A generic protocol for the purification and characterization of water-soluble complexes of affinity-tagged proteins and lipids.
Maeda, K., Poletto, M., Chiapparino, A. & Gavin, A.C.
Nat Protoc. 2014 Sep;9(9):2256-2266. doi: 10.1038/nprot.2014.148. Epub 2014 Aug28.
Interactions between lipids and proteins in the aqueous phases of cells contribute to many aspects of cell physiology. Here we describe a detailed protocol to systematically characterize in vivo-assembled complexes of soluble proteins and lipids. Saccharomyces cerevisiae strains expressing physiological amounts of a protein of interest fused to the tandem-affinity purification (TAP) tag are first lysed in the absence of detergent to capture intact protein-lipid complexes. The affinity-purified complexes (typically 30-50 kDa) are subjected to analytical size-exclusion chromatography (SEC) to remove contaminating lipids that elute at the void volume (>600 kDa), in order to achieve sufficient signal-to-background lipid ratios. Proteins in the SEC fractions are then analyzed by denaturing gel electrophoresis. Lipidomics techniques such as high-performance thin-layer chromatography or gas or liquid chromatography-mass spectrometry can then be applied to measure the elution profiles of lipids and to pinpoint the true interactors co-eluting with the TAP fusions. The procedure (starting from cell lysis) requires 2 d, and it can easily be adapted to other organisms.
A quantitative liposome microarray to systematically characterize protein-lipid interactions.
Saliba, A.E., Vonkova, I., Ceschia, S., Findlay, G.M., Maeda, K., Tischer, C., Deghou, S., van Noort, V., Bork, P., Pawson, T., Ellenberg, J. & Gavin, A.C.
Nat Methods. 2014 Jan;11(1):47-50. doi: 10.1038/nmeth.2734. Epub 2013 Nov 24.
Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds and signaling molecules, but they are still largely under-represented in known biological networks. Here we describe a liposome microarray-based assay (LiMA), a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed and high-throughput manner.
Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.
Maeda, K., Anand, K., Chiapparino, A., Kumar, A., Poletto, M., Kaksonen, M. & Gavin, A.C.
Nature. 2013 Sep 12;501(7466):257-61. doi: 10.1038/nature12430. Epub 2013 Aug 11.
The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.
Structural and functional characterization of a phosphatase domain within yeast general transcription factor IIIC.
Taylor, N.M., Glatt, S., Hennrich, M.L., von Scheven, G., Grotsch, H., Fernandez-Tornero, C., Rybin, V., Gavin, A.C., Kolb, P. & Muller, C.W.
J Biol Chem. 2013 May 24;288(21):15110-20. doi: 10.1074/jbc.M112.427856. Epub2013 Apr 8.
Saccharomyces cerevisiae tau55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (tau55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of tau55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phosphoserine- and phosphotyrosine-containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phosphoproteomic study identified additional phosphopeptides as possible targets that show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify tau55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities, and provide a small set of regulated phosphosite targets in vivo.
Detection and characterization of protein interactions in vivo by a simple live-cell imaging method.
Gallego, O., Specht, T., Brach, T., Kumar, A., Gavin, A.C. & Kaksonen, M.
PLoS One. 2013 May 1;8(5):e62195. doi: 10.1371/journal.pone.0062195. Print 2013.
Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).
Systematic identification of proteins that elicit drug side effects.
Kuhn, M., Al Banchaabouchi, M., Campillos, M., Jensen, L.J., Gross, C., Gavin, A.C. & Bork, P.
Mol Syst Biol. 2013;9:663. doi: 10.1038/msb.2013.10.
Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug-target relations to identify overrepresented protein-side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations.
Characterization of drug-induced transcriptional modules: towards drug repositioning and functional understanding.
Iskar, M., Zeller, G., Blattmann, P., Campillos, M., Kuhn, M., Kaminska, K.H., Runz, H., Gavin, A.C., Pepperkok, R., van Noort, V. & Bork, P.
Mol Syst Biol. 2013;9:662. doi: 10.1038/msb.2013.20.
In pharmacology, it is crucial to understand the complex biological responses that drugs elicit in the human organism and how well they can be inferred from model organisms. We therefore identified a large set of drug-induced transcriptional modules from genome-wide microarray data of drug-treated human cell lines and rat liver, and first characterized their conservation. Over 70% of these modules were common for multiple cell lines and 15% were conserved between the human in vitro and the rat in vivo system. We then illustrate the utility of conserved and cell-type-specific drug-induced modules by predicting and experimentally validating (i) gene functions, e.g., 10 novel regulators of cellular cholesterol homeostasis and (ii) new mechanisms of action for existing drugs, thereby providing a starting point for drug repositioning, e.g., novel cell cycle inhibitors and new modulators of alpha-adrenergic receptor, peroxisome proliferator-activated receptor and estrogen receptor. Taken together, the identified modules reveal the conservation of transcriptional responses towards drugs across cell types and organisms, and improve our understanding of both the molecular basis of drug action and human biology.
In vivo profiling and visualization of cellular protein-lipid interactions using bifunctional fatty acids.
Haberkant, P., Raijmakers, R., Wildwater, M., Sachsenheimer, T., Brugger, B., Maeda, K., Houweling, M., Gavin, A.C., Schultz, C., van Meer, G., Heck, A.J. & Holthuis, J.C.
Angew Chem Int Ed Engl. 2013 Apr 2;52(14):4033-8. doi: 10.1002/anie.201210178.Epub 2013 Feb 28. Europe PMC
Network Biology editorial 2013.
Marcotte, E., Boone, C., Babu, M.M. & Gavin, A.C.
Mol Biosyst. 2013 Jul 4;9(7):1557-8. doi: 10.1039/c3mb90018e. Epub 2013 May 28. Europe PMC
Cell type-specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP.
