16th Protein Kinase Meeting & FEBS/ESF Workshop on
Dynamics of Cell Signal Systems
25-28 September 2008
Oslo, Norway

Organisers:

Kjetil Taskén: The Biotechnology Centre of Oslo, University of Oslo, Norway
Miles Houslay: IBLS, University of Glasgow, UK
Johannes Bos: University Medical Centre Utrecht, The Netherlands
Jacques Pouyssegur: Institute of Signalling, Developmental Biology and Cancer Research, Nice, France
Peter Downes: School of Life Sciences, University of Dundee, UK
John Scott : The Biotechnology Centre of Oslo, University of Oslo, Norway

Draft Report

Summary

This FEBS/ESF workshop was arranged at Soria Moria Hotel and Conference Centre, Oslo Norway , September 25 th -28 th 2008. The workshop consisted of lectures and poster presentations with discussions, facilitating interactions between participants at all levels.

Scientific Content

Dr. Kjetil Tasken (Univ. of Oslo, Norway) opened the workshop Thursday September 25th with Session I: Dynamics of Signal Systems and gave a brief overview of the history of the meeting and an outline of the lectures. The session continued with two distinguished key note speakers, Dr. Tony Pawson (Samuel Lunenfeld Research Institute, Canada) presenting 'SH2 domains and tyrosine kinases - back to the future' and Dr. John Scott (Univ. of Washington, USA) introducing 'Signaling in time and space'. Dr. Pawson addressed the mechanisms underlying intracellular signal transduction with focus on modular proteins that mediate protein-protein interactions and presented a historical perspective on the SH2 domain function and the regulation of tyrosine kinases in signalling events. Dr. John Scott discussed AKAP signaling mechanisms by describing structural and functional aspects of AKAP function and adressed AKAP signaling under pathophysiological conditions. In particular, Dr. Scott described the effects of knocking out AKAP150 in mice. AKAP150 brings PKA, PP2B and PKC to postsynaptic membranes, and loss of AKAP150 disturbs neuronal processes in mice.

Friday September 26th
Session II: Signal Systems for Immune Cell Activation was chaired by Dr. John Scott (Univ. of Washington, USA) and opened with Dr. Art Weiss (UCSF, US) speaking on 'Initiation of T cell receptor Signaling'. Dr. Weiss gave an introduction to the complex activation mechanisms of T cells and described features of the regulatory events involved in the initiation of intracellular signalling processes following stimulation of the T cell receptor, covering initiation of signalling via ITAM phosphorylation and the sequential interactions between ITAMs and intracellular protein tyrosine kinases. The talk was followed by a presentation on 'SAP family adaptors in cell signaling' by Dr. Andre Veillette (Institut de Recherches de Cliniques de Montreal, Canada) who introduced the SAP and EAT-2 adaptor proteins and their functions in NK cells. These adaptors are composed almost exclusively of a single SH2 domain. Through this SH2 domain, the SAP adaptors may interact with tyrosine-based motifs of SLAM receptors involved in immunoregulation by promoting the recruitment of Fyn to the receptor. Dr. Veillette presented new data on the function of the SLAM-family receptor CRACC expressed in human NK cells and how ligand triggering of these receptors potentiates NK cytotoxicity. The next speaker was Dr. Klaus Okkenhaug (Babraham Institute, UK) who talked about 'PI3K in lymphocyte signaling and development'. Dr. Okkenhaug demonstrated that p110d is the key class IA PI3K isoform in mouse T cells and that p110d is continuously required for PIP3 accumulation in the immunological synapse. Furthermore, Dr. Okkenhaug talked about the role of PI3K in T cell activation and CD28 co-stimulation showing that its role depends on the nature of the stimulus. Dr. Okkenhaug concluded that CD28 potentiates the capacity of TCR to engage the PI3K pathway and not vice versa.

