Báo cáo khoa học: E1 – Protein kinases pptx

Additionally, Western blots demonstrated that NNK treatments result in epider- mal growth factor receptor, EGFR, and mitogen-activated protein kinase, Erk1/2 phosphorylation.. Upon flage

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@fini.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’, J Zhang'”, C.J HupfeldỶ, J.M OlefskyỶ,

K Kain",M H Ginsbergf, Y Wang” and § Chien”

’ Pharmacology, UCSD, La Jolla, CA, United States of America,

?Pharmacology, Johns Hopkins, Baltimore, MD, United States of

America, 7 Endocrinology & Metabolism, UCSD, La Jolla, CA,

United States of America, 4Medicine, UCSD, La Jolla, CA, Uni- ted States of America, ° 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, FHAI 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 f-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 «481 or «581 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 Sre activity from the bead References

1 Zhang et al PNAS 2001; 98: 14997-15002

2 Ting et al PNAS 2001; 98: 15003-15008.

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', V Filonenko' and I Gout?

"Institute of Molecular Biology and Genetics, Kiev, Ukraine,

?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 iv vitro kinase assay The N-

terminal region contains only two Ser/Thr sites and one of them,

Serl7, 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 SI17E 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!, S Wiesner, T Guettler*?, C MackerethỶ,

L L Remsing Rix!?, D Görlich”, M Sattler?, G Superti-Furga!*

"Center for Molecular Medicine, Austrian Academy of Sciences, Vienna, Austria, 7European Molecular Biology Laboratory, Heidel- berg, Germany, >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 Ber-Abl inhibition However, the major- ity of the Ber-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 Ber-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’, M S Tsao” and H M Schuller!

"Department of Pathobiology, Experimental Oncology Laboratory, University of TN, Knoxville, Tennessee, United States of America,

?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 im 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

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 (AGI478) and Erkl/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 PKCf- and JNK expression

E Bodur' and P G Layer?

"Department of Biochemistry, Faculty of Medicine, Hacettepe Uni-

versity, Ankara, Turkey , “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 CKIl 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 o) subunits and two regulatory (B) subunits

The B subunit mediates tetramer formation through the B-B homo-

dimerization and o-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; Lysl47Met) 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 Biologia Molecular y Celular del Cancer, 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

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 PKC§ isoforms were suggested

to play significant roles in T-cell activation Using the antisense technology, we previously investigated the function of PKC« [1]

in the activation of Jurkat cells, a leukaemia T-lymphocyte line

Following a similar approach, we now study the involvement of

PKC 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 (PKC6,¢,4,0 and ©) 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 TNF secretion or the

activation of the kinases ERK 1/2, 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 Lopez-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' and P G Layer’

"Department of Biochemistry, Faculty of Medicine, Hacettepe Uni-

versity, Ankara, Turkey , ?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 48h 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 72h 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-B8 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 Mészaros!, A Helfer”, S Peck?, V Bardéczy! and L Bégre”

Department of Biochemistry and Food Technology, Budapest Uni-

versity of Technology and Economics, Budapest, Hungary, “School

of Biological Sciences, Royal Holloway University of London,

Egham, United Kingdom ,*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 AtLMPK3/4/6 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 Atmkkl mutant plants confirmed the protoplast results The phenotype analysis implies a pathogen response specific function of At- MKK 1, 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-38 (GSK-3)

A Baki!, G I Szendrei? and G M Keseri!

'CADD&HTS Laboratory, Gedeon Richter Ltd, Budapest, Hun- gary, “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-3§ 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 GSK3-8 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

E1-015P

Covalent modification of hexokinase by

biologically active quinones

T Bozic', I Novakovic!”, M Gasic! and D Sladic!

"Department of Organic Chemistry, Laboratory of Bioorganic

Chemistry, University of Belgrade, Belgrade, Serbia and

Montenegro, “Institute of Chemistry Technology and Metallurgy,

Department of Chemistry, Belgrade, Serbia and Montenegro,

E-mail: thozic@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

pl 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>-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

Herceptin® resistant breast cancer xenografts

can be influenced by early trastuzumab

treatment

M Barok’, I Juh4sz*, M Balazs’, J W Park*, J Isola’,

Z Fazekas’®, G Vereb', J Szollésil®

"Department of Biophysics and Cell Biology, University of Debre-

cen, Debrecen, Hungary, *Department of Dermatology, University

of Debrecen, Debrecen, Hungary, * Department of Hygiene and

Epidemiology, University of Debrecen, Debrecen, Hungary,

“Department of Medicine, Division of Haematology/Oncology, San

Francisco, United States of America, °Laboratory of Cancer Gen-

etics, University of Tampere, Tampere, Finland, °Cell Biophysics

Research Group of Hungarian Academy of Sciences, University of

Debrecen, Debrecen, Hungary E-mail: barokm@dote.hu

Trastuzumab (Herceptin®) — 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.

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’, D Ferreira’, S B McMahon? and F Cruz)?

"Institute of Histology and Embryology, University of Porto,

Porto, Portugal, °Department of Urology, University of Porto,

Porto, Portugal, *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 HO) 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 RIIo 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 vive, we tagged Renilla luciferase (Rluc) or green fluorescent protein (GFP*) to the putative-binding partners hRIo, hRIJ« and PrKX as well hRIo 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 RI in vive Holo- enyzme formation with wildtype and mutant forms of hRIo 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 RIo resulted in the highest BRET level, and cAMP did not release the catalytic sub- unit from this holoenzyme complex

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 CAI 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

induction is based on the assumption that the direction of

change in synaptic efficacy is determined by the level of post-

synaptic Ca?* Activation of NMDA receptors by the glutam-

ate and subsequent strong depolarization by the backpropagat-

ing spike leads to an intensive Ca** influx High level of Ca?*

leads to the formation of a Ca/calmodulin complex, which

results in the autophosphorylation of a Ca/calmodulin-depend-

ent protein kinase I] (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" 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 PPl 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** Additional mechanisms, possibly dependent on a

dynamics of Ca?‘ 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!, B Kaminska-Kaczmarek? and

L Konarska!'

‘Department of Biochemistry and Clinical Chemistry, Medical Uni-

versity of Warsaw, Warsaw, Poland, *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, CB1l 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:

WINSS5,212-2 (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!, Y Sawano!, 9 Kosugel, Y Yamano’, K Kuroki? and K Ohtsuki!

"Laboratory of Genetical Biochemistry and Signal Biology, Gradu- ate School of Medical Sciences, University of Kitasato, Sagamiha-

ra, Kanagawa, Japan, *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 (RIo and RII«) of «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 vive; 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 Balfagén and M Ferrer Departamento de Fisiologia, Universidad Autonoma 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 (CENOS 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

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”-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!” and D Pignatelli!