Bonn, S., Zinzen, R.P., Perez-Gonzalez, A., Riddell, A., Gavin, A.C. & Furlong, E.E.
Nat Protoc. 2012 Apr 26;7(5):978-94. doi: 10.1038/nprot.2012.049.
This protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (BiTS-ChIP) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. Embryos expressing a cell type-specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using FACS on the basis of the cell type-specific nuclear marker. Tissue-specific chromatin is extracted, sheared by sonication and used for ChIP-seq or other analyses. The key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of FACS of nuclei, yielding very high purity. The protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. The full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.
Transcription start site associated RNAs in bacteria.
Yus, E., Guell, M., Vivancos, A.P., Chen, W.H., Lluch-Senar, M., Delgado, J., Gavin, A.C., Bork, P. & Serrano, L.
Mol Syst Biol. 2012 May 22;8:585. doi: 10.1038/msb.2012.16.
Here, we report the genome-wide identification of small RNAs associated with transcription start sites (TSSs), termed tssRNAs, in Mycoplasma pneumoniae. tssRNAs were also found to be present in a different bacterial phyla, Escherichia coli. Similar to the recently identified promoter-associated tiny RNAs (tiRNAs) in eukaryotes, tssRNAs are associated with active promoters. Evidence suggests that these tssRNAs are distinct from previously described abortive transcription RNAs. ssRNAs have an average size of 45 bases and map exactly to the beginning of cognate full-length transcripts and to cryptic TSSs. Expression of bacterial tssRNAs requires factors other than the standard RNA polymerase holoenzyme. We have found that the RNA polymerase is halted at tssRNA positions in vivo, which may indicate that a pausing mechanism exists to prevent transcription in the absence of genes. These results suggest that small RNAs associated with TSSs could be a universal feature of bacterial transcription.
Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium.
van Noort, V., Seebacher, J., Bader, S., Mohammed, S., Vonkova, I., Betts, M.J., Kuhner, S., Kumar, R., Maier, T., O'Flaherty, M., Rybin, V., Schmeisky, A., Yus, E., Stulke, J., Serrano, L., Russell, R.B., Heck, A.J., Bork, P. & Gavin, A.C.
Mol Syst Biol. 2012 Feb 28;8:571. doi: 10.1038/msb.2012.4.
Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.
Deciphering a global network of functionally associated post-translational modifications.
Minguez, P., Parca, L., Diella, F., Mende, D.R., Kumar, R., Helmer-Citterich, M., Gavin, A.C., van Noort, V. & Bork, P.
Mol Syst Biol. 2012 Jul 17;8:599. doi: 10.1038/msb.2012.31.
Various post-translational modifications (PTMs) fine-tune the functions of almost all eukaryotic proteins, and co-regulation of different types of PTMs has been shown within and between a number of proteins. Aiming at a more global view of the interplay between PTM types, we collected modifications for 13 frequent PTM types in 8 eukaryotes, compared their speed of evolution and developed a method for measuring PTM co-evolution within proteins based on the co-occurrence of sites across eukaryotes. As many sites are still to be discovered, this is a considerable underestimate, yet, assuming that most co-evolving PTMs are functionally associated, we found that PTM types are vastly interconnected, forming a global network that comprise in human alone >50 000 residues in about 6000 proteins. We predict substantial PTM type interplay in secreted and membrane-associated proteins and in the context of particular protein domains and short-linear motifs. The global network of co-evolving PTM types implies a complex and intertwined post-translational regulation landscape that is likely to regulate multiple functional states of many if not all eukaryotic proteins.
Structural analyses of the Slm1-PH domain demonstrate ligand binding in the non-canonical site.
Anand, K., Maeda, K. & Gavin, A.C.
PLoS One. 2012;7(5):e36526. Epub 2012 May 4.
BACKGROUND: Pleckstrin homology (PH) domains are common membrane-targeting modules and their best characterized ligands are a set of important signaling lipids that include phosphatidylinositol phosphates (PtdInsPs). PH domains recognize PtdInsPs through two distinct mechanisms that use different binding pockets on opposite sides of the beta-strands 1 and 2: i) a canonical binding site delimited by the beta1-beta2 and beta3-beta4loops and ii) a non-canonical binding site bordered by the beta1-beta2 and beta5-beta6loops. The PH domain-containing protein Slm1 from budding yeast Saccharomyces cerevisiae is required for actin cytoskeleton polarization and cell growth. We recently reported that this PH domain binds PtdInsPs and phosphorylated sphingolipids in a cooperative manner. PRINCIPAL FINDINGS: To study the structural basis for the Slm1-PH domain (Slm1-PH) specificity, we co-crystallized this domain with different soluble compounds that have structures analogous to anionic lipid head groups of reported Slm1 ligands: inositol 4-phosphate, which mimics phosphatidylinositol-4-phosphate (PtdIns(4)P), and phosphoserine as a surrogate for dihydrosphingosine 1-phosphate (DHS1-P). We found electron densities for the ligands within the so-called non-canonical binding site. An additional positively charged surface that contacts a phosphate group was identified next to the canonical binding site. CONCLUSIONS: Our results suggest that Slm1-PH utilizes a non-canonical binding site to bind PtdInsPs, similar to that described for the PH domains of beta-spectrin, Tiam1 and ArhGAP9. Additionally, Slm1-PH may have retained an active canonical site. We propose that the presence of both a canonical and a non-canonical binding pocket in Slm1-PH may account for the cooperative binding to PtdInsPs and DHS-1P.
Quantification of mRNA and protein and integration with protein turnover in a bacterium.
Maier, T., Schmidt, A., Guell, M., Kuhner, S., Gavin, A.C., Aebersold, R. & Serrano, L.