Session III: Spatiotemporal Organization of Ras-Raf-MAP Kinase Signaling Cascades was introduced by chair Dr. Peter Downes (Univ. of Dunde , UK). The first lecture was given by Dr. Christopher Marshall (Institute of Cancer Research, UK) talking about 'Kinase and GTPase Signalling Pathways Controlling Cell Movement'. Dr. Marshall described a signalling pathway leading to increased cell motility centred on expression of uPAR (urokinase-type plasminogen activator receptor). uPAR drives the formation of the p130Cas-CrkII complex together with beta3 integrin complexes, resulting in Rac activation. uPAR expression is consequently linked to increased cell motility. The talk was followed by Dr Richard Marais (Institute of Cancer Research, UK) presenting 'RAF signalling in melanoma'. He introduced the oncogenic role of b-Raf and b-Raf mutations in melanoma cells and showed how mutated b-Raf may affect signalling pathways leading to ERK hyperactivation. Dr. Marais also talked about regulation of the transcription factor MITF. Oncogenic B-Raf balances MITF expression to secure growth and survival of melanomas by both stimulating protein degradation as well as increasing transcription through the transcription factor BRN2. In contrast, melanocytes with wild type b-Raf do not have this regulatory pathway as BRN2 is absent. The session ended with a presentation on 'Hypoxia Signaling and Tumor Progression' by Dr. Jaques Pouyssegur (Univ. of Nice , France). 1-2 % of the genome is most likely regulated by the transcription factor HIF1alpha, and Dr. Pouyssegur showed that hypoxia increases HIF1alpha levels in the cells by stabilizing the protein through hydroxylation. Re-oxygenation, however, results in ubiquitination and degradation of HIF1alpha. High levels of HIF1alpha in tumors give the patients a bad prognosis because the tumors get resistant to radiation and chemotherapy and because it becomes a site of selection for metastatic variants.

Session IV: Dynamic Regulation of Trafficking, Sorting and Differentiation was chaired by Dr. Johannes Bos (Univ. of Utrecht, The Netherlands) and the first speaker in this session was Dr. Ivan Dikic (Univ. of Frankfurt, Germany) who talked on the subject 'Dynamics of ubiquitin signaling networks'. Dr. Dikic focused on molecular mechanisms, and in particular, he described the identification and characterization of Rpn13 as a bona fide ubiquitin receptor in the proteasome. Dr. Dikic also described molecular mechanisms of autophagy. Dr. Harald Stenmark (Norwegian Radium Hospital, Norway) followed with ' Endosomal sorting complexes and spatial control of receptor signaling' where he described different ESCRT (endosomal sorting complexes required for transport) complexes. ESCRTs are required for ligand-mediated down-regulation of EGF receptors and for sorting of EGFRs into the lumen. Dr. Stenmark concluded that the human ESCRT complexes mediate degradation of ubiquitinated receptors such as EGFR and that they contain 3 distinct ubiquitin binding domains. Dr. Ruth Palmer (Univ. of Umeå , Sweden) then gave a talk on the subject 'Anaplastic lymphoma kinase (Alk) signaling in Drosophila'. Dr. Palmer showed that Alk, a receptor tyrosine kinase, is required for visceral muscle development and that Alk activates ERK specifically in the founder cells of developing gut. The same pathway but at a different time, seems to be involved in Alk-mediated signaling that drives Duf/Kirre in founder cells to bind to Sns in fusion competent cells. Dr. Palmer also described that Alk activity in brain cells induces neurite outgrowth. In conclusion, Dr. Palmer described a key mechanism for coordination of muscle fusion and gut constriction via repeated use of the same signaling pathways in space and time in vivo.

Session V: Signal Initiation and Termination in Physiological Systems was chaired by Dr. Jacques Pouyssegur (Univ. of Nice, France), and a small rearrangement in the program was made. Dr. Manuela Zaccolo (Univ. of Glasgow, UK) opened this session by giving a talk on the subject 'Mechanisms underlying compartmentalized cAMP/PKA signaling'. Dr. Zaccolo described how it is possible to achieve specificity in cells and cell compartments although there are multiple receptors and targets all using cAMP as a second messenger. In particular, Dr. Zaccolo described cAMP/PKA signaling in cardiomyocytes showing that PKA is targeted to specific subcellular compartments through interaction with AKAPs whereas phosphodiesterases (PDEs) block diffusion of cAMP. Different PDEs that are regulated and located differently can thus degrade cAMP from compartments where cAMP is not necessary. Dr. Zaccolo was followed by Dr. Finn Olav Levy (Univ. of Oslo, Norway) speaking on 'Cross-talk between cAMP and cGMP signalling pathways in the failing heart'. Dr. Levy described effects of altered levels of the second messengers cAMP and cGMP in congestive heart failure (CHF) and changes to the natriuretic peptide (NP) system. He also described approaches that might be beneficial in treatment of CHF, such as enhancing function of the NP system through inhibitors of NPs and cGMP degradation (e.g. via PDE inhibitors). The last speaker in this session was Dr. George Baillie (Univ. of Glasgow, UK) who stepped in on short notice on behalf of Dr. Miles Houslay. Dr. Baillie talked about 'Ubiquitination of PDE4D5' where he demonstrated that PDE4D5 is transiently ubiquitinated in respons to ß-agonists. MDM2 is required for ubiquitination of PDE4D5, and MDM2 is recruited to the ß-arrestin/PDE4D5 complex by interacting with ß-arrestin.

