Roman Körner

Max Planck Institute of Biochemistry, München, Bavaria, Germany

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Publications (56)398.2 Total impact

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    ABSTRACT: When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.
    Cell 02/2014; 156(5):975-85. · 31.96 Impact Factor
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    ABSTRACT: Maternal Embryonic Leucine zipper Kinase (MELK) was recently shown to be involved in cell division of Xenopus embryo epithelial cells. The cytokinetic furrow of these cells ingresses asymmetrically and is developmentally regulated. Two subpopulations of xMELK, the mMELK (for "mitotic" xMELK) and iMELK ("interphase" xMELK), which differ in their spatial and temporal regulation, are detected in Xenopus embryo. How cells regulate these two xMELK populations is unknown. In this study we show that, in epithelial cells, xMELK is present at a higher concentration at the apical junctional complex, in contrast to mesenchyme-like cells, which have uniform distribution of cortical MELK. Interestingly, mMELK and iMELK also differ by their requirements towards cell-cell contacts to establish their proper cortical localization both in epithelial and mesenchyme-like cells. Receptor for Activated protein Kinase C (RACK1), which we identified as an xMELK partner, co-localizes with xMELK at the tight junction. Moreover, a truncated RACK1 construct interferes with iMELK localization at cell-cell contacts. Collectively, our results suggest that iMELK and RACK1 are present in the same complex and that RACK1 is involved in the specific recruitment of iMELK at the apical junctional complex in epithelial cells of Xenopus embryos.
    Biology open. 10/2013; 2(10):1037-48.
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    ABSTRACT: The mitotic spindle is an essential molecular machine involved in cell division, whose composition has been studied extensively by detailed cellular biology, high-throughput proteomics, and RNA interference experiments. However, because of its dynamic organization and complex regulation it is difficult to obtain a complete description of its molecular composition. We have implemented an integrated computational approach to characterize novel human spindle components and have analysed in detail the individual candidates predicted to be spindle proteins, as well as the network of predicted relations connecting known and putative spindle proteins. The subsequent experimental validation of a number of predicted novel proteins confirmed not only their association with the spindle apparatus but also their role in mitosis. We found that 75% of our tested proteins are localizing to the spindle apparatus compared to a success rate of 35% when expert knowledge alone was used. We compare our results to the previously published MitoCheck study and see that our approach does validate some findings by this consortium. Further, we predict so-called "hidden spindle hub", proteins whose network of interactions is still poorly characterised by experimental means and which are thought to influence the functionality of the mitotic spindle on a large scale. Our analyses suggest that we are still far from knowing the complete repertoire of functionally important components of the human spindle network. Combining integrated bio-computational approaches and single gene experimental follow-ups could be key to exploring the still hidden regions of the human spindle system.
    PLoS ONE 03/2012; 7(3):e31813. · 3.53 Impact Factor
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    ABSTRACT: Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.
    Proceedings of the National Academy of Sciences 01/2012; 109(1):149-154. · 9.81 Impact Factor
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    ABSTRACT: Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation. Quantitation of less than 10 fmol of protein standards with errors below 10% has been demonstrated using this method.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 828:115-20. · 1.29 Impact Factor
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    ABSTRACT: 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. 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. 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.
    PLoS ONE 04/2011; 6(4):e18793. · 3.53 Impact Factor
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    ABSTRACT: Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.
    Molecular &amp Cellular Proteomics 01/2011; 10(1):M110.004457. · 7.25 Impact Factor
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    ABSTRACT: Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5'-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5' phosphate of the small RNA.
    Nucleic Acids Research 11/2010; 39(6):2330-43. · 8.81 Impact Factor
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    ABSTRACT: Reversible protein phosphorylation is a key regulatory mechanism of mitotic progression. Importantly, protein kinases themselves are also regulated by phosphorylation-dephosphorylation processes; hence, phosphorylation dynamics of kinases hold a wealth of information about phosphorylation networks. Here, we investigated the site-specific phosphorylation dynamics of human kinases during mitosis using synchronization of HeLa suspension cells, kinase enrichment, and high resolution mass spectrometry. In biological triplicate analyses, we identified 206 protein kinases and more than 900 protein kinase phosphorylation sites, including 61 phosphorylation sites on activation segments, and quantified their relative abundances across three specific mitotic stages. Around 25% of the kinase phosphorylation site ratios were found to be changed by at least 50% during mitotic progression. Further network analysis of jointly regulated kinase groups suggested that Cyclin-dependent kinase- and mitogen-activated kinase-centered interaction networks are coordinately down- and up-regulated in late mitosis, respectively. Importantly, our data cover most of the already known mitotic kinases and, moreover, identify attractive candidates for future studies of phosphorylation-based mitotic signaling. Thus, the results of this study provide a valuable resource for cell biologists and provide insight into the system properties of the mitotic phosphokinome.