‘Institute of Histology Embryology, Porto, Portugal, ^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’-/HCO3° 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; 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,-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; exchanger in the rat intestinal epithelial IEC-6 cells Functional data confirmed the presence of a Na” -independent,

Cl -dependent and DIDS-sensitive HCO; 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,-like receptor agonist SKF 38393 pro- duced a concentration-dependent inhibition of Cl /HCO;— exchanger activity, which was antagonized by the D, selective antagonist SKF 83566 SKF 38393 (1 uM)-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, 347% reduction) Both PKA and PKC signaling pathways participate in D,-like receptor-mediated inhibition of the Cl/HCO; exchanger activ- ity Pre-treatment of cells with heparin (1 4M), a non-selective

G protein coupled-receptor kinase (GRK) inhibitor prevented the loss of D; receptor-mediated inhibitory effect on Cl/HCO3— exchanger activity after 15 min exposure to SKF 38393 1 uM (347% to 26+49% of inhibition) Overnight pre-treatment of IEC-6 cells with anti-GRK 4 antibody was ineffective in pre- venting loss of D,-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 2145% of reduction, respectively) It is concluded that dopamine D, receptors in IEC-6 rapidly desensi- tize to D,-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 Fundagao para a Ciéncia e

a Tecnologia (Portugal)

E1-027P The role of arachidonic acid in protein kinase

C alpha activation

J C Gomez-Fernandez, M J Lopez-Andreo, C Marin-Vicente,

R Lopez-Nicolas, A de Godos and S Corbalan-Garcia Departamento de Bioquimica y Biologia Molecular A, Universidad

de Murcia, Murcia, Spain E-mail: jcegomez@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?~ for full activity From experiments using site directed mutagenesis of Cl 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 Cl 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

E1-028P

5-aminoimidazole-4-carboxamide riboside

(AICAR) and metformin inhibit hepatic

mitochondrial oxidative phosphorylation by

two different AMPK-independent mechanisms

B Guigas', B Viollet, N Taleux!, M Foretz’, S Vaulont? and

L Hue!

"Hormone and Metabolic Research Unit, Institute of Cellular

Pathology, Brussels, Belgium, 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 « subunits (total «l—/— and liver

specific «#2—/-) AICAR and Met dose-dependently inhibited

oxygen consumption rate (JO2) in isolated hepatocytes from

wild type mice Pre-incubation of cells with 5mm 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 « 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 I], 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-Ispg) 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-Ispg 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-

la (COC]H; treatment) whereas f-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 Hernandez and P L.-Z Taracena Instituto de Biologia Molecular y Celular del Cancer, 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 BIR 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 API 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-I(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 $63 and S73 Both VRKI 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), H M Chen’, C T Lin’, R H Juang!

"Department of Biochemical Science and Technology, and Institute

of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan ROC, “Department of Life Science, Catholic Fu-Jen University, Taipei, Taiwan ROC, * 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

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 Jiménez’ and M Montiel”

"Biochemistry and Molecular Biology, Malaga, Spain, ° 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**-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 Sre 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 PK C-zeta, PI3K and

Sre family of tyrosine kinases, but not intracellular-free Ca?”

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 Juhász!, C Matta!, Z Szijgyártó”, G Czifra*, T Bird’,

P Gergely”, L Módis!, R Zákány!

"Department of Anatomy, University of Debrecen, Debrecen, Hun-

gary, “Department of Medical Chemistry, University of Debrecen,

Debrecen, Hungary, *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 im 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 1M 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 Jarv 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-

out serum in growth media Cells were treated with insulin

(1-100 nM) in the presence and absence of rapamycin (20 nM) In

HepG2 cells, the phosphorylation of mTOR was stimulated four-

fold by insulin and this was abolished in the presence of rapamycin

In HepG2-CA Akt/PKB cells, phosphorylation of mTOR was not

significantly changed in the presence of insulin + rapamycin The

phosphorylation of p70 S6 kinase was almost completely abolished

by rapamycin in both types of cells The reason for differential

effects of rapamycin on the phosphorylation of mTOR and p70 S6

kinase in HepG2 and HepG2-CA Akt/PKB cells requires further

investigation but it can be concluded that rapamycin can regulate

p70 S6 kinase (and possibly protein synthesis) both in parental and

overexpressing Akt/PKB hepatoma HepG2 cells irrespective of

their levels of phosphorylated PKB and survival characteristics

’ Department of Biochemistry, Tel Aviv University, Tel Aviv, Israel,

?Department of Plant Pathology and Microbiology, The Hebrew

University of Jerusalem, Rehovot, Israel, * Department of Cell

Research and Immunology, Tel Aviv University, Tel Aviv, Israel

E-mail: kolott@post.tau.ac.il

Coliphage | and its close relative, phage HK022, insert and excise

their circularized DNA into and out of the Escherichia coli chro-

mosome The enzyme that catalyzes this site-specific recombination

reaction is the phage-encoded Integrase (Int) protein that belongs

to the Tyr family of site-specific recombinases Protein phosphory-

lation 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 HK 022 Int pro-

tein, or Int overexpressed in FE coli is phosphorylated in Tyrosine

and Serine/Threonine residues Immunblot analyses with anti-

phosphotyrosine has shown that only catalytic C-terminal domain

of Int is phosphorylated whereas the arm-binding N-terminal Int

domain is not We also find that the Wzc tyrosine kinase of E coli

can phosphorylate tyrosine residues of Int and that the Wzb

protein phosphatase of E coli can dephosphorylate Int in vitro

Preliminary results have shown that in an E coli strain that over-

expresses Wzb phosphatase the Int-promoted site-specific excision

activity is reduced However in an £ coli strain that overexpresses

the Wzc kinase this activity is unaffected Experiments are under

way to test the role of Int-phosphorylation in the regulation of the

site-specific recombination process

E1-037P

Sequencing analysis of mutant allele cdc28-

srm of protein kinase CDC28 and molecular

dynamics study of glycine-rich loop in wild

type and mutant allele G16S of CDK2 as model

N A Koltovaya!, A.S Guerasimova’, D A Kretov! and

K T Kholmurodov!

"Department of Radiation and Radiobiological Research, Joint

Institute for Nuclear Research, Dubna, Moscow Region, Russian

Federation, *Max-Planck-Institute for Molecular Genetics, Berlin,

Germany E-mail: mirzo@jinr.ru

The central role that cyclin-dependent kinases play in the timing

of cell division and the high incidence of genetic alteration of

CDKs or deregulation of CDK inhibitors in a number of cancers make CDC28 of yeast Saccharomyces cerevisiae very attractive as

a model for studies of mechanisms of CDK regulation We have found that certain gene mutations including cdc28-srm affect maintenance of different genetic structures (Devin et al., 1990) and increased cell sensitivity to ionizing radiation (Koltovaya et al., 1998) A cde28-srm mutation is not temperature-sensitive mutation and differs from known cdc28-ts mutations because it has the evident phenotypic manifestations at 30 °C Sequencing analysis of cdc28-srm revealed a single nucleotide substitution G20S in the G-rich loop Despite its demonstrated importance, the role of the G-loop has remained unclear The crystal struc- ture of human CDK2 has served as a model for the catalytic core

of other CDKs, including CDC28 Nanoseconds long molecular dynamics (MD) trajectories of several human CDK2 complexes (inactive CDK2/ATP, fully active pT160-CDK2/cyclin A/ATP, inhibited pT14, PY15, pT160-CDK2/cyclin A/ATP and pT160- CDK2/cyclin A/ATP/substrate) were compared The MD simula- tions of substitution G16S (G20S in CDC28) in these complexes show conformational changes of CDK2 structure leading to the moving of the G-loop away from ATP and opening of the CDK2 substrate-binding box

