Báo cáo khoa học: E1 – Protein kinases pptx

E1 – Protein kinases E1-001 Protein interactions in cell signaling and polarity T. Pawson Samuel Lunenfeld Research Institute, University of Toronto, Toronto, Ontario, Canada. E-mail: pawson@mshri.on.ca A majority of human proteins have a modular composition, and are constructed of domains that mediate biomolecular inter- actions, and in some cases have catalytic functions. Interaction domains (i.e. SH2 domains) can regulate catalytic activity, direct enzymes to their substrates, and mediate the formation of multi-protein regulatory complexes, and of extended protein net- works. The functions of such modular protein interactions in phosphotyrosine-based signaling pathways, cytoskeletal organ- ization and cell polarity will be explored. The ability of aber- rant or artificial interactions to re-wire cell signaling will be discussed. E1-002 Regulation and function of the proto-oncogene protein kinase B/Akt B. A. Hemmings Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. E-mail: brian.hemmings@fmi.ch The proto-oncogene protein kinase B (PKB), also known as c-Akt, is a member of the second-messenger regulated sub- family of protein kinases. PKB is the major downstream target of receptor tyrosine kinases that signal via the phosphoinosi- tide (PI) 3-kinase. Receptor-activated PI 3-kinase produces the lipid second messenger PI-3,4,5-trisphosphate, leading to mem- brane attachment and subsequent phosphorylation and activa- tion of PKB. Activated PKB is implicated in glucose metabolism, transcriptional control, and in the regulation of apoptosis in many different cell types, including neurones. Fur- thermore, it is a central player in a signaling pathway of which many components (including PTEN, a negative regulator of this pathway) have been linked to tumorigenesis. Recent developments regarding the regulation of this signaling cassette will be presented. E1-003 Genetically encoded fluorescent reporters reveal spatiotemporal dynamics of kinase activities R. Y. Tsien 1 , J. Zhang 1,2 , C. J. Hupfeld 3 , J. M. Olefsky 3 , K. Kain 4 , M. H. Ginsberg 4 , Y. Wang 5 and S. Chien 5 1 Pharmacology, UCSD, La Jolla, CA, United States of America, 2 Pharmacology, Johns Hopkins, Baltimore, MD, United States of America, 3 Endocrinology & Metabolism, UCSD, La Jolla, CA, United States of America, 4 Medicine, UCSD, La Jolla, CA, Uni- ted States of America, 5 Bioengineering, UCSD, La Jolla, CA, Uni- ted States of America. E-mail: rtsien@ucsd.edu Genetically encoded fluorescent reporters of kinase/phosphatase activities result from tandem fusion of cyan fluorescent protein (CFP), a phosphoaminoacid-binding domain (SH2 for pTyr, FHA1 for pThr), a substrate sequence optimized for a given kinase, and yellow fluorescent protein (YFP). Phosphorylation of the substrate sequence causes it to bind intramolecularly to the phosphoaminoacid-binding domain, changing the distance and orientation between the CFP and YFP, and dynamically altering fluorescence resonance energy transfer [1, 2]. Improved reporters for protein kinase A reveal that in adipocytes, chronic insulin pre- treatment inhibits b-adrenergic but not other cAMP-elevating stimuli from activating protein kinase A (PKA), although chronic insulin meanwhile enhances b-adrenergic production of total cAMP. Disruption of PKA scaffolding mimics insulin’s preferen- tial interference with b-adrenergic signaling. Chronic insulin may disrupt close apposition of b-adrenergic receptors and PKA, exemplifying a novel mechanism for cross talk between heterolo- gous signal transduction pathways. In fibroblasts migrating during wound healing, a membrane-targeted PKA reporter shows that PKA is activated preferentially at the leading edge. This local acti- vation results from ligation of either a4b1ora5b1 integrins and is necessary for efficient motility. In human umbilical vein endothel- ial cells mechanically stimulated by laser tweezer traction on adherent fibronectin-coated beads, an improved Src reporter localized to the plasma membrane showed rapid Src activation in remote areas behind the direction of pull, sometimes accompanied by slower wave propagation of Src activity from the bead. References 1. Zhang et al. PNAS 2001; 98: 14997–15002. 2. Ting et al. PNAS 2001; 98: 15003–15008. Abstracts 298 E1-004 Biological functions of the Raf-1 ‘‘kinase’’ K. Ehrenreiter, D. Piazzolla, I. Sobczak, K. Meissl and M. Baccarini Departments of Microbiology and Genetics, Max F Perutz Laboratories at the Vienna Biocenter, University of Vienna, Vienna, Austria. E-mail: manuela.baccarini@univie.ac.at The Raf kinases have been mostly studied for their ability to relay signals from the small GTPase Ras to the MEK-ERK sign- aling module, which is activated in several human cancers. There- fore, these enzymes are considered promising therapeutic targets, and major efforts towards the development of kinase inhibitors have been/are being made. However, the biological functions of these molecules in the context of the whole organisms are as yet unclear, and efforts in this direction have been frustrated by the fact that ablation of the genes encoding the two major Raf kinas- es, B-Raf and Raf-1, is embryonic lethal. We are using condi- tional mutagenesis to circumvent embryonic lethality and to investigate the function of B-Raf and Raf-1 in the mouse. By this means, we have been able to show that the essential functions of Raf-1 in the context of embryonic development and in the adult animal are to promote cell survival and migration, rather than proliferation. These functions do not depend on the enzymatic activity of Raf-1, but are mediated via the inhibition of kinase targets, which will be discussed. E1-005 Casein kinase 2 interacts with and regulates subcellular localization of S6K1 G. Panasyuk 1 , V. Filonenko 1 and I. Gout 2 1 Institute of Molecular Biology and Genetics, Kiev, Ukraine, 2 Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom. E-mail: panasyuk_g@imbg.org.ua The ribosomal protein S6 kinase (S6K) belongs to the AGC fam- ily of Ser/Thr protein kinases which includes the protein kinase C’s, protein kinase B’s, SGKs, and 90-kDa ribosomal S6 kinases. Two forms of S6K have been identified (S6K1 and S6K2). Both kinases are activated in response to mitogenic stimuli and nutri- ents via PI3-K and mTOR signaling pathways, respectively. Bio- chemical and genetic studies provided the evidence for the involvement of S6K in the regulation of cell growth, size and proliferation. It is believed that conformational changes induced by multiple S/T phosphorylations open the structure of S6K1, making both N- and C-terminal regulatory domains available for protein–protein interactions. In this study, we describe the identi- fication of Casein Kinase 2 (CK2) as a physiological-binding partner of S6K1. Screening of a HeLa cDNA library with an activated version of S6K1 (T412D mutant) bait construct allowed us to isolate three clones corresponding to beta subunit of Casein Kinase 2 (CK2). The specificity of interaction between S6K1 and both CK2 alfa and beta subunits was further confirmed in mam- malian cells using immunoprecipitation studies with transiently overexpressed and native proteins. Bioinformatic analysis of S6K1 sequence revealed the presence of three potential phos- phorylation sites for CK2. The localization of CK2 phosphoryla- tion site was narrowed down to the N-terminal region of S6K1 with the use of deletion mutants in in vitro kinase assay. The N- terminal region contains only two Ser/Thr sites and one of them, Ser17, is in the CK2 phopshorylation motif. Mutation analysis of S17 clearly showed that it is the major in vitro phosphorylation site for CK2. Fluorescent microscopy study indicated that phos- phorylation-mimicking mutant of S6K1 (S17E) does not translo- cate to the nucleus in serum-stimulated cells. Treatment of cell with nuclear export inhibitor Leptomycin B demonstrated that S6K1 S17E mutant accumulates in the nucleus. These results indicate that nuclear import of S17E mutant is not affected while the export is significantly enhanced. In summary, this study shows for the first time that S6K1 interacts with and is phos- phorylated by CK2 in mammalian cells. The phosphorylation of S6K1 at S17 enhances its nuclear import, causing the accumula- tion of S6K1 in the cytoplasm. E1-006 Structure–function analysis on the mechanism of BCR-ABL/C-ABL localization O. Hantschel 1,2 , S. Wiesner 2 , T. Guettler 3 , C. Mackereth 2 , L. L. Remsing Rix 1,2 ,D.Go ¨ rlich 3 , M. Sattler 2 , G. Superti-Furga 1,2 1 Center for Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria, 2 European Molecular Biology Laboratory, Heidel- berg, Germany, 3 ZMBH, University of Heidelberg, Heidelberg, Germany. E-mail: