Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

Abnormal prostaglandin E2 production blocks myogenic differentiation in myotonic dystrophy.

The congenital form of myotonic dystrophy type 1 (DM1) is the most severe type of the disease associated with CTG expansions over 1500 repeats and delayed muscle maturation. The mechanistic basis of the congenital form of DM1 is mostly unknown. Here, we show that muscle satellite cells bearing large CTG expansions (>3000) secrete a soluble factor that inhibits the fusion of normal myoblasts in culture. We identified this factor as prostaglandin E2 (PGE(2)). In these DM1 cells, PGE(2) production is increased through up-regulation of cyclooxygenase 2 (Cox-2), mPGES-1 and prostaglandin EP2/EP4 receptors. Elevated levels of PGE(2) inhibit myogenic differentiation by decreasing the intracellular levels of calcium. Exogenous addition of acetylsalicylic acid, an inhibitor of Cox enzymes, abolishes PGE(2) abnormal secretion and restores the differentiation of DM1 muscle cells. These data indicate that the delay in muscle maturation observed in congenital DM1 may result, at least in part, from an altered autocrine mechanism. Inhibitors of prostaglandin synthesis may thus offer a powerful method to restore the differentiation of DM1 muscle cells.

Impact of Oral Targeted Therapy on the Economic Burden of Chronic Lymphocytic Leukemia in Canada.

Background: Continuous oral targeted therapy (OTT) for chronic lymphocytic leukemia (CLL) represents an effective therapy but also a major economic burden on the healthcare system. This study aimed to estimate future direct costs, along with the prevalence, of CLL in the era of continuous OTT in Canada. Methods: The economic burden of OTT was modelled and compared to chemoimmunotherapy (CIT), for CLL treatment. The burden was assessed/projected from 2011 to 2025. For the OTT scenario, CIT was considered the standard of care before 2015, while OTT was considered standard of care for patients with either unmutated immunoglobulin heavy-chain variable (IGHV) or del(17p)/TP53 mutations starting in 2015 and, from 2020 onwards, for all first-line treatments except for patients with mutated IGHV. A Markov model was developed including four health states: watchful-waiting, first-line treatment, relapse and death. Costs of therapy, follow-up/monitoring and adverse events were included. Key clinical parameters were extracted from pivotal clinical trials. Results: As incidence rates and rate of survival are increasing, the prevalence of CLL in Canada is projected to increase 1.8-fold, from 8301 patients in 2011 to 14,654 by 2025. Correspondingly, the total annual costs of CLL management are predicted to increase 15.7-fold, from $60.8 million to $957.5 million during that same period. Conclusions: Although OTT enhances survival for patients with CLL, it is nonetheless associated with an important economic burden due to the projected vast increase in costs from 2011 to 2025. Changes in clinical strategies, such as implementation of a fixed OTT treatment duration, could help alleviate financial burden.

BUBR1 Pseudokinase Domain Promotes Kinetochore PP2A-B56 Recruitment, Spindle Checkpoint Silencing, and Chromosome Alignment.

The balance of phospho-signaling at the outer kinetochore is critical for forming accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. A major player in determining this balance is the PP2A-B56 phosphatase, which is recruited to the kinase attachment regulatory domain (KARD) of budding uninhibited by benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This unleashes a rapid, switch-like phosphatase relay that reverses mitotic phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase. Here, we demonstrate that the C-terminal pseudokinase domain of human BUBR1 is required to promote KARD phosphorylation. Mutation or removal of the pseudokinase domain results in decreased PP2A-B56 recruitment to the outer kinetochore attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. Our data, therefore, elucidate a function for the BUBR1 pseudokinase domain in ensuring accurate and timely exit from mitosis.

Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint.

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a "prone-to-autophosphorylate" Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells.

Searching for Biosignatures in Exoplanetary Impact Ejecta.