References

1 Devin AB, Prosvirova Tyu, Peshekhonov VT, Chepurnaya

OV, Smirnova ME, Koltovaya NA, et al Yeast 1990; 6: 231-

243

2 Koltovaya NA, Arman IP, Devin AB Yeast 1998; 14: 133-

146

E1-038P The binding and phosphorylation at T231P motif in Tau is critical for tau sequential phospohrylation

P.-J F Lu, C.-Y Ko and L.-C Liang Laboratory of Signal Transduction, Department of Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan ROC E-mail: pjfranklu@isca.vghks.gov.tw Neurofibrillary tangle is a pathological hallmarker of Alzhei- mer’s disease that is composed of hyperphosphorylated Tau Hyperphosphorylation on Tau affects the binding to tubulin and capacity to promote microtubule assembly Glycogen Syn- thase Kinase-3a (GSK-3a) has been demonstrated to be the Tau protein kinase I im vivo It is important to understand the phosphorylation mechanism of Tau by GSK-3a In this study, wild type of Tau and its mutants including Tau-N, Tau-C, T231A, T231E, Taul54~441, S396A, S400A, S404A, S413A and S396A S400A were prepared and subjected for kinase assay and tubulin assembly assay The results have suggested that the binding and phosphorylation at T231P motif is critical for Tau sequential phosphorylation Interestingly, the priming phosphorylation is not required for the T231 and S404 phos- phorylation whereas the priming phosphorylation at S404 is critical for the sequential phosphorylation of $396 and S400 Taken together with the results of competitive binding and phosphorylation using tautide that contains T231P motif, a model of Tau phosphorylation by GSK-3a is proposed More- over, we used T231E, S396E, S400E and S404E mutants of Tau to study the functional regulation of Tau by GSK3a Our

in vivo and in vitro data suggested that hyperphosphorylation

of Tau can negatively regulate Tau’s ability to promote micro- tubule assembly whereas single phosphorylation might promote Tau’s function Thus, Phosphorylation of T231 by GSK-3a plays important role in result of hyperphosphorylation and functional regulation of Tau

E1-039P

HwSln1†1p, a putative sensor protein of the

HOG signalling pathway in the halophilic black

yeast Hortaea werneckii

M Lenassi and Plemenitas

Institute of Biochemistry, Faculty of Medicine, University of Ljub-

liana, Ljubljana, Slovenia E-mail: metka.lenassi@mf.uni-lj.si

Hortaea werneckii was isolated from solar salterns as one of the

predominant species of a group of halophilic and halotolerant

melanized yeast-ike fungi It can grow at salinities ranging from

0% to saturated solution of NaCl One of the responses to

increased salinity in the environment is activation of high osmo-

larity glycerol (HOG) signalling pathway resulting in glycerol-3-

phosphate dehydrogenase gene expression activation and conse-

quently in glycerol accumulation Recently, a homologue gene of

the key Saccharomyces cerevisiae mitogen-activated protein kin-

ase (MAPK) HOGI and two gene copies of a homologue

upstream scaffold MAPK kinase PBS2 were isolated from H we-

rneckii In this study, we present the homologue of a sensor histi-

dine kinase SLNI also isolated from H werneckii Slnip is a

negative regulator of the HOG pathway and becomes mainly de-

phosphorylated upon hyperosmotic shift Consequently, the

Ssk2p and Ssk22p MAPK kinase are activated and the signal is

transmitted through Pbs2p MAPK kinase to Hoglp MAPK,

which is translocated to the nucleus From the nucleotide

sequence of the isolated HwSLNI1 gene, the analysis of the pro-

moter region was performed and the amino acid sequence was

deduced The protein contains typical eukaryotic hybrid kinase

domains like kinase and receiver domain with conserved His and

Asp, and also a transmembrane domain that directs this protein

to the membrane Hybridization studies indicate that H we-

rneckii genome contains only one copy of the gene From the

expression studies, a model of action was proposed in which the

expression of this protein increases as a response to hypoosmotic

shock and decreases with hyperosmotic shock

E1-040P

Characterization of a heme-regulated

eukaryotic initiation factor 2x kinase

M Miksanova, J Igarashi, M Masahiro, H Kurokawa and

T Shimizu

Institute of Multidisciplinary Research for Advanced Materials,

Tohoku University, Sendai, Japan

E-mail: mmiksanova@ yahoo.com

Heme-regulated inhibitor (HRI) is a member of eukaryotic initi-

ation factor 2% kinase family HRI regulates globin synthesis in

reticulocytes in response to heme availability Heme deficiency

activates HRI that phosphorylates Ser51 of eIF2a, resulting in

inhibition of protein synthesis HRI contains a tightly bound

heme at the N-terminal domain Our earlier study suggests that

activation of HRI by nitric oxide (NO) is induced by the forma-

tion of a five-coordinate NO-heme complex in terms of optical

absorption, electron spin resonance and resonance Raman spec-

tra of purified full-length HRI-NO complex However, detailed

regulation mechanism of HRI upon heme-binding is not well

understood We measured the heme-binding affinity of HRI in

different phosphorylation state We also made several mutants

near the heme-binding sites to study the effect of phosphoryla-

tion on the activity and spectroscopic properties Moreover we

focused on regulation (inhibition or stimulation) of HRI Based

on our findings, we will discuss the relationships between heme

binding and catalysis of this enzyme

Acknowledgment: This work was supported in part by Japan

Society for the Promotion of Science Fellowship 16-04110 to M.M

E1-041P Alternative splicing as a new possible regulator of the enzymatic activity of

6-phosphofructo-2-kinase/fructose-2,6-

biphosphatase 4

D O Minchenko and O H Minchenko Molecular Biology, Palladin Institute of Biochemistry, Kiev, Ukraine E-mail: xanrok@yahoo.com