Mol Syst Biol. 2011 Jul 19;7:511. doi: 10.1038/msb.2011.38.
Biological function and cellular responses to environmental perturbations are regulated by a complex interplay of DNA, RNA, proteins and metabolites inside cells. To understand these central processes in living systems at the molecular level, we integrated experimentally determined abundance data for mRNA, proteins, as well as individual protein half-lives from the genome-reduced bacterium Mycoplasma pneumoniae. We provide a fine-grained, quantitative analysis of basic intracellular processes under various external conditions. Proteome composition changes in response to cellular perturbations reveal specific stress response strategies. The regulation of gene expression is largely decoupled from protein dynamics and translation efficiency has a higher regulatory impact on protein abundance than protein turnover. Stochastic simulations using in vivo data show how low translation efficiency and long protein half-lives effectively reduce biological noise in gene expression. Protein abundances are regulated in functional units, such as complexes or pathways, and reflect cellular lifestyles. Our study provides a detailed integrative analysis of average cellular protein abundances and the dynamic interplay of mRNA and proteins, the central biomolecules of a cell.
SnapShot: Protein-Protein Interaction Networks.
Seebacher, J. & Gavin, A.C.
Cell. 2011 Mar 18;144(6):1000-1000.e1. Europe PMC
Recent advances in charting protein-protein interaction: mass spectrometry-based approaches.
Gavin, A.C., Maeda, K. & Kuhner, S.
Curr Opin Biotechnol. 2011 Feb;22(1):42-9. doi: 10.1016/j.copbio.2010.09.007.Epub 2010 Oct 9.
Cellular functions are the result of the coordinated action of groups of proteins interacting in molecular assemblies or pathways. The systematic and unbiased charting of protein-protein networks in a variety of organisms has become an important challenge in systems biology. These protein-protein interaction networks contribute comprehensive cartographies of key pathways or biological processes relevant to health or disease by providing a molecular frame for the interpretation of genetic links. At a structural level protein-protein networks enabled the identification of the sequences, motifs and structural folds involved in the process of molecular recognition. A rapidly growing choice of technologies is available for the global charting of protein-protein interactions. In this review, we focus on recent developments in a suite of methods that enable the purification of protein complexes under native conditions and, in conjunction with protein mass spectrometry, identification of their constituents.
Mass Spectrometry Reveals Stable Modules in holo and apo RNA Polymerases I and III.
Lane, L.A., Fernandez-Tornero, C., Zhou, M., Morgner, N., Ptchelkine, D., Steuerwald, U., Politis, A., Lindner, D., Gvozdenovic, J., Gavin, A.C., Muller, C.W. & Robinson, C.V.
Structure. 2011 Jan 12;19(1):90-100.
RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.
A systematic screen for protein-lipid interactions in Saccharomyces cerevisiae.
Gallego, O., Betts, M.J., Gvozdenovic-Jeremic, J., Maeda, K., Matetzki, C., Aguilar-Gurrieri, C., Beltran-Alvarez, P., Bonn, S., Fernandez-Tornero, C., Jensen, L.J., Kuhn, M., Trott, J., Rybin, V., Muller, C.W., Bork, P., Kaksonen, M., Russell, R.B. & Gavin, A.C.
Mol Syst Biol. 2010 Nov 30;6:430. doi: 10.1038/msb.2010.87.
Protein-metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein-lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein-lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.
3rd US-EU workshop: systems level understanding of DNA damage responses.
Sander, M., Begley, T.J., Desaintes, C., Gavin, A.C., Pelroy, R., Pothof, J., Shiloh, Y., van Gent, D., Van Houten, B., Yaffe, M. & Mullenders, L.
Mutat Res. 2010 Oct 13;692(1-2):53-60. Epub 2010 Aug 19.
The 3rd US-EU Workshop on systems level understanding of DNA damage responses was held from March 30 to April 1, 2009 in Egmond aan Zee, The Netherlands. Objectives of the workshop were (1) to assess the current science of the DDR, in particular network level responses to chemotherapeutic and environmentally induced DNA damage; and (2) to establish the basis for a reciprocal scientific exchange program between the EU and US in the relevant areas of DDR research. Here, we report the highlights of the meeting program and conclude that this third meeting in 2009 refined the role of DDR networks in human disease.
Visualizing biological data - now and in the future.
O'Donoghue, S.I., Gavin, A.C., Gehlenborg, N., Goodsell, D.S., Heriche, J.K., Nielsen, C.B., North, C., Olson, A.J., Procter, J.B., Shattuck, D.W., Walter, T. & Wong, B.
Nat Methods. 2010 Mar;7(3 Suppl):S2-4.
Methods and tools for visualizing biological data have improved considerably over the last decades, but they are still inadequate for some high-throughput data sets. For most users, a key challenge is to benefit from the deluge of data without being overwhelmed by it. This challenge is still largely unfulfilled and will require the development of truly integrated and highly useable tools.
Visualization of omics data for systems biology.
Gehlenborg, N., O'Donoghue, S.I., Baliga, N.S., Goesmann, A., Hibbs, M.A., Kitano, H., Kohlbacher, O., Neuweger, H., Schneider, R., Tenenbaum, D. & Gavin, A.C.
Nat Methods. 2010 Mar;7(3 Suppl):S56-68.
High-throughput studies of biological systems are rapidly accumulating a wealth of 'omics'-scale data. Visualization is a key aspect of both the analysis and understanding of these data, and users now have many visualization methods and tools to choose from. The challenge is to create clear, meaningful and integrated visualizations that give biological insight, without being overwhelmed by the intrinsic complexity of the data. In this review, we discuss how visualization tools are being used to help interpret protein interaction, gene expression and metabolic profile data, and we highlight emerging new directions.
