Protein-tyrosine kinases are important not only for the development of malignanttumors but also for the regulation of growth and function of normal cells. Our cur-rent interest is to characterize cell signaling downstream of protein-tyrosinekinases that are relevant respectively to cancer development and to neuronalfunction. We are also interested in protein kinase signaling and chromosome dy-namics that are involved in the regulation of cell cycle progression, particularlythat of mitosis.

�The biological role of Tob family proteinsand CCR4/Not complex

Toru Suzuki, Junko Tsuzuku, MitsuhiroYoneda, Masahiro Morita, Yan Zhang, Ken-taro Ito, Shinichiro Ogawa, Akinori Takahashi,Yusuke Nakajima and Tadashi Yamamoto

By screening a cDNA expression library usingErbB-2 protein as a probe, we identified Tob, a45kDa protein as an ErbB2-interacting protein.Tob was homologous to the growth suppressingproteins, Btg1 and Btg2/PC3. We also clonednovel genes, tob2 and ana that are homologousto tob. These proteins compose a functionally re-lated anti-proliferative protein family, called theTob/Btg family.

To elucidate the physiological function of theTob/Btg family proteins, we generated micelacking tob, tob2 , or ana. By characterizing thempathologically and molecularly, we found thefollowing. (i) tob-deficient mice (tob－/－) had agreater bone mass resulting from increasednumber of osteoblasts in comparison with wild-type mice. We also found that aged tob－/－ mice

develop a variety of tumors. (ii) In contrast,mice lacking tob2 had decreased bone mass, andthe number of osteoclasts differentiated frombone marrow cells was increased. Furthermore,expression of RANKL mRNA in stromal cellswas increased in the absence of Tob2 and de-creased in the presence of Tob2. Tob2 interactedwith vitamin D3 receptor (VDR), suggesting itsinvolvement in VDR-mediated regulation oftranscription. In conclusion, Tob2 negativelyregulates formation of osteoclasts by suppress-ing RANKL expression through its interactionwith VDR. (iii) Unlike the other Tob family pro-teins, Ana is specifically expressed in type II al-veolar epithelial cells. Since lung adenocarci-noma is thought to be mainly derived fromtype II alveolar epithelial cells, Ana may be in-volved in development of lung tumor. Indeed,ana-deficient mice developed spontaneous tu-mors including lung adenocarcinoma. We alsofound that expression of ana gene was largelyreduced in almost all of the lung cancer celllines and clinical samples of the lung adenocar-cinoma examined. These data suggested thatdownregulation of ana gene might be responsi-

Department of Cancer Biology

Division of Oncology癌細胞シグナル研究分野

Professor Tadashi Yamamoto, Ph.DAssociate Professor Miho Ohsugi, Ph.D.Assistant Professor Toru Suzuki, Ph.D.Assistant Professor Takanobu Nakazawa, Ph.D.Project Assistant Professor Kazumasa Yokoyama, Ph.D.

教 授 理学博士 山 本 雅准教授 理学博士 大 杉 美 穂助 教 理学博士 鈴 木 亨助 教 医学博士 中 澤 敬 信特任助教 理学博士 横 山 一 剛

35

ble for lung adenocarcinoma progression. Cur-rently, we are investigating the molecularmechanism by which Ana regulates progressionof lung tumor.

Recently, we found that Tob is involved inDNA damage response. UV-induced stress pro-motes proteasome-dependent degradation ofTob, triggering an apoptotic signal. Tob witheither short deletion or a tag sequence at the C-terminus is resistant to UV-induced degradation.Introduction of the degradation-resistant Tobimpaired UV-induced apoptosis. Reciprocally,suppression of Tob by small-interfering RNA re-sulted in frequent induction of apoptosis irre-spective of the presence of functional p53 evenat UV doses that do not promote Tob degrada-tion. Thus, proteosomal clearance of Tob pro-vides a novel p53-independent pathway for UV-induced apoptosis.

Other studies to establish biological signifi-cance of the tob family members are following.First, we purified Tob-containing complexesfrom HeLa cells that stably express the Flagepitope-tagged Tob by immunoprecipitationwith anti-Flag antibody. Mass spectrometricanalysis showed that CNot1, CNot2, CNot3,CNot6/CCR4, CNot7 and CNot9 were includedin the Tob complex. We also showed that Tobsuppresses the deadenylase activity of CCR4/Not complex at least in vitro . Although theCCR4/Not complex possesses mRNA deadeny-lase activity, and thus regulates the stability ofmRNAs, biological roles of the complex inmammals remain unknown. Second, we per-formed yeast two-hybrid screening using Tob asa bait, and identified a serine/threonine kinaseNdr. Interestingly, a yeast homolog of thekinase is reported to associate with the CCR4/Not complex. Third, we newly identified CCR4b/CNot6L, which is homologous to yeast CCR4deadenylase. CCR4b forms a multi-subunit com-plex similar to the yeast CCR4/Not complex.Suppression of CCR4b by RNA interference(RNAi) results in growth retardation of NIH3T3cells accompanied by elevation of both p27Kip1

mRNA and p27Kip1 protein. Reintroduction ofwild-type CCR4b but not mutant CCR4b lackingdeadenylase activity restores the growth ofCCR4b-depleted NIH3T3 cells. The data suggestthat CCR4b regulates the cell growth in a man-ner dependent on its deadenylase activity. Ourfindings suggest that CCR4b regulates the turn-over rate of specific target mRNAs. It should beelucidated how CCR4/Not deadenylase com-plex recognize the specific mRNAs. Fourth, weexamined the effect of RNAi-mediated knockdown of the other components of the CCR4/Notcomplex on cell proliferation and differentiation.So far, we found that depletion of several com-

ponents of the complex results in cell growth ar-rest or in extensive cell death. The cell death oc-curred in a manner dependent on the caspaseactivity. We are now examining the mechanismsby which cell growth or apoptosis was inducedby down-regulation of the components of theCCR4/Not complex. Finally, to examine the bio-logical functions of CCR4/Not complex, we gen-erated cnot3, cnot6, cnot6L, cnot8, cnot9 and ndr-deficient mice. Our data suggest that most ofthe components in the complex are involved inembryonic development and energy homeosta-sis. To understand the underlying mechanismby which each molecule in the complex regu-lates such biological events, we are in search ofspecific mRNAs targeted by the CCR4/Notcomplex. Several approaches such as microarrayanalysis, Real-time PCR, and Northern blotanalysis have been undertaken.

�Roles of protein kinases in the centralnervous system

Takanobu Nakazawa, Kazumasa Yokoyama,Takeshi Inoue, Naosuke Hoshina, and TadashiYamamoto

The Src-family protein-tyrosine kinases areimplicated in various neural functions such asformation of neural network, myelination, andsynaptic plasticity. To analyze the roles of Srcand Fyn, we have been characterizing brain spe-cific substrates of these kinases, including N -methyl-D-aspartate (NMDA) type of ionotropicglutamate receptors. Our own studies haveshown that GluN2A and GluN2B subunits ofNMDA receptors, which play important roles inlearning, memory formation, and emotional be-haviors, are the major substrates of Fyn and Src.We identified Tyr-1472 phosphorylation onGluN2B and Tyr-1325 phosphorylation onGluN2A as the major tyrosine phosphorylationsite of GluN2B and GluN2A, respectively. Usingthe knock-in mouse lines expressing mutantGluN2B with a Tyr-1472-Phe (Y1472F) mutationor expressing mutant GluN2A with a Tyr-1325-Phe (Y1325F) mutation, we have shown thatTyr-1472 phosphorylation is a key mediator offear-related learning and emotional behavior inthe amygdala and that Tyr-1325 regulatesdepression-related behavior. We are now charac-terizing molecular mechanisms underlying thesebehavioral abnormalities.

In parallel of these studies, to uncover Src-and Fyn-mediated signaling pathways, we havebeen trying to identify binding partners andsubstrates of these kinases in the brain usingsolid-phase phosphorylation screening, yeasttwo-hybrid screening, and proteomic ap-

36

proaches. As a result, we have identified a num-ber of putative mediators of Fyn- and Src-mediated signaling, including NYAP, FAK,p250GAP, TCGAP, Nogo-A, and RhoGEFs.Among these proteins, we demonstrated thatNYAP family proteins are the most heavilytyrosine-phosphorylated proteins, accounting forabout half of tyrosine phosphorylation reactionsin the developing neuron. We generated NYAPfamily knockout mice and demonstrated thatthe NYAP family plays a pivotal role as the cen-tral scaffold in the PI3K signaling pathway. Weare now studying the physiological roles ofp250GAP, TCGAP, and FAK as well as NYAPin the neuronal functions.

In database search for novel protein-tyrosinekinases, we identified a kinase that is highly ex-pressed in the brain and termed it as BREK(Brain-Enriched Kinase). It turned out thatBREK is the same as AATYK2/LMTK2 found atthe almost same time by others. BREK is amember of a family consisting of AATYK1/LMTK1, AATYK2/BREK/LMTK2, AATYK3/LMTK3. BREK has also been termed as KPI-2/CPRK. All the family members are predomi-nantly expressed in the brain. We showed thatBREK plays a role in NGF signaling in PC12cells, suggesting that BREK is involved in neuraldevelopment and function in early postnatal de-velopment. To investigate the physiological roleof BREK, we generated mice with targeted dis-ruption of brek. While brek is highly expressedin the brain, it is also expressed in the testis. Wehave found that brek－/－ male mice are infertilewith azoospermia. We are currently studyingthe roles of BREK in the central nervous systemusing the knockout mice.