The PFKFB4 gene encoded isoenzyme of 6-phosphofructo-2-kin- ase/fructose-2,6-biphosphatase, which originally was found in the testes Constitutive levels of PFKFB4 mRNA were found in the mouse testis as well as in other organs (liver, lung and brain) and

in DB-1 melanoma cells Moreover, we observed additional bands of PFKFB4 mRNA in several mouse organs and DB-1 melanoma cells Sequence analysis of PFKFB4 cDNA from mouse testis and other organs and human DB-1 melanoma cells revealed several alternative splice isoforms There are three groups of splice isoforms One group of PFKFB4 alternative splice isoforms in the mouse and human melanoma cells has shorter amino-terminal part but has all kinase and biphosphatase domains Second group of PFKFB4 alternative splice isoforms in the mouse and human melanoma cells has shorter or modified carboxyl-terminal part, has all kinase domains and did not have one or more biphosphatase domains Third group of PFKFB4 alternative splice isoforms possibly produces one or two proteins: 6-phosphofructo-2-kinase and/or fructose-2,6-biphosphatase Thus, our results indicate that PFKFB4, which originally was found in the testis, is also expressed in the lung, liver and brain

as well as in DB-1 melanoma cells Moreover, we found several alternative splice isoforms of PFKFB4 in DB-1 melanoma cells and in mouse tissues, which have shorter amino- or carboxyl-ter- minal part These alternative splice isoforms of 6-phosphofructo- 2-kinase/fructose-2,6-biphosphatase-4 are similar in the human melanoma cells and mouse tissues and possibly have different enzymatic and regulatory properties Alternative splicing can be

a new possible regulator of the PFKFB4 enzymatic activity

E1-042P STAT5A phosphorylation: A novel regulatory signaling pathway mediated by the mu-opioid

receptor

G Mazarakou and Z Georgoussi Laboratory of Cellular Signaling and Molecular Pharmacology, Institute of Biology, National Center for Scientific Research

“Demokritos”’, Athens, Attiki, Greece

E-mail: gmazar@bio.demokritos.gr Many G protein-coupled receptors have been reported to activate not only conventional G protein signaling pathways, but also to increase tyrosine phosphorylation of the STAT family members

in various cell lines Signal Transducers and Activators of Tran- scription (STATs) are transcriptional factors that mediate many

of the effects of the cytokines All three opioid receptor subtypes (mu, delta, kappa) contain motifs, in their C-terminal regions, known to be critical for STATS binding In this regard, we dem- onstrate, in pull down experiments, using glutathione S-transf- erase fusion proteins containing either the rat mu-opioid receptor carboxyl-terminal tail, or the third intracellular loop that, STATSA, binds to these regions in an agonist independent man- ner Moreover, we demonstrate that exposure to opioid agonists

of COS-7 cells, expressing the rat mu-opioid receptor and STATSA, induces STATSA tyrosine phosphorylation in a time-dependent manner This phosphorylation was blocked by naloxone In addition, activation of the mu-opioid receptor, with

opioid agonist, stimulated the expression of a STATSA respon-

sive reporter gene Treatment of COS-7 cells, transiently expres-

sing the mu-opioid receptor and STATSA with the Src-specific

inhibitor PP1, abolished opioid agonist-mediated STATSA phos-

phorylation This finding is in accord with Sre kinase phosphory-

lation observed in COS-7 cells upon mu-opioid agonist

stimulation Our data reveal novel signaling pathways through

which the mu-opioid receptor regulates transcriptional activity,

probably altering gene expression in specific target neurons and

thereby inducing tolerance and dependence

Acknowledgment: This project was supported by the State

Scholarship Foundation of Greece to G.M and EPAN grant to

Z.G

E1-043P

Regulation of protein kinase Cé (PKCS) in rat

cardiomyocytes

T Markou, P H Sugden and A Clerk

Laboratory of Myocardial Systems Biology, NHLI Division,

Imperial College London, London, United Kingdom

E-mail: t.markou@imperial.ac.uk

PKC6 requires lipids (diacylglycerol, PtdSer) for activation and is

also modified by phosphorylation of Thr505, Ser643 and multiple

Tyr-residues Phosphorylation of Thr505/Ser643 is associated with

maturation to a form in which activation is facilitated, but some

studies in cardiomyocytes (CMs) suggest these events more

directly associate with activation Effects of endothelin-1 (ET),

PMA or PDGF on PKC6 translocation/phosphorylation (immu-

noblotting) or activity (AKRKRKGSFFYGG as selective sub-

strate, (+lipids) were studied in CMs For total PKC8, ET or

PMA increased P-Thr505 (~5x), P-Ser643 (~3x) and P-Tyr (~5x)

from ~1 min PDGF particularly increased P-Tyr (~7x) ET tran-

siently translocated (0.5—1 min) soluble PKC65 to membranes but

this rapidly reversed (3 min) Thr505/Ser643 phosphorylation of

PKC6 reappearing in the soluble fraction increased On MonoQ

FPLC using detergent-treated extracts, PKCS immunoreactivity

eluted as 2 major peaks Peak | (0.15 M NaCl was not phosphor-

ylated and was inactive The leading edge of peak 2 (0.21-0.25 M

NaCl) contained phospho-PKC6 with lipid-dependent activity

The trailing edge of peak 2 (0.25-0.30 M NaCl) contained lipid-

independent activity but relatively less phospho-PKC3/total

PKCŠð ET or PMA (10 min) promoted migration of PKC6 immu-

noreactivity from peak | to 2, and increased lipid-independent

activity A third (largely lipid-independent) peak eluted at 0.30-

0.36 M NaCl but contained little PKCS immunoreactivity Thus,

CMs contain an immature PKC6 pool (peak 1), which becomes

phosphorylated (P-Thr505/P-Ser643) in response to ET or PMA

resulting in maturation Other events render PKC6 activity lipid-

independent implying that, although PKC6 is transiently mem-

brane-bound following stimulation, it remains active after release

E1-044P

The role of protein kinase C isoenzymes

chondrogenesis of micro-mass cell cultures

C Matta!, T Juhasz!', Z Szíjgyártó”, G Czifra*, T Bird’,

P Gergely”, L Modis! and R Zákány!

Department of Anatomy, University of Debrecen, Debrecen,

Hungary, °Department of Medical Chemistry, University of

Debrecen, Debrecen, Hungary, *Department of Physiology,

University of Debrecen, Debrecen, Hungary

E-mail: mattacs@chondron.anat.dote.hu

High-density cell cultures (HDCs), established from chondrogenic

mesenchymal cells of limb buds from 4-day-old chicken embryos,

are a well-known model of im vitro cartilage differentiation We have detected the presence of various PKC isoenzymes in HDCs Although the overall PKC activity was slightly reduced, the expression and activity of PKCa were significantly increased dur- ing chondrogenesis The effects of different inhibitors of PKC isoenzymes and oxidative stress induced by hydrogen peroxide were also studied Cartilage formation was followed by detection

of metachromatic staining of the sulphated proteoglycan content

of the cartilage matrix; cartilage differentiation was determined

by RT-PCR reactions of the mRNA of the core protein of aggre- can We have demonstrated that under the effect of PKC inhibi- tors affecting the activity of more than one PKC isoenzymes, the amount of cartilage was only slightly reduced, whereas the prolif- eration of cells was significantly decreased Administration of rottlerin, a specific inhibitor of PKC6, resulted in a significant decrease of cartilage formation Under the effect of resveratrol,

an inhibitor of PKCa, chondrogenesis was also significantly reduced PKC isoenzymes also regulate cartilage differentiation via the modulation of the ERK1/2 pathway When ERK1/2 is activated, chondrogenesis is reduced In our experiments, parallel

to the inhibition of PKCs, the amount of phosphorylated ERK1/2 was increased, which resulted in a reduced chondrogenesis When HDCs were exposed to hydrogen peroxide, cartilage formation was significantly inhibited, while the expression and activity of PKCa were increased The inhibition of PKCa significantly reduced cartilage formation, and increased amount of phospho- ERK was detected by Western blots in resveratrol-treated colon- ies Our results suggest that PKCa is an important regulator of chondrogenesis and PKC« is one of the key elements responsible for reduced chondrogenesis under oxidative stress exerting its effect via phosphorylation of ERK 1/2