Saturday September 27th
Session VI: Control of Migration, Adhesion and Polarity was chaired by Dr. Margaret Frame (Univ. of Edinburgh, UK), and the first speaker was Dr. Philippe Bastiaens (Max Planck Institute for Molecular Physiology Dortmund, Germany) who gave a talk on the subject ' The spatial organization of Ras signalin ''. In his talk, Dr. Bastiaens addressed the questions of how spatial organization of Ras signaling systems is maintained in the cell and how spatial organization of Ras signaling systems affect signal responses. The next speaker was Dr. Johannes Bos (Univ. of Utrecht, The Netherlands) presenting 'Regulation and function of Epac'. In particular, Dr. Bos described the crystal structure of active Epac2 in complex with a cAMP analogue and Rap1B and how this structure is very different from that of the inactive form. Dr. Bos also described how Epac is regulated in time and space by cAMP and that endogenous cAMP induces a rapid translocation of Epac to the plasma membrane where it can activate Rap1. Rap1 activation is, however, not required for translocation. This was followed by Dr. Peter Downes (Univ. of Dundee, UK) who talked about 'PI3 kinase/PTEN signaling in cancer'. The balance of PTEN and SHIP determines the amount of PIP3 in the membrane and the activation status of PH domain-containing proteins, and inhibition of PTEN amplifies signals through the PI3K pathway. In particular, Dr. Downes showed that loss of PTEN blocks polarization and cyst formation in MDCK epithelial cell 3D culture and that PTEN knock-down affects polarization of NMuMG cells in matrigel. He also demonstrated that PTEN tumor suppressor function might require both lipid and protein phosphatase activities. The session closed with Dr. Roger Daly (Garvan Institute, Australia) presenting 'Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein'. Dr. Daly addressed the regulation and function of the adaptor protein Gab2 in cancer. Overexpression of Gab2, which is found in a subset of breast cancers, increases not only the proliferation of cancer cells but also their invasive properties. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. He presented results demonstrating that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2(S210A/T391A) exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Based on this he presented a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex.

Session VII: Signal Systems and Networks. Session was chaired by Dr. Philippe Bastiaens from the Max Planck Institute, Dortmund, Germany. Dr. Marian Walhout (Univ. of Massachusetts Medical School, USA) opened the session presenting 'Gene centered regulatory networks'. Dr. Walhout is taking a system level approach to study the principles that control differential gene expression using the nematode Caenorhabditis elegans as a model system. Her lab employs high-throughput technologies to identify protein-DNA interactions between transcription factors (TFs) and their target genes and protein-protein interactions between TFs (i.e. homo- and heterodimers). Through a DNA-protein interactome analysis based on Y1H screens she described results strongly supporting the existence of hubs in the protein-DNA networks. These are focal points with high in and out degree of signals suggesting that they may be global regulators of gene expression. They are further characterized by a higher degree of connectivity and flux capacity. Specifically, this approach also made it possible to identify 23 feedback loops and regulation by miRNA exemplified by LIN26/mir-43 which was verified in vivo in C.elegans. The second speaker was Dr. Karen Sachs (Stanford University, USA) presenting 'Elucidating signaling pathway interactions from single cell data using Bayesian network analysis'. Her introduction focused on the inherit problem of interpreting multidimensional signal data and the need to develop algorithms to be able to model the dynamic nature of signal system including feedback loops. Her approach is to use Bayesian network analysis to identify conditional dependencies to create signalling networks. Central in this approach is to include as many dimensions as possible in your analysis combined with a systematic use of perturbations. The potential of this method was illustrated by results where Bayesian analysis where applied on signal data from flowcytometry (phopho-flow) which included eleven dimensions at the single cell level. Dr. Ian M. Donaldson (Univ. of Oslo, Norway) continued the session with his talk entitled 'iRefindex. A meta-database of biomolecular interactions and complexes'. The aim of his approach is to tackle the large problem of redundancies of protein interaction data being spread across multiple databases. They have created a unifying index called iRefIndex based on data from primary interaction databases including BIND, BioGRID, DIP, HPRD, IntAct, MINT, MPact, MPPI and OPHID. iRefIndex uses the Sequence Global Unique Identifier (SEGUID) to group proteins and interactions into redundant groups. This method allows users to integrate their own data with the iRefIndex in a way that ensures proteins with the exact same sequence will be represented only once.