hantschel@embl.de In the past years, work in several laboratories, including our own, has led to important structural and functional insight into the mechanisms of c-Abl and Bcr-Abl inhibition. However, the major- ity of the Bcr-Abl and c-Abl proteins remains uncharacterized in a structural and mechanistic sense. In particular, no structural infor- mation for the proposed subcellular localization determinants of Bcr-Abl is available. The Bcr-Abl tyrosine kinase has acquired paradigmatic status as one of the first successful cases of modern targeted cancer therapy, but drug resistance and patient relapse remain problematic. For efficient transformation, cytoplasmic localization and binding to the F-actin cytoskeleton are required, whereas enhanced or forced nuclear localization of Bcr-Abl effi- ciently offsets the transforming capability by promoting apoptosis instead. An evolutionarily conserved region at the C-terminus of Bcr-Abl mediates binding to the F-actin cytoskeleton and also con- tains a consensus sequence for a leucine-rich nuclear export signal. We have embarked on a detailed structure functional analysis of this C-terminal region. Progress in the elucidation of the mechanism of F-actin binding and nuclear export will be presented. The data may bear implications for potential therapeutic intervention strat- egies complementary to inhibition of Bcr-Abl catalytic activity. E1-007P The tobacco-specific carcinogen, NNK, stimulates proliferation of pancreatic duct epithelia through beta-adrenergic transactivation of EGF receptors M. D. Askari 1 , M. S. Tsao 2 and H. M. Schuller 1 1 Department of Pathobiology, Experimental Oncology Laboratory, University of TN, Knoxville, Tennessee, United States of America, 2 Department of Pathology, Division of Cellular and Molecular Biology, University of Toronto, Toronto, Ontario, Canada. E-mail: maskari@utk.edu Pancreatic ductal adenocarcinoma is an aggressive smoking-associ- ated human cancer. The nicotine-derived 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK) is thought to contribute to the development of these neoplasms. An NNK has been identified as an agonist for both beta 1- and 2-adrenergic receptors. Binding of NNK to these receptors stimulates proliferation of pancreatic a- denocarcinoma cells in vitro and in hamster models. The goal of this study is to elucidate the NNK effects on the signal transduc- tion pathways downstream of beta-adrenergic receptors in immor- talized normal human pancreatic HPDE6-c7 cell, the putative cells of origin of pancreatic ductal adenocarcinoma. Previously, we had shown that NNK and beta-adrenergic agonist, isoproterenol, both Abstracts 299 increase cell proliferation in HPDE6-c7 cells and also result in an increase in intracellular cyclic AMP accumulation. Additionally, Western blots demonstrated that NNK treatments result in epider- mal growth factor receptor, EGFR, and mitogen-activated protein kinase, Erk1/2 phosphorylation. Here, we further report that in these cells stimulation of the b-adrenergic receptors by NNK results in a cross talk with EGFR, followed by phosphorylation of ERK 1/2. These responses are inhibited by beta-blockers (propran- olol), and kinase inhibitors of EGFR (AG1478) and Erk1/2 (PD98059). These data suggest that NNK binding to b-receptors might permit simultaneous transduction of two independent signa- ling pathways, the EGFR transactivation and/or the G-protein- cyclic AMP pathway. This interpretation is supported by the fact that the cyclic AMP inhibitor, SQ22536 and EGFR inhibitor, AG1478, each significantly, block the proliferative responses to NNK. These findings further suggest mechanisms that might con- tribute to the development of tobacco-associated pancreatic carcin- ogenesis all of which require further investigations. E1-008P Knockdown of BChE by sequence-specific siRNAs changes PKCb- and JNK expression E. Bodur 1 and P. G. Layer 2 1 Department of Biochemistry, Faculty of Medicine, Hacettepe Uni- versity, Ankara, Turkey , 2 Developmental Biology and Neurogenet- ics, Darmstadt Technical University, Darmstadt, Germany. E-mail: bodurebru@yahoo.com During development cholinesterase expression shows a co-regula- tion between acetylcholinesterase (AChE) and butyrylcholinest- erase (BChE). BChE is related to proliferation while AChE is expressed in differentiated cell types. In the present study, the interaction of cholinesterases with signaling pathways in the rat precursor retinal cell line R28 was investigated through RNA gene silencing. SiRNAs against BChE was designed and used successfully to knockdown BChE expression. This resulted in a differential expression of protein kinase C-b (PKC) and the c-jun N-terminal kinase (JNK) proteins as observed by Western blot analysis. BChE knockdown caused a significant increase in JNK2 expression both with BchE-specific siRNAs and control siRNAs on 48 h PT. This increase in JNK2 expression was observed with both sequence-specific siRNAs and the scrambled/mismatch siR- NAs as opposed to control cells. The control cells in this study were treated with only the transfection agent. With mock trans- fections, an increase on 72 h post-transfection was seen but the reason for such an increase is unclear. Overall, the pattern of increase in both JNK forms was parallel to each other. It can be argued that the scrambled siRNA pair by not binding to a speci- fic target caused a stress in R28 cells. The expression of PKCb- isoforms on the other hand was significantly decreased as opposed to both mock transfections and control cells. E1-009P Characterization of protein kinase CKII beta mutants defective for homodimerization T H. Kim, J Y. Lee, B. S. Kang and Y S. Bae Department of Biochemistry, Kyungpook National University, Daegu, South Korea. E-mail: ysbae@mail.knu.ac.kr Protein kinase CKII (CKII) is a ubiquitous and highly conserved Ser/Thr kinase, which is found in various subcellular compart- ments in all eukaryotes examined. CKII plays a critical role in cell growth and proliferation; however, its complete physiological role and regulatory mechanism remain obscure. CKII is composed of two catalytic (a or a’) subunits and two regulatory (b) subunits. The b subunit mediates tetramer formation through the b-b homo- dimerization and a–b heterodimerization. Recently, by using the reverse two-hybrid system, we have isolated CKIIb point mutants defective for binding to ribosomal protein L41 and homodimeriza- tion. Of these mutants, R26 (Leu39Pro; Lys147Met) and R75 (Asn67Ile; Lys139Arg; Met195Val; Thr213Ala) have been further characterized in vitro in this study. Purified R26 and R75 had abil- ity to bind to CKIIa, however, they had defective binding ability to wild-type CKIIb. R26 did little stimulate the catalytic activity of CKIIa, whereas R75 effectively stimulated the catalytic activity of CKIIa. Poly-L-lysine further increased the stimulation of the cata- lytic activity by R26 or R75. Circular dichroism and intrinsic fluor- escence data indicated different conformational changes of R26 and R75. Based on the molecular models of those mutants, we could explain the difference of their capability for b–b interaction and CKIIb activation. E1-010P Phosphorylation of p53 by the Vaccinia-related Kinase-2 (VRK2), a new human kinase S. Blanco, L. Klimcakova and P. Lazo Instituto de Biologı ´ a Molecular y Celular del Ca ´ ncer, CSIC-Uni- versity of Salamanca, Salamanca, Spain. E-mail: sblano@usal.es In the human kinome, the vaccinia-related kinase (VRK) family of Ser-Thr kinases represents a new branch diverging early from the group of casein kinases I. The family is composed of three mem- bers that appear to have a ubiquitous expression in adults, but the levels vary depending on the cell line type, and in murine embryos it is implicated in the early phases of the hematopoietic develop- ment. We now report that the human VRK2 gene generates, by alternative splicing of exon 13, two isoforms A and B of 508 and 397 amino acids, respectively. The two isoforms are expressed in many cell types, with a predominance of isoform A. VRK2A has a C-terminal hydrophobic region that anchors the protein to the endoplasmic reticulum and the nuclear membrane, whereas VRK2B is detected diffusively in both cytoplasm and nucleus. The subcellular location is likely to be a major determinant of their bio- logical activity. The substrate specificities in vitro of both isoforms are similar, including specific substrates, such as p53. Both VRK2 isoforms phosphorylate in vitro p53 in Thr18. The consequence is a disruption of p53 interaction with Mdm2, since this residue is essential for maintaining the structure of the p53-binding region, thus by reducing p53 ubiquitination, which contributes in part