Conrad von Schubert

Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.

Background Members 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 Findings Here, 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/Significance hMps1 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.

Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells.

Equal mitotic chromosome segregation is critical for genome integrity and is monitored by the spindle assembly checkpoint (SAC). We have previously shown that the consensus phosphorylation motif of the essential SAC kinase Monopolar spindle 1 (Mps1) is very similar to that of Polo-like kinase 1 (Plk1). This prompted us to ask whether human Plk1 cooperates with Mps1 in SAC signaling. Here, we demonstrate that Plk1 promotes checkpoint signaling at kinetochores through the phosphorylation of at least two Mps1 substrates, including KNL-1 and Mps1 itself. As a result, Plk1 activity enhances Mps1 catalytic activity as well as the recruitment of the SAC components Mad1:C-Mad2 and Bub3:BubR1 to kinetochores. We conclude that Plk1 strengthens the robustness of SAC establishment at the onset of mitosis and supports SAC maintenance during prolonged mitotic arrest.

Polo-like kinases

What is Polo and what are Plks? Plk stands for Polo-like kinase. In the 1980s, genetic screens in budding yeast and Drosophila identified several key regulators of mitosis, including the founding member of the Polo kinase family. Since then, a total of five mammalian paralogs of the Drosophila Polo gene have been discovered. These exhibit largely non-redundant functions and are differently expressed, localized, and regulated. The Polo homolog Plk1 is common to all eukaryotes, apart from plants and certain protozoan parasites. Plk4 is also present in most vertebrates and invertebrates and probably arose early on, in a first round of gene duplication. The evolutionarily ‘younger’ Plk2 sub-family is only found in some bilaterian animals and comprises two genuine kinases, Plk2 and Plk3, as well as the kinase-deficient Plk5.Is Plk1 the leader of the pack? Plk1 is a wizard of both mitosis and meiosis (M phase of the cell cycle). It is expressed in proliferating cells and regulates many aspects of M-phase progression — notably mitotic entry, spindle architecture and positioning, sister-chromatid separation, and cytokinesis. Hence, inactivation of Plk1 in cultured cells leads to cell-cycle arrest in early mitosis, followed by apoptosis. In addition, Plk1 is also involved in key processes, such as release of amphibian eggs from cell-cycle arrest upon fertilization, recovery of mammalian cells from DNA damage, and RNA polymerase III-dependent transcription. At this point in time, it seems fair to state that Plk1 is top dog of the family. But Plk4, another key regulator of cell division (see below), is increasingly challenging Plk1’s leadership position. In contrast, the roles of the younger Plk2 family members still remain sketchy.How does Plk1 manage all these different functions? The answer lies in the structure. Plk1, like all other family members, has a topology with two conserved domains: an amino-terminal serine/threonine kinase domain and a carboxy-terminal substrate-binding domain, known as the polo-box domain (PBD). Plk1 is a very busy kinase and several recent phospho-proteomics studies indicate that it targets a large number of physiological substrates. To perform its various tasks, Plk1 is targeted to distinct subcellular sites, such as centrosomes during G2 phase and kinetochores, spindle poles and the spindle midzone/midbody during M phase (Figure 1Figure 1). Plk1 localization is governed by docking of the PBD to specific motifs (Ser–Ser/Thr–Pro) that have been primed by phosphorylation. This beautiful mechanism confers both temporal and spatial control over Plk1 activity. For example, Plk1 docking to early mitotic interaction partners is often primed by cyclin-dependent kinase 1 (Cdk1). Concomitantly, this same kinase prevents Plk1 from binding to late mitotic interaction partners through inhibitory phosphorylation adjacent to PBD-binding motifs. When Cdk1 activity is reduced at the onset of mitotic exit, this suppression is relieved and Plk1 primes its own recruitment to proteins that are important for the initiation of cytokinesis. The spectrum of Plk1 regulation also includes more conventional mechanisms, notably activation-loop phosphorylation by Aurora kinases and proteasomal degradation at the hands of the major mitotic ubiquitin ligase known as anaphase-promoting complex/cyclosome (APC/C).Figure 1Immunofluorescence analysis of human hTERT-RPE1 cells showing Plk1 (green, left panel) at kinetochores as well as spindle poles and Plk4 (green, right panel) at centrosomes. Microtubules are shown in red, DNA in blue. Scale bars represent 5 μm. (Image: C. von Schubert; A.I. Ferrand, IMCF Biozentrum.)View Large Image | View Hi-Res Image | Download PowerPoint SlideI’ve heard that Plk4 is a master regulator of centriole and basal body formation — is this true? Yes, Plk4 indeed plays a key role in the