�Roles of chromokinesin Kid and mitotickinases in execution of cell division

Miho Ohsugi, Noriko Tokai-Nishizumi, NaokiOshimori, Natsuko Masuda, Kenji Iemura, XueLi, Shu-Jen Shiu, and Tadashi Yamamoto

Mitosis is a process whereby a complete copyof the genetic information is distributed to eachdaughter cell. This process is critically impor-tant, with even small errors leading to aneu-ploidy or cell death. The chromosomal and/orcentrosomal abnormalities are often observed intumor cells and those abnormalities may oftenbe the first events in the development of a can-cer. It is well known that microtubule-based mo-tor proteins are involved in spindle formationand chromosome movements in mitosis. In ad-dition, orchestrated regulation by mitotickinases is important for the progression of eachstep of mitosis.

i) The chromokinesin KidThe human chromokinesin Kid/Kinesin-10 is

a member of the chromosome-associated kinesinfamily identified in our lab in 1996. Kid hasbeen implicated in generating the polar ejectionforce that pushes the chromosome arms awayfrom the spindle poles toward the spindle equa-tor, as well as in the maintenance of spindlelength during prometaphase and metaphase. Wepreviously showed that chromosome localiza-tion of Kid during prometaphase and metaphaserequires Cdc2/cyclin B-mediated phosphoryla-tion on Thr463. In addition, Importin α directlyassociates with Kid via nuclear localization sig-nals (NLSs) and Ran-GTP-mediated dissociationof importin α from Kid promotes the accumula-tion of Kid on chromosomes. During anaphaseand telophase, Kid is localized in the intersticesbetween adjacent chromosomes and contributesto the tight clustering of anaphase chromosomes(anaphase chromsome compaction). Kid defi-ciency often leads to micro- or multinuclear for-mation at oocyte meiosis II and the first coupleof mitoses after fertilization, causing embryonicdeath in mice. Later somatic mitoses are not fa-tally affected by the absence of Kid, suggestingthat cell divisions under significant influence ofthe maternal factor specifically require Kid toprevent formation of multinucleated cells. Wefurther analyzed the chromosome dynamicsduring anaphase II and pronuclear formationprocess in wild type and Kid-deficient oocytes.Our data suggest that Kid is important for bothestablishment and maintenance of anaphase/te-lophase chromosome cluster especially duringfemale pronuclear formation process.ii) Mitotic kinase Plk1 and its substrates

Plk1 (polo-like kinase 1) is a highly conservedserine/threonine kinase that plays multiple piv-otal roles in mitosis, meiosis, and also in onco-genesis. However, the exact mechanisms ofPlk1’s actions remain to be elucidated. Throughthe solid-phase phosphorylation screening, wepreviously identified several Plk1 substrates in-cluding Kizuna (Kiz) which when depletedcauses fragmentation and dissociation of thepericentriolar material (PCM) from centrioles atprometaphase, resulting in multipolar spindles.Using a yeast two-hybrid screen for Kiz-interacting proteins, we identified an uncharac-terized centrosome protein Cep72. RNAi experi-ments demonstrate that Cep72 is essential for lo-calization of CG-NAP, a large coiled-coil proteinforming the structural framework of the PCM,as well as Kiz. Cep72 is also involved in γ-tubulin ring complexes (γ TuRCs) recruitment tothe centrosome and CG-NAP confers the micro-tubule nucleation activity on the γ TuRCs. Dur-ing mitosis, the Cep72-mediated centrosomal

37

MTOC activity helps connect spindle microtu-bules to the centrosome so that forces generatedby chromosome movement along microtubulesconverge on the PCM. Cep72-recruited Kiz en-sures structural integrity of the PCM to endurethe microtubule-mediated forces. Our data fur-ther suggest that Cep72-mediated spindle poleformation is essential to align chromosomes atthe metaphase plate and to generate tensions be-tween kinetochores. We are currently investigat-ing other newly identified substrates of Plk1,which will uncover the molecular mechanismsunderlying the Plk1-mediated control of cell di-visions and oncogenesis.iii) Chromosome dynamics in early mice em-

bryoIn vertebrates, oocytes are arrested at meta-

phase of the second meiosis, and fertilizationtriggers the anaphase onset and emission of thesecond polar body. Then, nuclear envelopes areassembled around maternal and paternal chro-

mosomes, forming separate haploid male andfemale pronuclei. In mouse embryo, this topo-logical genome separation appears to be pre-served up to the four-cell embryo stage andthen gradually disappears. Maternal proteinsand transcripts stored in oocytes control embry-onic development, until zygotic gene activationbegins. In mice, minor gene activation begins atthe 1-cell stage that is followed by a major geneactivation at the 2-cell stage. Therefore, comple-tion of oocyte meiosis II and first couple of mi-tosis after fertilization are almost exclusively un-der maternal control. We are interested in howthe structure and behavior of maternal and pa-ternal chromosomes change around the transi-tion point from maternal to embryonic control.We are addressing these issues by several ap-proaches including time-lapse imaging of chro-mosomes and some nuclear proteins in earlymouse embryos.

Publications

Taniguchi S, Nakazawa T, Tanimura A, KiyamaY, Tezuka T, Watabe AM, Katayama N, Yok-oyama K, Inoue T, Izumi-Nakaseko H, KakutaS, Sudo K, Iwakura Y, Umemori H, Inoue T,Murphy PN, Hashimoto K, Kano M, ManabeT, Yamamoto T. Involvement of NMDAR2Atyrosine phosphorylation in depression-relatedbehavior. EMBO J . 28: 3817-3829, 2009

Futatsumori-Sugai M, Abe R, Watanabe M, Ku-dou M, Yamamoto T, Ejima D, Arakawa T,Tsumoto K. Utilization of Arg-elution methodfor FLAG-tag based chromatography. ProteinExpr Purif . 67: 148-155, 2009

Matsumura T, Kawamura-Tsuzuku J, YamamotoT, Semba K and Inoue J. TRAF-interactingprotein with a forkhead-associated domain B(TIFAB) is a negative regulator of the TRAF6-induced cellular functions. J Biochem 146: 375-381, 2009

Oshimori N, Li X, Ohsugi M, Yamamoto T.Cep72 regulates localization of key centroso-mal proteins and proper bipolar spindle for-mation. EMBO J 28: 2066-2076

Kawa S, Matsushita H, Ohbayashi H, Semba K,and Yamamoto T. A novel mouse monoclonalantibody targeting ErbB2 suppresses breastcancer growth. Biochem Biophys Res Commun384: 329-333, 2009

Miyai K, Yoneda M, Hasegawa U, Toida S, IzuY, Hiroaki H, Hayata T, Ezura Y, Mizutani S,Miyazono K, Akiyoshi K, Yamamoto T, NodaM, ANA deficiency enhances BMP-induced

ectopic bone formation via transcriptionalevents. J Biol Chem 284: 10593-10600, 2009

Yoneda M, Suzuki T, Nakamura T, Ajima R,Yoshida Y, Kakuta S, Sudo K, Iwakura Y,Shibutani M, Mitsumori K, Yokota J andYamamoto T. Deficiency of antiproliferativefamily protein Ana correlates with develop-ment of lung adenocarcinoma. Cancer Sci 100:225-232, 2009

Horiuchi M, Takeuchi K, Noda N, Muroya N,Suzuki T, Nakamura T, Kawamura-Tsuzuku J,Takahasi T, Yamamoto T and Inagaki F,Structural basis for the antiproliferative activ-ity of the Tob-hCaf1 complex. J Biol Chem 284:13244-13255, 2009

Kakugawa S, Shimojima M, Goto H, HorimotoT, Oshimori N, Neumann G, Yamamoto T,and Kawaoka Y, Mitogen-Activated ProteinKinase-Activated Kinase RSK2 Plays a Role inInnate Immune Responses to Influenza VirusInfection. J Virol 83: 2510-2517, 2009

Suzuki T, Tsuzuku J, Kawakami K, Miyasaka Tand Yamamoto T. Proteasome-mediated deg-radation of Tob is pivotal for triggering UV-induced apoptosis. Oncogene 28: 401-411. 2009

Oyama M, Kozuka-Hata H, Tasaki S, Semba K,Hattori S, Sugano S, Inoue J and Yamamoto T.Temporal perturbation of tyrosine-phosphoproteome dynamics reveals thesystem-wide regulatory networks. Mol CellProt 8: 226-231, 2009

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Membrane proteins and their ligands including signaling molecules and extracellu-lar matrix proteins mediate complex arrays of cell signaling. Fate and activities ofthese proteins are often regulated by proteases in the extracellular space. We arecurrently focused on studying biological roles of membrane-anchored type of ma-trix metalloproteinases (MT-MMPs) which are potent modulators of pericellular mi-croenvironment and eventually regulate varieties of cellular functions such as pro-liferation, migration, apoptosis, and differentiation. Since uncontrolled expression ofMT-MMPs in tumors contributes to their malignant characteristics, they are poten-tial targets for cancer therapy.

1. High throughput analysis of proteins asso-ciating with a proinvasive MT1-MMP in hu-man malignant melanoma A375 cells

Taizo Tomari, Naohiko Koshikawa, TakayukiUematsu1, Takashi Shinkawa, Daisuke Ho-shino, Nagayasu Egawa1, Toshiaki Isobe andMotoharu Seiki: 1Department of Chemistry,Graduate School of Science, Tokyo Metropoli-tan University, Hachioji, Tokyo 192-0397, Ja-pan

Membrane-type 1 matrix metalloproteinase(MT1-MMP), a powerful modulator of thepericellular environment, promotes migration,invasion, and proliferation of cells. To performits potent proteolytic activity in a controlledmanner, MT1-MMP has to be regulated pre-cisely. However, our knowledge about sub-strates and regulatory proteins is still very lim-ited. In this study we identify a catalog of pro-teins that directly or indirectly interact withMT1-MMP. We expressed a FLAG-tagged MT1-MMP stably in human malignant melanoma

A375 cells. We prepared cell lysate using Brij98and MT1-MMP was affinity purified togetherwith associating proteins using an anti-FLAGantibody. A distinct set of membrane proteinswas found to copurify with MT1-MMP whenbiotin-labeled proteins were monitored. The pro-teins were analyzed with an integrated systemcomposed of nano-flow liquid chromatographyand tandem mass spectrometry. We identified158 proteins including several previously re-ported to bind MT1-MMP, although most hadnot previously been identified. Six of thesemembrane proteins, including one previouslyshown to interact with MT1-MMP, were co-expressed with MT1-MMP in HT1080 cells. Fiveof the latter were found to associate with MT1-MMP in an immunoprecipitation assay. Im-munostaining of cells expressing each of thesetest proteins revealed that one colocalized withMT1-MMP at the ruffling membrane and theother at the perinuclear vesicles. In contrast, an-other protein which did not coprecipitate withMT1-MMP showed no colocalization. Recombi-nant MT1-MMP cleaved two of the tested pro-

Department of Cancer Biology

Division of Cancer Cell Research腫瘍細胞社会学分野

Professor Motoharu Seiki, D.M.ScLecturer Naohiko Koshikawa, Ph.D.Assistant Professor Takeharu Sakamoto, Ph.D.Assistant Professor Daisuke Hoshinao, Ph.D.