E1-045P

Synthesis and biological evaluation of

4-phenylamino-6-phenyl pyrimidine derivatives

as peptidomimetic inhibitors of protein

kinases

G Németh!Ý, Z GreffT, Z Vargal*, D Hafenbradl°, B Klebl°,

Z Székelyhidi!*, N Breza!, J Pato*, G Kéri!?*4 and

L Orfi!?*

‘Rational Drug Design Laboratory, Cooperation Research Centre, Semmelweis University, Budapest, Hungary, ?Department of Med- ical Chemistry, Pathobiochemistry and Molecular Biology, Sem- melweis University, Budapest, Hungary, *Department of

Pharmaceutical Chemistry, Semmelweis University, Budapest,

Hungary, ‘Vichem Chemie Ltd., Budapest, Hungary, ? Axxima Pharmaceuticals AG, Munich, Germany

E-mail: gnemeth@vichem.hu Cyclin-dependent kinases play a central role in the initiation, ordering and completion of cell-cycle events Human tumour development is associated with numerous alterations of CD-type kinases and their regulators The importance of these kinases in cell-cycle regulation, and their links with differentiation and apoptosis have stimulated an active search for peptidomimetic inhibitors of CDKs Substituted 4-phenylamino-6-phenyl pyrimi- dines constitute an important group of cyclin-dependent protein kinase inhibitors The general synthetic route to these interesting heterocyclic compounds, using 4,6-dichloropyrimidine and the related boronic acids as the starting materials, is well known The drawback to this reaction is that only certain boronic acids are accessible We developed an alternative way to prepare 4-phe- nylamino-6-phenyl pyrimidine derivatives The key intermediating

to desired compounds, 4-chloro-6-substituted phenyl pyrimidine, can be synthesized in two different ways from substituted aceto- phenones via 3-(2 or 3-substituted-phenyl)-3-oxo-propionic acid

ethyl esters Scale up of these reactions is facile and high diversity

of derivatives is available as compared with the general procedure

(Suzuki-coupling) Our compounds were examined for inhibiting

activity in selective CDK-1/2, -4/6 and -9 bichemical kinase

assays and their ICS0 values were also determined A series of

compounds were selective as a potent CDK inhibitor, e.g for

CDK9 in the nanomolar range, for other CDKs in the sub-

micromolar range

E1-046P

Specific interaction between S6K1 and CoA

synthase

I Nemazanyy’, A Zhyvoloup', I Gout? and V Filonenko!

Department of Structure and Functions of Nucleic Acids, Institute

of Molecular Biology and Genetics, Kiev, Ukraine, Department of

Biochemistry and Molecular Biology, University College London,

London, United Kingdom E-mail: nemazanyy@imbg.org.ua

Ribosomal protein S6 kinase (S6K) is a key regulator of cell size

and growth It is regulated via phosphoinositide 3-kinases (PI3K)

and the mammalian target of rapamycin (mTOR) signaling path-

ways The yeast two-hybrid screen was used to isolate binding

partners towards S6K1 One of the interacting molecules was

found to encode a novel protein, termed CoA synthase In our

initial studies, we focused on molecular cloning, biochemical and

functional characterization of this protein We found that CoA

synthase mediates the last two steps in CoA biosynthesis via 4’-

phosphopantetheine adenylyltransferase and dephospho-CoA

kinase activities and termed it CoA synthase Furthermore, we

demonstrated that CoA synthase is localized on the outer mit-

ochondrial membrane and that its activity is strongly activated

by phospholipids Molecular cloning and characterization of

CoA synthase provided us with necessary reagents required to

study the specificity of interaction with S6K1 and its functional

consequence in mammalian cells Here we demonstrate for the

first time specific interaction between CoA synthase and S6K1 by

co-immunoprecipitation studies in mammalian cells and by BIA-

core analysis in vitro The C-terminal regions of CoA synthase

and S6Ks mediate the interaction between both proteins CoA

synthase is not a substrate for S6K in vitro and its activity is not

affected by rapamycin or LY294002 in vivo The physiological

relevance of the identified interaction is currently under investiga-

tion

E1-047P

6-Phosphofructo-2-kinase (pfkfb3) gene

promoter contains hypoxia-inducible factor-1

binding sites necessary for transactivation in

response to hypoxia

M Obach!, A Navarro-Sabatel, J Caro’, X Kong”, J Duran!,

M Gomez!, J.C Perales’, F Ventural, J L Rosa! and

R Bartrons!

"Department of Ciencies Fisiologiques Il, Laboratory of Bioquimi-

ca, University of Barcelona, Hospitalet De Llobregat, Spain,

°Department of Medicine, University of Thomas Jefferson,

Philadelphia, Pennsylvania, United States of America

E-mail: mobach@ub.edu

The upregulation of glycolysis to enhance the production of

energy under reduced pO, is a hallmark of the hypoxic

response A key regulator of glycolytic flux is fructose-2,6-bis-

phosphate and its steady-state concentration is regulated by the

action of different isozymes product of four genes (pfkfb1-4) Pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia To understand the organization of cis-act- ing sequences that are responsible for oxygen-regulated pfkfb3 gene, we have studied its S’flanking region Extensive analysis

of S’pfkfb3 promoter sequence revealed the presence of putative consensus-binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation These DNA-consensus sequences included estrogen receptor (ER), hypoxia response element (HRE), early growth response (EGR- 1) and specific protein (Spl) putative-binding sites Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit con- structs, demonstrates that the sequence located from —1269 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 -1279 and —1288 was corroborated by electrophoretic mobil- ity shift assay and biotinylated oligonucleotide pull-down In addition, HIF-lalpha null mouse embryo fibroblasts transfected with a full length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene Altogether, these results demonstrate that pfkfb3 is a hypoxia inducible gene that

is stimulated through HIF factor interaction with the consensus HRE site in its promoter region

E1-048P

Contribution of intracellular signaling cascades

to the cardioprotective effect of poly(ADP- ribose) polymerase inhibitors in myocardial ischemia-reperfusion

A Palfi'?, A Toth’, K Hanto!”, E Hocsak?, R Halmosi!,

E Szabados', K Hideg*, B Sumegi* and K Toth!