Impact of genome reduction on bacterial metabolism and its regulation.
Yus, E., Maier, T., Michalodimitrakis, K., van Noort, V., Yamada, T., Chen, W.H., Wodke, J.A., Guell, M., Martinez, S., Bourgeois, R., Kuhner, S., Raineri, E., Letunic, I., Kalinina, O.V., Rode, M., Herrmann, R., Gutierrez-Gallego, R., Russell, R.B., Gavin, A.C., Bork, P. & Serrano, L.
Science. 2009 Nov 27;326(5957):1263-8.
To understand basic principles of bacterial metabolism organization and regulation, but also the impact of genome size, we systematically studied one of the smallest bacteria, Mycoplasma pneumoniae. A manually curated metabolic network of 189 reactions catalyzed by 129 enzymes allowed the design of a defined, minimal medium with 19 essential nutrients. More than 1300 growth curves were recorded in the presence of various nutrient concentrations. Measurements of biomass indicators, metabolites, and 13C-glucose experiments provided information on directionality, fluxes, and energetics; integration with transcription profiling enabled the global analysis of metabolic regulation. Compared with more complex bacteria, the M. pneumoniae metabolic network has a more linear topology and contains a higher fraction of multifunctional enzymes; general features such as metabolite concentrations, cellular energetics, adaptability, and global gene expression responses are similar, however.
Transcriptome complexity in a genome-reduced bacterium.
Guell, M., van Noort, V., Yus, E., Chen, W.H., Leigh-Bell, J., Michalodimitrakis, K., Yamada, T., Arumugam, M., Doerks, T., Kuhner, S., Rode, M., Suyama, M., Schmidt, S., Gavin, A.C., Bork, P. & Serrano, L.
Science. 2009 Nov 27;326(5957):1268-71.
To study basic principles of transcriptome organization in bacteria, we analyzed one of the smallest self-replicating organisms, Mycoplasma pneumoniae. We combined strand-specific tiling arrays, complemented by transcriptome sequencing, with more than 252 spotted arrays. We detected 117 previously undescribed, mostly noncoding transcripts, 89 of them in antisense configuration to known genes. We identified 341 operons, of which 139 are polycistronic; almost half of the latter show decaying expression in a staircase-like manner. Under various conditions, operons could be divided into 447 smaller transcriptional units, resulting in many alternative transcripts. Frequent antisense transcripts, alternative transcripts, and multiple regulators per gene imply a highly dynamic transcriptome, more similar to that of eukaryotes than previously thought.
Proteome organization in a genome-reduced bacterium.
Kuhner, S., van Noort, V., Betts, M.J., Leo-Macias, A., Batisse, C., Rode, M., Yamada, T., Maier, T., Bader, S., Beltran-Alvarez, P., Castano-Diez, D., Chen, W.H., Devos, D., Guell, M., Norambuena, T., Racke, I., Rybin, V., Schmidt, A., Yus, E., Aebersold, R., Herrmann, R., Bottcher, B., Frangakis, A.S., Russell, R.B., Serrano, L., Bork, P. & Gavin, A.C.
Science. 2009 Nov 27;326(5957):1235-40. doi: 10.1126/science.1176343.
The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.
Meeting report on the 7th World Congress of the Human Proteome Organization (HUPO) in Amsterdam: Proteome Biology.
Gavin, A.C., Aebersold, R. & Heck, A.J.
Mol Cell Proteomics. 2008 Nov;7(11):2288-91.
The 7th World Congress of the Human Proteome Organization HUPO was held in Amsterdam, the Netherlands, from August 16 to 20, 2008. The event offered a very dense 5-day agenda consisting of an exciting scientific program documenting the tremendous progress, the current challenges, and the major recent accomplishments of proteomics, an exhibition in which all the major vendors in the field of proteomics from around the globe showcased their products, technologies, and services, educational and training events in which new proteomic technologies were introduced and taught, and a series of workshops and discussion forums of all HUPO sanctioned initiatives, including a potential Human Proteome project. Around 1200 scientific abstracts were received, 80 companies signed up for and supported the exhibition, and the total number of registrations was just above 1700, with close to 700 also present for the weekend's Pre-Program. More than 60 nationalities were represented at the meeting.
Drug target identification using side-effect similarity.
Campillos, M., Kuhn, M., Gavin, A.C., Jensen, L.J. & Bork, P.
Science. 2008 Jul 11;321(5886):263-6.
Targets for drugs have so far been predicted on the basis of molecular or cellular features, for example, by exploiting similarity in chemical structure or in activity across cell lines. We used phenotypic side-effect similarities to infer whether two drugs share a target. Applied to 746 marketed drugs, a network of 1018 side effect-driven drug-drug relations became apparent, 261 of which are formed by chemically dissimilar drugs from different therapeutic indications. We experimentally tested 20 of these unexpected drug-drug relations and validated 13 implied drug-target relations by in vitro binding assays, of which 11 reveal inhibition constants equal to less than 10 micromolar. Nine of these were tested and confirmed in cell assays, documenting the feasibility of using phenotypic information to infer molecular interactions and hinting at new uses of marketed drugs.
Interaction networks for systems biology.
Bader, S., Kuhner, S. & Gavin, A.C.
FEBS Lett. 2008 Apr 9;582(8):1220-4. Epub 2008 Feb 20.
Cellular functions are almost always the result of the coordinated action of several proteins, interacting in protein complexes, pathways or networks. Progress made in devising suitable tools for analysis of protein-protein interactions, have recently made it possible to chart interaction networks on a large-scale. The aim of this review is to provide a short overview of the most promising contributions of interaction networks to human biology, structural biology and human genetics.
