Mitotic phosphorylation of tumor suppressor DAB2IP maintains spindle assembly checkpoint and chromosomal stability through activating PLK1-Mps1 signal pathway and stabilizing mitotic checkpoint complex

Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP’s interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.

Spatiotemporal coordination of Greatwall-Endos-PP2A promotes mitotic progression

Mitotic entry involves inhibition of protein phosphatase 2A bound to its B55/Tws regulatory subunit (PP2A-B55/Tws), which dephosphorylates substrates of mitotic kinases. This inhibition is induced when Greatwall phosphorylates Endos, turning it into an inhibitor of PP2A-Tws. How this mechanism operates spatiotemporally in the cell is incompletely understood. We previously reported that the nuclear export of Greatwall in prophase promotes mitotic progression. Here, we examine the importance of the localized activities of PP2A-Tws and Endos for mitotic regulation. We find that Tws shuttles through the nucleus via a conserved nuclear localization signal (NLS), but expression of Tws in the cytoplasm and not in the nucleus rescues the development of tws mutants. Moreover, we show that Endos must be in the cytoplasm before nuclear envelope breakdown (NEBD) to be efficiently phosphorylated by Greatwall and to bind and inhibit PP2A-Tws. Disrupting the cytoplasmic function of Endos before NEBD results in subsequent mitotic defects. Evidence suggests that this spatiotemporal regulation is conserved in humans.

Mitotic H3K9ac is controlled by phase-specific activity of HDAC2, HDAC3 and SIRT1

Mitosis comprises multiple changes, including chromatin condensation and transcription reduction. Intriguingly, while histone acetylation levels are reduced during mitosis, the mechanism of this reduction is unclear. We studied the mitotic regulation of H3K9ac by using inhibitors of histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during mitotic stages and cytokinesis by immunofluorescence and immunoblots. We identified HDAC2, HDAC3 and SIRT1 as modulators of the mitotic levels of H3K9ac. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition, increased H3K9ac levels in metaphase. Next, we performed ChIP-seq in mitotic cells following targeted inhibition of these histone deacetylases. While the genomic areas impacted by HDAC2 and HDAC3 were mostly concordant, a subset of loci were unique to each enzyme. Interestingly, HDAC3-specific targets were enriched for genes involved in mitosis regulation. Our results support a model in which H3K9 deacetylation is a stepwise process – at prophase HDAC2 modulates most transcription-associated H3K9ac-marked loci and at metaphase HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

Physiological and Pathological Roles of Mammalian NEK7

NEK7 is the smallest NIMA-related kinase (NEK) in mammals. The pathological and physiological roles of NEK7 have been widely reported in many studies. To date, the major function of NEK7 has been well documented in mitosis and NLRP3 inflammasome activation, but the detailed mechanisms of its regulation remain unclear. This review summarizes current advances in NEK7 research involving mitotic regulation, NLRP3 inflammasome activation, related diseases and potential inhibitors, which may provide new insights into the understanding and therapy of the diseases associated with NEK7, as well as the subsequent studies in the future.

Characterization of a novel separase-interacting protein and candidate new securin, Eip1p, in the fungal pathogen Candida albicans

Proper chromosome segregation is crucial for maintaining genomic stability and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete Candida albicans, an important pathogen of humans, is essential for chromosome segregation . However, C. albicans lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed Esp1p-Interacting Protein1 (Eip1p)/Orf19.955p, an uncharacterized protein specific to Candida species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APCCdc20-dependent manner, similar to securins. Eip1p is strongly enriched in response to methyl methanesulfate (MMS) or hydroxyurea (HU) treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes.

Understanding the basis of CYP26 mediated regulation of lens regeneration using ex vivo eye cultures and 4-oxo-RA

PURPOSEXenopus has the remarkable ability to regenerate a lens from the basal cornea epithelial cells in response to signals from the retina. Previous work demonstrated that the Retinoic Acid (RA) metabolizing enzyme CYP26 is expressed in the cornea, and that its activity is required for lens regeneration. Gaps remain in our knowledge as to whether CYP26 is needed only to attenuate RA signaling via RA elimination, or whether it also acts to generate retinoid metabolites, such as 4-oxo-RA, to act as signaling ligands. Other key questions are why CYP26 antagonism, but not exogenous retinoids, can reduce cell division in the cornea, and when during regeneration CYP26 is important.MATERIALS AND METHODSEx vivo cultures supplemented with RA, 4-oxo-RA, or the CYP26 inhibitor Liarozole were used to assay the effects of these compounds on lens regeneration. Similarly, corneas were explanted, cultured in the presence of these compounds, and assayed for mitotic changes by counting anti-Histone H3 positive nuclei. qPCRs validated responsiveness to these compounds.RESULTSEx vivo cultures showed that when the media was supplemented with the RA metabolite 4-oxo-RA in addition to Liarozole, lens regeneration was still inhibited. 4-oxo-RA also does not rescue the loss of cell division in the cornea that is observed upon CYP26 antagonism. Liarozole inhibited regeneration when added 12 hours after lentectomy, but not when added 48 hours after.CONCLUSIONSThese data show that the necessity of CYP26 is not explained as a generator of 4-oxo-RA for regeneration. Moreover, Liarozole-induced mitotic reduction is not explained by 4-oxo-RA deficiency. These results support a model of RA-independent mitotic regulation by CYP26, though other retinoid metabolites may be active. Finally, CYP26 activity is only needed between 12 and 48 hours post-surgery, showing that its action is required only during the earliest stages of lens regeneration.Financial interestsThe authors declare no competing financial interests.

Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila

The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis

Mitosis is controlled by reversible protein phosphorylation involving specific kinases and phosphatases. A handful of major mitotic protein kinases, such as the cyclin B–CDK1 complex, the Aurora kinases, and Polo-like kinase 1 (PLK1), cooperatively regulate distinct mitotic processes. Research has identified proteins and mechanisms that integrate these kinases into signaling cascades that guide essential mitotic events. These findings have important implications for our understanding of the mechanisms of mitotic regulation and may advance the development of novel antimitotic drugs. We review collected evidence that in vertebrates, the Aurora kinases serve as catalytic subunits of distinct complexes formed with the four scaffold proteins Bora, CEP192, INCENP, and TPX2, which we deem “core” Aurora cofactors. These complexes and the Aurora-PLK1 cascades organized by Bora, CEP192, and INCENP control crucial aspects of mitosis and all pathways of spindle assembly. We compare the mechanisms of Aurora activation in relation to the different spindle assembly pathways and draw a functional analogy between the CEP192 complex and the chromosomal passenger complex that may reflect the coevolution of centrosomes, kinetochores, and the actomyosin cleavage apparatus. We also analyze the roles and mechanisms of Aurora-PLK1 signaling in the cell and centrosome cycles and in the DNA damage response.