Spatial/temporal organisation and regulation of the mapk pathway (MAPK SIGNALLING)

In our part of the project we analysed cells expressing either conditional forms of Raf or cells with deleted raf (KO cells) using proteomic methods for differentially expressed/modified proteins. Next to conditional Raf we also used serum or TPA. We detected more than 90 Raf regulated phosphoproteins in total. From these we could identify 28 using mass spectrometry. 8 of theses proteins are known to be localised in the nucleus, 4 can shuttle between nucleus and cytoplasm, 15 are found in the nucleus and 1 in the mitochondria. 7 of the proteins are involved in nuclear structure and heterochromatin assembly, 7 in signal transduction, 3 in mRNA transport or stabilisation, 4 have a role in the cytoskeleton, 3 in mRNA transport, 2 are involved in translation the rest have various functions, like e.g. in the proteasome. Using Raf inhibitors U0126/PD98059 we detected several proteins that are phosphorylated after Raf activation independent of the MEK/ERK part of the pathway. The substrates that we identified so far can, after evaluation, be valuable targets for identifying the activation state of the Raf/ERK pathway or for inhibition or augmentation of the pathway in therapeutically interventions. We also investigated the mitochondrial proteome under Raf/ERK stimulation. Using newly developed methods for mitochondrial isolation, we could detect diversity in mitochondrial populations. The mitochondria are divided into 7 different subpopulations, four of which are apparent in exponentially growing cells, while in cells with acute stimulation of the Raf/ERK pathway three different forms arise, which over the period of some hours change into the formerly observed populations. Their apparent isoelectrical point defines these populations. Therefore, we assume that charges or compositions of proteins integral to the outer membrane, or associated with it, change. In minor projects we analysed the proteomes of haematopoietic cells. Using different cell lines and blood samples we have identified some three proteins that might be over expressed in cells from acute myeloid leukaemia as opposed to normal CD34+ cells. Using 2D Gel Difference Electrophoresis (DiGE) we have compared alterations in protein expression and modification in Raf-1 KO fibroblasts and cells reconstituted with Raf-1 or a kinase negative Raf-1 mutant under conditions of serum starvation and stimulation with serum. Most changes in the Raf-1 KO cells were reversed by expression of either wt Raf-1 or kinase negative Raf-1. The main changes observed included changes in proteins that regulate apoptosis and the cytoskeleton. These are consistent with the role of Raf-1 in apoptosis and cell migration that is observed on the cellular and organismal level. The main value of the result obtained is of scientific nature, and resides in the knowledge acquired on the function of the proteins of the MAPK cascade and its organisation in terms of protein complexes and subcellular compartmentalisation. The descriptions of the result have been/will be published in a series of publication from the partner labs, and thus disseminated to the scientific community. The result is being/will be used by the partners themselves, to carry out specific scientific projects, and by other scientists without the consortium who are interested in exploiting our models to ask a specific question in their system. We have already received such requests, and have complied with all of them. Biomedical/pharma companies value the expertise and the knowledge we are accumulating on these eminently druggable kinase module. In particular, the results has already revealed alternative targets of the kinases in the pathway; and it has predictive value in the context of the possible side effects of therapeutic approaches aimed at interfering with a given component of the pathway. Both issues are eminently relevant when evaluating the suitability of a given protein as a therapeutic target.

