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Karin Grosstessner-Hain,
Björn Hegemann,
Maria Novatchkova,
Jonathan Rameseder,
Brian A Joughin, Otto Hudecz,
Elisabeth Roitinger,
Peter Pichler,
Norbert Kraut,
Michael B Yaffe,
Jan-Michael Peters,
Karl Mechtler
[show abstract]
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ABSTRACT: Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.
Molecular & Cellular Proteomics 08/2011; 10(11):M111.008540. · 7.40 Impact Factor
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Björn Hegemann,
James R A Hutchins, Otto Hudecz,
Maria Novatchkova,
Jonathan Rameseder,
Martina M Sykora,
Sihan Liu,
Michael Mazanek,
Péter Lénárt,
Jean-Karim Hériché,
Ina Poser,
Norbert Kraut,
Anthony A Hyman,
Michael B Yaffe,
Karl Mechtler,
Jan-Michael Peters
[show abstract]
[hide abstract]
ABSTRACT: Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes, the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as "switchboards" to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase-substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes with the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Polo-like kinase (PLK1) inhibitor, BI 4834, we determined the kinase dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on six proteins as direct candidate substrates of AURKB or PLK1.
Science Signaling 01/2011; 4(198):rs12. · 7.50 Impact Factor
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Jes Alexander,
Daniel Lim,
Brian A Joughin,
Björn Hegemann,
James R A Hutchins,
Tobias Ehrenberger,
Frank Ivins,
Fabio Sessa, Otto Hudecz,
Erich A Nigg,
Andrew M Fry,
Andrea Musacchio,
P Todd Stukenberg,
Karl Mechtler,
Jan-Michael Peters,
Stephen J Smerdon,
Michael B Yaffe
[show abstract]
[hide abstract]
ABSTRACT: The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.
Science Signaling 01/2011; 4(179):ra42. · 7.50 Impact Factor
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Michael H A Schmitz,
Michael Held,
Veerle Janssens,
James R A Hutchins, Otto Hudecz,
Elitsa Ivanova,
Jozef Goris,
Laura Trinkle-Mulcahy,
Angus I Lamond,
Ina Poser,
Anthony A Hyman,
Karl Mechtler,
Jan-Michael Peters,
Daniel W Gerlich
[show abstract]
[hide abstract]
ABSTRACT: When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A-B55alpha complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A-B55alpha functions downstream of Cdk1 inactivation. PP2A-B55alpha isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-beta1, and RNAi depletion of importin-beta1 delayed mitotic exit synergistically with PP2A-B55alpha. This demonstrates that PP2A-B55alpha and importin-beta1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.
Nature Cell Biology 09/2010; 12(9):886-93. · 19.49 Impact Factor
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Michael H. A. Schmitz,
Michael Held,
Veerle Janssens,
James R. A. Hutchins, Otto Hudecz,
Elitsa Ivanova,
Jozef Goris,
Laura Trinkle-Mulcahy,
Angus I. Lamond,
Ina Poser,
Anthony A. Hyman,
Karl Mechtler,
Jan-Michael Peters,
Daniel W. Gerlich
Nature Cell Biology 08/2010; 12(9):886-893. · 19.49 Impact Factor
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James R A Hutchins,
Yusuke Toyoda,
Björn Hegemann,
Ina Poser,
Jean-Karim Hériché,
Martina M Sykora,
Martina Augsburg, Otto Hudecz,
Bettina A Buschhorn,
Jutta Bulkescher, [......],
Steffen Lawo,
Laurence Pelletier,
Holger Stark,
Kim Nasmyth,
Jan Ellenberg,
Richard Durbin,
Frank Buchholz,
Karl Mechtler,
Anthony A Hyman,
Jan-Michael Peters
[show abstract]
[hide abstract]
ABSTRACT: Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.
Science 04/2010; 328(5978):593-9. · 31.20 Impact Factor
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Sandra Morandell,
Karin Grosstessner-Hain,
Elisabeth Roitinger, Otto Hudecz,
Thomas Lindhorst,
David Teis,
Oliver A Wrulich,
Michael Mazanek,
Thomas Taus,
Florian Ueberall,
Karl Mechtler,
Lukas A Huber
[show abstract]
[hide abstract]
ABSTRACT: Signaling networks regulate cellular responses to external stimuli through post-translational modifications such as protein phosphorylation. Phosphoproteomics facilitate the large-scale identification of kinase substrates. Yet, the characterization of critical connections within these networks and the identification of respective kinases remain the major analytical challenge. To address this problem, we present a novel approach for the identification of direct kinase substrates using chemical genetics in combination with quantitative phosphoproteomics. Quantitative identification of kinase substrates (QIKS) is a novel-screening platform developed for the proteome-wide substrate-analysis of specific kinases. Here, we aimed to identify substrates of mitogen-activated protein kinase/Erk kinase (Mek1), an essential kinase in the mitogen-activated protein kinase cascade. An ATP analog-sensitive mutant of Mek1 (Mek1-as) was incubated with a cell extract from Mek1 deficient cells. Phosphorylated proteins were analyzed by LC-MS/MS of IMAC-enriched phosphopeptides, labeled differentially for relative quantification. The identification of extracellular regulated kinase 1/2 as the sole cytoplasmic substrates of MEK1 validates the applicability of this approach and suggests that QIKS could be used to identify substrates of a wide variety of kinases.