to its stabilization and might promote other protein interactions, such as the co-activator p300, which promotes its stabilization and transactivation by acetylation. Only overexpression of VRK2B, which is located in the nucleus, contributes to p53 stabilization by a post-translational mechanism. On the other hand, VRK2A, which has a different subcellular location, does not colocalize with p53, is not able to induce its stabilization. E1-011P Use of antisense RNA to study the function of PKCb in T-cell activation M. C. Cervin ˜ o and P. Barja Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, Santiago de Compostela, Spain. E-mail: bnprimi@usc.es The members of the protein kinase C (PKC) family have been involved in signal transduction pathways regulating growth, prolif- eration and apoptosis. More specifically, the calcium-dependent and structurally similar PKCa and PKCb isoforms were suggested to play significant roles in T-cell activation. Using the antisense technology, we previously investigated the function of PKCa [1] Abstracts 300 in the activation of Jurkat cells, a leukaemia T-lymphocyte line. Following a similar approach, we now study the involvement of PKCb in that process by transfecting Jurkat cells with the pREP3 episomal vector containing a copy of the corresponding gene in the antisense orientation. Transfected cells were further selected with hygromycin and cloned by limit dilution. Immunoblotting analysis of several stably transfected antisense clones showed consistent and specific reductions in PKCb levels when compared with control clones containing the pREP3 vector alone, without changes in the levels of PKCa and other isoenzymes (PKCd,e,k,h and f) expressed in Jurkat cells. Stimulation of PKCb-reduced clones with phorbol 12-myristate 13-acetate (PMA) alone or in the presence of ionomy- cin significantly increased IL-2Ra expression, decreased CD69 lev- els and IL-8 production but did not affect TNFa secretion or the activation of the kinases ERK1/2, p38 and SAPK and the transcrip- tional factor ATF-2. Similarly, tyrosine phosphorylation pattern and the proliferative activity were not altered in Jurkat clones. Thus, the findings of this study demonstrate the role that PKCb plays in specific events of the T-lymphocyte activation process. Reference 1. Lo ´ pez-Lago et al. Eur J Immonol 1999; 29: 466–476. E1-012P Knockdown of BChE by sequence specific siRNAs changes PKCb and JNK expression E. Bodur 1 and P. G. Layer 1 1 Department of Biochemistry, Faculty of Medicine, Hacettepe Uni- versity, Ankara, Turkey , 2 Developmental Biology and Neurogenet- ics, Technical University of Darmstadt, Darmstadt, Germany. E-mail: bodurebru@yahoo.com During development, cholinesterase expression shows a co-regu- lation between acetylcholinesterase (AChE) and butyrylcholinest- erase (BChE). BChE is related to proliferation while AChE is expressed in differentiated cell types. In the present study, the interaction of cholinesterases with signaling pathways in the rat precursor retinal cell line R28 was investigated through RNA gene silencing. SiRNAs against BChE was designed and used successfully to knockdown BChE expression. This resulted in a differential expression of protein kinase C-b (PKC-b) and the c- jun N-terminal kinase (JNK) proteins as observed by Western blot analysis. BChE knockdown caused a significant increase in JNK2 expression both with BchE-specific siRNAs and control siRNAs on 48 h PT. This increase in JNK2 expression was observed with both sequence-specific siRNAs and the scrambled/ mismatch siRNAs as opposed to control cells. The control cells in this study were treated with only the transfection agent. With mock transfections, an increase on 72 h post-transfection was seen but the reason for such an increase is unclear. Overall, the pattern of increase in both JNK forms was parallel to each other. It can be argued that the scrambled siRNA pair by not binding to a specific target caused a stress in R28 cells. The expression of PKC-b isoforms on the other hand was significantly decreased as opposed to both mock transfections and control cells. E1-013P MAPKs modules in plant pathogen perception T. Me ´ sza ´ ros 1 , A. Helfer 2 , S. Peck 3 , V. Bardo ´ czy 1 and L. Bo ¨ gre 2 1 Department of Biochemistry and Food Technology, Budapest Uni- versity of Technology and Economics, Budapest, Hungary, 2 School of Biological Sciences, Royal Holloway University of London, Egham, United Kingdom , 3 The Sainsbury Laboratory, John Innes Centre, Norwich, United Kingdom. E-mail: bardoczy@mail.bme.hu Mitogen-activated protein kinase (MAPKs) cascades are funda- mental modules of signal transduction in all eukaryotes. The plant MAPK research has seen an accelerating broadening of the field for the last decade. Plant proteins involved in these cascades outnumber the mammalian counterparts indicating the existence of a sophisticated MAPK networks in plants. Though the involvement of MAPKs in plant pathogen responses is amongst the most intensively studied research fields, our knowledge is far from complete. In our work, we focused on the bacterial-derived elicitor–flagellin-induced responses and demonstrated the func- tion of AtMPK3/4/6 in this pathway. Upon flagellin treatment, rapid and transient activation of these kinases was detected by measuring the kinase activity of immuno-affinity purified protein complexes. In the next step, the upstream-activating partners of these kinases were described by application of protoplast tran- sient expression system. To provide in planta validation of these putative interaction, mutant plant carrying T-DNA insertion in the upstream kinase (AtMKK1) was isolated. The decreased kin- ase activity of AtMPK3/4/6 in the flagellin-treated Atmkk1 mutant plants confirmed the protoplast results. The phenotype analysis implies a pathogen response specific function of At- MKK1, since the only obvious difference is the higher pathogen susceptibility of Atmkk1 plants. The microarray data presented further evidence of the above-mentioned function showing reduced level of pathogen-related gene expression in flagellin- challenged Atmkk1 mutant plants. E1-014P A new, non-radioactive method for screening against glycogen synthase kinase-3b (GSK-3b) A. Baki 1 , G. I. Szendrei 2 and G. M. Keseru ˆ 1 1 CADD&HTS Laboratory, Gedeon Richter Ltd, Budapest, Hun- gary, 2 Department. of Synthetic Laboratory II, Gedeon Richter Ltd, Budapest, Hungary. E-mail: a.baki@richter.hu Glycogen synthase kinase-3b (GSK-3b) is a serine/threonine protein kinase; its activity is inhibited by a variety of extracel- lular stimuli including insuline, growth factors and cell adhe- sion. Inhibition of GSK-3b activity has been proposed to play a role in the regulation of numerous signalling pathways and pathological conditions. Compounds selectively inhibiting GSK- 3b activity may be therapeutically useful in diseases associated with elevated GSK3-b activity, such as non-insulin dependent diabetes mellitus and neurodegenerative disease. We have devel- oped a new, non-radioactive method for measuring the GSK- 3b activity based on Kinase-GloTM protocol (Promega). This is a homogeneous method of measuring kinase activity by quantifying the amount of ATP remaining in solution follow- ing the kinase reaction. Reagent for the ATP detection is the Kinase-GloTM reagent, which relies on the properties of a thermostabile luciferase (UltraGlowTM recombinant luciferase) that is formulated to generate a stabile ‘‘glow-type’’ lumines- cent signal. In order to get the best performance employing the Kinase-GloTM, we have optimized the kinase reaction con- ditions with the respect to the amount of ATP, kinase and substrate. The new assay has been compared with the radioact- ive method, determining the amount of [32P]-ATP incorporated into the substrate during the kinase reaction. The two methods have the same sensibility and were equally suitable for deter- mining GSK-3b activity. The Kinase-GloTM luminescent assay (Promega) is a homogeneous, non-radioactive, sensible method for measuring GSK-3b activity and it could be used in high- throughput screening (HTS) application. Abstracts 301 E1-015P Covalent modification of hexokinase by biologically active quinones T. Bozic 1 , I. Novakovic 1,2 , M. Gasic 1 and D. Sladic 1 1 Department of Organic Chemistry, Laboratory of Bioorganic Chemistry, University of Belgrade, Belgrade, Serbia and Montenegro, 2 Institute of Chemistry Technology and Metallurgy, Department of Chemistry, Belgrade, Serbia and Montenegro, E-mail: tbozic@chem.bg.ac.yu It is well known that antitumor agents with quinone moiety inhi- bit certain glycolytic enzymes such as hexokinase and phospho- fructokinase. Avarone/avarol quinone/hydroquinone