A portal for managing 2D gel data, Ashwin Kotwaliwale, Keith Vass and Walter Kolch Summary: Signalling pathways lie at the core of information transmission from extracellular environment to the nucleus, producing specific biological responses. Using 2D gels we have attempted to identify a large number of proteins ideal for protein expression studies. Managing 2D gel data to understand protein interactions and compare results using computational methods is the aim of our study. We have developed a portal coupled to a relational database for managing 2D gel data. Introduction: The overall aim of our project is to model the MAPK pathway using high throughput proteomic data such as 2D gels and understand the properties of protein interactions within the MAPK pathway. We use data from 2 different gel systems (a) the DiGE, which employs the 3 fluro-dye-detection system (b) P32 labelled gels. DiGE detects difference in protein expression between samples using a common master gel and identifying each spot on the master as a single protein and mapping it across to all the gels. Quantitation is obtained by comparing against the master and so to each other. Using the DeCyder software the results can be saved as XML documents achieving compatibility on various computer operating systems. These XML documents store raw and processed values, aiding in analytical work. Tools to query XML documents using XPATH and XSLT exist, however, those methods are difficult and not appropriate for storage and comparing results. As an alternative to the XML-based methods we have designed a relational database management system to store DiGE data and Meta information about experiments as recommended in the Pedro model. As proteomics experiments are expensive and time consuming, success of the project in part would depend on efficient data management. The user is served by a rich portal interface developed in .NET platform. The portal is a modification of the fully content managed, database driven IBuySpy portal which is freely available over the internet. We have written custom modules to add, edit and retrieve experimental information and integrated them into the portal. .NET was a chosen platform as it offers extended support for XML documents and object oriented programming style for designing web pages. The web pages are an actual implementation of ASP.NET data model with VB.NET as code behind. The basic modules within the IBuySpy portal are used for storing protocols, contacts lists and discussion boards etc which is often needed by collaborating labs and proteomics community users. These modules prove useful to manage large projects wherein different bits of the experiment are performed in different geographic locations. The database is implemented in Microsoft SQL Server 2000. The database consists of a basic schema and a gel specific schema. The gel specific schema address tables used to store DiGE gel information such as spots, spotsets, spotratios and proteinID s. Meta information about the experiments is stored in other relevant tables. We have taken a modular programming approach and have written stored procedures to access data. Data flow is implemented in 3 distinct layers (a) User interface performing client-side validation (b) Business object layer which is an implementation of classes to extract data from XML documents. For DiGE data we have written a parser which implements methods of the input-output and file reader classes of the .NET framework. We use the XML streamreader which is a forward, read only implementation of the XML streamreader class. As opposed to this the P32 gel data is parsed within the database server using the Data Transformation Services. This ensures even greater efficiency and speed for data handling. (c) Data access layer which comprises of stored procedures and in some case SQL commands to interact with the database. Querying the database is a possible by various means. Firstly, we have a set of standard queries built into the database; one such example is the one that transforms DiGE data so as to compare protein expression in a set of gels. Secondly, there is often a need to connect the database directly to 3rd party software. Our database is tested with standard tools such as MATLAB and R. Lastly, we are working on an interactive query tool where the non expert users can design queries for reuse later.

Our experiments have involved the detection of hERK1 (human ERK1) localization, cytoplasmic-nuclear translocation, and dimerization in live cells using high-resolution bio-imaging methods. Specific fluorescent tagging of hERK1 in vivo has been achieved through the use of eGFP (enhanced green fluorescent protein ) fusion proteins. This eGFP-hERK1 has been shown to respond to stimulation in the same way as wild type hERK1, indicating that the presence of the GFP tag does not inhibit its function. A fusion protein with photoactivatable GFP (paGFP) has also been generated. EGFP-hERK14, a mutant of hERK1 in which the four amino acids necessary for homodimer formation are deleted, has also been characterized. The creation of these constructs and their successful transfection into ERK1-knock-out cells (provided by WP3) allowed us to study the real-time dynamics of ERK in the native environment of the cell; these investigations are still in progress. We have monitored real-time detection of eGFP-hERK1 translocation to the nucleus upon activation and compared this with the quiescent state, the kinetics of EGFP-hERK14 and the effects of nuclear export inhibitors (leptomycin B). Detection and characterization of translocation to and from the nucleus is essential for interpreting the role of ERK in signal transduction. We determined hERK1 translocation rates under different conditions using various FRAP/FLIP protocols in which the cytoplasm, nucleus or parts of the nucleus were bleached. We determined that the GFP-hERK1 was in continuous exchange between cytoplasm and nucleus and mobile within the nucleus. Dimerization of ERK has been proposed to be a requirement for nuclear translocation. However, GFP-hERK1Δ4 also accumulated in the nucleus upon stimulation with an apparent rate similar to wild type. This result was consistent with parallel real-time homoFRET measurements (a technique which detects interactions between like fluorophores) indicating that the wild type GFP-hERK1 does not dimerize before moving to the nucleus. Comparison of the rates determined with those predicted by different models is currently underway. Studies to detect interactions with other signalling cascade partners can now be designed using FRET with eGFP-ERK1 as a donor or acceptor. In addition, new derivatives of the bisarsenylated FlAsH probe have been developed in order to facilitate biochemical identification and fractionation of the intracellular partners of ERK1. The generation of a hERK1 construct containing a new and improved FlAsH tag (a hexapeptide motif including 4 cysteines) at the C-terminus has shown positive labeling results with a reduction in the background signal, which was a problem present earlier in the project. Deliverable achieved: 2.2 Spatio-temporal description of (lack of) homo-association of ERK1 Milestones achieved: M14: Constuction and expression of GFP-ERK1.