With the number of confirmed rocky exoplanets increasing steadily, their characterization and the search for exoplanetary biospheres are becoming increasingly urgent issues in astrobiology. To date, most efforts have concentrated on the study of exoplanetary atmospheres. Instead, we aim to investigate the possibility of characterizing an exoplanet (in terms of habitability, geology, presence of life, etc.) by studying material ejected from the surface during an impact event. For a number of impact scenarios, we estimate the escaping mass and assess its subsequent collisional evolution in a circumstellar orbit, assuming a Sun-like host star. We calculate the fractional luminosity of the dust as a function of time after the impact event and study its detectability with current and future instrumentation. We consider the possibility to constrain the dust composition, giving information on the geology or the presence of a biosphere. As examples, we investigate whether calcite, silica, or ejected microorganisms could be detected. For a 20 km diameter impactor, we find that the dust mass escaping the exoplanet is roughly comparable to the zodiacal dust, depending on the exoplanet's size. The collisional evolution is best modeled by considering two independent dust populations, a spalled population consisting of nonmelted ejecta evolving on timescales of millions of years, and dust recondensed from melt or vapor evolving on much shorter timescales. While the presence of dust can potentially be inferred with current telescopes, studying its composition requires advanced instrumentation not yet available. The direct detection of biological matter turns out to be extremely challenging. Despite considerable difficulties (small dust masses, noise such as exozodiacal dust, etc.), studying dusty material ejected from an exoplanetary surface might become an interesting complement to atmospheric studies in the future. Key Words: Biosignatures-Exoplanets-Impacts-Interplanetary dust-Remote sensing. Astrobiology 17, 721-746.

Mitotic phosphotyrosine network analysis reveals that tyrosine phosphorylation regulates Polo-like kinase 1 (PLK1).

Tyrosine phosphorylation is closely associated with cell proliferation. During the cell cycle, serine and threonine phosphorylation plays the leading role, and such phosphorylation events are most dynamic during the mitotic phase of the cell cycle. However, mitotic phosphotyrosine is not well characterized. Although a few functionally-relevant mitotic phosphotyrosine sites have been characterized, evidence suggests that this modification may be more prevalent than previously appreciated. Here, we examined tyrosine phosphorylation in mitotic human cells including those on spindle-associated proteins.? Database mining confirmed ~2000 mitotic phosphotyrosine sites, and network analysis revealed a number of subnetworks that were enriched in tyrosine-phosphorylated proteins, including components of the kinetochore or spindle and SRC family kinases. We identified Polo-like kinase 1 (PLK1), a major signaling hub in the spindle subnetwork, as phosphorylated at the conserved Tyr217 in the kinase domain. Substitution of Tyr217 with a phosphomimetic residue eliminated PLK1 activity in vitro and in cells. Further analysis showed that Tyr217 phosphorylation reduced the phosphorylation of Thr210 in the activation loop, a phosphorylation event necessary for PLK1 activity. Our data indicate that mitotic tyrosine phosphorylation regulated a key serine/threonine kinase hub in mitotic cells and suggested that spatially separating tyrosine phosphorylation events can reveal previously unrecognized regulatory events and complexes associated with specific structures of the cell cycle.

Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation.

During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation.

Transcription regulation by the noncoding RNA SRG1 requires Spt2-dependent chromatin deposition in the wake of RNA polymerase II.

Spt2 is a chromatin component with roles in transcription and posttranscriptional regulation. Recently, we found that Spt2 travels with RNA polymerase II (RNAP II), is involved in elongation, and plays important roles in chromatin modulations associated with this process. In this work, we dissect the function of Spt2 in the repression of SER3. This gene is repressed by a transcription interference mechanism involving the transcription of an adjacent intergenic region, SRG1, that leads to the production of a noncoding RNA (ncRNA). We find that Spt2 and Spt6 are required for the repression of SER3 by SRG1 transcription. Intriguingly, we demonstrate that these effects are not mediated through modulations of the SRG1 transcription rate. Instead, we show that the SRG1 region overlapping the SER3 promoter is occluded by randomly positioned nucleosomes that are deposited behind RNAP II transcribing SRG1 and that their deposition is dependent on the presence of Spt2. Our data indicate that Spt2 is required for the major chromatin deposition pathway that uses old histones to refold nucleosomes in the wake of RNAP II at the SRG1-SER3 locus. Altogether, these observations suggest a new mechanism of repression by ncRNA transcription involving a repressive nucleosomal structure produced by an Spt2-dependent pathway following RNAP II passage.