The social network of a cell: Recent advances in interactome mapping.
Charbonnier, S., Gallego, O. & Gavin, A.C.
Biotechnol Annu Rev. 2008;14:1-28.
Proteins very rarely act in isolation. Biomolecular interactions are central to all biological functions. In human, for example, interference with biomolecular networks often lead to disease. Protein-protein and protein-metabolite interactions have traditionally been studied one by one. Recently, significant progresses have been made in adapting suitable tools for the global analysis of biomolecular interactions. Here we review this suite of powerful technologies that enable an exponentially growing number of large-scale interaction datasets. These new technologies have already contributed to a more comprehensive cartography of several pathways relevant to human pathologies, offering a broader choice for therapeutic targets. Genome-wide scale analyses in model organisms reveal general organizational principles of eukaryotic proteomes. We also review the biochemical approaches that have been used in the past on a smaller scale for the quantification of the binding constant and the thermodynamics parameters governing biomolecular interaction. The adaptation of these technologies to the large-scale measurement of biomolecular interactions in (semi-)quantitative terms represents an important challenge.
The minimum information required for reporting a molecular interaction experiment (MIMIx).
Orchard, S., Salwinski, L., Kerrien, S., Montecchi-Palazzi, L., Oesterheld, M., Stumpflen, V., Ceol, A., Chatr-Aryamontri, A., Armstrong, J., Woollard, P., Salama, J.J., Moore, S., Wojcik, J., Bader, G.D., Vidal, M., Cusick, M.E., Gerstein, M., Gavin, A.C., Superti-Furga, G., Greenblatt, J., Bader, J., Uetz, P., Tyers, M., Legrain, P., Fields, S., Mulder, N., Gilson, M., Niepmann, M., Burgoon, L., Rivas Jde, L., Prieto, C., Perreau, V.M., Hogue, C., Mewes, H.W., Apweiler, R., Xenarios, I., Eisenberg, D., Cesareni, G. & Hermjakob, H.
Nat Biotechnol. 2007 Aug;25(8):894-8.
A wealth of molecular interaction data is available in the literature, ranging from large-scale datasets to a single interaction confirmed by several different techniques. These data are all too often reported either as free text or in tables of variable format, and are often missing key pieces of information essential for a full understanding of the experiment. Here we propose MIMIx, the minimum information required for reporting a molecular interaction experiment. Adherence to these reporting guidelines will result in publications of increased clarity and usefulness to the scientific community and will support the rapid, systematic capture of molecular interaction data in public databases, thereby improving access to valuable interaction data.
New perspectives on an old disease: proteomics in cancer research.
Gallego, O. & Gavin, A.C.
Genome Biol. 2007 Apr 30;8(4):303.
: A report on the American Association for Cancer Research Conference 'Advances in Proteomics in Cancer Research', Amelia Island, USA, 27 February-2 March 2007.
Towards quantitative analysis of proteome dynamics.
Kuhner, S. & Gavin, A.C.
Nat Biotechnol. 2007 Mar;25(3):298-300. Europe PMC
Casein kinase 2-dependent serine phosphorylation of MuSK regulates acetylcholine receptor aggregation at the neuromuscular junction.
Cheusova, T., Khan, M.A., Schubert, S.W., Gavin, A.C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H.R. & Hashemolhosseini, S.
Genes Dev. 2006 Jul 1;20(13):1800-16.
The release of Agrin by motoneurons activates the muscle-specific receptor tyrosine kinase (MuSK) as the main organizer of subsynaptic specializations at the neuromuscular junction. MuSK downstream signaling is largely undefined. Here we show that protein kinase CK2 interacts and colocalizes with MuSK at post-synaptic specializations. We observed CK2-mediated phosphorylation of serine residues within the kinase insert (KI) of MuSK. Inhibition or knockdown of CK2, or exchange of phosphorylatable serines by alanines within the KI of MuSK, impaired acetylcholine receptor (AChR) clustering, whereas their substitution by residues that imitate constitutive phosphorylation led to aggregation of AChRs even in the presence of CK2 inhibitors. Impairment of AChR cluster formation after replacement of MuSK KI with KIs of other receptor tyrosine kinases correlates with potential CK2-dependent serine phosphorylation within KIs. MuSK activity was unchanged but AChR stability decreased in the presence of CK2 inhibitors. Muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function. This is the first description of a regulatory cross-talk between MuSK and CK2 and of a role for the KI of the receptor tyrosine kinase MuSK for the development of subsynaptic specializations.
Proteome survey reveals modularity of the yeast cell machinery.
Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dumpelfeld, B., Edelmann, A., Heurtier, M.A., Hoffman, V., Hoefert, C., Klein, K., Hudak, M., Michon, A.M., Schelder, M., Schirle, M., Remor, M., Rudi, T., Hooper, S., Bauer, A., Bouwmeester, T., Casari, G., Drewes, G., Neubauer, G., Rick, J.M., Kuster, B., Bork, P., Russell, R.B. & Superti-Furga, G.
Nature. 2006 Mar 30;440(7084):631-6. Epub 2006 Jan 22.
Protein complexes are key molecular entities that integrate multiple gene products to perform cellular functions. Here we report the first genome-wide screen for complexes in an organism, budding yeast, using affinity purification and mass spectrometry. Through systematic tagging of open reading frames (ORFs), the majority of complexes were purified several times, suggesting screen saturation. The richness of the data set enabled a de novo characterization of the composition and organization of the cellular machinery. The ensemble of cellular proteins partitions into 491 complexes, of which 257 are novel, that differentially combine with additional attachment proteins or protein modules to enable a diversification of potential functions. Support for this modular organization of the proteome comes from integration with available data on expression, localization, function, evolutionary conservation, protein structure and binary interactions. This study provides the largest collection of physically determined eukaryotic cellular machines so far and a platform for biological data integration and modelling.