‘First Department of Medicine, University of Pécs, Medical School, Pécs, Hungary, °Department of Biochemistry and Medical Chemistry, University of Pécs, Medical School, Pécs, Hungary, Institute of Organic and Medicinal Chemistry, University of Pécs, Medical School, Pécs, Hungary E-mail: anita.palfi@aok.pte.hu Objective: We tested the hypothesis whether poly(ADP-ribo- syljation of nuclear proteins and its inhibition might modulate intracellular signaling cascades in myocardial ischemia-reper- fusion Since the activation of Akt and mitogen-activated protein kinase (MAPK) cascades can play a pivotal role in the survival

of cardiomyocytes during ischemia-reperfusion, we studied the effect of PARP inhibition on these signaling pathways

Methods: A novel PARP inhibitor (L-2286) was administered during ischemia-reperfusion in Langendorff perfused rat hearts and in isoproterenol-induced myocardial infarction Subse- quently, the cardiac energy metabolism, oxidative damage and the phosphorylation state of Akt and MAPK cascades were mon- itored

Results: L-2286 exerted significant protective effect against ische- mia-reperfusion-induced myocardial injury in both experimental models More importantly, L-2286 facilitated the ischemia-reper- fusion-induced activation of Akt, extracellular signal-regulated kinase (ERK) and p38-MAPK in both isolated hearts and in vivo cardiac injury By contrast, isoproterenol-induced rapid c-jun N- terminal kinase (JNK) activation was repressed by L-2286 Conclusion: We first provided evidence that PARP inhibition in

ex vivo and in vivo model systems beneficially modulated the intracellular signaling, which may contribute to the cardioprotec- tive properties of PARP inhibitors

E1-049P

Regulation of protein kinase B by

DNA-dependant protein kinase

J Feng!, J Park”, P Cron‘, M ShongỶ, D Hess! and

B A Hemmings!

’Freidrich Miescher Institute, Basel, Switzerland, ? Department of

Pharmacology, Medical School, Chungnam National University,

Daejeon, South Korea, *Department of Internal Medicine, Medical

School, Chungnam National University, Daejeon, South Korea

E-mail: insulin@cnu.ac.kr

A 350-kDa protein corresponding to a major Ser-473 kinase

activity (S473K) was purified from the membrane fraction of

HEK293 cells and identified as DNA-PK in vitro, DNA-PK

phosphorylated PKB on Ser-473, resulting in a ~10-fold

enhancement of PKB activity Knockdown of DNA-PKcs by si-

RNA impaired Ser-473 phosphorylation in response to insulin

and pervanadate stimulation Extracts from DNA-PKces-deficient

cells carrying mutations in the Prkde gene had a reduced level of

Ser-473 phosphorylation; this effect was restored by complemen-

tation with human Prkdc gene DNA-PKcs associated and co-

localized with PKB at the plasma membrane We conclude that

DNA-PK is in part responsible for Ser-473 phosphorylation of

PKB Identification of the S473K may help define the detailed

mechanism of PKB/Akt activation

E1-050P

A vector suite approach to baculovirus

expression

S C Pengelley', M Abbott? and I M Jones!

'School of Animal and Microbial Sciences, University of Reading,

Reading, Berkshire, United Kingdom, °EST-Bio, AstraZeneca,

Macclesfield, Cheshire, United Kingdom

E-mail: sarO02scp@rdg.ac.uk

To improve the possibilities of rapid and successful baculovirus

expression, we have developed a suite of vectors based around

the shared cloning site and reading frame We used differential

sequences for the restriction enzyme Sfil to provide directional

cloning sites that would be unlikely in the majority of target

ORFs The suite includes vectors providing the solubility enhan-

cer 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, extracel-

lular matrix proteins, have been cloned into each vector In the

case of the kinases, we found that fusion to MBP rescued expres-

sion of the otherwise non-expressible kinase Cot but made little

difference to well-expressed kinases IKK and Lek Fusion to

GFP was also beneficial and provided a rapid and quantitative

readout of expression level using flow cytometry The GFP tag

was also useful in mapping the most expressible domains While

full length IKK fused to GFP was abundantly expressed, reduc-

tion of the ORF to a sequence encoding only the catalytic

domain led to a substantial loss of expression However, cell-free

coupled transcription and translation of the same vectors showed

equivalent expression of both the truncated protein and the full-

length ORF, suggesting that cell-dependent protein degradation

following synthesis rather than inherent non-translatability was

most probably responsible for differential expression in S/9 cells

Confocal microscopy was used to investigate the cellular location

of the expressed kinases and ADAMTs proteins in relation

to markers of both lysosomes and proteasomes and will be

presented

E1-051P Activation of signal transduction pathways in chondrocytes under hyperosmotic conditions

B Racz', B Gasz', A Tamas’, D Reglédi’, E Réth' and

B Borsiczky!

"Department of Surgical Research and Techniques, University of Pécs, Pécs, Hungary, *Department of Anatomy, University of Pécs, Pécs, Hungary E-mail: tikis5@freemail.hu

Introduction: Several studies indicated that osmotic stress acti- vates intracellular secondary messenger signals that may contrib- ute to the pathogenic alterations of different cells Surprisingly, there is little data regarding the action of such pathways in chondrocytes that are frequently exposed to increased osmotic loading during articular diseases Our study aimed to investigate the activation of stress-activated protein kinase pathways and their possible role in chondrocyte damage

Materials and methods: Isolated swine chondrocytes were incubated for 6h under normal (300 mOsm) and hyperosmotic (600 mOsm) conditions The phosphorylation of p38 mitogen- activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (ERK1/2), cAMP responsive element-binding tran- scription factor (CREB) and protein kinase B (Akt1) was investi- gated by Western blot analysis The cellular damage was characterized via the ratio of viable cells and the percentage of apaptotic chondrocytes detected by annexin-v flow cytometry Results: Increased phosphorylation of p38 MAPK, ERK1/2, CREB and Aktl was found in the chondrocytes treated by hyper- osmotic media (23192pixel; 2113/5585pixel; 21900pixel; 792pixel) compared with the control cells (11580pixel; 949/1436pixel; 5195pixel; 283pixel) Meanwhile, elevated proportion of apoptotic cells (6.6% vs 11.09%) and decreased number of viable chondro- cytes (92.43% vs 69.02%) was found in the osmotic stress group Conclusion: Our results show that several intracellular signal transduction pathways are activated in the chondrocytes under osmotic stress, which may be responsible for the apoptotic changes

in chondrocytes Since increased osmotic loading often accompan- ies articular diseases, it is suggested that the mentioned pathways play a role in the patomechanism of such clinical conditions Acknowledgment: Supported By OTKA F046504 and ETT 596/2003

E1-052P

Involvement of protein kinase CK2 in the MDR phenotype of CEM cells

G Di Maira!2, F Brustolon!”, V Cenni’, L A Pinna!?,

S Marmiroli? and M Ruzzene!2

‘Department of Biological Chemistry, University of Padova, Pad- ova, Italy, °Venetian Institute of Molecular Medicine (VIMM), University of Padova, Padova, Italy, 7Department of Anatomy and Histology, University of Modena and Reggio Emilia, Modena, Italy E-mail: maria.ruzzene@unipd it