    Molecular &amp Cellular Proteomics 06/2010; 9(6):1167-81. · 7.25 Impact Factor
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    ABSTRACT: MS has become a method-of-choice for proteome analysis, generating large data sets, which reflect proteome-scale protein-protein interaction and PTM networks. However, while a rapid growth in large-scale proteomics data can be observed, the sound biological interpretation of these results clearly lags behind. Therefore, combined efforts of bioinformaticians and biologists have been made to develop strategies and applications to help experimentalists perform this crucial task. This review presents an overview of currently available analytical strategies and tools to extract biologically relevant information from large protein lists. Moreover, we also present current research publications making use of these tools as examples of how the presented strategies may be incorporated into proteomic workflows. Emphasis is placed on the analysis of Gene Ontology terms, interaction networks, biological pathways and PTMs. In addition, topics including domain analysis and text mining are reviewed in the context of computational analysis of proteomic results. We expect that these types of analyses will significantly contribute to a deeper understanding of the role of individual proteins, protein networks and pathways in complex systems.
    Proteomics 03/2010; 10(6):1270-83. · 3.97 Impact Factor
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    ABSTRACT: During mitosis, phosphorylation of spindle associated proteins is a key regulatory mechanism for spindle formation, mitotic progression, and cytokinesis. In the recent past, mass spectrometry has been applied successfully to identify spindle proteomes and phosphoproteomes, but did not address their dynamics. Here, we present a quantitative comparison of spindle phosphoproteomes prepared from different mitotic stages. In total, we report the identification and SILAC based relative quantitation of 1940 unique phosphorylation sites and find that late mitosis (anaphase, telophase) is correlated with a drastic alteration in protein phosphorylation. Further statistical cluster analyses demonstrate a strong dependency of phosphorylation dynamics on kinase consensus patterns, thus, linking subgroups of identified phosphorylation sites to known key mitotic kinases. Surprisingly, we observed that during late mitosis strong dephosphorylation occurred on a significantly larger fraction of phospho-threonine than phospho-serine residues, suggesting a substrate preference of phosphatases for phospho-threonine at this stage. Taken together, our results constitute a large quantitative data resource of phosphorylation abundances at distinct mitotic stages and they provide insight into the systems properties of phosphorylation dynamics during mitosis.
    Journal of Proteome Research 09/2009; 8(10):4553-63. · 5.06 Impact Factor
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    ABSTRACT: Comprehensive phosphorylation site mapping is the central goal of phosphoproteome studies, but complete protein sequence coverage is rarely obtained using one single protease. In this study, we have evaluated the use of elastase, in comparison to trypsin, to increase phosphorylation site coverage of mitotic spindle proteins enriched from cultured human cells. We took advantage of the high mass accuracy of Orbitrap mass spectrometers and optimized the database search specificity by analyzing both elastase cleavage preferences and employing a dedicated two-step database search strategy. Through this approach, we have approximately doubled the number of detectable phosphorylation sites from elastase digested samples. Remarkably, phosphorylation sites detected by trypsin and elastase were highly complementary with an overlap of less than 10%. In total, we identified 1068 phosphorylation sites using trypsin and 467 phosphorylation sites using elastase. Approximately 30% of the phosphorylation sites were exclusively identified after digestion by elastase, demonstrating the value of this enzyme for phosphoproteome studies.
    Analytical Chemistry 12/2008; 80(24):9526-33. · 5.83 Impact Factor
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    ABSTRACT: Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.
    Molecular cell 09/2008; 31(3):438-48. · 14.46 Impact Factor
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    Rainer Malik, Erich A Nigg, Roman Körner
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    ABSTRACT: A key challenge in phosphoproteomic studies is to distinguish functionally relevant phosphorylation sites from potentially 'silent' phosphorylation. Considering that relevant phosphorylation sites are expected to be better conserved during evolution than overall Serine, Threonine and Tyrosine (S/ T/ Y) residues, we asked whether this can be directly demonstrated through statistic analysis, using a large experimental dataset. Analyzing phosphoproteomic data derived from the human mitotic spindle apparatus, we found that 95.2% of 1744 phosphorylation sites are conserved in at least one of six other vertebrate species. Using a new score, termed conservation Z-score (CZ-score), we demonstrate that phosphorylation sites are significantly better conserved than other S/T/Y sites, a conclusion validated from several kinase consensus motifs. Most importantly, phosphorylation sites with experimentally verified biological functions were significantly better conserved than other phosphorylation sites, indicating that analysis utilizing evolutionary conservation may constitute a powerful basis for the development of improved phosphorylation site predictors. malik@biochem.mpg.de Supplementary data are available at Bioinformatics online.