Session VIII: Spatiotemoral Dynamics of Cyclic Nucleotide Signalling was chaired by Dr. John Scott (Univ. Washington, USA) standing in for Dr. Miles Houslay (Univ. of Glasgow, UK). This session opened with Dr. Susan Taylor (UCSD, USA) presenting 'Dynamic Scaffolds for Assembly of PKA Proteomes'. Dr. Taylor showed structures of the PKA catalytic subunit complexed to the regulatory subunit RIalpha to describe how RI undergoes a large conformational change upon binding to the catalytic unit. This change involves uncoupling of the cAMP-binding domains and a wrapping around the C unit large lobe. Furthermore, mutagenesis of critical residues in the cAMP-binding sites, revealed a molecular mechanism for cAMP-mediated activation of PKA. This provided an excellent example on how complete complex structures, not only kinase cores, are essential for fully understanding the assembly and organisation of dynamic processes. Dr. Marco Conti (UCSF, USA) continued the session with 'A novel macromolecular complex organizing cAMP signaling that control flagellar motility'. Dr Conti introduced the subject by describing how cAMP plays a critical role in regulation of flagellar motility and showed how the second messenger load influence flagellar waveform and beat. Defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility. The session continued with Dr. Kjetil Taskén (Univ. of Oslo, Norway) presenting 'Spatiotemoral control of cyclic AMP signaling processes'. Dividing his presentation in two, Dr. Taskén spoke first on the cAMP-mediated regulation of T-cell immune responses via the Ezrin-PKA-Csk pathway and a complex that also involves EBP50 and Cbp/PAG. When T-cells and antigen-presenting cells make contact, this complex is initially located to the immunological synapse and then translocates to the distal pole. Dr. Taskén continued his lecture with cAMP-mediated signalling processes in adipocytes, describing a novel regulatory pathway involving OPA1 (optic atrophy 1), a component of the mitochondrial matrix in lipolysis. Closing this session Dr. Wolfgang Dostmann (Univ. of Vermont, USA) gave a short talk on 'Differential patterning of intracellular cGMP' focussing on the development of cGMP-indicators, cygnets, as a means of investigation the spatial dynamics of intracellular cGMP levels in real time.

Sunday September 28th
Session IX: Dynamic Regulation of Metabolic Control Systems
The session was chaired by Dr. Knut Martin Torgersen (Univ. of Oslo, Norway). The first speaker was Dr. Carol MacKintosh (Univ. of Dundee, Scotland) presenting ' 14-3-3s as dimeric digital devices that convert AGC and CAMK signals into actions '. Based on the use of 14-3-3-affinity capture combined with mass spectrometry her group aim to identify phosphorylation sites for 14-3-3 binding and define how the global 14-3-3-binding phosphoproteome responds to different extracellular stimuli and drugs. Results illustrate how the 14-3-3-linked circuitry of cells shifts from one state to another. This was exemplified in the characterization of two related Rab-GTPase activating proteins (RabGAPs) implicated in trafficking of GluT4 glucose transporters from intracellular vesicles to the cell surface to mediate influx of glucose. We have found that a RabGAP named AS160 binds 14-3-3s in response to insulin, whereas the related TBC1D1 binds 14-3-3s when cells are treated with growth factors and activators of the energy-sensing kinase AMPK. They are now using cell biology and transgenic approaches to further understand the complementary regulation of AS160 and TBC1D1, and its relevance to cancer and diabetes. The second presentation was by Dr. Anders Tengholm (Uppsala Univ., Sweden). His talk entitled 'Second messenger oscillations in the control of pulsatile insulin secretion' focused on the interplay between the second messengers cAMP and Ca2+ in the regulation of insulin signalling in pancreatic beta-cells. By using a ratiometric evanescent-wave-microscopy to measure cAMP concentration beneath the plasma membrane, he provided evidence for marked cAMP oscillations in addition to Ca2+ oscillations in the temporal control of insulin secretion. Simultaneous measurements of intracellular Ca2+ concentration revealed that the two messengers are interlinked and reinforce each other. Moreover, cAMP oscillations are capable of inducing rapid on-off Ca2+ responses, but only sustained elevation of cAMP concentration induces nuclear translocation of the catalytic subunit of the cAMP-dependent protein kinase. Together these results suggest a new signaling mode for cAMP that differentially regulates downstream cellular targets. Last speaker of this session was Dr. Debra Nathan (Pfizer System Biology, MA, USA) presenting 'Signaling dynamics in adipocytes transitioning between insulin-sensitive and insulin-resistant states' . She presented the broad scope of Pfizer system biology to elucidate mechanisms of insulin resistance in adipocytes and to identify key regulatory points that may be sensitive for drug intervention. This is part of the Insulin Resistance Pathways Project which include three main activities: i) to collect targeted dynamic data about insulin pathways within specific human cell types; ii) to use mathematical models to better understand the biological mechanisms of these pathways; and, iii) to translate findings to predict human clinical response. Their goal is to understand this network and how it changes in diseases like diabetes.