The multidrug resistance (MDR) phenotype of cancer cells is the main reason for failures of anti-tumour therapy; several inde- pendent factors have been reported as responsible for MDR oc- curance, but their mechanisms of action are still largely unknown CK2 is a ubiquitous and constitutively active Ser/Thr protein kinase, whose substrates are involved in many important cell processes, and whose role as an anti-apoptotic kinase has been recently described Here, we report data supporting a con- tribution of CK2 in the MDR phenotype We analyzed a pair of tumour cell lines: CEM-S (the parental lymphoblastoid cell line, normally sensitive to chemotherapeutic agent-induced apoptosis), and its MDR derivative CEM-R (resistant to a panel of pro- apoptotic agents) We disclosed major differences between these

two variants in terms of CK2 catalytic subunit expression and

activity; on the contrary, the CK2 regulatory subunit is equally

expressed As a consequence, the phosphorylation pattern of

endocellular CK2 substrates is significantly different in the two

cell lines We also found that treatment of CEM-R with highly

specific CK2 inhibitors induces apoptosis and allows a higher

intracellular accumulation of chemotherapeutic agents We there-

fore suggest that CK2 can be considered one of the important

targets in the attempt to counteract the MDR phenomenon

E1-053P

Glivec inhibits proliferation and reduces

invasive potential of dedifferentiated human

thyroid cancer cells by downregulating Wnt/

beta-catenin signalling

A S Rao!, N Kremenevskaja!, J Resch!, R von Wasielewski*

and G Brabant!

'Department of Gastroenterology, Hepatology and Endocrinology,

Hannover Medical School, Hannover, Lower Saxony Germany,

?Institute of Pathology, Hannover Medical School, Hannover,

Lower Saxony, Germany E-mail: srinivas86(@ yahoo.com

Aberrant Wnt/beta-catenin signalling plays a critical role in thy-

roid cancer Moreover, tyrosine phosphorylation of beta-catenin

disrupts adherens junctions by inhibiting its binding to E-cadher-

in and favors invasiveness of thyroid cancer cells Since beta-cate-

nin is known to be linked to tyrosine kinases such as c-abl,

PDFR and c-kit in leukemic cells, we tested the hypothesis that

beta-catenin is linked to these kinases in neoplastic thyrocytes

and that this pathway may be modulated by the selective tyrosine

kinase inhibitor, Glivec Tissue microarray of histologically pro-

ven primary anaplastic thyroid carcinomas (ATC, n = 12) and

three cell lines derived from ATC demonstrated positive immuno-

staining for c-abl in 25% and for PDGF-R and c-kit in 8.3%,

respectively Coimmunoprecipitation experiments with c-abl and

beta-catenin revealed a direct functional link Treatment of c-abl-

positive ATC cell lines with Glivec drastically reduced beta-cate-

nin protein expression and redistributed beta-catenin from a nuc-

lear localization to the cell membrane Further, Glivec increased

membranous E-cadherin expression, increased adherens junction

stability and decreased matrigel invasion Proliferation of c-abl-

positive ATC cell lines was dose-dependently reduced by approxi-

mately 50% without affecting apoptosis (caspase 3/7 assay) This

appeared to be a beta-catenin-mediated effect as shown by the

results of reporter gene assay for beta-catenin (TOP/FOP flash)

and rt-PCR for CyclinDl, a known target of beta-catenin

involved in proliferation In contrast, neoplastic thyrocytes negat-

ive for Glivec targets showed no effect Collectively, our data

provide a novel molecular mechanism for the anti-tumour activ-

ity of Glivec and open new therapeutic options for ATC patients

expressing Glivec sensitive tyrosine kinases

E1-054P

The effect of a novel PARP inhibitor HO-3089

on the LPS stimulated RAW 264,7 murine

macrophage cells

B Radnai!, B Veres!, K Hanto!”, P Jakus', G Varbiro!,

Z Bognar!, A Tapodi', S Veto! and B Sumegi!

‘Department of Biochemistry and Medical Chemistry, University

of Pécs, Medical School, Pécs, Hungary, *First Department of

Medicine, University of Pécs, Medical School, Pécs, Hungary

E-mail: balazs.radnai@aok.pte.hu

Activation of the nuclear enzyme poly-(ADP-ribose)-polymerase

(PARP) is involved in numerous pathophysiological conditions

We have previously shown that 4-hydroxyquinazoline, (4-HQN)

a potent PARP inhibitor, has protective effect on LPS-induced endotoxic shock by modulation of kinase cascades and regulation

of transcription factors in mice Since blood peripheral mononu- clear cells and macrophages play key role in endotoxin-induced inflammatory processes, we previously investigated the role of 4- HQN on LPS-stimulated murine macrophages In this study, we investigated the effect of a novel PARP inhibitor the HO-3089 HMG-1 is a late mediator of the inflammatory processes Since our results indicated that HO-3089 has not any effect on HMG-1 activation, we decided to study early mediators of inflammation, such as P-Akt, P-Gsk-3-B, MAP kinases and TNF-a Following exposure to LPS, we detected phosphorylation and thereby acti- vation of several kinases (P-ERK1/2, P-Gsk-3-B and P-p38) HO-3089 treatment decreased the level of LPS-induced activation

of Gsk-3-B, ERK1/2 and p38 In our experimental model of LPS-induced murine macrophages, we did not find any activation

of JNK and Akt We measured the highest level of TNF-o 5 h after LPS treatment, which was significantly attenuated by HO-3089 Finally, we found the induction and suppression of numerous genes, mediated by LPS and HO-3089, which are pre- sumably involved in the protection against LPS-mediated endotoxic shock Our experiments showed that HO-3089 signifi- cantly attenuated the LPS-induced activation of several kinases (Gsk-3-B, ERK1/2, p38) and TNF-a HO-3089 induced and sup- pressed numerous genes, which are presumably involved in the protection against LPS-mediated endotoxic shock

E1-055P Imaging of PKB phosphorylation as an in vivo

readout for PTEN inhibition studies

E Rosivatz', R Vilar? and R Woscholski!

