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The chromosomal passenger complex (CPC) is a heterotetrameric regulator of eukaryotic cell division, consisting of an Aurora-type kinase and a scaffold built of INCENP, Borealin, and Survivin. While most CPC components are conserved across eukaryotes, orthologs of the chromatin reader Survivin have previously only been found in animals and fungi, raising the question of how its essential role is carried out in other eukaryotes. By characterizing proteins that bind to the Arabidopsis Borealin ortholog, we identified BOREALIN RELATED INTERACTOR 1 and 2 (BORI1 and BORI2) as redundant Survivin-like proteins in the context of the CPC in plants. Loss of BORI function is lethal and a reduced expression of BORIs causes severe developmental defects. Similar to Survivin, we find that the BORIs bind to phosphorylated histone H3, relevant for correct CPC association with chromatin. However, this interaction is not mediated by a BIR domain as in previously recognized Survivin orthologs but by an FHA domain, a widely conserved phosphate-binding module. We find that the unifying criterion of Survivin-type proteins is a helix that facilitates complex formation with the other two scaffold components and that the addition of a phosphate-binding domain, necessary for concentration at the inner centromere, evolved in parallel in different eukaryotic groups. Using sensitive similarity searches, we find conservation of this helical domain between animals and plants and identify the missing CPC component in most eukaryotic supergroups. Interestingly, we also detect Survivin orthologs without a defined phosphate-binding domain, likely reflecting the situation in the last eukaryotic common ancestor.

Cdk1-mediated phosphorylation of threonine and serine residues on cell cycle regulators needs to be removed after mitosis. Hein et al. show that the known preference of the PP2A–B55 phosphatase for threonine provides temporal regulation of mitotic exit. Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit 1 , 2 , 3 , 4 , 5 . Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains unexplored. Here we demonstrate that the phosphothreonine preference of PP2A–B55 provides an essential regulatory element of mitotic exit. To allow rapid activation of the anaphase-promoting complex/cyclosome (APC/C) co-activator Cdc20, inhibitory phosphorylation sites are conserved as threonines while serine substitutions delay dephosphorylation and Cdc20 activation. Conversely, to ensure timely activation of the interphase APC/C co-activator Cdh1, inhibitory phosphorylation sites are conserved as serines, and threonine substitutions result in premature Cdh1 activation. Furthermore, rapid translocation of the chromosomal passenger complex to the central spindle is prevented by mutation of a single phosphorylated threonine to serine in inner centromere protein (INCENP), leading to failure of cytokinesis. Altogether, the findings of our work reveal that the inherent residue preference of a protein phosphatase can provide temporal regulation in biological processes.

The kinase Aurora B forms the chromosomal passenger complex (CPC) together with Borealin, INCENP, and Survivin to mediate chromosome condensation, the correction of erroneous spindle-kinetochore attachments, and cytokinesis. Phosphorylation of histone H3 Thr3 by Haspin kinase and of histone H2A Thr120 by Bub1 concentrates the CPC at the centromere. However, how the CPC is recruited to chromosome arms upon mitotic entry is unknown. Here, we show that asymmetric dimethylation at Arg2 on histone H3 (H3R2me2a) by protein arginine methyltransferase 6 (PRMT6) recruits the CPC to chromosome arms and facilitates histone H3S10 phosphorylation by Aurora B for chromosome condensation. Furthermore, in vitro assays show that Aurora B preferentially binds to the H3 peptide containing H3R2me2a and phosphorylates H3S10. Our findings indicate that the long-awaited key histone mark for CPC recruitment onto mitotic chromosomes is H3R2me2a, which is indispensable for maintaining appropriate CPC levels in dynamic translocation throughout mitosis. The proteins of the chromosomal passenger complex help chromosomes condense before cell division, but how this complex arrives at chromosomes was not known. Here the authors show that PRMT6 methylates histone H3 to recruit the chromosomal passenger complex.

A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.

HORMAD1 expression is usually restricted to germ-line cells but is also aberrantly expressed in 60% of triple-negative breast cancers (TNBCs), where it is bi-modally expressed and associated with genomic instability. Here, we show that out-of-context HORMAD1 expression in mitotic cells perturbs mitotic arrest and generates aneuploidy. These phenotypes are caused by out-of-context HORMAD1 expression driving a weakening of the spindle assembly checkpoint (SAC) and/or in kinetochore-microtubule error correction. These mitotic effects of HORMAD1 are MAD2L1-independent, but instead caused by a HORMAD1/Aurora B interaction and defective Aurora B/INCENP signalling. Consistent with this mechanism, aberrant HORMAD1 expression causes sensitivity to MPS1, Aurora B or BUB1 inhibitors currently being investigated as cancer treatments. Our data suggests how out-of-context expression of a meiotic gene imparts genomic instability upon tumour cells and also identifies several associated dependencies as mechanism-based therapeutic targets for a large, biomarker-defined, subset of cancers. HORMAD1 expression is typically restricted to germline cells where it has an important role in meiotic recombination but has been shown to be upregulated in triple negative breast cancer (TNBC). Here, the authors report that aberrant HORMAD1 expression weakens the spindle assembly checkpoint, driving sensitivity to AURORA kinase inhibition.