教 授 医学博士 清 木 元 治講 師 理学博士 越 川 直 彦助 教 医学博士 坂 本 毅 治助 教 医学博士 星 野 大 輔

39

teins at least in vitro. Thus, we provide a valu-able resource to identify substrates and regula-tors of MT1-MMP in tumor cells.

2. Identification and characterization of Lu-theran blood group glycoprotein as a newsubstrate of membrane-type 1 matrix met-alloproteinase 1 (MT1-MMP): a systemicwhole cell analysis of MT 1-MMP-associating proteins in A431 cells

Daigo Niiya, Nagayasu Egawa, TakeharuSakamoto1, Yamato Kikkawa2, Takashi Shink-awa2, Toshiaki Isobe, Naohiko Koshikawa, andMotoharu Seiki: 1Laboratory of Clinical Bio-chemistry, Tokyo University of Pharmacy andLife Sciences, Hachioji 192-0392, Japan, 2De-partment of Chemistry, Graduate School ofScience , Tokyo Metropolitan University ,Hachioji, Tokyo 192-0397, Japan

Membrane-type 1 matrix metalloproteinase 1(MT1-MMP) is a potent modulator of thepericellular microenvironment and regulates cel-lular functions in physiological and pathologicalsettings in mammals. MT1-MMP mediates itsbiological effects through cleavage of specificsubstrate proteins. However, our knowledge ofMT1-MMP substrates remains limited. To iden-tify new substrates of MT1-MMP, we purifiedproteins associating with MT1-MMP in humanepidermoid carcinoma A431 cells and analyzedthem by mass spectrometry. We identified 163proteins including membrane proteins, cytoplas-mic proteins, and functionally unknown pro-teins. Sixty-four membrane proteins were identi-fied and they included known MT1-MMP sub-strates. Of these, eighteen membrane proteinswere selected and we confirmed their associa-tion with MT1-MMP using an immunoprecipita-tion assay. Co-expression of each protein to-gether with MT1-MMP revealed that nine pro-teins were cleaved by MT1-MMP. Lutheranblood group glycoprotein (Lu) is one of the pro-teins cleaved by MT1-MMP and we confirmedthe cleavage of the endogenous Lu protein byendogenous MT1-MMP in A431 cells. Mutationof the cleavage site of Lu abrogated processingby MT1-MMP. Lu protein expressed in A431cells bound to laminin-511 and knockdown ofMT1-MMP in these cells increased both theirbinding to Laminin-511 and the amount of Luprotein on the cell surface. Thus, we have iden-tified new physiological substrates of MT1-MMPby analysis of the associating proteins withMT1-MMP and demonstrated that the list ofmembrane proteins is an enriched source ofMT1-MMP substrates.

3. The cytoplasmic tail of MT1-MMP regulatesmacrophage motility independently fromits protease activity

Takeharu Sakamoto and Motoharu Seiki

MT1-MMP is a proinvasive protease thatregulates various cellular functions as evidencedby myriad defects in different types of cells andtissues in MT1-MMP-deficient (MT1－/－) mice.Here we demonstrate that MT1－/－ mice exhibitfewer infiltrating macrophages into sites of in-flammation. MT1－/－ macrophages exhibited areduced ability to invade reconstituted basementmembrane (Matrigel) and invasion by wild type(WT) macrophages was inhibited by a syntheticMMP inhibitor (BB94) to a level similar to thatof MT1－/－ cells. The rate of migration of MT1－/－

macrophages was also low compared to that ofthe WT cells and re-expression of MT1-MMP inMT1－/－ macrophages reconstituted their migra-tory activity. Unexpectedly, however, BB94 didnot inhibit the migration of WT macrophages.The migration-boosting activity of MT1-MMP isretained in a mutant that lacks most of the ex-tracellular portion including the catalytic andhemopexin-like domains. In contrast, deletion ofthe cytoplasmic tail (CP) abolished the activitycompletely. Thus, we have demonstrated thatMT1-MMP regulates macrophages via itsinvasion-promoting protease activity as well asits CP-dependent non-proteolytic activity toboost cell migration.

4. Mint3 enhances the activity of hypoxia-inducible factor-1 (HIF-1) in macrophagesby suppressing the activity of factor inhib-iting HIF-1

Takeharu Sakamoto and Motoharu Seiki

Hypoxia Inducible Factor-1 (HIF-1) is a keytranscription factor regulating cellular responsesto hypoxia and is composed of α and βsubunits. During normoxia, Factor InhibitingHIF-1 (FIH-1) inhibits the activity of HIF-1 bypreventing HIF-1α binding to p300/CBP viamodification of the Asn803 residue. However, it isnot known whether FIH-1 activity can be regu-lated in an oxygen-independent manner. In thisstudy, we survey possible binding proteins toFIH-1 and identify Mint3/APBA3, which hasbeen reported to bind Alzheimer’s beta-amyloidprecursor protein (APP). Purified Mint3 bindsFIH-1 and inhibits the ability of FIH-1 to modifyHIF-1α in vitro. In a reporter assay, the activityof HIF-1α is suppressed due to endogenousFIH-1 in HEK293 cells and expression of Mint3antagonizes this suppression. Macrophages are

40

known to depend on glycolysis for ATP produc-tion due to elevated HIF-1 activity. FIH-1 activ-ity is suppressed in macrophages by Mint3 so asto maintain HIF-1 activity. FIH-1 forms a com-plex with Mint3 and these two factors co-localize within the perinuclear region. Knock-down of Mint3 expression in macrophages leadsto redistribution of FIH-1 to the cytoplasm anddecreases glycolysis and ATP production. Thus,Mint3 regulates the FIH-1-HIF-1 pathway,which controls ATP production in macrophages,and therefore represents a potential new thera-peutic target to regulate macrophage-mediatedinflammation.

5. A novel protein associated with MT1-MMPbinds p27kip1, and regulates RhoA activa-tion, actin remodelling and matrigel inva-sion

Daisuke Hoshino, Taizo Tomari, Makoto Na-gano, Naohiko Koshikawa and Motoharu Seiki

Pericelllular proteolysis by membrane-type 1matrix metalloproteinase (MT1-MMP) plays apivotal role in tumour cell invasion. Localizationof MT1-MMP at the invasion front of cells, e.g.on lamellipodia and invadopodia, has to beregulated in co-ordination with reorganizationof the actin cytoskeleton. However, little isknown about how such invasion-related actin

structures are regulated at the sites where MT1-MMP localizes. During analysis of MT1-MMPassociated proteins, we identified a heretoforeuncharacterized protein. This protein, which wecall p27RF-Rho, enhances activation of RhoA byreleasing it from inhibition by p27kip1 andthereby regulates actin structures, though MT1-MMP does not play a role in this process. p27kip1

is a well-known cell cycle regulator in the nu-cleus. In contrast, cytoplasmic p27kip1 has beendemonstrated to bind GDP-RhoA and inhibitGDP-GTP exchange mediated by guanine nu-cleotide exchange factors (GEFs). p27RF-Rhobinds p27kip1 and prevents p27kip1 from binding toRhoA, thereby freeing the latter for activation.Knockdown of p27RF-Rho expression renderscells resistant to RhoA activation stimuli,whereas overexpression of p27RF-Rho sensitizescells to such stimulation. p27RF-Rho exhibits apunctate distribution in invasive human tumourcell lines. Stimulation of the cells with LPA in-duces activation of RhoA and induces the for-mation of punctate actin structures within fociof p27RF-Rho localization. Some of the punctateactin structures co-localize with MT1-MMP andcortactin. Down-regulation of p27RF-Rho pre-vents both redistribution of actin into the punc-tate structures and tumour cell invasion. Thus,p27RF-Rho is a new potential target for cancertherapy development.

Publications

Hoshino, D., T. Tomari, M. Nagano, N. Ko-shikawa, and M. Seiki. A novel protein associ-ated with membrane-type 1 matrix metallo-proteinase binds p27 (kip1) and regulatesRhoA activation, actin remodeling, and ma-trigel invasion. J Biol Chem . 284: 27315-26.2009

Miller, M.C., H.B. Manning, A. Jain, L. Troeberg,J. Dudhia, D. Essex, A. Sandison, M. Seiki, J.Nanchahal, H. Nagase, and Y. Itoh. Mem-brane type 1 matrix metalloproteinase is acrucial promoter of synovial invasion in hu-man rheumatoid arthritis. Arthritis Rheum . 60:686-97. 2009

Niiya, D., N. Egawa, T. Sakamoto, Y. Kikkawa,T. Shinkawa, T. Isobe, N. Koshikawa, and M.Seiki. Identification and characterization ofLutheran blood group glycoprotein as a newsubstrate of membrane-type 1 matrix metallo-

proteinase 1 (MT1-MMP): a systemic wholecell analysis of MT1-MMP-associating proteinsin A431 cells. J Biol Chem . 284: 27360-9. 2009

Sakamoto, T., and M. Seiki. Cytoplasmic tail ofMT1-MMP regulates macrophage motility in-dependently from its protease activity. GenesCells . 14: 617-26. 2009

Sakamoto, T., and M. Seiki. Mint3 enhances theactivity of hypoxia-inducible factor-1 (HIF-1)in macrophages by suppressing the activity offactor inhibiting HIF-1. J Biol Chem . 284:30350-9. 2009

Tomari, T., N. Koshikawa, T. Uematsu, T. Shink-awa, D. Hoshino, N. Egawa, T. Isobe, and M.Seiki. High throughput analysis of proteinsassociating with a proinvasive MT1-MMP inhuman malignant melanoma A375 cells. Can-cer Sci . 100: 1284-90. 2009

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Human cancers develop and progress toward malignancy through accumulation ofmultiple genetic alterations. Elucidation of these alterations is essential to providemolecular targets for prevention, diagnosis, and treatment of cancer. Our currentinterest is to understand the role of cell adhesion in cancer invasion and metasta-sis, as well as in mast cell function. To this end, an immunoglobulin superfamilycell adhesion molecule, CADM1/TSLC1, and its cascade were identified and arebeing characterized. Genetic and epigenetic abnormalities in human tumors arealso being investigated.