‘Department of Biological Sciences, Imperial College London, London, UK, *Department of Chemistry, Imperial College London, London, UK E-mail: e.rosivatz@ic.ac.uk

Tensin homologue deleted on chromosome 10 (PTEN) is a major tumor suppressor protein that dephosphorylates membrane phos- phatidylinositol lipids such as PI(3)P, PI(3,4)P2 and PI(3,4,5)P3, thus, counteracting the action of PI3-kinases and its downstream targets PTEN prevents the activation of the survival signaling kinase Akt/PKB, which can be monitored by detecting phos- phorylation at two sites (Thr308 and Ser473) Bisperoxovanadi-

um (bpV) molecules were recently shown to specifically inhibit PTEN In order to test the ability of vanadium complexes on PTEN inhibition im vivo, we established an imaging method using Akt/PKB phosphorylation as readout for cellular PI(3,4,5)P3 lev- els Starved NIH 3T3 fibroblasts pre-treated with 500 nm of var- ious vanadium complexes were stimulated with non-activatory concentrations of insulin or a novel synthetic compound C, respectively Cells were phalloidin stained and/or immunostained with a monoclonal phospho-specific Ser473 Akt/PKB antibody Actin polymerization and PKB phosphorylation were monitored with fluorescence microscopy As expected, the tested compounds induced the phosphorylation of PKB under PI 3-kinase primed conditions In addition, inhibition of PTEN correlates with stress fiber breakdown and cortical actin accumulation Interestingly, bpV treatment, which leads to increased PI(3,4,5)P3 levels, gener- ated by PI3-kinase, indirectly inhibits PKB activation after com- pound C challenge This is consistent with our findings that compound C inhibits PKB phosphorylation in stimulated cells, but induces Ser473 phosphorylation in starved cells In summary,

we have shown that the tested vanadium complexes inhibit PTEN at nanomolar concentrations and are useful research tools for investigations analyzing PI3-kinase-mediated signaling in vivo bpV molecules helped reveal a novel PKB activatory pathway, which is independent of PI3-kinase activation

E1-056P

Novel VEGF family defines the differential

signals for angiogenesis and vascular

permeability

M Shibuya, H Takahashi, S Yamaguchi and Y Sakurai

Division of Genetics, Institute of Medical Science, University of

Tokyo, Tokyo, Japan E-mail: shibuya@ims.u-tokyo.ac.jp

Vascular endothelial growth factor (VEGF)-A consists of a ho-

modimer, and binds to two receptors, VEGFRI(FIt-1) and VEG-

FR2(KDR) to exert signals for angiogenesis and vascular

permeability However, the precise mechanisms are not fully

understood We found that a new type of VEGF, VEGF-E, acti-

vates only VEGFR2 and carries a potent angiogenic activity sim-

ilar to VEGF-A Also, two intramolecular loops formed by S—S

bonds, loop-1 and loop-3, in VEGF-E interact with each other to

generate a structure that binds and activates VEGFR2 with a

high affinity K14-VEGF-E transgenic mice demonstrated a

strong angiogenic response with very low edema, suggesting that

activation of only VEGFR2 induces mainly the signals for angio-

genesis, whereas simultaneous activation of VEGFRI and VEG-

FR2 promotes both angiogenesis and permeability Recently, we

have obtained a strong supportive evidence for this model We

identified another new VEGF-related protein, Tf-svWEGF, pre-

sent in the snake venom of Habu, a snake at southern Japan

Interestingly, Tf-svVEGF binds tightly to VEGFRI1 but weakly

to VEGFR2, and generates a strong signal for vascular permeab-

ility with less angiogenic activity These results indicate that the

tertiary structure formed by loop-1 and -3 in VEGF determines

the degree of the activation of VEGFR2 and angiogenic

response, and other portions determine the affinity to VEGFRI1

important for vascular permeability For the signaling toward an-

giogenesis, we have found that a single autophosphorylated tyro-

sine in VEGFR-2 (1175-Tyr) is crucial for unique signaling via

PLCg-PKC-MAP kinase pathway toward angiogenesis These

results propose new molecular targets for artificial modulation of

angiogenesis and vascular permeability

E1-057P

Opposite roles of protein kinase C in the

control of HGF- or phorbol ester-induced cell

scattering

S Sipeki, L Buday and A Farago

Medical Chemistry, Molecular Biology and Pathobiochemistry,

Semmelweis University, Budapest, Hungary

E-mail: sipeki@ puskin.sote.hu

Understanding how cell scattering is regulated has important

applications in developmental biology, tissue regeneration and

cancer research Hepatocyte growth factor/scatter factor (HGF)

acting on the c-Met receptor tyrosine kinase is a physiological

inductor of cell scattering In our experiments, the HepG2 human

hepatoma cell line provides a model for investigating the control

of cell scattering The HepG2 cells respond with more intensive

migration to phorbol ester (PMA) than to HGF We found that

protein kinase C (PKC) was responsible for the PMA-induced

scattering but detained the HGF-initiated cell migration The sus-

tained 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 cells In the signalling system of HGF, the pho-

spatidylinositol 3-kinase (PI3K) contributed to the activation of

the Erk1/2 MAP kinases PKC decreased the duration of PI3K

activation The HGF-induced actin cytoskeleton changes leading

to cell migration are known to be initiated by the PI3K through the activation of the Rac GTPase Thus, two of the pathways mediating the signal from HGF-treatment to migratory response can be suppressed by PKC To the contrary, the PMA-induced cell scattering did not require the activity of the PI3K Neither the Rac GTPase nor the Sre tyrosine kinase was involved in the transmission of the signal from the PMA-activated PKC to the actin cytoskeleton changes The mechanism that result in cell migration through actin cytoskeleton changes seems to involve a cell-specific substrate of the PMA-activated PKC

E1-058P FAK and PI-3K are activated in micrometastatic

human breast cancer cells

G Kallergi!, D Mavroudisˆ, A GravanisỶ, V Georgoulias” and

C Stournaras'

"Department of Biochemistry, University of Crete, Heraklion,

71110 Greece, *Department of Clinical Oncology, University of Crete, Heraklion, 71110 Greece, *Department of Pharmacology, University of Crete, Heraklion, 71110 Greece

E-mail: cstourn@med.uoc.gr

We have previously reported that actin cytoskeleton dynamics, migration and growth of MCF7 human breast cancer cells are under the control of a specific-signaling cascade involving phos- phorylation of focal adhesion kinase (FAK), PI-3 kinase, and activation of the small GTPase Racl We have also documented the expression of cytokeratins in circulating human breast cancer micrometastatic cells, and their value as specific-micrometastatic cell markers In the present study, we report that in micrometa- static cell preparations from breast cancer patients (i) actin microfilaments were reduced, showing an impaired distribution, (ii) expression of cytokeratin-19 and phosphorylated FAK was strongly correlated, suggesting that p-FAK, in addition to CK-

19, may represent a novel and reliable marker for the detection

of circulating micrometastatic cells in breast cancer patients and (iii) both FAK and PI-3K kinase were phosphorylated on tyro- sine in cultures of isolated micrometastatic cells, indicating acti- vation of a specific-signaling cascade similar to that identified in MCH?7 cells In these cell preparations, we have also assessed the expression pattern of pro- and anti-apoptotic Bcl-2 and Fas/FasL proteins as well as their sensitivity to apoptotic signals Our find- ings 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 in human peripheral blood

mononuclear cells

J Siednienko, M Ciuman, H Witwicka, E Kurowska and

W A Gorezyca Laboratory of Signalling Proteins, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland E-mail: siednienko@immuno.iitd.pan.wroc.pl

Nuclear factor-kappaB (NF-kappaB) is a key transcription factor regulating expression of genes important for inflammatory response It has been also reported that constitutive activity of NF-kappaB prevents spontaneous apoptosis of circulating immune cells In its inactive form, NF-kappaB is associated with inhibitory protein IkappaB and is maintained in the cytoplasm as

a trimeric complex Activation leads to dissociation and