couple, isolated from marine sponge Dysidea avara, shows significant antitumor activity due to two principal mechanisms of action: generation of oxygen radicals and alkylation of cellular nucleo- philes. In this work, inhibition of sulfhydryl containing glycolytic enzyme hexokinase by avarone and its derivatives was studied. The enzyme contains lysine and sulfhydryl residues, which can be modified by the quinone moiety. Our results show that avarone and its derivatives are capable of covalent modification of hexok- inase. Shift of pI of the enzyme upon modification toward lower pI values indicated that lysine amino groups are the targets in the reaction with quinones. Also, polymerization of hexokinase occurs. The most likely reaction is double Michael addition of lysine NH 2 -groups on quinone nucleus, as shown previously for polymerization of b-lactoglobulin [1]. The inhibition of hexokin- ase, and consequently of glycolytic metabolism is probably not the principal mechanism of antitumor action of avarone but the fact that it reduces the energy available to cells has an additive effect to other modes of action of avarone. Reference 1. Novakovic I, Vujcic Z, Bozic T, et al. J Serb Chem Soc 2003; 68: 243–248. E1-016P HerceptinÒ resistant breast cancer xenografts can be influenced by early trastuzumab treatment M. Barok 1 , I. Juha ´ sz 2 , M. Bala ´ zs 3 , J. W. Park 4 , J. Isola 5 , Z. Fazekas 1,6 , G. Vereb 1 , J. Szo ¨ llo ˜ si 1,6 1 Department of Biophysics and Cell Biology, University of Debre- cen, Debrecen, Hungary, 2 Department of Dermatology, University of Debrecen, Debrecen, Hungary, 3 Department of Hygiene and Epidemiology, University of Debrecen, Debrecen, Hungary, 4 Department of Medicine, Division of Haematology/Oncology, San Francisco, United States of America, 5 Laboratory of Cancer Gen- etics, University of Tampere, Tampere, Finland, 6 Cell Biophysics Research Group of Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary. E-mail: barokm@dote.hu Trastuzumab (HerceptinÒ) – a humanized monoclonal antibody against erbB2 – has anticancer effect and when is given as a sin- gle agent for first line treatment of ErbB2 only 40% of patients show responses, mechanisms that underlie resistance to it, are still not fully understood. In order to examine the development of trastuzumab resistance and to find its possible molecular back- ground, we inoculated JIMT-1 cells (an erbB2 positive breast cancer cell line established by Jorma Isola) subcutaneously into young female SCID mice. We started the trastuzumab treating of the first mice group when the tumors began to grow. The second mice group was inoculated with the tumor cells and trastuzumab at the same time. All mice were treated with 5 mg/kg trast- uzumab weekly. The tumors in first group could grow but slower than tumors in the control group. Tumors in second group could start to grow, then the growing was stopped, moreover the tumor size decreased. Then, from the week 4, the tumors started to grow exponentially. SCID mice with JIMT-1 xenograft were sac- rificed, the whole blood and the bone marrow from the femurs were taken. The tumors was taken out and used for immunohist- ochemistry and cytogenetics. Tumor cell lines were re-established from the xenografts. For detection of circulating tumor cells (CTC), the blood and the bone marrow were centrifuged on den- sity gradient and cells were collected from the buffy coat then stained for erbB2, HLA class I and erbB1. We could detect circu- lating CTCs both in blood and bone marrow and we were able to detect real micrometastasis consisting of 5–10 tumor cells. Our results suggest that erbB2-positive breast cancer patents should have trastuzumab treatment as soon as possible, although the resistance to trastuzumab may develop later. E1-017P Regulation of the DNA-binding ability of P53 protein by phosphorylation and by monoclonal antibodies V. Brazda, E. Jagelska, P. Pecinka and E. Palecek Academy of Sciences, Brno, Czech Republic. E-mail: vaclav@ibp.cz Tumour suppressor protein p53 represents one of the most important factors regulating cell proliferation, differentiation and programmed cell death. P53 triggers growth arrest or apoptosis in response to different cellular stress signals and its biochemical function is associated with the ability to operate as a transcription factor. The way of p53 activation seems to be the critical event responsible for the p53 response to DNA damage. The most likely physiological mechanism of p53 acti- vation to sequence-specific DNA binding is its post-transla- tional modification. Using non-radioactive EMSA, we show that phosphorylation of p53 protein with CKII at Ser392, PKC at Ser378 and cdk2/cyclinA at Ser315 can efficiently activate p53 to sequence-specific DNA binding in vitro. In addition, p53 co-phosphorylation with cdk2/cyclinA and PKC or cdk2/cyclin- A and CKII increases the DNA-binding activity of p53 but, in contrast, co-phosphorylation with PKC and CKII results in the decrease of the p53-DNA binding. To study activation of p53 to sequence-specific binding, we used also supershifting of p53/ DNA complexes by monoclonal antibodies. C-terminal specific antibodies Bp53-6.1, Bp53-10.1, Bp53-30.1 activated the sequence-specific binding of p53 to consensus sequence in a selective manner, while other MAbs DO-l, DO-13, ICA 9 did not influence the p53/DNA binding. We have developed a rapid and reliable method for analyzing binding of p53 protein to DNA using a modified enzyme-linked immunosorbent assay. Our results indicate the crucial role of post-translational modifi- cations in the regulation of p53-DNA-binding activity but also suggest that antagonistic relations exist between different stress signalling pathways. GACR 301/04/P025 and GAAV B500040502. Abstracts 302 E1-018P Phosphorylation of extracellular signal regulated kinases (ERK) in the spinal cord depends on vanilloid-insensitive primary afferents as shown by intravesical instillation of resiniferatoxin C. D. Cruz 1,3 , D. Ferreira 1 , S. B. McMahon 3 and F. Cruz 1,2 1 Institute of Histology and Embryology, University of Porto, Porto, Portugal, 2 Department of Urology, University of Porto, Porto, Portugal, 3 Neurorestoration group, King’s College, London, United Kingdom. E-mail: ccruz@med.up.pt ERK belong to the MAPK family of intracellular signalling cas- cades. Recent studies showed that noxious stimulation of somatic and visceral structures led to the activation of ERK signalling cascade in the spinal cord. In particular, this cascade seems to play a major role in spinal neuronal response to bladder stimula- tion. However, the nature of the primary afferent fibres, which convey the sensitive input leading to the phosphorylation of ERK (phosphoERK), is currently poorly understood. In this study, adult female Wistar rats were treated intravesically with RTX 100 nm or its vehicle. On the following day, animals were divided in groups according to the type of stimulation (intravesi- cal application of acetic acid 1% or 3% and urinary bladder dis- tension at 60 cm H 2 O). After being deeply anesthetized, animals were stimulated for 15 min. Two hours afterwards, animals were again stimulated. Spinal cords were collected and processed for c-Fos (a marker of neuronal activity of RTX-sensitive fibres) and phosphoERK imunocytochemistry. Preliminary results indicate that animals treated with RTX 100 nm showed a markedly diminished c-Fos expression while phosphoERK levels were sim- ilar to those observed in controls, despite the type of stimulation. Our results suggest that ERK phosphorylation in the spinal cord is not dependant upon sensitive input conveyed by RTX-sensitive fibres, contrary to c-Fos expression. This is surprising since ERK activation can control c-Fos expression in several systems. Alter- natively, in spinal cord neurons, c-Fos expression may be regula- ted through other signalling pathways. Acknowledgment: Grant SFRH/BD/2001/5826, FCT/PGDB; FCT project POCTI/1999/NSE/32466, Portugal. E1-019P Protein phosphatase 4: a multifunctional protein serine/threonine phosphatase sensitive to antitumour drugs P. T. Cohen Medical Research Council Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, Tayside, Scotland. E-mail: p.t.w.cohen@dundee.ac.uk Most cellular functions are regulated by reversible protein phos- phorylation. Protein phosphatase 4 (Ppp4/PP4) is a ubiquitous protein phosphatase that dephosphorylates serine and threonine residues. Although Ppp4 is most closely related to PP2A in the PPP family, it is an essential enzyme in higher eukaryotes required for the maturation of centrosomes and is now known to regulate a variety of other cellular functions independently of PP2A. Ppp4 is located not only at centrosomes, but also