Session X: Spatiotemporal Control of Signaling Events in Cancer Development.
The session was chaired by Dr. Richard Marais ( Institute of Cancer Research , UK ). First speaker was Dr. Margaret Frame ( Univ. of Edinburgh , Scotland ) presenting ' Tyrosine kinase signalling in space and time ' where she focused on the FERM domain in the regulation of focal adhesion kinase (FAK). FAK acts as a molecular scaffold integrating signals downstream of integrin receptors. Binding partners to the FERM domain was identified by a proteomic approach and included RACK1 and Arp2/3. The interaction between FAK and RACK1 was mapped in detail and further incorporated results describing the binding to PDE4b5 and how these interactions were necessary for cells invasive behaviour including wound healing assays. Together results were used to illustrate how complex protein networks control cell behaviour related to cancer. The session was continued by Dr. Jorge Moscat (Univ. of Cincinatti, Ohio, US) presenting 'The atypical PKC pathways in inflammation and cancer'. The main goal of his lab is to elucidate the precise mechanisms of action of the atypical PKC-p62 and Par-4 complexes in cell growth and differentiation in cancer, obesity and asthma. His focus in this talk was the role of atypical PKCs in the regulation of NF-kB activation and how this was integrated by p62 and further explained some of the phenotype observed in p62-/- mice. He next discussed how p62 is involved in signalling from transformed Ras and how p62 could act as a link between Ras and NF-kB and the development of lung cancer. Next presentation was by Dr. Bjørn Tore Gjertsen (Univ. of Bergen , Norway) entitled 'p53 beta/gamma isoform expression identify chemotherapy responsive acute myeloid leukaemia patients'. AML is considered a stem cell disease and by analyzing the p53 status in each patient the goal of his group is to use this as a marker to identify disease variants insensitive to chemotherapy. Although p53 mutations are rare in AML, expression of different splice variants combined with the large amount of posttranslational modifications can provide a tool to identify subclasses of patients that will respond to certain treatment. p53 can modulate growth factor signalling and play an important role in the split between cell cycle arrest and cell death. Through a detailed characterization of p53 from AML patients after chemotherapy data was presented arguing for a positive correlation between the expression of the beta/gamma subunit and responsiveness to chemotherapy. The last talk of the meeting was by Dr. Michael Leitges ( Univ. of Oslo , Norway ) entitled 'Role of individual PKCs in the APC/Min model '. His group aims to dissect the role of different PKCs through the phenotyping of knock out mice. Except for PKCiota-/-, which are embryonic lethal, mice lacking PKC variants are viable and productive. However, by crossing these strains into different disease models the goal is to identify dinstinct functionality between PKC familiy members. One of his current focuses is to address the role of PKCs in the APC/Min mice which provide a model for colon cancer.

Poster session:
A total of 51 abstracts had been submitted for the poster session. The first evening of the meeting Thursday 25th, these posters were displayed in the room next to the conference room where all the lectures were held.

The day after, a two hour long period had been assigned for poster viewing while the poster presenters were present and available for questions. During this time, there was no other official program, and therefore, most of the conference participants took part. There were a lot of good discussions, and it was especially inspiring for the poster presenters because all the distinguished speakers of the meeting showed great interest in the posters. Such discussions and feedback from experts in the field are always highly appreciated. Also important, the content of the different posters reflected the different topics covered by the lectures, meaning that is was a good match between the interests of the young researchers and the principal investigators attending the meeting.

In the evening the same day, the poster award committee consisting of Manuela Zaccolo (University of Glasgow), Richard Marais (Institute of Cancer Research London), and Torkel Vang (The Biotechnology Centre of Oslo) announced the three winners of the poster awards (receiving gift cards of values 600, 350, and 350 Norwegian kroner, respectively). The quality of the posters was high, and it was a challenging task to decide the three winners.

Prize for best poster went to Choel Kim (Howard Hughes Medical Institute, University of California, San Diego) and co-workers for their presentation entitled 'The first crystal structure of cyclic GMP-dependent protein kinase 1 b dimerization domain reveals the molecular features of an extended leucine/isoleucine zipper'. The two second prizes were given to Giuletta De Benedetto (Venetian Institute of Molecular Medicine, Padova) and co-workers for their poster 'Protein kinase A type 1 and type 2 define distinct intracellular signaling compartments', and to Alessia Perino (Molecular Biotechnology Center, University of Torino) and co-workers for their work entitled 'PI3K g as a new AKAP controlling PDE3B activity in a PKA-dependent manner' .