Protein co-membership and biochemical affinity purifications.
Gavin, A.C. & Hopf, C.
Drug Discovery Today: Technology 2006
Expanding the frontiers of proteomics.
Gavin, A.C. & Heck, A.
Drug Discovery Today: Technology 2006
Keystone symposia: proteomics and bioinformatics and systems and biology.
Expert Rev Proteomics. 2005 Jun;2(3):291-3. Europe PMC
Structure-based assembly of protein complexes in yeast.
Aloy, P., Böttcher, B., Ceulemans, H., Leutwein, C., Mellwig, C., Fischer, S., Gavin, A.C., Bork, P., Superti-Furga, G., Serrano, L. & Russell, R.B.
Science 2004 Mar 26;303(5666):2026-9.
Images of entire cells are preceding atomic structures of the separate molecular machines that they contain. The resulting gap in knowledge can be partly bridged by protein-protein interactions, bioinformatics, and electron microscopy. Here we use interactions of known three-dimensional structure to model a large set of yeast complexes, which we also screen by electron microscopy. For 54 of 102 complexes, we obtain at least partial models of interacting subunits. For 29, including the exosome, the chaperonin containing TCP-1, a 3'-messenger RNA degradation complex, and RNA polymerase II, the process suggests atomic details not easily seen by homology, involving the combination of two or more known structures. We also consider interactions between complexes (cross-talk) and use these to construct a structure-based network of molecular machines in the cell.
A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.
Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P.O., Bergamini, G., Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., Hopf, C., Huhse, B., Mangano, R., Michon, A.M., Schirle, M., Schlegl, J., Schwab, M., Stein, M.A., Bauer, A., Casari, G., Drewes, G., Gavin, A.C., Jackson, D.B., Joberty, G., Neubauer, G., Rick, J., Kuster, B. & Superti-Furga, G.
Nat Cell Biol 2004 Feb;6(2):97-105. Epub 2004 Jan 25.
Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.
Protein complexes and proteome organization from yeast to man.
Gavin, A.C. & Superti-Furga, G.
Curr Opin Chem Biol 2003 Feb;7(1):21-7.
Protein complexes may well be the most relevant molecular units of cellular function. The activities of protein complexes have to be regulated both in time and space to integrate within the overall cell programs. The cell can be compared to a factory orchestrating individual assembly lines into integrated networks fulfilling particular and superimposed tasks. Recent proteome-wide studies provide insight into the properties of cellular protein complexes, their modular nature, their interaction with other complexes and the resulting preliminary organization chart of the proteome.
90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors.
Grandi, P., Rybin, V., Bassler, J., Petfalski, E., Strauss, D., Marzioch, M., Schafer, T., Kuster, B., Tschochner, H., Tollervey, D., Gavin, A.C. & Hurt, E.
Mol Cell 2002 Jul;10(1):105-15.
We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.
A complex prediction: three-dimensional model of the yeast exosome.
Aloy, P., Ciccarelli, F.D., Leutwein, C., Gavin, A.C., Superti-Furga, G., Bork, P., Böttcher, B. & Russell, R.B.
EMBO Rep 2002 Jul;3(7):628-35.
We present a model of the yeast exosome based on the bacterial degradosome component polynucleotide phosphorylase (PNPase). Electron microscopy shows the exosome to resemble PNPase but with key differences likely related to the position of RNA binding domains, and to the location of domains unique to the exosome. We use various techniques to reduce the many possible models of exosome subunits based on PNPase to just one. The model suggests numerous experiments to probe exosome function, particularly with respect to subunits making direct atomic contacts and conserved, possibly functional residues within the predicted central pore of the complex.
Functional organization of the yeast proteome by systematic analysis of protein complexes.
Gavin, A.C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J.M., Michon, A.M., Cruciat, C.M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M.A., Copley, R.R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G. & Superti-Furga, G.
Nature 2002 Jan 10;415(6868):141-7.
Most cellular processes are carried out by multiprotein complexes. The identification and analysis of their components provides insight into how the ensemble of expressed proteins (proteome) is organized into functional units. We used tandem-affinity purification (TAP) and mass spectrometry in a large-scale approach to characterize multiprotein complexes in Saccharomyces cerevisiae. We processed 1,739 genes, including 1,143 human orthologues of relevance to human biology, and purified 589 protein assemblies. Bioinformatic analysis of these assemblies defined 232 distinct multiprotein complexes and proposed new cellular roles for 344 proteins, including 231 proteins with no previous functional annotation. Comparison of yeast and human complexes showed that conservation across species extends from single proteins to their molecular environment. Our analysis provides an outline of the eukaryotic proteome as a network of protein complexes at a level of organization beyond binary interactions. This higher-order map contains fundamental biological information and offers the context for a more reasoned and informed approach to drug discovery.
Perspectives: signal transduction. Cell survival demands some Rsk.
Nebreda, A.R. & Gavin, A.C.
Science. 1999 Nov 12;286(5443):1309-10. Europe PMC
A p90(rsk) mutant constitutively interacting with MAP kinase uncouples MAP kinase from p34(cdc2)/cyclin B activation in Xenopus oocytes.
Gavin, A.C., Ni Ainle, A., Chierici, E., Jones, M. & Nebreda, A.R.
Mol Biol Cell 1999 Sep;10(9):2971-86.
The efficient activation of p90(rsk) by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90(rsk). The MAP kinase p42(mpk1) can associate with p90(rsk) in G(2)-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90(rsk) mutant named D2 constitutively interacts with p42(mpk1). In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42(mpk1) docking site. D2 expression uncouples the activation of p42(mpk1) and p34(cdc2)/cyclin B in response to progesterone but does not prevent signaling through p90(rsk). Instead, D2 interferes with a p42(mpk1)-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42(mpk1) and Plx1 during oocyte maturation is independent of p34(cdc2)/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42(mpk1) can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.