present at low levels in the cytoplasm with a predominant location in the nucleus. Recently, Ppp4 has been shown to play a role in several nuclear functions, including the activities of chromatin and spliceosomal assembly via interaction with the survival of motor neurons (SMN) complex. Ppp4 participates in cellular signalling pathways in the cytoplasm, including the TOR pathway. A vari- ety of antitumour agents and toxins potently inhibit Ppp4. The catalytic subunit of Ppp4 (Ppp4c) exists in high molecular mass complexes. Regulatory subunits (R1 and R2) have been identified in mammals that interact with Ppp4c, control its activity and allow interaction with a third ‘‘variable subunit’’. The roles and interplay of these different regulatory complexes in cellular func- tions will be discussed. Orthologues of some mammalian Ppp4 subunits can be identified in Saccharomyces cerevisiae and their sensitivities to the anticancer, DNA-binding drug, cisplatin, will be considered. E1-020P Investigating human protein kinase X holoenzyme formation using bioluminescence resonance energy transfer (BRET) M. Diskar, F. W. Herberg and A. Prinz Department of Biochemistry, Laboratory of F.W. Herberg, Univer- sity of Kassel, Kassel, Germany. E-mail: diskar@gmx.de The human X chromosome encoded protein kinase (PrKX) is a catalytic subunit of type I cAMP-dependent protein kinase (PKA). The function of PrKX is unclear and discussed contro- versial. It was described as a lineage specific protein kinase, cru- cial for macrophage and granulocyte maturation, possibly involved in cellular morphogenesis. PrKX phosphorylates the PKA-regulatory (R) subunit RIIa in vitro. High affinity binding to the heat stable protein kinase inhibitor (PKI) and RI-subunit was demonstrated by surface plasmon resonance-binding studies [Zimmermann et al. (1999) J. Biol. Chem. 274(9):5370–5378]. To investigate holoenzyme formation of PrKX in vivo, we tagged Renilla luciferase (Rluc) or green fluorescent protein (GFP 2 )to the putative-binding partners hRIa, hRIIa and PrKX as well hRIa that was mutated in the cAMP binding pocket(s). After co-transfection of COS7 cells, we monitored BRET between the fusion proteins. The results show that in contrast to Ca, PrKX does not form a holoenzyme complex with RIIa in vivo. Holo- enyzme formation with wildtype and mutant forms of hRIa leads to a 2–2.5-fold increase in BRET signal. The rise of intracellular cAMP or incubation with 8-Br-cAMP leads to a dose-dependent signal reduction up to 75%. Incubation with the PKA antagonist Rp-8-Br-cAMPS, however, is followed by signal increase, indica- ting stabilization of the holoenzyme complex. Co-expression of PrKX and a dominant negative mutant of RIa resulted in the highest BRET level, and cAMP did not release the catalytic sub- unit from this holoenzyme complex. Acknowledgment: Funding: EU-RTD-QLRT-2001-02149; DFG-He-1818/4. E1-021P Possible mechanisms underlying spike-timing dependent synaptic plasticity in the CA1 field of rat hippocampus A. A. Denisov and S. N. Cherenkevich Biophysics Department, Belarus State University, Minsk, Belarus. E-mail: denisov@bsu.by Many modern models of brain functions are based on a spike- timing dependent form of synaptic plasticity (STDP). STDP means that the induction of a long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission depends on precise timing of pre- and post-synaptic activity. Our investi- gations of a timing dependence of associative synaptic plasticity in the CA1 field of rat hippocampal slices in vitro showed that a long-term decrease in synaptic efficacy, caused by a pre-syn- aptic activation after a post-synaptic depolarization, depends on post-synaptic complex spike bursting. Standard model of STDP Abstracts 303 induction is based on the assumption that the direction of change in synaptic efficacy is determined by the level of post- synaptic Ca 2+ . Activation of NMDA receptors by the glutam- ate and subsequent strong depolarization by the backpropagat- ing spike leads to an intensive Ca 2+ influx. High level of Ca 2+ leads to the formation of a Ca/calmodulin complex, which results in the autophosphorylation of a Ca/calmodulin-depend- ent protein kinase II (CaMKII). The autophosphorylated CaM- KII phosphorylates AMPA receptors. Phosphorylation of the protein leads to increase of probability of high conduction states of the AMPA receptor channel and accordingly to induc- tion of LTP. Post-synaptic spike followed by the activation of NMDA receptors causes small influx of Ca 2+ . Slight increase of the post-synaptic calcium level activates calcineurin via Ca/ calmodulin complex. Activation of the calcineurin dephosphory- lates inhibitor I-1 and results in activation of the protein phos- phatase PP1 and subsequent LTD by dephosphorylation of AMPA receptors. Our data show that LTD dependence on a type of post-synaptic bursting is difficult to explain in terms of the standard model that based on the peak level of post-synap- tic Ca 2+ . Additional mechanisms, possibly dependent on a dynamics of Ca 2+ level, must be accounted for. E1-022P Inhibition of Akt kinase and p42/44 MAP kinase (Erk1/2) pathways during cannabinoid- induced apoptosis of human glioma cells A. Ellert-Miklaszewska 1 , B. Kaminska-Kaczmarek 2 and L. Konarska 1 1 Department of Biochemistry and Clinical Chemistry, Medical Uni- versity of Warsaw, Warsaw, Poland, 2 Department of Cell Biology, Laboratory of Transcription Regulation, Nencki Institute, Warsaw, Poland. E-mail: aellert@nencki.gov.pl Cannabinoids are known to induce apoptosis of glioma cells in vitro and in vivo via their specific receptors, CB1 and/or CB2. Studying their molecular mechanisms of action, we have shown previously that the level of active protein kinase B (Akt) and p42/44 MAP kinase (Erk1/2) decreased under exposure of rat C6 glioma cells to cannabinoid treatment. It leads to activation of bad function and induction of apoptotic death via mitochondrial pathway. In our recent experiments, four human glioma cell lines with different p53 and PTEN status: T98 (p53mut, PTENmut), U373MG and U87MG (p53mut, PTENmut) and LN229 (p53mut, PTENwt) were exposed to synthetic cannabinoids: WIN55,212-2 (non-selective CB1/CB2 agonist) and JWH133 (CB2-selective). Susceptibility of human glioma cells to cannabi- noid treatment correlated with cannabinoid receptor expression, analyzed by RT-PCR. The percentage of surviving cells, as meas- ured by MTT test, was, however, independent from p53 and PTEN status. Both compounds, once effective, triggered a decrease of mitochondrial membrane potential, induced a clea- vage of caspase-9, -3 and -7 and PARP proteolysis. Similarly to the effects observed previously in C6 cells, cannabinoid treatment produced downregulation of the Akt and Erk signalling pathways in human cell lines prior to appearance of any signs of apoptosis. However, time and strength of response differed between the lines. We conclude that cannabinoids are efficient inhibitors of human glioma cells growth but the execution phase of cell death depends on the molecular background of glioma cells. Concomit- ant inhibition of both affected survival pathways seems to be cru- cial for glioma cell death. Acknowledgment: Supported by the Grant No. 006/2002 from the Polish Pharmacy and Medicine Development Foundation by Polpharma S.A. E1-023P Biochemical characterization and the biological significance of the CK2-activated a-type cAMP-dependent protein kinases (CK2-aPKAs) in vitro M. Enomoto 1 , Y. Sawano 1 , S. Kosuge 1 , Y. Yamano 2 , K. Kuroki 2 and K. Ohtsuki 1 1 Laboratory of Genetical Biochemistry and Signal Biology, Gradu- ate School of Medical Sciences, University of Kitasato, Sagamiha- ra, Kanagawa, Japan, 2 Laboratory of Molecular and Cellular Biology, Cancer Research Institute, University of Kanazawa, Kan- azawa, Ishikawa, Japan. E-mail: mm04010y@st.kitasato-u.ac.jp Object: It is well known that cAMP-dependent protein kinase (PKA) is activated through the binding of cAMP to the regula- tory (R) subunit of PKA in vitro and in vivo. Recently, we repor- ted that (i) the R-subunits (RIa and RIIa)ofa-type PKAs are effectively phosphorylated by casein kinase 2 (CK2) in vitro; and (ii) this full phosphorylation in the absence of cAMP results in an activation of PKA activity in vitro [1]. Therefore, the present study was carried out to biochemically characterize the CK2-acti- vated PKA (CK2-aPKA) using sperm protamine and hepatitis B virus (HBV) core protein (HCP) as its substrates in vitro. Results and discussion: The phosphorylation of PKA R-sub- units by CK2 resulted in an activation of PKA activity and this activation was a similar level to that of cAMP-activated PKA (cAMP-aPKA) using histone H2B as a substrate in