1. The biological and pathological functionsof CADM1/TSLC1 protein in epithelialstructure

Mika Sakurai-Yageta, Yumi Tsuboi, MiwakoIwai, Shigefumi Murakami, Takeshi Ito, Aki-hiko Ito, and Yoshinori Murakami

CADM1/TSLC1 is an immunoglobulin super-family cell adhesion molecule and primarily in-volved in epithelial cell adhesion. We have pre-viously shown that CADM1 associates with anactin-binding protein, 4.1B/DAL-1, and a mem-ber of scaffold proteins, membrane proteinpalmitoylated 3 (MPP3). We have newly demon-strated that MPP1/p55 and MPP2/DLG2, aswell as MPP3, interact with both CADM1 and4.1B, forming a tripartite-complex. Among MPP1-3, MPP2 is recruited to the CADM1-4.1B com-plex in the early process of adhesion in HEK293cells. By suppressing CADM1 expression usingsiRNA, HEK293 cells lose epithelia-like structureand show flat morphology with immature celladhesion. 4.1B and MPP2, as well as E-cadherinand ZO-1, are mislocalized from the membraneby depletion of CADM1 in HEK293. These find-

ings suggest that CADM1 is involved in the for-mation of epithelia-like cell structure with 4.1Band MPP2, while loss of its function could causemorphological transformation of cancer cells. Tofurther investigate the function of this cascade,additional binding proteins to CADM1 are beingidentified by proteomics approaches. Dynamicregulation of CADM1 protein on the cell mem-brane is also being analyzed using photo-bleaching assay. Furthermore, molecular mecha-nism of transcriptional regulation of the CADM1gene is investigated.

2. Patho-physiological function of theCADM1 protein in animal models

Masayoshi Nagata, Taketo Kawai, Hiromi Ichi-hara, Tomoko Masuda, Takeshi Ito and Yoshi-nori Murakami

To understand the physiological role ofCADM1 protein in animal models, Cadm1-deficient mice were generated. We have previ-ously reported that Cadm1－/－ mice showed maleinfertility due to disruption of adhesion of sper-matocytes to Sertoli cells in the seminiferous tu-

Department of Cancer Biology

Division of Molecular Pathology人癌病因遺伝子分野

Professor Yoshinori Murakami, M.D., Ph.D.Associate Professor Akihiko Ito, M.D., Ph.D.Assistant Professor Mika Sakurai-Yageta, Ph.D.

教 授 医学博士 村 上 善 則准教授 医学博士 伊 藤 彰 彦助 教 医学博士 櫻 井 美 佳

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bules. Genetic complementation study of theCadm1－/－ mice by crossing the transgenic miceexpressing human CADM1 is being investi-gated. We have also found that both Cadm1－/－

and Cadm1＋/－ mice show significant increase inthe incidence of spontaneous development oflung adenomas and adenocarcinomas, indicatingthat the CADM1 is a potent suppressor gene inlung tumor. Additional molecular alterations inlung tumors are being investigated to under-stand the molecular mechanisms of lung tumori-genesis.

3. Functional analyses of CADM1 overex-pression in adult T-cell leukemia -lym-phoma (ATL) and a subset of other humantumors.

Mari Masuda1, Miwako Iwai, Mika Sakurai-Yageta , Shigefumi Murakami , TomokoMasuda, Akihiko Ito, and Yoshinori Mu-rakami: 1Chemotherapy Division , NationalCancer Center Research Institute

We have previously reported in collaborationwith others that CADM1 is overexpressed inadult T-cell leukemia-lymphoma (ATL) cells. Ithas been suggested that the expression ofCADM1 protein promotes infiltration of leuke-mic cells into various organs which is one of thefrequent clinical manifestations of ATL. Wehave newly demonstrated that the cytoplasmicdomain of CADM1 directly interacts with T-lymphoma invasion and metastasis 1 (Tiam1), aRac-specific guanine nucleotide exchange factor(RacGEF), through its PDZ domain. This interac-tion induces lamellipodia formation through Racactivation in HTLV-I transformed cell lines aswell as ATL cell lines. These results indicate thatTiam1 integrates signals from CADM1 to regu-late the actin cytoskeleton through Rac activa-tion. It is probable, therefore, that the CADM1-Tiam1 interaction promotes cell motility leadingto tissue infiltration of leukemic cells in ATL pa-tients. Since overexpression of CADM1 is alsoobserved in a subset of human tumors withhigh metastatic activity, its pathological signifi-cance is also being analyzed. Furthermore, pos-sible cross-talk of CADM1 cascade with otherknown signal transduction pathways is exam-ined by screening the inhibitors of CADM1-mediated cell adhesion and motility.

4. Analyses of genetic and epigenetic abnor-malities in human tumors

Yasuhiro Ebihara, Mika Takahashi, MasayoshiNagata, Taketo Kawai, Miwako Iwai, andYoshinori Murakami

To understand the molecular features of mul-tistage carcinogenesis in human, aberrations ofCADM1 and the molecules in its cascade, aswell as other key molecules in human tumori-genesis, were examined in various cancers. In 40renal clear cell carcinoma (RCCC), loss ofCADM4 expression was observed in 28 (70％)tumors and associated with vascular infiltration.Moreover, introduction of CADM4 into RCCCcells, 786-O, suppressed tumor formation innude mice. These results suggest that CADM4functions as a tumor suppressor in RCCC. Aber-rations of the CADM1 cascade in other humantumors, including those from the lung, head andneck, breast, and bladder are being investigated.

5. CADM1 is a key regulator of nerve-mastcell interaction: possible mechanism in-volving CADM1 mRNA splicing

Akihiko Ito, Mitsuru Hagiyama, Naoki Ichiy-anagi, Yusuke Nagara, Yuki Ikeda, KeikoKimura

CADM1 plays an important role not only intumor suppression but also in cellular adhesionthat mediates cell and tissue type-specific func-tions. Among a variety of adhesions mediatedby CADM1, we have mainly investigated physi-ological and pathological aspects of cellular ad-hesion between nerve and mast cells. Close ap-position of nerve and mast cells in vivo isviewed as a functional unit of neuro-immunemechanisms, and is sustained mainly by trans-homophilic binding of CADM1. Cerebral nerve-mast cell interaction may be developmentallymodulated, because neonatal mouse cerebrumcontained abundant transcripts for two splicingisoforms, CADM1b and CADM1c, while adultcerebrum contained mainly CADM1d tran-scripts. To probe how individual isoforms areinvolved in nerve-mast cell interaction, Neuro2a,a neuroblastoma cell line expressing CADM1cendogenously, was modified to express addi-tionally either GFP-tagged CADM1b or CADM1d, and was cocultured on their neurite networkswith wild-type mouse bone marrow-derivedmast cells (BMMCs), which expressed CADM1cexclusively. Crosslinking experiments revealedthat CADM1d preferred to cluster as a homodi-mer along Neuro2a neurites. Interestingly, whenintracellular Ca2＋ concentrations were monitoredin neurite-attendant BMMCs after Neuro2a cellswere specifically activated by histamine, theCADM1d assembly site was found to be moreeffective in evoking mast cell response againstneurite activation. These results indicated thatCADM1d is a specific neuronal isoform that en-hances nerve-mast cell interaction, and sug-

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gested that nerve-mast cell interaction might bereinforced in adult brains due to greater CADM1d expression.

6. Physical aspects of cellular adhesion: es-tablishment of a non-contact measurementsystem assisted by femtosecond laser

Akihiko Ito, Mitsuru Hagiyama, Naoki Ichiy-anagi, Takahiro Mimae, Keiko Kimura

Although our understandings on adhesionmolecules have lately made remarkable ad-vances at genetic and molecular levels, physicalaspects of cellular adhesion have not been fullyexamined yet. Currently, we developed a newmethod to estimate intercellular adhesivestrength by focusing an intense femtosecond la-ser pulse in cell culture medium through an ob-

jective lens. Since this laser shot generates atransient stress, which propagates like “Tsu-nami” from the laser focal point with a few tensµm diameter, cell-cell detachment is induced asa function of the local magnitude of the “Tsu-nami”, which depends on the laser intensity andthe distance between the laser focal point andthe cell-cell contact. For example, in monolayerculture of polarized epithelial MDCK cells, lat-eral membrane-lateral membrane dissociationbetween neighboring cells progressed graduallyas intense laser shots were repeated. We esti-mated the force of the “Tsunami” by measuringthe bending movement of an atomic force mi-croscope cantilever placed instead of target cellsin culture medium, and found that the forceloaded on the detached cells was in a µN order,indicating that MDCK cells adhered to eachother in a µN order of force.

Publications

Masuda M, Maruyama T, Ohta T, Ito A, Haya-shi T, Tsukasaki K, Kamihira S, Yamaoka S,Hoshino H, Yoshida T, Watanabe T, Stan-bridge EJ and Murakami Y. CADM1 interactswith Tiam1 and promotes invasive phenotypeof human T-cell leukemia virus type I (HTLV-I) transformed cells and adult T-cell leukemia(ATL) cells. Journal of Biological Chemistry , inpress.

Sakurai-Yageta M, Masuda M, Tsuboi Y, Ito A

and Murakami Y. Tumor suppressor CADM1is involved in epithelial cell structure. BiochemBiophys Res Commun , 390: 977-982, 2009.

Hagiyama M, Ichiyanagi N, Kimura BK, Mu-rakami Y and Ito A. Expression of a solubleisoform of cell adhesion molecule 1 in thebrain and its involvement in directional neu-rite outgrowth. Am J Pathol , 174: 2278-89,2009.

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Gene expression is largely regulated by signal transduction triggered by variousstimulations. Several lines of evidence indicate that genetic defects of moleculesinvolved in the signal transduction or the gene expression lead to abnormal celldifferentiation or tumor formation. Our goal is to understand the molecular mecha-nisms of disease pathogenesis and oncogenesis by elucidating normal regulationof intracellular signal transduction and gene expression involved in cell prolifera-tion and differentiation. We have identified and been interested in Tumor necrosisfactor receptor-associated factor 6 (TRAF6), which transduces signal emanatingfrom the TNFR superfamily and the TLR/IL-1R family leading to activation of tran-scription factor NF-κB and AP-1. By generating TRAF6-deficient mice, we foundthat TRAF6 is essential for osteoclastogenesis, self-tolerance, lymph node organo-genesis and formation of skin appendices. We are currently focusing on molecularmechanisms underlying TRAF6-mediated activation of signal transduction path-ways and how TRAF6 is involved in osteoclastogenesis and self-tolerance. In ad-dition, NF-κB is thought to be constitutively activated in cancer cells and this acti-vation could be involved in the malignancy of tumors. Thus, we are also investi-gating the molecular mechanisms and target genes of the constitutive activation ofNF-κB.