A MAP kinase docking site is required for phosphorylation and activation of p90(rsk)/MAPKAP kinase-1.
Gavin, A.C. & Nebreda, A.R.
Curr Biol 1999 Mar 11;9(5):281-4.
Activation of the various mitogen-activated protein (MAP) kinase pathways converts many different extracellular stimuli into specific cellular responses by inducing the phosphorylation of particular groups of substrates. One important determinant for substrate specificity is likely to be the amino-acid sequence surrounding the phosphorylation site; however, these sites overlap significantly between different MAP kinase family members. The idea is now emerging that specific docking sites for protein kinases are involved in the efficient binding and phosphorylation of some substrates    . The MAP kinase-activated protein (MAPKAP) kinase p90 rsk contains two kinase domains : the amino-terminal domain (D1) is required for the phosphorylation of exogenous substrates whereas the carboxy-terminal domain (D2) is involved in autophosphorylation. Association between the extracellular signal-regulated kinase (Erk) MAP kinases and p90(rsk) family members has been detected in various cell types including Xenopus oocytes   , where inactive p90(rsk) is bound to the inactive form of the Erk2- like MAP kinase p42(mpk1). Here, we identify a new MAP kinase docking site located at the carboxyl terminus of p90(rsk). This docking site was required for the efficient phosphorylation and activation of p90(rsk) in vitro and in vivo and was also both necessary and sufficient for the stable and specific association with p42(mpk1). The sequence of the docking site was conserved in other MAPKAP kinases, suggesting that it might represent a new class of interaction motif that facilitates efficient and specific signal transduction by MAP kinases.
Regulation of human c-Abl tyrosine kinase activity in Xenopus oocytes and acceleration of progesterone-induced G2/M transition by oncogenic forms.
Dorey, K., Barila, D., Gavin, A.C., Nebreda, A.R. & Superti-Furga, G.
Biol Chem. 1999 Feb;380(2):223-30.
Deregulated activity of the Abl protein tyrosine kinase is oncogenic in humans and in animals. The normal cellular form of the enzyme is maintained at a low state of activity by mechanisms that have not yet been entirely elucidated. In particular, little is known about the trans-acting cellular factors involved. We have tested the activity of human c-Abl microinjected into oocytes of Xenopus laevis. In contrast to versions of Abl capable of transforming mammalian cells, which were highly active when introduced into oocytes, the activity of wild type c-Abl was inhibited. Oncogenic forms of Abl efficiently enhanced the ability of Xenopus oocytes to enter M phase following stimulation by progesterone. Abl-enhanced maturation was normal as judged by accumulation of Mos as well as activation of MAP kinase and Cdc2/CyclinB (MPF). Concomitant with maturation and activation of these kinases, Abl became extensively phosphorylated. Altogether, this suggests that an SH3 domain-dependent Abl regulation mechanism similar to the one observed in mammalian cells operates in Xenopus oocytes. Maturation enhancement by microinjection into Xenopus oocytes represents a useful novel assay for analyzing Abl activity. Moreover, the Xenopus oocyte may be a convenient source of trans-acting Abl regulators for biochemical studies.
A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.
Palmer, A., Gavin, A.C. & Nebreda, A.R.
EMBO J 1998 Sep 1;17(17):5037-47.
M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.
Ribosomal S6 kinase p90rsk and mRNA cap-binding protein eIF4E phosphorylations correlate with MAP kinase activation during meiotic reinitiation of mouse oocytes.
Gavin, A.C. & Schorderet-Slatkine, S.
Mol Reprod Dev 1997 Mar;46(3):383-91.
During meiotic reinitiation of the mouse oocyte, entry into M-phase is regulated by changes of protein phosphorylation and by the stimulation of selective mRNA translation following the nuclear membrane dissolution. Our results reveal that M-phase kinases (MAP kinase and histone H1 kinase) are being activated together with S6 kinase and with the phosphorylation of eIF4E, the cap-binding subunit of the initiation factor eIF-4F. In order to test which signaling pathway(s) is(are) involved, okadaic acid and cycloheximide have been used as tools for differentially modulating MAP and histone H1 kinase activities. A role for MAP kinases in the phosphorylation of eIF4E and the activation of S6 kinase is suggested. The possible implication of p90rsk and/or of p70s6k in the overall increase in S6 kinase activity has been examined. p70s6k does not appear to be involved since phosphorylated forms are found in prophase and maturing oocytes. In contrast, p90rsk is phosphorylated and activated in maturing oocytes. p90rSk phosphorylation correlates with the activation of S6 kinase. These results suggest that the overall increase of S6 kinase activity is mostly due to p90rsk activation. The roles of eIF4E phosphorylation and S6 kinase activation in the physiological induction of M-phase and in the okadaic acid-induced premature mitotic events are discussed.
An accumulation of p34cdc2 at the end of mouse oocyte growth correlates with the acquisition of meiotic competence.
de Vant'ery, C., Gavin, A.C., Vassalli, J.D. & Schorderet-Slatkine, S.
Dev Biol 1996 Mar 15;174(2):335-44.
