The cardiac pathological response to sustained pressure overload involves myocyte hypertrophy and dysfunction along with interstitial changes such as fibrosis and reduced capillary density. These changes are orchestrated by mechanical forces and factors secreted between cells. One such secreted factor is TGF-β, which is generated by and interacts with multiple cell types. Here we have shown that TGF-β suppression in cardiomyocytes was required to protect against maladaptive remodeling and involved noncanonical (non-Smad-related) signaling. Mouse hearts subjected to pressure overload and treated with a TGF-β-neutralizing Ab had suppressed Smad activation in the interstitium but not in myocytes, and noncanonical (TGF-β-activated kinase 1 [TAK1]) activation remained. Although fibrosis was greatly reduced, chamber dysfunction and dilation persisted. Induced myocyte knockdown of TGF-β type 2 receptor (TβR2) blocked all maladaptive responses, inhibiting myocyte and interstitial Smad and TAK1. Myocyte knockdown of TβR1 suppressed myocyte but not interstitial Smad, nor TAK1, modestly reducing fibrosis without improving chamber function or hypertrophy. Only TβR2 knockdown preserved capillary density after pressure overload, enhancing BMP7, a regulator of the endothelial-mesenchymal transition. BMP7 enhancement also was coupled to TAK1 suppression. Thus, myocyte targeting is required to modulate TGF-β in hearts subjected to pressure overload, with noncanonical pathways predominantly affecting the maladaptive hypertrophy/dysfunction.

The transforming-growth-factor-beta-activated kinase TAK1 is a member of the mitogen-activated protein kinase kinase kinase family, which couples extracellular stimuli to gene transcription. The in vivo function of TAK1 is not understood. Here, we investigated the potential involvement of TAK1 in cardiac hypertrophy. In adult mouse myocardium, TAK1 kinase activity was upregulated 7 days after aortic banding, a mechanical load that induces hypertrophy and expression of transforming growth factor beta. An activating mutation of TAK1 expressed in myocardium of transgenic mice was sufficient to produce p38 mitogen-activated protein kinase phosphorylation in vivo, cardiac hypertrophy, interstitial fibrosis, severe myocardial dysfunction, 'fetal' gene induction, apoptosis and early lethality. Thus, TAK1 activity is induced as a delayed response to mechanical stress, and can suffice to elicit myocardial hypertrophy and fulminant heart failure.

SummaryThe BubR1 checkpoint protein performs multiple functions in mitosis. We have carried out a functional analysis of conserved motifs of human BubR1 (also known as BUB1B) and demonstrate that spindle assembly checkpoint (SAC) and chromosome attachment functions can be uncoupled from each other. Mutation of five proline-directed serine phosphorylation sites, identified in vivo by mass spectrometry, essentially abolishes attachment of chromosomes to the spindle but has no effect on SAC functionality. By contrast, mutation of the two conserved KEN boxes required for SAC function does not impact chromosome congression. Interestingly, the contribution of the two KEN-box motifs is not equal. Cdc20 associates with the N-terminal but not C-terminal KEN box, and mutation of the N-terminal KEN motif results in more severe acceleration of mitotic timing. Moreover, the two KEN motifs are not sufficient for maximal binding of Cdc20 and APC/C, which also requires sequences in the BubR1 C-terminus. Finally, mutation of the GLEBS motif causes loss of Bub3 interaction and mislocalization of BubR1 from the kinetochore; concomitantly, BubR1 phosphorylation as well as SAC activity and chromosome congression are impaired, indicating that the GLEBS motif is strictly required for both major functions of human BubR1.

Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of the Aspergillus nidulans NIMA kinase. MAD1 interacts with NEK2A in vitro and in vivo via a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling.Chromosome movements during mitosis are governed by the interaction of spindle microtubules with a specialized chromosome domain located within the centromere. This specialized region, called the kinetochore (1, 2), is the site for spindle microtubule-centromere association. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore has an active function in chromosomal segregation through microtubule motors and spindle checkpoint sensors located at or near it (3-5).Several lines of evidence have implicated the kinetochore in generation of a diffusible checkpoint signal that can block cell cycle progression into anaphase until all kinetochores have successfully attached to spindle microtubules. Delayed attachment of one or more chromosomes to the spindle is correlated with a corresponding delay in the onset of anaphase. For mutants that fail to arrest the cell cycle in mitosis after disassembly of microtubules in budding yeast, genetic screen has identified three MAD (mitotic arrest deficiency) and three BUB (budding uninhibited by benomyl) genes (7). Vertebrate homologues of MAD1 (8), MAD2 (9 -10), BUB3 (11-12), BUB1, and BUBR1 (13-15) are spindle checkpoint components transiently associated with kinetochore. Expression of the kinetochore binding domain of murine BUB1 (13) or injection of antibodies against BUBR1 (15) results in premature onset of anaphase, presumably by replacement of the endogenous proteins at kinetochores. Collectively, these data indicate that binding of these spindle checkpoint components at the kinetochores may generate a signal in response to spindle defects and/or aberrant kinetochore protein-protein interactions.Mitosis is orchestrated by signaling cascades that coordinate mitotic p...

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zeste White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZW10 kinetochore localization. In addition, depletion of Zwint-1 affects no mitotic arrest but causes aberrant premature chromosome segregation. These Zwint-1-suppressed cells display chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP-F and dynamitin but not BUB1 with the kinetochore. Finally, our studies show that Zwint-1 is a new component of the mitotic checkpoint, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding interphase cells with multinuclei. As ZW10 and Zwint-1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.Chromosome movements during mitosis are governed by the interaction of spindle microtubules with a specialized chromosome domain located within the centromere. This specialized region, called the kinetochore (1, 2), is the site for spindle microtubule-centromere association. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore has an active function in chromosomal segregation through microtubule motors and spindle checkpoint sensors located at or near it (3-5).Eukaryotic organisms require extraordinary fidelity in chromosome segregation during meiosis and mitosis as aberrant chromosome segregation can be catastrophic to an organism or its progeny. One of the evolutionarily conserved multiprotein complexes essential for the fidelity of chromosome segregation contains several proteins, including ZW10 1 (Zeste White 10) and ROD (Rough Deal) (6 -8). Mutations in the Drosophila ZW10 or ROD genes cause similar defects, most noticeably in lagging chromatids that remain at the metaphase plate late in anaphase, leading to high levels of aneuploidy among daughter cells.ZW10 and ROD proteins display remarkable dynamics in their intracellular location during cell division (7, 9, 10). Both proteins accumulate strongly at the outer kinetochore plates during prometaphase. At metaphase, ZW10 and ROD depart from the kinetochores and relocate onto spindle microtubules. During anaphase, the proteins are no longer found on kinetochore microtubules and instead localize exclusively to the kinetochores of the separating chromosomes.Besides binding to ROD, ZW10 is responsible for localization of cytoplasmic dynein to kinetochores (10 -12) via a direct contact with dynamitin, a...

Background: HJURP is a molecular chaperone essential for the deposition of the centromere marker CENP-A. Results: Mis18␤ binds with and specifies the centromere localization of HJURP. Conclusion: Mis18␤ governs centromere assembly via the Mis18␤-HJURP-CENP-A axis. Significance: Our finding reveals a novel mechanism underlying CENP-A incorporation into the centromere.

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

BackgroundMembers of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.Methodology/Principal FindingsHere, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.Conclusions/SignificancehMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.