In the course of the project, we have developed genetically manipulated mice where components of the MAPK/ERK pathway are constitutively or conditionally ablated. The main value of the result obtained is of scientific nature, and resides in the knowledge acquired on the function of the proteins of the MAPK cascade in the context of the whole organism. The descriptions of the result have been/will be published in a series of publication from the partner labs, and thus disseminated to the scientific community. The result is being/will be used by the partners themselves, to carry out specific scientific projects, and by other scientists without the consortium who are interested in exploiting our models to ask a specific question in their system. We have already received a large number of such requests, and have complied with most of them. Biomedical/pharma companies value the expertise and the knowledge we are accumulating on these eminently druggable kinase module. In particular, the results has the potential, and has already, revealed alternative targets of the kinases in the pathway; and it has predictive value in the context of the possible side effects of therapeutic approaches aimed at interfering with a given component of the pathway. Both issues are eminently relevant when evaluating the suitability of a given protein as a therapeutic target.

In this part of the project we isolated and characterised Raf-1 signalling complexes and identified their components by mass spectrometry. We used three strategies to identify functionally important proteins: - Comparison of complexes formed by Raf-1 with complexes formed by mutant Raf-1 proteins that induce interesting biological phenotypes. For instance, the Raf-1 S259A point mutant efficiently stimulates the ERK pathway as well as proliferation, but fails to induce transformation in fibroblasts and differentiation in PC12 cells. The use of this mutant led to the identification of PP5 a protein phosphatase that selectively removes an activating phosphorylation in Raf-1 after mitogen stimulation. - Comparison of complexes formed by Raf-1 under different conditions of cellular stimulation, e.g. serum starvation versus mitogen stimulation. This approach led to the identification of MST2 which selectively associates with Raf-1 in serum starved cells and induces apoptosis in response to stress signals. Raf-1 inhibits MST2 activation. - Comparison of complexes formed by Raf-1 in different subcellular compartments. Density gradient fractionation of Raf-1 complexes allowed the separation of at least 5 different Raf-1 complexes. The proteomic characterisation of these complexes led to the identification of synaptojanin2 a protein that is involved in receptor endocytosis and also has a splice variant with mitochondrial localisation. Thus, all three strategies were applied successfully and can serve as a design template for the analysis of signalling pathways by proteomics. The main value of the result obtained is of scientific nature, and resides in the knowledge acquired on the function of the proteins of the MAPK cascade and its organisation in terms of protein complexes and subcellular compartmentalisation. The descriptions of the result have been/will be published in a series of publication from the partner labs, and thus disseminated to the scientific community. The result is being/will be used by the partners themselves, to carry out specific scientific projects, and by other scientists without the consortium who are interested in exploiting our models to ask a specific question in their system. We have already received such requests, and have complied with all of them. Biomedical/pharma companies value the expertise and the knowledge we are accumulating on these eminently druggable kinase module. In particular, the results has already revealed alternative targets of the kinases in the pathway; and it has predictive value in the context of the possible side effects of therapeutic approaches aimed at interfering with a given component of the pathway. Both issues are eminently relevant when evaluating the suitability of a given protein as a therapeutic target.

In the course of the project, we have developed new mouse models for human diseases, including neuronal degeneration, multistage carcinogenesis and tumour angiogenesis. They arise from the genetically manipulated mice where components of the MAPK/ERK pathway are constitutively or conditionally ablated, described in result 26965. The main value of the result obtained is of scientific nature, and resides in the knowledge acquired on the function of the proteins of the MAPK cascade in the context of cancer development and neuronal development. The descriptions of the result will soon be published in a series of publication from the partner labs, and thus disseminated to the scientific community. The result is being/will be used by the partners themselves, to carry out specific scientific projects, and by other scientists without the consortium who are interested in exploiting our models to ask a specific question in their system. We have already received a large number of such requests, and have complied with most of them. Biomedical/pharma companies value the expertise and the knowledge we are accumulating on these eminently druggable kinase module. In particular, the results has the potential, and has already, revealed alternative targets of the kinases in the pathway; and it has predictive value in the context of both the efficacy and the possible side effects of therapeutic approaches aimed at interfering with a given component of the pathway. Both issues are eminently relevant when evaluating the suitability of a given protein as a therapeutic target.

Deliverables

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