Growing incompetent mouse oocytes released from follicular cells are unable to spontaneously resume meiosis in vitro. To identify the reasons for meiotic incompetence in these cells, the levels of p34cdc2/cyclin B kinase and p42MAPK between incompetent and competent oocytes were compared. p34cdc2 was present at very low levels in incompetent oocytes and accumulated abruptly at the time of meiotic competence acquisition. By contrast, cyclin B and p42MAPK were present at similar concentrations in both types of oocytes. Okadaic acid induced centrosome phosphorylation and meiotic reinitiation in incompetent oocytes, without inducing an increase in p34cdc2 concentration. However, the p34cdc2 present in incompetent oocytes was activated and all events following germinal vesicle breakdown were induced up to the formation of a metaphase I spindle including p42MAPK activation, sustained increase in p34cdc2 kinase activity, and translational activation of a dormant mRNA. We suggest that a threshold level of p34cdc2 has to be reached for meiotic reinitiation to be spontaneously triggered: competence is restricted at a point preceding MPF activation. Whatever the mechanism involved in this restriction point, i.e., subthreshold concentration of p34cdc2 and/or lack of an activator or presence of an inhibitor, it is bypassed by okadaic acid. Downstream of this point meiosis progresses up to metaphase 1, even though p34cdc2 concentration remains low.
Histone H1 kinase activity, germinal vesicle breakdown and M phase entry in mouse oocytes.
Gavin, A.C., Cavadore, J.C. & Schorderet-Slatkine, S.
J Cell Sci 1994 Jan;107 ( Pt 1):275-83.
Meiotic reinitiation of the mouse oocyte is characterized by a slow entry into metaphase I, beginning with germinal vesicle breakdown and ending with spindle formation. It is accompanied by a cascade of protein kinases and phosphatases increasing protein phosphorylation. The activation of histone H1 kinase and that of the mitogen-activated protein kinase p42 have been compared during spontaneous or okadaic acid-induced meiotic reinitiation. In spontaneously maturing oocytes, histone H1 kinase activity increases before germinal vesicle breakdown (2-fold), in a protein synthesis-independent manner. It is associated with the disappearance of the upper migrating form of p34cdc2, which, in our system, seems to represent the tyrosine phosphorylated form. Following germinal vesicle breakdown, histone H1 kinase activity culminates (8-fold) in metaphase I and requires protein synthesis. Activation by phosphorylation of p42MAPK is observed as a permanent shift upward-migrating form and by its myelin basic protein kinase activity. It occurs after germinal vesicle breakdown and depends on protein synthesis. In contrast, no increase of histone H1 kinase is detectable in oocytes induced to reinitiate meiosis by a transient inhibition of okadaic acid-sensitive phosphatase(s), either before germinal vesicle breakdown or during the following 7 hours of culture. A slight increase is nevertheless evident after 17 hours, when oocytes are arrested with an abnormal metaphase I spindle. The upper migrating form of p34cdc2 is present for 8 hours. The activation of p42MAPK begins before germinal vesicle breakdown.(ABSTRACT TRUNCATED AT 250 WORDS)
Okadaic acid and p13suc1 modulate the reinitiation of meiosis in mouse oocytes.
Gavin, A.C., Vassalli, J.D., Cavadore, J.C. & Schorderet-Slatkine, S.
Mol Reprod Dev 1992 Nov;33(3):287-96.
Short-term exposure to okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, induced resumption of meiosis, including metaphase spindle formation, in mouse oocytes treated with a phosphodiesterase inhibitor, while long incubations with OA arrested oocyte maturation at a step prior to spindle formation. To explore the basis for this difference, the overall patterns of protein synthesis and phosphorylation and the production of tissue-type plasminogen activator (tPA), the synthesis of which is induced after germinal vesicle breakdown (GVBD), were analyzed under various OA treatments. Short-term exposure to OA led to tPA production and did not greatly affect the maturation-associated changes in protein phosphorylation. By contrast, a long application of OA did not result in tPA production and induced more marked changes in protein phosphorylation. Microinjection into prophase oocytes of the product of the fission yeast gene p13suc1, known to inhibit p34cdc2 kinase activation and/or activity, prevented meiotic reinitiation. This effect was overcome by microinjection of OA, at concentrations higher than those required for induction of maturation in the absence of p13suc1. These observations suggest that inhibition of phosphatase 1 or 2A or both triggers meiotic resumption by acting at the same site or at a site proximal to the p13suc1-sensitive step of cdc2 kinase activation.
Induction of M-phase entry of prophase-blocked mouse oocytes through microinjection of okadaic acid, a specific phosphatase inhibitor.
Gavin, A.C., Tsukitani, Y. & Schorderet-Slatkine, S.
Exp Cell Res 1991 Jan;192(1):75-81.
We report that a specific inhibitor of types 1 and 2A phosphatases, okadaic acid (OA), induces germinal vesicle break down (GVBD) and chromosome condensation when microinjected into denuded mouse oocytes maintained in prophase block by analogs of cAMP, inhibitors of phosphodiesterase, or a tumor-promoting phorbol ester. GVBD and chromosome condensation are also observed when incompetent oocytes are similarly injected with OA, this effect being dependent on the oocyte diameter. Marked changes in cell shape, cytoskeletal organization, and chromosome condensation with abnormal or abortive spindle formation are associated with such injections. The polar body is not formed. These results led to the conclusions that in mouse oocytes, OA acts distal to both the cAMP-modulated pathway involved in meiotic arrest and the inhibitory action exerted by tumor-promoting phorbol esters.
The interaction of lithium with forskolin-inhibited meiotic maturation of denuded mouse oocytes.
Gavin, A.C. & Schorderet-Slatkine, S.
Exp Cell Res 1988 Nov;179(1):298-302.
Lithium reverses at millimolar concentrations and in a dose-dependent manner the inhibition of meiotic reinitiation (GVBD) induced by an activator of adenylate cyclase, forskolin, and does this independently of exogenous myo-inositol. These results argue for a pivotal role of adenylate cyclase in the regulation of meiotic resumption and for a possible action of lithium on G proteins rather than at the level of phosphatidylinositol resynthesis.