    Bioinformatics 07/2008; 24(12):1426-32. · 4.62 Impact Factor
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    ABSTRACT: Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles.
    Nature Biotechnology 03/2008; 26(2):164-7. · 39.08 Impact Factor
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    ABSTRACT: Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.
    Journal of Biological Chemistry 02/2008; 283(2):816-23. · 4.60 Impact Factor
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    ABSTRACT: We identify PICH (Plk1-interacting checkpoint "helicase"), a member of the SNF2 ATPase family, as an interaction partner and substrate of Plk1. Following phosphorylation of PICH on the Cdk1 site T1063, Plk1 is recruited to PICH and controls its localization. Starting in prometaphase, PICH accumulates at kinetochores and inner centromeres. Moreover, it decorates threads that form during metaphase before increasing in length and progressively diminishing during anaphase. PICH-positive threads connect sister kinetochores and are dependent on tension, sensitive to DNase, and exacerbated in response to premature loss of cohesins or inhibition of topoisomerase II, suggesting that they represent stretched centromeric chromatin. Depletion of PICH causes the selective loss of Mad2 from kinetochores and completely abrogates the spindle checkpoint, resulting in massive chromosome missegregation. These data identify PICH as a novel essential component of checkpoint signaling. We propose that PICH binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores.
    Cell 02/2007; 128(1):101-14. · 33.12 Impact Factor
  • GBM Fall meeting Hamburg 2007. 01/2007; 2007(Fall).
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    ABSTRACT: UDP-glucose is the universal activated form of glucose, employed in all organisms for glucosyl transfer reactions and as precursor for various activated carbohydrates. In animal and fungal metabolism, UDP-glucose is required for utilization of galactose and for the synthesis of glycogen, the major carbohydrate storage polymer. The formation of UDP-glucose is catalyzed by UDP-glucose pyrophosphorylase (UGPase), which is highly conserved among eukaryotes. Here, we present the crystal structure of yeast UGPase, Ugp1p. Both in solution and in the crystal, Ugp1p forms homooctamers, which represent the enzymatically active form of the protein. Ugp1p subunits consist of three domains, with the active site presumably located in the central SpsA GnT I core (SGC) domain. The association in the octamer is mediated by contacts between left-handed beta-helices in the C-terminal domains, forming a toroidal solenoid structure in the core of the complex. The catalytic domains attached to this scaffold core do not directly contact each other, consistent with simple Michaelis-Menten kinetics found for Ugp1p. Conservation of hydrophobic residues at the subunit interfaces suggests that all fungal and animal homologs form this quarternary structure arrangement in contrast to monomeric plant UGPases, which have charged residues at these positions. Implications of this oligomeric arrangement for regulation of UGPase activity in fungi and animals are discussed.
    Journal of Molecular Biology 01/2007; 364(4):551-60. · 3.96 Impact Factor
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    ABSTRACT: Formation of a bipolar mitotic spindle in somatic cells requires the cooperation of two assembly pathways, one based on kinetochore capture by centrosomal microtubules, the other on RanGTP-mediated microtubule organization in the vicinity of chromosomes. How RanGTP regulates kinetochore-microtubule (K-fiber) formation is not presently understood. Here we identify the mitotic spindle protein HURP as a novel target of RanGTP. We show that HURP is a direct cargo of importin beta and that in interphase cells, it shuttles between cytoplasm and nucleus. During mitosis, HURP localizes predominantly to kinetochore microtubules in the vicinity of chromosomes. Overexpression of importin beta or RanT24N (resulting in low RanGTP) negatively regulates its spindle localization, whereas overexpression of RanQ69L (mimicking high RanGTP) enhances HURP association with the spindle. Thus, RanGTP levels control HURP localization to the mitotic spindle in vivo, a conclusion supported by the analysis of tsBN2 cells (mutant in RCC1). Upon depletion of HURP, K-fiber stabilization is impaired and chromosome congression is delayed. Nevertheless, cells eventually align their chromosomes, progress into anaphase, and exit mitosis. HURP is able to bundle microtubules and, in vitro, this function is abolished upon complex formation with importin beta and regulated by Ran. These data indicate that HURP stabilizes K-fibers by virtue of its ability to bind and bundle microtubules. Our study identifies HURP as a novel component of the Ran-importin beta-regulated spindle assembly pathway, supporting the conclusion that K-fiber formation and stabilization involves both the centrosome-dependent microtubule search and capture mechanism and the RanGTP pathway.
    Current Biology 05/2006; 16(8):731-42. · 9.92 Impact Factor

Publication Stats

4k Citations
398.20 Total Impact Points

Institutions

  • 2002–2014
    • Max Planck Institute of Biochemistry
      • Department of Cellular Biochemistry
      München, Bavaria, Germany
  • 2012
    • Research Institute of Influenza
      Sankt-Peterburg, St.-Petersburg, Russia
    • Technical University of Denmark
      • Center for Biological Sequence Analysis
      Copenhagen, Capital Region, Denmark
  • 2000–2004
    • University of Southern Denmark
      • Department of Biochemistry and Molecular Biology
      Odense, South Denmark, Denmark
  • 1998–2000
    • Odense University Hospital
      • Molecular biology laboratory
      Odense, South Denmark, Denmark