vitro. Interest- ingly, the phosphorylation of protamines (salmines AI and AII) by CK2-aPKA was about 10-fold higher than that determined with cAMP-aPKA in vitro. No significant effects of cAMP and DNA on the CK2-aPKA-mediated phosphorylation of salmines were observed in vitro. The CK2-aPKA-induced high phosphory- lation was observed with HCP, like protamine, containing at least three PKA phosphorylation sites (R-R-R-S/T) on its C-terminal region in vitro. As expected, recombinant HCP (rHCP) was about 10-fold phosphorylated at Ser-residues by CK2-aPKA and this was about threefold higher than that determined with cAMP-aPKA in vitro. These results strongly suggest that (i) sperm protamines as well as rHCP may function preferential targets of CK2-aPKAs without cAMP in vivo; and (ii) the pref- erential phosphorylation of these DNA-binding functional pro- teins may be involved in the gene expression in fertilized eggs and in an assembly of HBV particle in the infected cells. Reference 1. Kosuge S, Sawano Y, Ohtsuki K. Biochem Biophys Res Common 2003; 310: 163–168. E1-024P Role of protein kinase C on the regulation of endothelial and neuronal nitric oxide synthase function in rat mesenteric artery J. Blanco-Rivero, G. Balfago ´ n and M. Ferrer Departamento de Fisiologı ´ a, Universidad Auto ´ noma de Madrid, Madrid, Spain. E-mail: mercedes.ferrer@uam.es The objective of the present study was to assess the regulatory effect of protein kinase C (PKC) on endothelial and neuronal nitric oxide synthase (eNOS and nNOS, respectively) functions. For this purpose, superior mesenteric arteries with and without endothelium from male Sprague–Dawley rats were used to ana- lyze if PKC regulates: (i) the activity of eNOS and nNOS, by incubating the arteries with the fluorescent probe 4,5-diamino- fluorescein, and (ii) the vasomotor function of endothelial and neuronal NO release induced by acetylcholine (Ach) and electri- cal field stimulation (EFS), respectively. In endothelium intact arteries, Ach induced an NO release that was not modified by Abstracts 304 pre-incubation with either the PKC activator phorbol 12,13-dib- utyrate (PDBu) or the PKC inhibitor calphostin C. In these arteries, pre-incubation with PDBu or calphostin C did not alter the relaxation induced by Ach. In endothelium-denuded mesen- teric arteries, the EFS-induced NO release was increased by PDBu and decreased by calphostin C pre-incubation. EFS induced a contractile response, which was increased by the addi- tion of the NOS inhibitor N w -nitro-arginine-methyl ester (L- NAME). Calphostin C increased the EFS-induced contraction, and further addition of L-NAME did not affect that response. PDBu reduced the EFS-induced contractions, and this effect was reversed by the further addition of L-NAME. These results show that while PKC does not affect eNOS function, it does activate nNOS function. Acknowledgment: Supported by grants from FIS (PI020335 and C03/01) and DGCYT (BFI2001-1324). E1-025P ACTH activates p38 MAPK in the adrenal gland in vivo J. G. Ferreira 1,2 and D. Pignatelli 1,2 1 Institute of Histology Embryology, Porto, Portugal, 2 IBMC, Porto, Portugal. E-mail: jferreir@med.up.pt ACTH released from the pituitary is thought to act through the activation of cAMP/PKA in the adrenal cortex cells hence stimu- lating steroidogenesis. Although ACTH was originally considered not to have proliferative effects on the adrenal, it has recently been described that it could also induce cell proliferation acting via other signalling cascades. The protein p38 is a member of the MAPK family of cascades. It is activated by stress stimuli and cytokines, becomes phosphorylated via MKK3/6 and regulates a diversity of cellular processes such as apoptosis, inflammation and differentiation. Our purpose was to study the effects of administration of ACTH or Dexamethasone (Dxm – a synthetic glucocorticoid) on the activation of p38 in vivo and how this acti- vation might be related to increased cell proliferation. Using rats submitted to both treatment protocols at variable dosage, we attempted to determine if ACTH could be related to p38 activa- tion. Blood was collected for hormonal analysis and the adrenals, after fixation with paraformaldehyde, were used to study the expression of phospho-p38. Double staining with PCNA and phospho-p38 was also performed to correlate the activation of p38 with increased levels of proliferation on the adrenal. Overall, we observed that ACTH increased adrenal weight as well as the levels of corticosterone. The treatment also increased the activa- tion of p38 when compared with control or Dxm-treated animals. We have also shown that ACTH increases PCNA expression in a dose-dependent manner and that p38 co-localizes with PCNA, suggesting that adrenal proliferation could be regulated by this pathway. E1-026P Dopamine D-like receptor-mediated inhibition of Cl – /HCO 3 – exchanger activity is regulated by G protein-coupled receptor kinase 6 (GRK 6) in rat intestinal epithelial IEC-6 cells S. Fraga and P. Soares-da-Silva Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal. E-mail: sfraga@med.up.pt The Cl – /HCO 3 – exchanger and the Na + /H + exchanger are extremely important in the maintenance of intracellular pH and cell volume in intestinal cells and essential for electroneutral NaCl absorption. Recently, it has been reported that dopamine D 1 -like receptor stimulation induces a concentration- and time- dependent inhibition of Na + /H + exchange at intestinal level. The aim of the present work was to evaluate the effect of acti- vation of this class of receptors upon the activity of the Cl – / HCO 3 – exchanger in the rat intestinal epithelial IEC-6 cells. Functional data confirmed the presence of a Na + -independent, Cl – -dependent and DIDS-sensitive HCO 3 – transport system in these cells. IEC-6 cells express the putative anion transporter SLC26A6 as confirmed by RT-PCR and immunoblotting stud- ies. The dopamine D 1 -like receptor agonist SKF 38393 pro- duced a concentration-dependent inhibition of Cl – /HCO 3 – exchanger activity, which was antagonized by the D 1 selective antagonist SKF 83566. SKF 38393 (1 lm)-induced inhibition was maximal at 5 min of exposure to the agonist (26±6%) and was progressively lost with the increase of the agonist exposure time (10 min, 18±6% reduction; 15 min, 3±7% reduction). Both PKA and PKC signaling pathways participate in D 1 -like receptor-mediated inhibition of the Cl – /HCO 3 – exchanger activ- ity. Pre-treatment of cells with heparin (1 lm), a non-selective G protein coupled-receptor kinase (GRK) inhibitor prevented the loss of D 1 receptor-mediated inhibitory effect on Cl – /HCO 3 – exchanger activity after 15 min exposure to SKF 38393 1 lm (3±7% to 26±4% of inhibition). Overnight pre-treatment of IEC-6 cells with anti-GRK 4 antibody was ineffective in pre- venting loss of D 1 -like dopamine receptor-mediated inhibition of the exchanger activity after 15 min exposure to SKF 38393. In contrast, pre-treatment with anti-GRK6A and anti-GRK6B antibodies effectively prevented receptor desensitization (from 4±6% to 18±2% and 21±5% of reduction, respectively). It is concluded that dopamine D 1 receptors in IEC-6 rapidly desensi- tize to D 1 -like receptor stimulation and that GRK 6 splice vari- ants are involved in agonist-mediated responsiveness and desensitization. Acknowledgment: Supported by Grant POCTI/CBO/42788/ 2001 and SFRH/BD/4595/2001 from Fundac¸ a ˜ o para a Cieˆ ncia e a Tecnologia (Portugal). E1-027P The role of arachidonic acid in protein kinase C alpha activation J. C. Go ´ mez-Ferna ´ ndez,M.J.Lo ´ pez-Andreo, C. Marı ´ n-Vicente, R. Lo ´ pez-Nicola ´ s, A. de Godos and S. Corbala ´ n-Garcı ´ a Departamento de Bioquı ´ mica y Biologı ´ a Molecular A, Universidad de Murcia, Murcia, Spain. E-mail: jcgomez@um.es Arachidonic acid may act as a signalling molecule and is gener- ated by the activity of phospholipase A2. We have observed that arachidonic acid is a good activator of protein kinase Ca (PKCa) requiring Ca 2+ for full activity. From experiments using site directed mutagenesis of C1 and C2 domains, we con- cluded that arachidonic acid activates PKCa through the cal- cium-binding site located at the C2 domain. In addition, the localization of the enzyme in the plasma membrane was observed to be also mediated by the lysine-rich cluster present in the C2 domain. Mutations of some C1 residues showed that arachidonic acid also activates through the C1A subdomain, but in an unspecific mode. It was also found that high concen- trations of arachidonic acid inhibits the activity of PKCa and this was attributed to their cooperation with diacylglycerol to produce non-lamellar phases in the lipid vesicles necessary to activate the enzyme. The