1. Molecular mechanism of the regulation ofRel/NF-κB transcription factor

Jin Gohda, Takayuki Matsumura1, KohsukeYamazaki, Yuko Hata2, Masaaki Oyama2, Ken-taro Semba1 and Jun-ichiro Inoue: 1Institutefor Biomedical Engineering, Consolidated Re-search Institute for Advanced Science andMedical Care, Waseda University, 2MedicalProteomics Laboratory, IMSUT

Transcription factor Rel/NF-κB binds specifi-cally to a decameric motif of nucleotide, κB site,and activates transcription. The activation ofRel/NF-κB has been demonstrated to be carried

out post-translationally upon extracellular stim-uli through membrane receptors such as mem-bers of the TLR/IL-1R family and of TNFR su-perfamily. Rel/NF-κB forms a complex withregulatory protein, IκB, and is sequestered inthe cytoplasm prior to stimulation. Upon stimu-lation, IκB is rapidly phosphorylated on twospecific serine residues by IκB kinase (IKK) com-plex followed by Lys48-linked ubiquitinationand proteasome-dependent degradation of IκB.Rel/NF-κB subsequently translocates to the nu-cleus to activate transcription of target genes.This project is to identify a molecule that trans-duces signal from membrane receptors to Rel/NF-κB/IκB complex. We have previously identi-

Department of Cancer Biology

Division of Cellular and Molecular Biology分子発癌分野

Professor Jun-ichiro Inoue, Ph.D.Associate Professor Taishin Akiyama, Ph.D.Research Associate Jin Gohda, Ph.D.Research Associate Noritaka Yamaguchi, Ph.D.

教 授 薬学博士 井 上 純一郎准教授 薬学博士 秋 山 泰 身助 教 薬学博士 合 田 仁助 教 薬学博士 山 口 憲 孝

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fied upstream activators of Rel/NF-κB, tumornecrosis factor receptor-associated factor (TRAF)5 and TRAF6. We have also found thatMAPKKK, TAK1 is downstream of TRAF6 andbecomes activated upon dimerization of TRAF6and that TRAF6, together with Ubc13/Uev1Acomplex, catalyses the formation of Lys63 (K63)-linked polyubiquitin chains that conjugateTRAF6 and NEMO, and mediates IKK activa-tion through unique proteasome-independentmechanism. Several lines of evidence suggestthat TRAF6 is able to activate some kinase otherthan TAK1. Although TAK1 and mitogen-activated protein kinase kinase kinase 3(MEKK3) are both crucial for IL-1-dependent ac-tivation of NF-κB, their potential functional andphysical interactions remain unclear. This year,we demonstrated that TAK1-mediated activationof NF-κB required the transient formation of asignaling complex that included TRAF 6,MEKK3, and TAK1. Site-specific, K63-linkedpolyubiquitination of TAK1 at Lys-209, likelycatalyzed by TRAF6 and Ubc13, was requiredfor the formation of this complex. After TAK1-mediated activation of NF-κB, TRAF6 subse-quently activated NF-κB through MEKK3 inde-pendently of TAK1, thereby establishing con-tinuous activation of NF-κB, which was requiredfor the production of sufficient cytokines. There-fore, we propose that the cooperative activationof NF-κB by two mechanistically and temporallydistinct MEKK3-dependent pathways that di-verge at TRAF6 critically contributes to immuneand inflammatory systems.

2. Molecular mechanism regulating thymicmicroenvironment to establish self-tolerance

Taishin Akiyama, Yusuke Shimo, Daisuke Oh-shima, Hiromi Yanai, and Jun-ichiro Inoue

Clonal deletion is one of the main mecha-nisms to maintain T-cell tolerance. T-cell clonesthat have high avidity for self-antigens areeliminated during their development in the thy-mus. Self-antigens are predominantly expressedand presented by epithelial cells (TECs) anddendritic cells. Medullary thymic epithelial cells(mTECs) have a unique property to promiscu-ously express peripheral tissue specific self-antigens (TSAs). Therefore, it has been proposedthat developing T-cells encounter TSAs in thethymic medullar for clonal deletion. This hy-pothesis is supported by the study of autoim-mune regulator (Aire), inactivation or deficiencyof which results in autoimmune polyendocrino-pathy-candidiasis ectodermal dystrophy. Aire ispreferentially expressed in mTECs and lack of

Aire in mice results in the defective expressionof some TSAs.

Even though an increasing body of evidenceindicates crucial roles of mTECs on establishingself-tolerance in thymus, signaling pathways un-derlying the differentiation and proliferation ofmTECs expressing Aire and TSAs are poorlyunderstood. We previously found that TNF re-ceptor family members RANK and CD40 coop-eratively regulate the development of mTECsexpressing Aire and TSAs. Ligation of RANK orCD40 is sufficient for inducing mature mTECsexpressing Aire and TSAs in fetal thymic stromain TRAF6 and NF-κB inducing kinase (NIK) de-pendent manner.

Another TNF receptor family member OPG,which is a decoy receptor of RANKL, is in-volved in controlling the mTEC development.OPG-deficient mice showed enhanced develop-ment of mTECs expressing Aire and severalTSAs. We also have found that ligation ofRANK but not CD40 induces OPG in fetal thy-mic stroma, suggesting that the expression ofOPG is not directly coupled with final matura-tion of mTECs. Consistently, the expression ofOPG is significantly reduced in RANKL-deficient mice. Deficiency of OPG resulted inslight but statistically significant increment inthe ratio of regulatory T cells in CD4 singlepositive T cells in thymus. Overall, these datasuggested that a negative feedback loop consisit-ing of RANKL-RANK-OPG in TECs control thenumber of mTECs and mTEC-dependent immu-nological tolerance induction.

3. Involvement of a novel TRAF6-interactingprotein in anti-viral signaling pathways

Hirohisa Shiraishi, Taishin Akiyama, and Jun-ichiro Inoue

TRAF6 regulates innate and acquired immuneresponses by activating NF-κB and MAPkinases. To elucidate molecular mechanisms un-derlying the activation of TRAF6-dependent sig-nal, we screened for proteins that bind TRAF6,by in vitro virus (IVV) method and subsequentco-immunoprecipitation assays. As a result, weidentified a novel TRAF6-binding protein. In or-der to address the role of this protein in theTRAF6-mediated activation of NF-κB, we per-formed a reporter analysis using NF-κB pro-moter conjugated with luciferase gene. Althoughenforced expression of this protein did not acti-vate NF-κB by itself, NF-κB activation inducedby TRAF6-overexpression was enhanced whenthis protein was co-expressed. We have recentlyreported that TRAF6 plays a critical role inRIG-I like helicase (RLH) signaling, which con-

46

trols the expression of type-I interferon and in-flammatory cytokines by responding againstRNA virus infection. Moreover, this protein alsobinds TRAF3, but not TRAF2 and TRAF5. Giventhat both TRAF3 and TRAF6 function down-stream of RIG-I, these results led us to checkwhether the identified protein is involved in theRIG-I pathway. The NF-κB reporter analysis re-vealed that RIG-I-induced NF-κB activation wasinhibited by co-expression of this protein, sug-gesting that this protein could be a negativeregulator for the RIG-I pathway. Taken together,our data leads to a hypothesis that the novelTRAF6-binding protein regulates the RLH path-way through modulating the function of TRAF6and TRAF3.

4. Hunting for NF-κB-inducing kinase (NIK)associated proteins regulating immune re-sponse

Miho Shinzawa, Hiromi Yanai, Taishin Aki-yama, and Jun-ichiro Inoue

Transcription factor NF-κB family plays animportant role in regulating immune develop-ments and immune responses. The activation ofNF-κB is mainly divided into classical and alter-native pathways. The alternative pathway re-quires NIK for the activation and regulates thedevelopment and organization of some lym-phoid organs. The alymphoplasia (aly) mice,which carry a dysfunctional mutation in NIKgene, and NIK-deficient mice completely lacklymph nodes and Payer’s patches, and exhibitdisturbed thymus architecture. In addition tolymphoid organogenesis, T cells from aly miceshow impaired proliferation and IL-2 produc-tion in response to anti-CD3 stimulation, indi-cating that a NIK-dependent signal regulates Tcell response. In order to clarify the molecularmechanism underlying the activation of theNIK-dependent signal in immune response, weexploit proteins interacting with NIK by “in vi-tro virus” screening and subsequent immuno-precipitation assays. As a result, we identified anovel NIK-associated protein, which is able tosuppress NF-κB activation induced by NIKoverexpression. We are currently investigatingthe physiological importance of the interactionbetween NIK and this identified NIK-associatedprotein in T cells or other immune competentcells.

5. Molecular mechanism of RANK signalingin osteoclastogenesis

Jin Gohda, Yuu Taguchi, Yoko Kiga, SayakaYamane, and Jun-ichiro Inoue

Osteoclasts are responsible for bone resorptionand play a crucial role in bone homeostasis inconcert with osteoblasts, which mediate boneformation. Excess formation or activity of osteo-clasts results in pathological bone resorption,such as postmenopausal osteoporosis and rheu-matoid arthritis. Therefore, precise elucidationof the regulatory mechanisms of osteoclasto-genesis is essential for understanding the onsetof skeletal diseases and for developing drugs totreat such diseases.

Osteoclastogenesis is tightly regulated by theRANK/RANKL-signaling in progenitor cells. In-tracellular signaling pathways of RANK are me-diated by an adaptor molecule, TRAF6. TheRANK/RANKL-signaling activates NF-κB andAP-1, and induces PLCγ2-mediated Ca2＋ oscilla-tion, which is required for induction of NFATc1,a master transcriptional factor in osteoclasto-genesis. However, the molecular mechanisms bywhich the RANK/RANKL-signaling mediatesosteoclastogenic signals remains to be eluci-dated. This year, we identified a novel domain,HCR (highly conserved domain in RANK).HCR-deficient RANK could not induce osteo-clast differentiation even in the presence ofTRAF6-binding site. This is because lack of HCRresulted in impaired activation of NF-κB andPLCγ2 at 24 h after stimulation although theiractivation was not affected during first 1 h afterstimulation. Addition of the HCR to the cyto-plasmic tail of CD40, which itself is unable toinduce osteoclastogenesis, resulted in long-termactivation of RANK-signaling and activation ofNFATc1 to induce osteoclast formation. Theseresults suggest that the HCR is capable of in-ducing osteoclastogenesis in concert with theTRAF6-binding site. Furthermore, we found thatthe HCR binds Gab2, which then associates withPLCγ2 in a stimulation dependent manner.Moreover, our experiment revealed that Gab2bound TRAF6. These results strongly suggestthat HCR provides a platform for forming anovel signal complex containing Gab2, PLCγ2,and TRAF6 upon RANKL stimulation, and con-tinuously activate RANK signaling. In additionwe showed that expression of HCR-peptides it-self severely inhibited formation of osteoclasts.This result indicates that HCR peptides itself isa potent inhibitor of osteoclastogenesis presum-ably by squelching signal complexes. These re-sults suggest that, HCR-signaling may be anovel therapeutic target for pathologic bone re-sorption.