biogenesis of centrioles and basal bodies. Centrioles are important for the assembly of centrosomes — the major microtubule-organizing centers of animal cells (Figure 1Figure 1) — and as basal bodies they trigger the formation of cilia and flagella. Depletion of Plk4 results in loss of centrioles, whereas its overexpression triggers excessive centriole formation. Murine Plk4–/– embryos die early in development, confirming that Plk4-deficient cells are defective in cell-cycle progression. In most cells, Plk4 levels are extremely low and this reflects a self-destruction mechanism based on trans-autophosphorylation within Plk4 dimers, followed by ubiquitylation and proteasomal degradation. How Plk4 regulates centriole duplication remains to be understood, but clearly the PBD is important for Plk4 localization to centrosomes. Interestingly, Plk1 also plays an important role in centrosome biology in that it contributes to restrict centriole duplication to once per cell cycle.Are Plk2 and Plk3 merely afterthoughts of vertebrate evolution? That’s a bit harsh! Perhaps it will simply take more time to better understand what these kinases are actually doing — but, obviously, they are not required in many species. Both Plk2 and Plk3 were originally identified as early-response genes that are upregulated following serum stimulation of quiescent murine fibroblasts. Subsequently, both genes were also assigned tumor-suppressor roles: Plk2 expression was reported to be downregulated in several types of cancer, whereas Plk3–/– mice are prone to tumor development. Remarkably, however, Plk2 as well as Plk5 are also expressed in non-proliferative tissue of the central nervous system and Plk2 was implicated in synaptic plasticity. Hence, although Plk2 family members may not be essential for life, they are likely to play important roles. This rings a cautionary bell with regard to the development of Plk1 inhibitors as anti-cancer drugs (see below).What about Plk5, the new kid on the block? Plk5 probably functions as a decoy kinase. In humans, for example, the Plk5 sequence contains a stop codon within the kinase domain, and a protein fragment is only resurrected thanks to a start codon downstream, resulting in expression of a catalytically inactive, truncated protein. Although expressed, Plk5 was initially given the cold shoulder because it shows the characteristics of a pseudo-gene. However, recent studies have now shown that Plk5 is expressed in non-proliferative tissues, mostly the brain. In addition, it was found to be upregulated in fibroblasts upon serum starvation or DNA damage, whereas overexpression triggered a G0/G1-like arrest. Thus, it has been proposed that Plk5 function is related to stress responses.Are Plks attractive drug targets for cancer treatment? Yes and no — the future will tell. So far, the focus has been on targeting Plk1: human Plk1 is highly expressed in proliferating tissues, often upregulated in tumors, and elevated expression in tumors is associated with poor prognosis. Furthermore, overexpression of Plk1 leads to transformation of cultured cells, likely via the stimulation of a mitotic transcription program involving the transcription factor FOXM1. In addition, it is in principle possible to interfere with Plk1 function not only via the usual route of ATP-competitive inhibitors (which of course raises concerns about specificity), but also by interfering with PBD binding to docking proteins. Several early cell-culture studies had suggested that tumor cells may be more sensitive to Plk1 inhibition than normal cells, but whether a sufficient therapeutic window can be found in a clinically relevant context remains to be determined. Several Plk1 inhibitors are presently in clinical trials and it will be interesting to see how these agents fare for the benefit of patients.

Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.

Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524

The kinase Bub1 functions in the spindle assembly checkpoint (SAC) and in chromosome congression, but the role of its catalytic activity remains controversial. Here, we use two novel Bub1 inhibitors, BAY-320 and BAY-524, to demonstrate potent Bub1 kinase inhibition both in vitro and in intact cells. Then, we compared the cellular phenotypes of Bub1 kinase inhibition in HeLa and RPE1 cells with those of protein depletion, indicative of catalytic or scaffolding functions, respectively. Bub1 inhibition affected chromosome association of Shugoshin and the chromosomal passenger complex (CPC), without abolishing global Aurora B function. Consequently, inhibition of Bub1 kinase impaired chromosome arm resolution but exerted only minor effects on mitotic progression or SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of Paclitaxel, impairing both chromosome segregation and cell proliferation. These findings are relevant to our understanding of Bub1 kinase function and the prospects of targeting Bub1 for therapeutic applications. DOI: http://dx.doi.org/10.7554/eLife.12187.001

Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments.