Proteomics 03/2010; 10(10):2015-25. · 4.43 Impact Factor
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[show abstract]
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ABSTRACT: Metal oxide affinity chromatography (MOAC) has become a prominent method to enrich phosphopeptides prior to their analysis by liquid chromatography-mass spectrometry. To overcome limitations in material design, we have previously reported the use of nanocasting as a means to generate metal oxide spheres with tailored properties. Here, we report on the application of two oxides, tin dioxide (stannia) and titanium dioxide (titania), for the analysis of the HeLa phosphoproteome. In combination with nanoflow LC-MS/MS analysis on a linear ion trap-Fourier transform ion cyclotron resonance instrument, we identified 619 phosphopeptides using the new stannia material, and 896 phosphopeptides using titania prepared in house. We also compared the newly developed materials to commercial titania material using an established enrichment protocol. Both titania materials yielded a comparable total number of phosphopeptides, but the overlap of the two data sets was less than one-third. Although fewer peptides were identified using stannia, the complementarity of SnO(2)-based MOAC could be shown as more than 140 phosphopeptides were exclusively identified by this material.
Analytical Chemistry 03/2010; 82(7):2726-33. · 5.86 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004; Larsen et al., 2005; Kweon and Hakansson, 2006; Cantin et al., 2007; Collins et al., 2007). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO(2)-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO(2) and ZrO(2) columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 02/2010; 878(5-6):515-24. · 2.78 Impact Factor
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[show abstract]
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ABSTRACT: Sample carryover is a significant problem that occurs in high-performance liquid chromatography (HPLC) analysis. Carryover effects cannot be tolerated in any high-performance liquid chromatography-mass spectroscopy (HPLC-MS) separation system, and proteomics analysis must be performed in a separation system with virtually no carryover. Several procedures have been tested for effective and fast removal of interfering peptides and proteins originating from previous analyses in the HPLC system. We have developed and optimized a cleaning method for eliminating carryover caused by the autosampler and the trap column. The new washing method uses an injection of trifluoroethanol into the injection path and onto the trap column to remove strongly bound peptides and proteins, and it includes trifluoroethanol as an additional solvent in the chromatographic mobile phase for enhanced cleaning of the separation column. By application of this method, a significant reduction in carryover was achieved without any loss in the amount of proteins and peptides identified by MS.
Analytical Chemistry 07/2009; 81(14):5955-60. · 5.86 Impact Factor
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Michael H A Schmitz,
Michael Held,
Veerle Janssens,
James R A Hutchins, Otto Hudecz,
Elitsa Ivanova,
Jozef Goris,
Laura Trinkle-Mulcahy,
Angus I Lamond,
Ina Poser,
Anthony A Hyman,
Karl Mechtler,
Jan-Michael Peters,
Daniel W Gerlich
[show abstract]
[hide abstract]
ABSTRACT: When vertebrate cells exit mitosis, they reorganize various cellular structures to build functional interphase cells1. This depends on Cdk1 inactivation and subsequent dephosphorylation of its substrates2-4. Members of PP1 and PP2A phosphatase families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5, 6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we used a live imaging assay to screen by RNAi a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identified a trimeric PP2A-B55α complex as a key factor for postmitotic reassembly of the nuclear envelope, the Golgi apparatus, and decondensed chromatin, as well as for mitotic spindle breakdown. Using a chemically-induced mitotic exit assay, we found that PP2A-B55α functions downstream of Cdk1 inactivation. PP2A-B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate histone H1 and it was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor Importin β1, and RNAi depletion of Importin β1 delayed mitotic exit synergistically with PP2A-B55α. This demonstrates that PP2A-B55α and Importin β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 12 (2010): 886-893, doi:10.1038/ncb2092. This work was supported by SNF research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation to DWG, and a MBL Summer Research Fellowship by the Evelyn and Melvin Spiegel Fund to DWG, a Roche Ph.D. fellowship to MHAS, and a Mueller fellowship of the Molecular Life Sciences Ph.D. program Zurich to MH. MH and MHAS are fellows of the Zurich Ph.D. Program in Molecular Life Sciences. VJ and JG were supported by grants of the ‘Geconcerteerde OnderzoeksActies’ of the Flemish government, the ‘Interuniversitary Attraction Poles’ of the Belgian Science Policy P6/28 and the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’. AIL is a Wellcome Trust Principal Research Fellow. AAH acknowledges funding by the Max Planck Society, the EU-FP6 integrated project MitoCheck, and the BMBF grant DiGtoP [01GS0859]. Work in the groups of KM and JMP was supported by the EU-FP6 integrated project MitoCheck, Boehringer Ingelheim and by the GEN-AU programme of the Austrian Federal Ministry of Science and Research (Austrian Proteomics Platform III), by MeioSys within the Seventh Framework Programme of the European Commission, and by Chromosome Dynamics, which is funded by the Austrian Science Foundation (FWF).
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James R A Hutchins,
Yusuke Toyoda,
Björn Hegemann,
Ina Poser,
Jean-Karim Hériché,
Martina M Sykora,
Martina Augsburg, Otto Hudecz,
Bettina A Buschhorn,
Jutta Bulkescher, [......],
Steffen Lawo,
Laurence Pelletier,
Holger Stark,
Kim Nasmyth,
Jan Ellenberg,
Richard Durbin,
Frank Buchholz,
Karl Mechtler,
Anthony A Hyman,
Jan-Michael Peters
[show abstract]
[hide abstract]
ABSTRACT: Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.
Science (New York, N.Y.), v.328, 593-599 (2010).