6. Molecular mechanisms of NF-κB activationin Adult T-cell leukemia

Jin Gohda, Yuri Shibata, Akihisa Hirosawa,

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and Jun-ichiro Inoue

Human T-cell leukemia virus type 1 (HTLV-1)infects and immortalizes CD4-positive T cells,and it is etiologically associated with Adult T-cell leukemia (ATL). NF-κB is constitutively acti-vated during development of ATL, and its acti-vation plays a crucial role in T-cell transforma-tion and survival of leukemic cells. Therefore,clarification of the molecular mechanisms ofconstitutive NF-κB activation may lead to devel-opment of drugs to treat ATL. Constitutive NF-κB activation in HTLV-1-infected cells is causedby HTLV-1-derived oncoprotein, Tax. Tax di-rectly associates with NEMO, a noncatalyticsubunit of IκB kinase (IKK) complex, and in-duces activation of IKK leading to NF-κB activa-tion. However, little is known about how Taxinduces activation of the IKK complex after itbinds to NEMO.

We have previously established a cell-free sys-tem to analyze the Tax mediated activation ofthe IKK complex. Addition of recombinant Taxprotein, which was produced by a vaculovirusprotein expression system, to cell extracts re-sulted in the phosphorylation of IκBα, indicatingthat Tax protein can activate endogenous IKKcomplex in cell lysates. Using this cell-free sys-tem, We demonstrated that Tax-induced IKK ac-tivation requires unidentified factors on additionto the components of IKK complex. To identifythe factors involved in the Tax-induced IKK ac-tivation, we purified the Tax-IKK protein com-plex from cell extracts. Proteomics analysis ofthe purified Tax-IKK complex showed that thecomplex includes ten novel components of theIKK complex. We are currently clarifying the in-volvement and functions of these protein factorsin the Tax-induced IKK activation.

7. Role of TRAF6 in T cell proliferation

Hidehiko Motegi, Taishin Akiyama and Jun-ichiro Inoue

Recently, we found that CD4-positive T cellsfrom TRAF6-deficient mice proliferated morerapidly than WT CD4-positive T cells in re-sponse to T-cell antigen receptor (TCR) stimula-tion. Furthermore, expression of protein andmRNA of Interleukin-2 (IL-2), which is essentialfor T cell proliferation, are also enhanced inTRAF6-deficient T cells. We are currently focus-ing on molecular mechanisms of such an abnor-mality in the absence of TRAF6.

8. Analysis of the molecular mechanism andthe functional significance of constitutiveNF-κB activation in basal-like subtypebreast cancer cells

Noritaka Yamaguchi, Taku Ito, MizukiYamamoto , Hiroyuki Takayama , MamiYokota, Kentaro Semba1 and Jun-ichiro Inoue

Recent studies of gene expression profileshave revealed that breast cancer is categorizedinto four subtypes: luminal, basal-like, ERBB2-positive, and normal breast-like. Among thesesubtypes, the basal-like subtype is the most ma-lignant form of breast cancer and resistant tocurrently available targeted therapeutic strate-gies, such as hormone therapy and Herceptin.Our past study has shown that NF-κB is consti-tutively activated and crucial for proliferation ofbasal-like subtype breast cancer cells. The pro-tein levels of NF-κB-inducing kinase (NIK) wereup-regulated, and knockdown of NIK reducedconstitutive NF-κB activation in these cells.These results suggested that up-regulation ofNIK is one of the major causes of constitutiveNF-κB activation and that the NIK-NF-κB path-way may be an attractive therapeutic target forthe basal-like subtype breast cancer. This year,we have analyzed a molecular mechanism lead-ing to up-regulation of NIK in the basal-likesubtype breast cancer cells and found that NIKmRNA expression levels were elevated in them.We have also found that NIK expression may beepigenetically regulated in breast cancer cellsand are currently analyzing details of this epige-netic mechanism regulating NIK expression. Tounderstand the molecular function of NF-κB inthe basal-like subtype cells, we have analyzedNF-κB target genes in these cells by expressionof the non-degradable IκBα super-repressor,which can specifically inhibit NF-κB activation.Several promising candidates , which arethought to be involved in cancer stem cells,bone metastasis, and immunosuppression, havebeen identified, and their functional significanceis currently analyzed in breast cancer xenocraftmodels.

9. NIK is involved in constitutive activation ofthe alternative NF-κB pathway and prolif-eration of pancreatic cancer cells

Takashi Nishina, Noritaka Yamaguchi, Jin Go-hda, Kentaro Semba1 and Jun-ichiro Inoue:

Pancreatic cancer has one of the poorest prog-noses among human neoplasms. Constitutive ac-tivation of NF-κB is frequently observed in pan-creatic cancer cells and is involved in their ma-

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lignancy. However, little is known about themolecular mechanism of this constitutive NF-κBactivation. This year, we showed that the alter-native pathway is constitutively activated andNIK is significantly expressed in pancreatic can-cer cells. SiRNA-mediated silencing of NIK ex-pression followed by subcellular fractionationrevealed that NIK is constitutively involved inthe processing of p100 and nuclear transport of

p52 and RelB in pancreatic cancer cells. In addi-tion, NIK silencing significantly suppressed pro-liferation of pancreatic cancer cells. These resultsclearly indicate that NIK is involved in the con-stitutive activation of the alternative pathwayand controls cell proliferation in pancreatic can-cer cells. Therefore, NIK might be a novel targetfor the treatment of pancreatic cancer.

Publications

Yamamoto, T., Yokota, Y., Mitsuki, Y., Mizuko-shi, F., Tsuchiya, T., Terahara, K., Inagaki, Y.,Yamamoto, N., Kobayashi, K., and Inoue, J.Selective transmission of R5 HIV-1 over X4HIV-1 at the dendritic cell-T cell infectioussynapse is determined by the T cell activationstate. PLos Pathogens 5(1): e1000279 (2009).

Oyama, M., Kozuka-Hata, H, Tasaki, S., Semba,K., Hattori, S., Sugano, S., Inoue, J. andYamamoto, T. Temporal perturbation oftyrosine-phosphoproteome dynamics revealsthe system-wide regulatory networks. Mol.Cell. Proteomics 8, 226-231 (2009)

Helmy, M., Gohda, J., Inoue, J., Tomita, M.,Tsuchiya, M., and Selvarajoo K. PredictingNovel Features of Toll-Like Receptor 3 Signal-ing in Macrophages. PLoS ONE 4(3): e4661(2009).

Konno, H., Yamamoto, T., Yamazaki, K., Gohda,J., Akiyama, T., Semba, K., Goto, H., Kato, A,Yujiri, T, Imai, T., Kawaguchi, Y., Su, B.,Takeuchi, O., Akira, S., Tsunetsugu-Yokota,Y., & Inoue, J. TRAF6 Establishes Innate Im-mune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA andDNA. PLoS ONE , 4(5): e5674 (2009).

Yamaguchi, N., Ito, T., Azuma, S., Ito, E.,Honma, R., Yanagisawa, Y., Nishikawa, A.,Kawamura, M., Imai, J., Watanabe, S., Semba,K. and Inoue, J. Constitutive activation of NF-κB is preferentially involved in the prolifera-tion of basal-like subtype breast cancer celllines. Cancer Sci . 100, 1668-1674 (2009).

Matsumura, T., Kawamura-Tsuzuku, J., Yama-moto, T., Semba, K., and Inoue, J. TRAF-interacting protein with a forkhead-associateddomain B (TIFAB) is a negative regulator ofthe TRAF6-induced cellular functions. J. Bio-chem . 146, 375-381 (2009).

Nishina, T., Yamaguchi, N., Gohda, J., Semba, K.

and Inoue, J. NIK is involved in constitutiveactivation of the alternative NF-κB pathwayand proliferation of pancreatic cancer cells.Biochem. Biophys. Res. Commun . 388, 96-101(2009).

Taguchi,Y., Gohda, J., Koga,T., Takayanagi, H.,and Inoue, J. A unique domain in RANK isrequired for Gab2 and PLCγ2 binding to es-tablish osteoclastogenic signals. Genes Cells 14,1331-1345 (2009).

Yamazaki, K., Gohda, J., Kanayama, A., Mi-yamoto, Y., Sakurai, H., Yamamoto, M., Akira,S., Hayashi, H., Su, B., and Inoue, J. TwoMechanistically and Temporally Distinct NF-κB Activation Pathways in IL-1 Signaling. Sci.Signal . 2, ra66 (2009).

Ishikawa, K., Akiyama, T., Ito, K., Semba, K.,and Inoue, J. Independent stabilizations ofpolysomal Drg1/Dfrp1 complex and non-polysomal Drg2/Dfrp2 complex in mammal-ian cells. Biochem. Biophys. Res. Commun . 390,552-556 (2009).

Nagatake, T., Fukuyama, S., Kim, D.Y., Goda,K., Igarashi, O., Sato, S., Nochi, T., Sagara, H.,Yokota, Y., Jetten, A.M., Kaisho, T., Akira, S.,Mimuro, H., Sasakawa, C., Fukui, Y., Fuji-hashi, K., Akiyama, T., Inoue, J., Penninger, J.M., Kunisawa, J., Kiyono, H. Id2-, RORγt-, andLTβR-independent initiation of lymphoid or-ganogenesis in ocular immunity. J Exp Med .206, 2351-2364 (2009).

Ando, K., Hasegawa, K., Shindo, K., Furusawa,T., Fujino, T., Kikugawa, K., Nakano, H.,Takeuchi, O., Akira, S., Akiyama, T., Gohda,J., Inoue, J., and Hayakawa, M. Human lac-toferrin activates NF-κB through Toll-like re-ceptor 4 (TLR4) pathway while it interfereswith the lipopolysaccharide-stimulated TLR4signaling. FEBS Journal (2010) in press.