formation of non-lamellar phases was detected through 31P-NMR. Abstracts 305 E1-028P 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin inhibit hepatic mitochondrial oxidative phosphorylation by two different AMPK-independent mechanisms B. Guigas 1 , B. Viollet 2 , N. Taleux 1 , M. Foretz 2 , S. Vaulont 2 and L. Hue 1 1 Hormone and Metabolic Research Unit, Institute of Cellular Pathology, Brussels, Belgium, 2 Department of Genetics, Develop- ment and Molecular Pathology, Institute Cochin, Paris, France. E-mail: bruno.guigas@horm.ucl.ac.be AMP-activated protein kinase (AMPK) is a metabolic sensor involved in cell energy homeostasis. In response to various metabolic stresses, AMPK phosphorylates multiple downstream targets leading to reciprocal inhibition and activation of anabo- lic and catabolic processes. Mitochondria are one of the main ATP producers in eukaryotic cells. While AMPK might be involved in mitochondrial biogenesis, the role of AMPK in the acute regulation of mitochondrial oxidative phosphorylation (OXPHOS) is not known. We investigated the effects of AI- CAR and metformin (Met), two well-known AMPK activators, on hepatic mitochondrial OXPHOS in newly engineered mice inactivated for the two AMPK a subunits (total a1–/– and liver specific a2–/–). AICAR and Met dose-dependently inhibited oxygen consumption rate (JO2) in isolated hepatocytes from wild type mice. Pre-incubation of cells with 5 mm Met or 1 mm AICAR led to the same inhibition of JO2 ( 30%, P < 0.05) by two different mechanisms, because the effect of AICAR but not of Met was reversed by DNP, an uncoupler of OXPHOS. Interestingly, in mice lacking both a subunits of AMPK, basal JO2 was drastically reduced compared with wild type (–45%, P < 0.05) but inhibition of OXPHOS by AICAR and Met was still present, indicating that both effects were AMPK-indepen- dent. In agreement with our previous results (El-Mir et al., 2000; Detaille et al., 2002; Guigas et al., 2004), the effect of Met persisted after hepatocytes permeabilization with digitonin and resulted exclusively from inhibition of the mitochondrial respiratory chain complex 1. Conversely, in these cells, the effect of AICAR was no longer present suggesting that it could be due to Z ribosides, which accumulate in cells. E1-029P Specific expression of pfkfb4 gene in spermatogonia germ cells and analysis of its 5¢-flanking region M. Gomez, A. Manzano, A. Navarro-Sabate, J. Duran, M. Obach, J. C. Perales and R. Bartrons Department of Ciencies Fisiologiques II, Laboratory of Biochemis- try, University of Barcelona, Hospitalet De Llobregat, Spain. E-mail: m.gomez@ub.edu The results presented demonstrate the expression of pfkfb4 gene in adult testis and in a mouse spermatogonia germ cell line (GC-1spg). The genomic organization of the human pfkfb4 gene shows the existence of 14 exons and 13 introns, spanning 45 kb. A detailed analysis of the 5¢flanking region by transient trans- fection assays with different 5¢-deletion promoter constructs in GC-1spg and mouse sertoli cells (TM-4) allows us to define the minimal promoter unit, containing several GC-rich and ETF sequences along the first-141 nucleotides involved in basal expression. This gene is activated by serum and chemical hypox- ia (COCl 2 treatment) whereas b-estradiol decreases its expres- sion. E1-030P Human vaccinia-related kinase1 (VRK1) cooperates with JNK in the transcriptional activity of ATF2 and c-Jun by N-terminal phosphorylation A. S. Herna ´ ndez and P. L Z. Taracena Instituto de Biologı ´ a Molecular y Celular del Ca ´ ncer, University of Salamanca, Salamanca, Spain. E-mail: sevilla@usal.es The vaccinia-related kinases (VRK) form a group of human kin- ases grouped together with the casein kinase I family within the human kinome but distantly related to them. They have a high homology with the B1R vaccinia virus kinase. These kinases phosphorylate transcription factors related to stress responses, such as p53, c-Jun and ATF2. In this report, we have studied the phosphorylation by VRK1 of ATF2 and c-Jun as substrates. (A) ATF2 regulates gene expression by forming dimmers with pro- teins with basic region-leucine zipper domains and recognizing cAMP-response elements or AP1 sequences. VRK1 phosphory- lates ATF2 mainly on T73 and S62 stabilizing the protein. Muta- genesis studies showed that T73 and S62 are also implicated in ATF2 transcriptional activation. VRK1 can activate the collage- nase gene promoter that is regulated by ATF2 in a dose-depend- ent manner. Loss of kinase activity VRK-1(K179E) mutant, or the T73A substitution in ATF2 prevents both its accumulation and activation of transcription. VRK1 and JNK have an additive effect on ATF2 dependent transcription at suboptimal doses. Therefore, two groups of amino acids in the ATF2 amino-ter- minal region can integrate different cellular signals mediated by at least five different kinases. (B) Phosphorylation of c-Jun occurs as a response to many different types of stress signals. VRK1 also phosphorylates c-Jun inducing the stabilization and accumulation of the protein. VRK1 activates c-Jun dependent transcription, which is dependent on phosphorylation of S63 and S73. Both VRK1 and JNK have an additive effect on the tran- scriptional activation of c-Jun indicating that they can cooperate when they are at suboptimal dose. E1-031P Site-specific phosphorylation of L-form starch phosphorylase by a protein kinase activity from sweet potato roots G. H. Young 1 , H. M. Chen 2 , C. T. Lin 3 , R. H. Juang 1 1 Department of Biochemical Science and Technology, and Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan ROC, 2 Department of Life Science, Catholic Fu-Jen University, Taipei, Taiwan ROC, 3 Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan ROC. E-mail: juang@ntu.edu.tw A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP). This insertion blocks the starch-binding site on the L-SP molecule, and decreases the binding affinity of L-SP toward starch. The computational analy- sis of the amino acid sequence on L78 predicts several phos- phorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from sweet potato roots. The kinase was parti- ally purified by liquid chromatography, and its native molecular mass was estimated as 338 kD. An expressed peptide (L78P) con- taining the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP Abstracts 306 lacking the L78 insertion) and the proteolytic-modified L-SP, which lost its L78 fragment, could not be phosphorylated. Fur- thermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. E1-032P Muscarinic acetylcholine receptors promote growth of human breast cancer cells E. Jime ´ nez 1 and M. Montiel 2 1 Biochemistry and Molecular Biology, Malaga, Spain, 2 Biochemis- try and Molecular Biology, Malaga, Spain. E-mail: ejg@uma.es In MCF-7 human breast cancer cells, muscarinic acetylcholine re- ceptors (mAChR) activation by carbachol (Cch) induces a time and dose-dependent increase in the MAPK/ERK phosphoryla- tion. Cells pre-treatment with U73122, a selective PLC inhibitor, or cells incubation in a Ca 2+ -free medium did not alter Cch-sti- mulated MAPK/ERK phosphorylation. Phosphorylation of MAPK/ERK was mimicked by the PKC activator PMA, but Cch-evoked MAPK/ERK activation was unaffected by down- regulation of PKC or cells pre-treatment with GF109203X, a PKC inhibitor. However, Cch-stimulated MAPK/ERK phos- phorylation was blocked by myristoylated PKC-zeta pseudosub- strate, a specific inhibitor of PKC-zeta, and high doses of staurosporine. Pre-treatment of the human breast cancer cells with wortmannin or LY294002, selective inhibitors of PI3K, diminished Cch-mediated MAPK/ERK phosphorylation. Similar results were observed when MCF-7 cells were pre-treated with genistein, a non-selective tyrosine kinase inhibitor, or with the specific Src tyrosine kinase inhibitor PP2. Moreover, in MCF-7 human breast cancer cells, mAChR stimulation induced an increase of protein synthesis and cell proliferation, and these effects were prevented by PD098059, a specific inhibitor of the mitogen-activated kinase kinase. In conclusion, analyses of mAChR downstream effectors revealed that PKC-zeta, PI3K and Src family of tyrosine kinases, but not intracellular-free Ca 2+ mobilization and conventional and novel PKC activation, are key molecules in the signal cascade leading to MAPK/ERK acti- vation, and that MAPK/ERK are involved in the regulation of growth and proliferation of MCF-7 human breast cancer cells. E1-033P Calcineurin regulates chondrogenesis via the modulation of ERK1/2 activity T. Juha ´ sz 1 , C. Matta 1 , Z. Szı ´ jgya ´ rto ´ 2 , G. Czifra 3 ,T.Bı ´ ro ´ 3 , P. Gergely 2 ,L.Mo ´ dis 1 ,R.Za ´ ka ´ ny 1 1 Department of Anatomy, University of Debrecen, Debrecen, Hun- gary, 