The multiplexing capabilities of isobaric mass tag-based protein quantification, such as Tandem Mass Tags or Isobaric Tag for Relative and Absolute Quantitation have dramatically increased the scope of mass spectrometry-based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample prefractionation methods allows for comprehensive studies of complex proteomes. However, reporter ion-based quantification has often been criticized for limited quantification accuracy due to interference from coeluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-to-implement remedy that relies on spiking a 6-protein calibration mixture to the samples. We evaluated our ratio adjustment approach using two large scale TMT 10-plex data sets derived from a human cancer and noncancer cell line as well as E. coli cells grown at two different conditions. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity-based label free quantification. Studying the protein set identified by both methods, we found that differentially abundant proteins were assigned dramatically higher statistical significance when quantified using TMT. Data are available via ProteomeXchange with identifier PXD003346.

The IKK Inhibitor BMS-345541 Affects Multiple Mitotic Cell Cycle Transitions

The IκB kinase (IKK) complex controls processes such as inflammation, immune responses, cell survival and the proliferation of both normal and tumor cells. By activating NFκB, the IKK complex contributes to G1/S transition and first evidence has been presented that IKKα also regulates entry into mitosis. At what stage IKK is required and whether IKK also contributes to progression through mitosis and cytokinesis, however, has not yet been determined. In this study, we use BMS-345541, a potent allosteric small molecule inhibitor of IKK, to inhibit IKK specifically during G2 and during mitosis. We show that BMS-345541 affects several mitotic cell-cycle transitions, including mitotic entry, prometaphase to anaphase progression and cytokinesis. Adding BMS-345541 to the cells released from arrest in S-phase blocked the activation of aurora A, B and C, Cdk1 activation and histone H3 phosphorylation. Additionally, treatment of the mitotic cells with BMS-345541 resulted in precocious cyclin B1 and securin degradation, defective chromosome separation and improper cytokinesis. BMS-345541 was also found to override the spindle checkpoint in nocodazole-arrested cells. In vitro kinase assays using BMS-345541 indicate that these effects are not primarily due to a direct inhibitory effect of BMS-345541 on mitotic kinases such as Cdk1, Aurora A or B, Plk1 or NEK2. This study points towards a new potential role of IKK in cell cycle progression. Since deregulation of the cell-cycle is one of the hallmarks of tumor formation and progression, the newly discovered level of BMS 345541 function could be useful for cell-cycle control studies and may provide valuable clues for the design of future therapeutics.

Abstract 2725: Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524

The maintenance of correct chromosome number (euploidy) during cell division is ensured by a highly conserved surveillance mechanism termed ‘spindle assembly checkpoint’ which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. The mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) was reported to contribute to both chromosome congression and checkpoint function, yet the role of BUB1 catalytic activity in these processes remains a matter of debate. To differentiate between catalytic and non-catalytic functions of BUB1 we compared phenotypes provoked by BUB1 protein depletion with specific BUB1 kinase inhibition using two novel small molecule inhibitors of BUB1, termed BAY-320 and BAY-524. BAY-320 and BAY-524 were highly potent and selective ATP-competitive inhibitors of BUB1 kinase activity with IC50 values in the single digit nanomolar range (at 10 micromolar ATP concentration). By monitoring phosphorylation of Thr120 in histone H2A, we showed that both compounds acted as potent BUB1 kinase inhibitors both biochemically and in human cells. We found that BUB1 inhibition substantially altered the chromosomal association of Shugoshin and the chromosomal passenger complex without major effects on global Aurora B function. Consequently, inhibition of BUB1 kinase clearly impaired chromosome arm resolution but, in stark contrast to depletion of BUB1 protein, only had a minor effect on cell cycle and SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of paclitaxel, synergistically affecting chromosome segregation and cell proliferation. These findings are highly relevant to both our understanding of BUB1 kinase function during mitosis and the prospects of BUB1 as a target of anti-cancer therapies. In this regard, BAY-320 and BAY-524 are first-in-class inhibitors of BUB1 kinase and their potential utility as anti-cancer agents is being explored. Citation Format: Anna P. Baron, Conrad von Schubert, Fabien Cubizolles, Gerhard Siemeister, Marion Hitchcock, Anne Mengel, Jens Schroder, Amaury Fernandez-Montalvan, Martin Lange, Franz von Nussbaum, Dominik Mumberg, Erich Nigg. Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2725.