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The major interest of this division is in molecular signals that regulate a variety ofcellular activities. Our aim is to address how deregulated cellular signals give riseto neoplastic, immune, neural, metabolic, or developmental disorders. Our goal isto understand the molecular bases of tumorigenesis and the development of otherintractable diseases as a path toward uncovering therapeutic targets. Currently,we are investigating regulatory mechanisms in protein-tyrosine kinase (PTK)-mediated signaling pathways and their pathophysiological roles in hematopoietictumors and myasthenia.

1. Activation of the receptor tyrosine kinaseMuSK by the cytoplasmic adaptor-like pro-tein Dok-7.

Inoue, A., Tezuka, T., Hamuro, J., Ikegami, T.,Higuchi, O., and Yamanashi, Y.:

Protein-tyrosine kinases (PTKs) play crucialroles in a variety of signaling pathways thatregulate proliferation, differentiation, motility,and other activities of cells. Therefore, deregu-lated PTK signals give rise to a wide range ofdiseases such as neoplastic disorders. To under-stand molecular bases of PTK-mediated signal-ings, we identified a common substrate of manyPTKs as Dok-1 in 1997. Since then, the Dok-family has been expanded to seven members,Dok-1 to Dok-7, which share structural similari-ties characterized by the NH2-terminal pleckstrinhomology (PH) and phosphotyrosine binding(PTB) domains, followed by the Src homology 2(SH2) target motifs in the COOH-terminal moi-ety, suggesting an adaptor function. Indeed, asmentioned later, Dok-1 and Dok-2 recruit p120rasGAP upon tyrosine phosphorylation to sup-

press Ras-Erk signaling. Unlike the other mem-bers of the Dok-family proteins, Dok-7 is prefer-entially expressed in muscle tissues. Further-more, we found that Dok-7 is specifically local-ized to the postsynaptic area of neuromuscularjunction (NMJ), suggesting its role in NMJ for-mation and/or maintenance.

The NMJ is a synapse between a motor neu-ron and skeletal muscle, where the motor nerveterminal is apposed to the endplate. The con-traction of skeletal muscle is controlled by theneurotransmitter acetylcholine (ACh), which isreleased from the presynaptic motor nerve ter-minal. To achieve efficient neuromuscular trans-mission, acetylcholine receptors (AChRs) mustbe densely clustered on the postsynaptic musclemembrane of the NMJ. Failure of AChR cluster-ing is associated with disorders of neuromuscu-lar transmission such as congenital myasthenicsyndromes (CMS) and myasthenia gravis, whichare characterized by fatigable muscle weakness.The formation of NMJs is orchestrated by themuscle-specific receptor tyrosine kinase MuSKand by neural Agrin, an extracellular activatorof MuSK. However, experimentally when motor

Department of Cancer Biology

Division of Genetics腫瘍抑制分野

Professor Yuji Yamanashi, Ph.D.Associate Professor Osamu Higuchi, Ph.D.Assistant Professor Ryuichi Mashima, Ph.D.Assistant Professor Tohru Tezuka, Ph.D.

教 授 理学博士 山 梨 裕 司准教授 理学博士 樋 口 理助 教 工学博士 真 嶋 隆 一助 教 理学博士 手 塚 徹

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nerves are ablated, AChRs form clusters in thecorrect, central region of muscle upon embryo-genesis, which is known as prepatterning of thereceptors, in a manner dependent on MuSK. Inaddition, in vivo overexpression of MuSKcauses neuromuscular synapse formation in theabsence of Agrin, suggesting that muscle-intrinsic, cell-autonomous activation of MuSKmay be adequate to trigger presynaptic andpostsynaptic differentiation in vivo. However,the mechanisms by which MuSK is activated in-dependently of nerve and Agrin have long beenunclear.

Because both MuSK and the adaptor-like cyto-plasmic protein Dok-7 are localized to the post-synaptic region of NMJ, we had previously ex-amined their interaction and found, to our sur-prise, that Dok-7 can induce MuSK activation incultured myotubes and plays an essential role inneuromuscular synaptogenesis, including pre-patterning of AChRs, although the mechanismsby which Dok-7 regulates MuSK activity andpromotes synapse formation had been unclear.In the current study, we found that Dok-7 di-rectly interacts with the cytoplasmic portion ofMuSK and activates the receptor tyrosine kinase,and that neural Agrin requires Dok-7 in order toactivate MuSK. Indeed, in vivo overexpressionof Dok-7 increased MuSK activation and pro-moted NMJ formation. Furthermore, Dok-7 wasrequired for the localization of MuSK in the cen-tral region of muscle, which is essential for thecorrect formation of NMJs in this region. Theseobservations indicate that Dok-7 positively regu-lates neuromuscular synaptogenesis by control-ling MuSK activity, its distribution, and respon-siveness to neural Agrin. The exceptional as-pects of Dok-7-MuSK-Agrin signaling lie in theunusual role of adaptor-like protein in the acti-vation of receptor tyrosine kinase; the adaptor-like cytoplasmic protein (Dok-7) activates the re-ceptor tyrosine kinase (MuSK) in the absence ofthe extracellular activator (Agrin), but activationof MuSK by the extracellular activator (Agrin)requires the cytoplasmic protein (Dok-7). Fur-thermore, the indispensability of Agrin for NMJformation in vivo strongly suggests that bothDok-7 and Agrin are required for full activationof MuSK to a level sufficient for NMJ formation.We are investigating the signaling mechanismsinvolving Dok-7, Agrin, MuSK, and LDLreceptor-related protein 4 (LRP4), which forms acomplex with MuSK and acts as an Agrin-binding module of the complex. In particular,we are interested in why LRP4, a receptor ofAgrin, appears to be essential for Agrin-independent, but Dok-7- and MuSK-dependentprepatterning of AChRs during embryogenesis.

2. Pathophysiological mechanisms underly-ing DOK7 myasthenia.

Higuchi, O., Inoue, A., Hamuro, J., Spearman,H.1, Beeson, D.1, and Yamanashi, Y.: 1Weather-all Institute of Molecular Medicine, Universityof Oxford.

As mentioned above, impaired clustering ofAChRs could underlie NMJ disorders that areboth autoimmune (anti-MuSK antibody-positivemyasthenia gravis) and genetic (congenital my-asthenic syndromes (CMS)) in origin. Therefore,our findings that Dok-7 activates MuSK to clus-ter AChRs and to form NMJs had suggestedthat DOK7 is a candidate gene for mutations as-sociated with CMS. Indeed, we previously dem-onstrated that biallelic mutations in DOK7 un-derlie a major subgroup of CMS with predomi-nantly proximal muscle weakness that did notshow tubular aggregates on muscle biopsy butwere found to have normal AChR function butabnormally small and simplified NMJs. Thisnew disease entity is termed as DOK7 myasthe-nia.

The overwhelming majority of patients withDOK7 myasthenia have at least one allele with aframeshift mutation that causes a truncation inthe COOH-terminal region of Dok-7 and affectsMuSK activation. We previously identified achromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES) in theCOOH-terminal moiety and demonstrated thatthe NES-mediated cytoplasmic location of Dok-7is essential for regulating the interaction withMuSK in cultured myotubes. The NH2-terminalPH domain is responsible for the nuclear importof Dok-7. We also demonstrated that the SH2target motifs in the COOH-terminal moiety ofDok-7 are active, and crucial for MuSK activa-tion in cultured myotubes. To further investigatepathophysiological mechanisms underlyingDOK7 myasthenia, we have established mice ec-topically expressing Dok-7 proteins that havemutations in the COOH-terminal moiety. Sincesome of these mutants lack major phosphoryla-tion sites and are indeed hardly tyrosine phos-phorylated by MuSK, this study may also ad-dress if tyrosine phosphorylation, thereby anadaptor function, of Dok-7 is required for NMJformation in vivo. Effects of the mutations invivo are under investigation.

3. Preparation of an Adeno-associated virusvector for Dok-7 expression.

Tezuka, T., Arimura, S., Okada, T.1, Takeda,S.,1, and Yamanashi, Y.: 1Department of Mo-lecular Therapy, National Institute of Neurosci-

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ence.

As mentioned above, DOK7 myasthenia is as-sociated with impaired neuromuscular forma-tion due to decreased ability of Dok-7 to activateMuSK in myotubes. Interestingly, in vivo over-expression of Dok-7 increased MuSK activationand promoted NMJ formation in the correct,central region of the skeletal muscle. Becausethese genetically manipulated mice did notshow any sign of disease, overexpression ofDok-7 in the skeletal muscle of patients withDOK7 myasthenia might ameliorate NMJ forma-tion and muscle weakness. As an initial step to-ward a potential therapy, we are generating anAdeno-associated virus-based vector that couldstrongly express Dok-7 in the skeletal muscle fora long period.

4. Negative regulation of PTK-mediated sig-naling by the Dok-family proteins in hema-topoietic cells.

Mashima, R., Honda, K.1, Morita, Y.2, Ema,H.2, Hishida, Y., Seed, B.3, Nakauchi, H.2, Oda,H.1, and Yamanashi, Y.: 1Department of Pa-thology, Tokyo Women’s Medical University,2Laboratory of Stem Cell Therapy, IMSUT,3Department of Molecular Biology, HarvardMedical School.

The Dok-family proteins can be classified intothree subgroups based on their structural simi-larities and expression patterns; namely, 1)Dok-1, -2, and -3, which are preferentially ex-pressed in hematopoietic cells, 2) Dok-4, -5, and-6, which are preferentially expressed in non-hematopoietic cells, 3) Dok-7, which is preferen-tially expressed in muscle cells. As mentionedabove, Dok-1 and its closest family Dok-2 recruitp120 rasGAP upon tyrosine phosphorylation tosuppress Ras-Erk signaling. Indeed we haddemonstrated that Dok-1 and Dok-2 are keynegative regulators of the hematopoietic growthand survival signaling as well as the TLR4-mediated innate immune signaling. In the cur-rent study, we found that the Dok-family pro-teins that are preferentially expressed in hema-topoietic cells negatively regulate PTK-mediatedsignaling and inhibit neoplastic transformationinto aggressive tumor cells in vivo. We are in-vestigating roles of these Dok-family proteinsusing genetically manipulated animals.