2 Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary, 3 Department of Physiology, University of Debrecen, Debrecen, Hungary. E-mail: juhaszt@chondron.anat.dote.hu The aim of our studies was to investigate the effects of oxidative stress on the in vitro formation of cartilage in high-density micro- mass cell cultures of chicken embryonic chondrogenic cells. The cartilage content of the cultures was estimated by metachromatic staining; differentiation of chondrogenic cells into chondrocytes was followed by RT-PCR reactions detecting the mRNA of the core protein of aggrecan. Protein phosphatase 2B (PP2B, calcineu- rin) plays a role in the regulation of chondrogenesis and this enzyme is sensitive to oxidative stress. Calcineurin was found as a positive regulator of chondrogenesis in chondrifying chicken micromass cultures, as cyclosporine A (CsA) reduced both the amount of cartilage and the expression of mRNAs of aggrecan and as it was expected the activity of calcineurin was also inhib- ited by CsA. Cartilage formation was inhibited with application of 0.1–4 mm hydrogen peroxide. Hydrogen peroxide inhibited the formation of cartilage in a concentration-dependent manner. Activities of MAP kinases are dependent on the phosphorylation level of these proteins. ERK1/2 is known to inhibit cartilage for- mation and calcineurin is a possible regulatory factor in the acti- vation of ERK1/2. Oxidative stress decreased the activity of calcineurin but the phosphorylation of ERK1/2 was extremely ele- vated either by 1 mM hydrogen peroxide or 2 lM CsA. The ERK inhibitor PD098059 attenuated the depletion of cartilage matrix in the cultures treated with hydrogen peroxide or CsA. Our results suggest that chondrogenesis inhibiting effect of hydrogen peroxide is mediated, at least partly, by inhibition of calcineurin and by activation of ERK1/2. Thus, our results indicate that the inhibi- tion of the activity of calcineurin in cells exposed to oxidative stress can be an important factor, which causes reduction of carti- lage formation via modulation of the ERK1/2 pathway. E1-034P Monofunctional and bifunctional inhibitors of cAMP-dependent protein kinase A. Kuznetsov and J. Ja ¨ rv Organic and Bioorganic Chemistry, Tartu University, Tartu, Estonia. E-mail: aleksei.kuznetsov@ut.ee Kinetic analysis of inhibition of phosphorylation of Kemptide (LRRASLG), catalyzed by the catalytic subunit of cAMP- dependent protein kinase, by different monofunctional and bi- functional inhibitors was studied at wide ATP and peptide con- centration interval, and a simple procedure was proposed for characterization of interaction of this inhibitor with the free enzyme and the enzyme–ATP and enzyme–peptide complexes. The second-order rate constants, calculated from the steady-state reaction kinetics, were used for this analysis to avoid the compli- cations related to the sophisticated catalytic mechanism of the protein kinase catalyzed reaction. Parameters characterizing structural factors, responsible for transfer of monofunctional inhibitors into the group of bifunctional inhibitors, are proposed. The role of these structural factors in effectiveness of cAMP- dependent protein kinase inhibitors is discussed. E1-035P Effect of rapamycin on mTOR and p70 S6 kinase phosphorylation in HepG2 cells with and without overexpression of constitutively active Akt/PKB S. Varma, D. Gupta, R. L. Khandelwal Department of Biochemistry, University of Saskatchewan, Saska- toon, Saskatchewan, Canada. E-mail: ramji.khandelwal@usask.ca mTOR (mammalian target of rapamycin) is a serine-threonine kin- ase, which is known to play an important role in the regulation of cell growth. It activates ribosomal p70 S6 kinase and inhibits elon- gation factor elF4E inhibitor (4E-BP). Rapamycin inhibits mTOR and its downstream signalling. The mechanism of action of rapa- mycin is that it forms a complex with immunophilin FK506-bind- ing protein 12 (FKBP12), which in turns complexes with mTOR thereby inhibiting its activity. mTOR phosphorylation levels are controlled by the activation of Akt/PKB. Therefore, the effects of rapamycin on phosphorylation of mTOR (Ser 2448) and p70 S6 kinase (Thr 389) were examined both in the absence and presence of insulin in parental HepG2 and HepG2 cells constitutively over- expressing Akt/PKB (HepG2-CA Akt/PKB). Constitutive expres- sion of Akt/PKB enabled HepG2 cells to survive up to 96 h with- Abstracts 307 [...]... (Int) protein that belongs to the Tyr family of site-specific recombinases Protein phosphorylation has been known for a long time in the eukaryotes to play an important role in signal transduction, metabolism and malignant transformation Protein phosphorylation by protein kinases and dephosphorylation by protein phosphatases have been recently demonstrated in bacteria We find that the purified HK022 Int protein, ... J Mol Biol 2001; 308: 1–8 ¨ ¨ 2 Torok Biochem Soc Trans 2002; 30: 5 5–6 1 ¨ ¨ 3 Torok et al Biochemistry 2001; 40: 1487 8–1 4890 4 Tzortzo¨ ¨ poulos and Torok Biochemistry 2004; 43: 640 4–6 414 ¨ ¨ E1- 069P The hypothalamic AMPK-mediated stimulation of food intake is lacking in Lou/C rats, a strain resistant to obesity N Taleux1,2, B Guigas1, G Lacraz2, R Favier2, X Leverve2 and L Hue1 1 Hormone and Metabolic... of p50RhoGAP significantly The fragments 4 9–4 39, 12 1–4 39 and 16 9–4 39 exhibited identical GAP activity, thus amino acids 4 9–1 68 seem not to influence the interaction of Rac with p50RhoGAP In contrast, removal of the next 20 amino acids (16 9–1 98) resulted in a further increase in the GAP activity When prenylated Rac expressed in Sf9 cells was used, the full-length protein and all the different constructs... (EGR1) and specific protein (Sp1) putative-binding sites Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs, demonstrates that the sequence located from –1 269 to – 1297 relative to the start site is required for HIF-1 induction The effective binding of HIF-1 transcription factor to the HREs at –1 279 and –1 288 was corroborated... databases However, at present, protein products corresponding to these mRNAs are not identified and their enzymatic and functional properties are not characterized In the present study, we identify the protein product of GenBank entry BC082842, as the major PDK isoform expressed in Xenopus oocytes at the protein level Product of this gene was annotated earlier as ‘unknown protein This protein was partially... stress-activated protein kinase pathways and their possible role in chondrocyte damage Materials and methods: Isolated swine chondrocytes were incubated for 6 h under normal (300 mOsm) and hyperosmotic (600 mOsm) conditions The phosphorylation of p38 mitogenactivated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (ERK1/2), cAMP responsive element-binding transcription factor (CREB) and protein. .. to HGF We found that protein kinase C (PKC) was responsible for the PMA-induced scattering but detained the HGF-initiated cell migration The sustained activation of the Erk1/2 MAP kinases was found to be indispensable for the cell migration triggered by either factor PKC accounted for the PMA-induced activation of the Erk1/2 MAP kinases, but decreased the activation of these MAP kinases in HGF-treated... assessed the expression pattern of pro- and anti-apoptotic Bcl-2 and Fas/FasL proteins as well as their sensitivity to apoptotic signals Our findings suggest that the above signaling kinases may represent novel targets for the detection and/or manipulation of micrometastatic breast cancer cells E1- 059P Cyclic GMP-dependent protein kinase is indispensable for activation of NF-kappaB by nitric oxide/cGMP... and in vivo model systems beneficially modulated the intracellular signaling, which may contribute to the cardioprotective properties of PARP inhibitors 311 Abstracts E1- 049P Regulation of protein kinase B by DNA-dependant protein kinase E1- 051P Activation of signal transduction pathways in chondrocytes under hyperosmotic conditions J Feng1, J Park2, P Cron1, M Shong3, D Hess1 and B A Hemmings1 1 Freidrich... vectors providing the solubility enhancer maltose-binding protein as an amino terminal fusion and both green fluorescence protein and poly histidine as carboxyl terminal fusion tags To validate the vector suite approach, 3 human kinase ORFs and 3 ORFs encoding ADAMTs, extracellular matrix proteins, have been cloned into each vector In the case of the kinases, we found that fusion to MBP rescued expression . E1 – Protein kinases E1- 001 Protein interactions in cell signaling and polarity T. Pawson Samuel. panasyuk_g@imbg.org.ua The ribosomal protein S6 kinase (S6K) belongs to the AGC fam- ily of Ser/Thr protein kinases which includes the protein kinase C’s, protein kinase B’s,