chromosomes reveals that an inactive centromere cannot maintain H3T3ph at metaphase, indicating that a functional centromere is required for H3T3 phosphorylation. H3T3ph locates at a newly formed centromeric region that lacks detectable CentC sequences and strongly reduced CRM and ZmBs repeat sequences at metaphase II. These results suggest that centromeric localization of H3T3ph is not dependent on centromeric sequences. In maize meiocytes, H3T3 phosphorylation occurs at the late diakinesis and extends to the entire chromosome at metaphase I, but is exclusively limited to the centromere at metaphase II. The H3T3ph signals are absent in the afd1 (absence of first division) and sgo1 (shugoshin) mutants during meiosis II when the sister chromatids exhibit random distribution. Further, we show that H3T3ph is mainly located at the pericentromere during meiotic prophase II but is restricted to the inner centromere at metaphase II. We propose that this relocation of H3T3ph depends on tension at the centromere and is required to promote bi‐orientation of sister chromatids.     

Regulation of mitotic chromosome cohesion by Haspin and Aurora B

In vertebrate mitosis, cohesion between sister chromatids is lost in two stages. In prophase and prometaphase, cohesin release from chromosome arms occurs under the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to prevent removal of centromeric cohesin until anaphase. The regulatory enzymes that act to sustain centromeric cohesion are incompletely described, however. Haspin/Gsg2 is a histone H3 threonine-3 kinase required for normal mitosis. We report here that both H3 threonine-3 phosphorylation and cohesin are located at inner centromeres. Haspin depletion disrupts cohesin binding and sister chromatid association in mitosis, preventing normal chromosome alignment and activating the spindle assembly checkpoint, leading to arrest in a prometaphase-like state. Overexpression of Haspin hinders cohesin release and stabilizes arm cohesion. We conclude that Haspin is required to maintain centromeric cohesion during mitosis. We also suggest that Aurora B regulates cohesin removal through its effect on the localization of Shugoshin.     

A small-molecule inhibitor of Haspin alters the kinetochore functions of Aurora B

By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint (SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the CPC without affecting the bulk of Aurora B activity in HeLa cells. Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 and SAC override at high nocodazole concentrations. 5-ITu also impaired kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed by concomitant inhibition of phosphatase activity. Forcing localization of Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but failed to rescue the SAC override. This result suggests that a target of 5-ITu, possibly Haspin itself, may further contribute to SAC signaling downstream of Aurora B.     

Ready,Set…Poised!: Polycomb target genes are bound by poised RNA polymerase II throughout differentiation

In embryonic stem cells (ESCs), silent genes with major developmental functions display a unique epigenetic state in which strong and broad binding by Polycomb repressive complexes (PRCs) is accompanied by the presence of poised RNA polymerase II (RNAPII) and activating histone marks (e.g. H3K4me3) (Azuara et al, 2006 ; Bernstein et al, 2006 ; Stock et al, 2007 ; Brookes et al, 2012 ). It has been suggested that the plasticity and broad differentiation potential of pluripotent cells might rely, at least partly, on this unique epigenetic state (Bernstein et al, 2006 ; Stock et al, 2007 ). In their recent study, Pombo and colleagues (Ferrai et al, 2017 ) show that a similar epigenetic state can be found at a subset of major developmental genes throughout the differentiation of ESCs into neurons, providing novel and exciting insights into the molecular basis of cellular plasticity in differentiated cells.     

Characterization of plant Aurora kinases during mitosis

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.†These authors equally contributed to this work     

PP1/Repo-man dephosphorylates mitotic histone H3 at T3 and regulates chromosomal aurora B targeting