5. Proteomic analyses.

Tezuka, T., Higuchi, O., Inoue, A., Iemura, S.1,Natsume, T.1, and Yamanashi, Y.: 1National In-stitute of Advanced Science and Technology,

Biological Information Research Center.

To gain insights into signaling mechanismsunderlying a variety of physiological and patho-physiological events, including NMJ formationand tumorigenesis, we have performed massspectrometry-based proteomic analyses. We areinvestigating roles of candidate proteins that ap-pear to be involved in each biological event.

6. Development of a method for evaluation ofserum autoantibodies in patients.

Higuchi, O., and Yamanashi, Y.

Autoimmune disorders are caused by anti-bodies generated in patients against self-antigens. However, pathogenic antibodies re-main to be identified in many cases, hamperingtheir diagnosis and clinical management. Toovercome this roadblock, we have developed aluminescence-based novel method to efficientlydetect serum autoantibodies in patients. We areinvestigating pathogenic involvement of patientautoantibodies that have been identified by thismethod.

7. Negative regulation of PTK-mediated sig-naling by the Cbl-family proteins.

Tezuka, T., Yamanashi, Y., and Yamamoto,T.1: 1Division of Oncology, Department of Can-cer Biology, IMSUT.

Since we have identified Cbl (Casitas B-lineage lymphoma) as a protein that interactswith Lyn, a member of the Src-family PTKs, wehave been investigating physiological roles ofthe Cbl-family proteins. In general, throughtheir RING-type ubiquitin ligase activity, theseproteins facilitate ubiquitination and degrada-tion of various tyrosine-phosphorylated proteinsto negatively regulate the signaling mediated byPTKs.

The mammalian Cbl-family is comprised ofCbl, Cbl-b, and Cbl-c/Cbl-3. Cbl-c was identi-fied by our and other groups as the latest mem-ber in humans and mice. Like other members,Cbl-c contains the conserved NH2-terminal tyro-sine kinase-binding (TKB) domain, linker, andRING finger motif. However, Cbl-c lacks an ex-tensive proline-rich sequence and an ubiquitin-associated leucine zipper motif that are presentin the COOH-terminal moieties of Cbl and Cbl-b. In addition, the conserved region (TKB-linker-RING finger) of Cbl-c has only 50％ identity tothose of Cbl and Cbl-b, but Cbl and Cbl-b have84％ identity to each other in this region. Thesecharacteristics as well as the phylogenetic stud-

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ies show that Cbl-c is a distinct member amongthe mammalian Cbl-family proteins. In the cur-rent study, we identified a single amino acidresidue that is essential for a characteristic func-

tion and structure of Cbl-c. The structure-function relationship of the other members ofthe Cbl-family is also under investigation.

Publications

Inoue, A., Setoguchi, K., Matsubara, Y., Okada,K., Sato, N., Iwakura, Y., Higuchi, O. andYamanashi, Y. Dok-7 activates the muscle re-ceptor kinase MuSK and shapes synapse for-mation. Science Signal., 2: ra7, 2009.

Mashima, R., Hishida, Y., Tezuka, T. andYamanashi, Y. The roles of Dok family adap-

tors in immunoreceptor signaling. Immunol.Rev., 232: 273-285, 2009.井上茜，瀬戸口喜代子，樋口理，山梨裕司．受容体型チロシンキナーゼの細胞内からの活性化：Dok-７による神経筋シナプス形成の分子機構．実験医学．２７�：１３８４―１３８７，２００９．

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Carbohydrates bound to the polypeptide chains widely occur in the body, and theirstructures change during development and differentiation of the cells and underpathological conditions. Our major research interest is to elucidate direct and indi-rect roles of the carbohydrates in cell-cell interaction. For this end, we are cur-rently focusing on structure and function of carbohydrate binding proteins and theirligands, regulation of protein structures and functions by glycosylation, and devel-opment of new methods and tools for protein-carbohydrate interaction.

1. Cell adhesion molecules involved in mam-malian fertilization

Etsuko Mori, Dong Cun Jin and Seiichi Taka-saki

Fertilization is the sum of the cellular mecha-nisms that pass the genome from one generationto the next and initiate development of a neworganism. An ovulated mammalian egg is en-closed by two layers: an outer layer of cumuluscells and an inner, thick extracellular matrix, thezona pellucida. Sperm penetrate both layers toreach the egg plasma membrane. Sperm alsobind transiently to the egg zona pellucida andthe egg plasma membrane and then fuse. Recog-nition of the zona pellucida by the sperm cellcan be subdivided in two phases: the primarybinding in which proteins that are situated onlyat the apical plasma membrane of the acrosome-intact sperm head interact with the zona pellu-cida and the secondary binding in which themolecules of acrosome-reacted sperm cell inter-act with the zona pellucida. Sperm-zona bindingresults almost instantaneously in the acrosomereaction. To solve the mechanism underlyingthese processes, identification of functionalmolecules on the surface of both gametes is es-sential. Its importance is also pointed out by

clinical studies of infertility. Male-factor infertil-ity is primarily diagnosed by abnormal semenvariables (concentration, motility, and morphol-ogy). However, it has been shown by clinical ex-amination that more than 50％ of couples withno fertilization during in vitro fertilization werenormal on semen analysis. Remarkably, 75％ ofthese men had sperm that poorly bound to thezona pellucida and 47％ had no sperm thatpenetrate the zona pellucida. In addition, 25％of infertile men with normal semen variablesand normal sperm binding to the zona pellucidahad a defective zona-induced acrosome reaction.Thus, defects in sperm binding and/or induc-tion of the acrosome reaction are importantcauses of sperm dysfunction in men with appar-ently normal semen. We are now focusing onthe adhesion mechanism working in fertiliza-tion, especially at the stage of sperm-zona pellu-cida binding. An effort has been made to definethe sperm surface protein(s) that binds to thezona. Many candidates have been proposed, butnone has found wide acceptance. For example,male mice obtained with gene knockout for thesperm surface proteins fertilin β, cyritestin orthe spermatogenesis-specific chaperone calmeginare infertile and produce sperm that cannot bindto the zona. pellucida. However, the calmegin-null sperm have been shown to lack fertilinβ.

Department of Cancer Biology

Division of Biochemistry腫瘍分子医学分野

Associate Professor Seiichi Takasaki, Ph.D. 准教授 医学博士 高 崎 誠 一

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Both fertilin β and cyritestin knockout sperm,through an unknown mechanism, lose not onlythe deleted gene product but other membraneproteins as well. Thus, the sperm protein(s) re-sponsible for the zona pellucida binding has notbeen identified. We have so far analyzed oligo-saccharides of boar zona pellucida glycoproteinsand found out that oligosaccharides containingN-acetyllactosamine structure and Lewis Xstructure are important for sperm binding.Based on the structural information about thefunctional ligands on the zona pellucida, wehave been investigating sperm molecules recog-nizing the oligosaccharides of zona pellucidaglycoproteins. Two kinds of affinity technologywere applied to detect or isolate carbohydratebinding proteins in detergent-solubilized boarsperm plasma membrane. The first was to detectproteins that bind to the plastic well coated withdextran-based multiple oligosaccharide with N-acetyllactosamine structure. The second was toisolate the proteins using the Sepharose beadscoupled with desialylated α1-acid glycoproteinthat contains N-acetyllactosamine -type oligosac-charides. Then, the two proteins with apparentmolecular masses of 70 kDa and 40 kDa weredetected as candidates for sperm carbohydratebinding proteins. Proteomics analysis suggestedthat the two proteins are members of ADAM (adisintegrin and metalloprotease). Both of the re-combinant proteins produced by yeast wereshown to have adhesion activity to cell surfaceintegrin molecules, and we are currently analyz-ing the carbohydrate binding activity of the pro-teins produce by mammalian cells. We also ana-lyzed recombinant ADAMs of mice that wereproduced by yeast, and found that ADAM4,ADAM5 an ADAM6 also bind to integrins.Their role in sperm-oviduct epithelial cell inter-action is being studied.

2. Analysis of cancer-related sialic acid andsialylated glycans

Hideo Asakawa1, Eiko Wada, and SeiichiTakasaki: 1Department of Internal Medicine,Kudanzaka Hospital.

Sialylated glycoconjugates seem to be in-volved in tumour biology, since aberrant glyco-sylation patterns are very common in humanand animal neoplasias. These tumor-associated

carbohydrate antigens are of prognostic value,since their expression frequently correlates withinvasiveness, metastasis and the tumor grade.The majority of sialylated tumor markers inman involve changes in the presentation of N-acetylneuraminic acid (NeuAc). However, evi-dence is accumulating to suggest that N-glycolylneuraminic acid (NeuGc) can appear incertain human cancers. Actually, we found thatNeuGc-containing glycoproteins are expressedin human hepatocarcinoma tissue and other tu-mor cell lines. In addition, we found that serafrom carcinoma patients contain antibodies rec-ognizing NeuGc-containing glycoconjugates .Their content was much higher than that of nor-mal humans. Thus, the quantification of NeuGcand antibody recognizing NeuGc may be ofclinical value. However, expression of NeuGc inman is a paradox in glycobiology, becauseCMP-NeuAc hydroxylase that is involved in thede novo synthesis of NeuGc is non-functional inman. The mechanism by which human cancercells synthesize NeuGc has to be elucidated.

Sialic acid is usually linked to terminal galac-tose residues, internal N-acetylglucosamine, orN-acetylgalactosamine residues of glycans in α-configulation. No β-linked sialic acids have sofar been found in glycoproteins and glycolipids.However, it is possible that β-linked sialic acidsare synthesized by the cells under pathologicalcondition. To address this issue, an antibodyraised against a synthetic β-linked sialic acidcontaining glycolipid was used. Interestingly,gangliosides from human cancer cell lines werestained with the antibody by the immunochemi-cal method, but those from normal cells werenot. The result suggests that β-linked sialic acidsare expressed on the glycoconjugates of cancercells. However, there is no chemical evidencethat the epitose is really sialylated by β-linkage.The previous search for materials which are re-active to the antibody suggested that the com-mercial pig pepsin preparation contains the epi-tope.Therefore, we tried to isolate epitope-containing materials in the pepsin preparationin combination of ethanol extraction of the ma-terial with reversed phase- and normal phase-HPLC, and found that several fractions are reac-tive to the antibody, MS/MS analysis supportedthat glycoconjugates containing β-linked sialicacids exists in nature.

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