Chromosomal instability is a prominent biological feature of myelodysplastic syndromes (MDS), with over 50% of patients with MDS harboring chromosomal abnormalities or a complex karyotype (CK). Despite this observation, the mechanisms underlying mitotic and chromosomal defects in MDS remain elusive. In this study, we identified ectopic expression of the transcription factor ONECUT3, which is associated with CKs and poorer survival outcomes in MDS. ONECUT3-overexpressing cell models exhibited enrichment of several notable pathways, including signatures of sister chromosome exchange separation and mitotic nuclear division with the upregulation of INCENP and CDCA8 genes. Notably, dysregulation of chromosome passenger complex (CPC) accumulation, besides the cell equator and midbody, during mitotic phases consequently caused cytokinesis failure and defective chromosome segregation. Mechanistically, the homeobox (HOX) domain of ONECUT3, serving as the DNA binding domain, occupied the unique genomic regions of INCENP and CDCA8 and transcriptionally activated these 2 genes. We identified a lead compound, C5484617, that functionally targeted the HOX domain of ONECUT3, inhibiting its transcriptional activity on downstream genes, and synergistically resensitized MDS cells to hypomethylating agents. This study revealed that ONECUT3 promoted chromosomal instability by transcriptional activation of INCENP and CDCA8, suggesting potential prognostic and therapeutic roles for targeting high-risk MDS patients with a CK.

Aurora kinases B and C (AURKB/AURKC) are activated by binding to the C-terminal domain of INCENP. Full activation requires phosphorylation of two serine residues of INCENP that are conserved through evolution, although the mechanism of this activation has not been explained. Here we present crystal structures of the fully active complex of AURKC bound to INCENP, consisting of phosphorylated, activated, AURKC and INCENP phosphorylated on its TSS motif, revealing the structural and biochemical mechanism of synergistic activation of AURKC:INCENP. The structures show that TSS motif phosphorylation stabilises the kinase activation loop of AURKC. The TSS motif phosphorylations alter the substrate-binding surface consistent with a mechanism of altered kinase substrate selectivity and stabilisation of the protein complex against unfolding. We also analyse the binding of the most specific available AURKB inhibitor, BRD-7880, and demonstrate that the well-known Aurora kinase inhibitor VX-680 disrupts binding of the phosphorylated INCENP TSS motif. The inner centromere protein (INCENP) activates Aurora kinase B (AURKB) and Aurora kinase C. Here the authors provide insights into the activation mechanism of AURKB/C by determining the crystal structure of fully active phosphorylated human AURKC bound to the phosphorylated C-terminal IN-box section of human INCENP.

Neoadjuvant chemotherapy (NACT), a key strategy for various cancers, markedly improves patient prognosis and 5-year survival rates. However, numerous patients develop resistance to NACT and thus fail to benefit from it. Therefore, identifying reliable biomarkers to predict patient responsiveness to NACT remains a critical challenge. Here, we demonstrate that elevated expression of INCENP and CDCA8 contributes to poor NACT responsiveness across multiple cancers. Mechanistically, the 5′UTR (GGACT at position 113) of INCENP and the 3′UTR (GGACT at position 1041) of CDCA8 undergo m⁶A methylation and are recognized by YTHDF3, which facilitates their translation through interaction with eIF3A, ultimately driving poor response to NACT. Moreover, inhibition of INCENP and CDCA8 enhances NACT sensitivity by promoting multipolar spindle formation. Collectively, our findings establish that INCENP and CDCA8 serve as crucial biomarkers for predicting NACT responsiveness and as potential therapeutic targets for combination therapy with NACT to improve patient survival. While neoadjuvant chemotherapy can improve outcomes in patients with esophageal squamous carcinoma, resistance often develops. Here, the authors identify elevated INCENP and CDCA8 via YTHDF3 mediated translation as markers of poor response to neoadjuvant chemotherapy in esophageal squamous carcinoma.

There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinome-wide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin β1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening. Identifying kinases responsible for specific phosphorylation events remains challenging. Here, the authors leverage kinase inhibitor profiles for the identification of kinase-substrate site pairs in cell extracts, developing a method that can identify the enzymes responsible for unassigned phosphorylation events.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei , the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique ‘two-in-one’ CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.