The transient mitotic histone H3 phosphorylation by various protein kinases regulates chromosome condensation and segregation, but the counteracting phosphatases have been poorly characterized [1-8]. We show here that PP1γ is the major histone H3 phosphatase acting on the mitotically phosphorylated (ph) residues H3T3ph, H3S10ph, H3T11ph, and H3S28ph. In addition, we identify Repo-Man, a chromosome-bound interactor of PP1γ [9], as a selective regulator of H3T3ph and H3T11ph dephosphorylation. Repo-Man promotes H3T11ph dephosphorylation by an indirect mechanism but directly and specifically targets H3T3ph for dephosphorylation by associated PP1γ. The PP1γ/Repo-Man complex opposes the protein kinase Haspin-mediated spreading of H3T3ph to the chromosome arms until metaphase and catalyzes the net dephosphorylation of H3T3ph at the end of mitosis. Consistent with these findings, Repo-Man modulates in a PP1-dependent manner the H3T3ph-regulated chromosomal targeting of Aurora kinase B and its substrate MCAK. Our study defines a novel mechanism by which PP1 counteracts Aurora B.     

It's a family act: the geminin triplets take center stage in motile ciliogenesis

The balance between proliferation and differentiation is a fundamental aspect of multicellular life. Perhaps nowhere is this delicate balance more palpable than in the multiciliated cells (MCCs) that line the respiratory tract, the ependyma, and the oviduct. These cells contain dozens to hundreds of motile cilia that beat in a concerted fashion to generate directed fluid flow over the tissue surface. Although MCCs have exited the cell cycle, remarkably, they retain the ability to duplicate their centrioles and to mature those centrioles into ciliary basal bodies—two features, which are known to be normally under strict cell cycle control (Firat‐Karalar & Stearns, 2014 ). How post‐mitotic MCCs retain this ability, remains unclear. In the past several months, four research articles, including one from Terré et al in this issue of The EMBO Journal, have described a vital role for the geminin coiled‐coil domain‐containing protein (Gemc1) in the MCC gene expression program in multiple tissues and organisms, that bring us closer to understanding this question (Kyrousi et al, 2015 ; Zhou et al, 2015 ; Arbi et al, 2016 ; Terré et al, 2016 ).     

Stretching the arms of the type VI secretion sheath protein

The bacterial type VI secretion system (T6SS) is a multicomponent complex responsible for the translocation of effector proteins into the external milieu. The T6SS consists of an external sheath, an internal rigid tube, a baseplate, and a T6SS‐specific membrane complex. Secretion is accomplished by the contraction of the sheath, which expels the effector‐loaded tube. In this issue of EMBO reports, Brackmann et al 1 show how modifications of the sheath subunits can lock the T6SS assembly in the extended state. These findings allowed Wang et al 2 and Nazarov et al 3 to purify the T6SS sheath–tube–baseplate complex in the extended pre‐secretion state and to analyze its structure using cryo‐electron microscopy (cryoEM).     

Retraction Statement: CXCL12/CXCR4 chemokine signaling in spinal glia induces pain hypersensitivity through MAPKs‐mediated neuroinflammation in bone cancer rats

‘CXCL12/CXCR4 chemokine signaling in spinal glia induces pain hypersensitivity through MAPKs‐mediated neuroinflammation in bone cancer rats’ by Hu X.‐M., Liu Y.‐N., Zhang H.‐L., Cao S.‐B., Zhang T., Chen L.‐P. and Shen W. The above article from Journal of Neurochemistry, published online on 26 January 2015 and in volume 132, issue 4, pages 452–463 (available through www.onlinelibrary.wiley.com ), and its subsequent Corrigendum, published online on 5 February 2015 and in volume 132, issue 4, p. 487, have been retracted by agreement between the Journal's Editor‐in‐Chief, Jörg Schulz, corresponding author Wen Shen on behalf of the authors, and John Wiley & Sons Ltd. The retraction has been agreed as the same GFAP immunostaining image was used to represent different experimental conditions in two different publications (Shen et al. [2014] in the Journal of Neuroinflammation and Hu et al. [2015] in the Journal of Neurochemistry), with apparent brightness changes between the images. Shen et al. (2014) show in the outer right panel of Figure 4a, as well as in Fig. 8A for the GFAP/sham condition, a GFAP immunostaining after treatment with TCI + Fluorocitrate. The same image, at a lower intensity, is used in Hu et al. (2015) in the first panel of figure 5b as a sham control. The shape of the tissue margins of the spinal cord section as well as several landmark epitopes that point towards identical images are encircled:

Shen et al. 2014 Figure 4a     

Novel aspects in the life cycle and biotrophic interactions in Pezizomycetes (Ascomycota,Fungi)

The ascomycete class Pezizomycetes (single order Pezizales) is known for its cup‐shaped fruit bodies and the evolution of edible truffles and morels, but little is known about the ontogeny and ecology of this large and ecologically diverse fungal group. In this issue of Molecular Ecology, Healy et al. ( 2013 ) make a great leap forward by describing and identifying asexual, anamorphic structures that produce mitotic spores in many ectomycorrhiza‐forming truffle and nontruffle species on soil surfaces worldwide (Fig.  1 ). Although such anamorphic forms have been reported sporadically from certain ectomycorrhizal and saprotrophic Pezizomycetes (e.g. Warcup 1990 ), Healy et al. ( 2013 ) demonstrate that these terricolous asexual forms are both taxonomically and geographically more widespread and, in fact, much more common than previously understood. We anticipate that deeper insight into other substrates, provided by molecular analyses of materials such as dead wood and seeds, is likely to reveal numerous anamorphs of saprotrophic and pathogenic Pezizomycetes as well (see Marek et al. 2009 ).     

Ajuba: a new microtubule-associated protein that interacts with BUBR1 and Aurora B at kinetochores in metaphase

Background information. The role of the LIM‐domain‐containing protein Ajuba was initially described in cell adhesion and migration processes and recently in mitosis as an activator of the Aurora A kinase. Results. In the present study, we show that Ajuba localizes to centrosomes and kinetochores during mitosis. This localization is microtubule‐dependent and Ajuba binds microtubules in vitro. A microtubule regrowth assay showed that Ajuba follows nascent microtubules from centrosomes to kinetochores. Owing to its contribution to mitotic commitment and its microtubule‐dependent localization, Ajuba could also play a role during the metaphase—anaphase transition. We show that Ajuba interacts with Aurora B and BUBR1 [BUB (budding uninhibited by benomyl)‐related 1], two major components of the mitotic checkpoint. Inhibition of BUBR1 by siRNA (small interfering RNA) disrupts chromosome alignment at the metaphase plate and modifies Ajuba localization due to premature mitotic exit. Conclusions. Ajuba is a microtubule‐associated protein that collaborates with Aurora B and BUBR1 at the metaphase—anaphase transition and this may be important to ensure proper chromosome segregation.     

EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B

The Aurora B kinase coordinates kinetochore–microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. We find that EB1, a microtubule plus end–tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). Our data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase.     

Haspin: a newly discovered regulator of mitotic chromosome behavior

The haspins are divergent members of the eukaryotic protein kinase family that are conserved in many eukaryotic lineages including animals, fungi, and plants. Recently-solved crystal structures confirm that the kinase domain of human haspin has unusual structural features that stabilize a catalytically active conformation and create a distinctive substrate binding site. Haspin localizes predominantly to chromosomes and phosphorylates histone H3 at threonine-3 during mitosis, particularly at inner centromeres. This suggests that haspin directly regulates chromosome behavior by modifying histones, although it is likely that additional substrates will be identified in the future. Depletion of haspin by RNA interference in human cell lines causes premature loss of centromeric cohesin from chromosomes in mitosis and failure of metaphase chromosome alignment, leading to activation of the spindle assembly checkpoint and mitotic arrest. Haspin overexpression stabilizes chromosome arm cohesion. Haspin, therefore, appears to be required for protection of cohesion at mitotic centromeres. Saccharomyces cerevisiae homologues of haspin, Alk1 and Alk2, are also implicated in regulation of mitosis. In mammals, haspin is expressed at high levels in the testis, particularly in round spermatids, so it seems likely that haspin has an additional role in post-meiotic spermatogenesis. Haspin is currently the subject of a number of drug discovery efforts, and the future use of haspin inhibitors should provide new insight into the cellular functions of these kinases and help determine the utility of, for example, targeting haspin for cancer therapy.