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者〉伊澤 大介\r\n〈著者所属〉英国Cancer Research UK Gurdon Institute\r\n〈著者email〉di222@cam.ac.uk（伊澤大介）\r\n\r\n〈対象論文〉\r\nHow APC/C-Cdc20 changes its substrate specificity in mitosis.\r\nDaisuke Izawa, Jonathon Pines\r\nNature Cell Biology, 13, 223-233 (2011)\r\n\r\n〈要 約〉\r\n　体細胞分裂の進行においては適切な時期に適切な制御タンパク質を分解する必要がある．ユビキチンリガーゼのひとつであるAPC/Cはさまざまな制御タンパク質の分解を適切な時期に誘導することにより体細胞分裂の進行を正確に制御している．スピンドルチェックポイントとよばれる機構はAPC/Cの活性化タンパク質Cdc20にはたらきかけて中期における制御タンパク質の分解を特異的に阻害するが，前中期においては制御タンパク質の分解を阻害しない．しかしながら，このような制御がどのようになされているかは不明であった．この研究では，Cdc20が前中期と中期とで異なるサブユニットを介してAPC/Cと相互作用することを発見した．さらに，APC10は中期における制御タンパク質の分解には必須であるが，前中期における制御タンパク質の分解には必要ないことを発見した．\r\n\r\nはじめに\r\n　体細胞分裂（有糸分裂）はDNA複製期において複製された2組の染色体を2つの娘細胞に等分に分配するステージである1)（図1）．哺乳類の細胞では，前期（prophase）には染色体が凝縮し，前中期（prometaphase）には核膜の崩壊が起こり中心体から伸びた紡錘体により姉妹染色体が捕捉され，中期（metaphase）にはすべての姉妹染色体が赤道面に配置される．後期（anaphase）には姉妹染色体がそれぞれ両極に分配され，終期（telophase）では染色体の脱凝縮と核膜の再形成ののち細胞質分裂が完了し2つの細胞が形成される．\r\n　体細胞分裂において分裂期の進行に必要な制御タンパク質は分裂期をおえるときには不活性化されなければならない．そのための方法のひとつとしてユビキチン-プロテアソーム系によるタンパク質分解という直接的な制御がある．ユビキチンリガーゼによりポリユビキチン化され標的となった制御タンパク質はプロテアソームにより認識されただちに分解される．たとえば，サイクリン依存性キナーゼ（cyclin-dependent kinase：CDK）とサイクリンB1との複合体であるCdk1-サイクリンB1は分裂期への進行に必須な制御タンパク質であるが，中期には分解され不活性化される．分解されないようなサイクリンB1の変異体を発現させると細胞は分裂期を終了できなくなり細胞周期を停止してしまう．後期の進行に必須な役割をはたすセパラーゼの阻害タンパク質であるセキュリンも中期に分解されるが，セキュリンの分解が起こらないと細胞周期は中期で停止する．\r\n　APC/C（anaphase promoting complex/cyclosome）はおもに体細胞分裂期にはたらくユビキチンリガーゼとして知られ，サイクリンB1やセキュリンの分解を制御するタンパク質複合体として発見された．のちの研究から，APC/Cが体細胞分裂期におけるほかのさまざまな制御タンパク質を特異的な時期に分解することが明らかになっている2)（図1）．前中期にはサイクリンAやNek2A，中期にはサイクリンB1やセキュリン，後期にはCdc20，終期にはPlk1やAurora Aなどが，時期に特異的にAPC/Cを介して分解される3)．これらの制御タンパク質が円滑に分解されないと，細胞周期は停止してしまうか不均等な体細胞分裂が起こるかして細胞は最終的に死にいたる．以上のことから，このような制御機構は非常に重要であるが，どのようにAPC/Cが時期に特異的に分解されるべき制御タンパク質を認識しているかは明確には解明されていない．\r\n　APC/Cの活性化タンパク質としてCdc20とCdh1の2つが知られている4)．Cdc20は前中期および中期においてAPC/Cを活性化し，Cdh1は後期からG1期までにおいてAPC/Cを活性化する．これらの活性化タンパク質は分解されるべき制御タンパク質を認識してAPC/Cへと運ぶ役割をはたしていると考えられている．前中期においては中心体から伸びた紡錘体が姉妹染色体を捕捉するが，1対の姉妹染色体がそれぞれ両極から捕捉されることが均等な染色体分配を行うために重要である．均等な染色体分配を保障する機構であるスピンドルチェックポイント機構は不均等な姉妹染色体の捕捉を検出して後期への進行を阻害するシグナルを発し，Cdc20の活性を制御していることが知られている4)．詳細な機構は議論の最中であるが，Cdc20がMad2，BubR1，Bub3からなるMCC（mitotic checkpoint complex）複合体もしくはそのいずれかと結合することで，Cdc20の活性が制限されるものと考えられている．しかし，スピンドルチェックポイント機構に機能するタンパク質がどのようにして直接にCdc20を阻害しているのかはいまのところ不明である．興味深いのは，前中期においてCdc20の活性は制限されているのにもかかわらず，サイクリンAやNek2Aなどの制御タンパク質はAPC/CとCdc20に依存して分解されていることである．\r\n　もっとも重要な疑問は，APC/CとCdc20が前中期に分解すべき制御タンパク質と中期に分解すべき制御タンパク質とをどのように区別しているかである．APC/Cは11個のサブユニットからなる巨大な複合体であり5)，ほかのユビキチンリガーゼと比べ独特な構成をもつ．ユビキチン付加反応を担うAPC2とAPC11，基質の認識に必要と考えられているAPC10，タンパク質相互作用ドメインとして知られるTPRモチーフを複数もつAPC3，APC6，APC7，APC8，そして，複合体構造の土台となるAPC1，APC4，APC5から構成されている．このように，APC/Cは複数のサブユニットから構成されているが個々のサブユニットの機能はほとんど解明されていない．筆者らは，機能が未解明なサブユニットに着目しAPC/C自体が制御タンパク質の分解における特異性になんらかの寄与をはたしている可能性を探索した．\r\n　ヒト細胞においてそれぞれのサブユニットに対するsiRNA処理を行って特定のサブユニットの発現をノックダウンした状況において制御タンパク質の分解がどのような影響をうけるかを解析した．モデルとなる制御タンパク質として，前中期に分解される制御タンパク質にはサイクリンA，中期に分解される制御タンパク質にはサイクリンB1を用いた．実験手法として，蛍光タンパク質とサイクリンAあるいはサイクリンB1との融合タンパク質をヒト細胞に発現させ，蛍光顕微鏡による経時観察を行いその蛍光強度を測定することによってその制御タンパク質が分解される時期と速度を解析した．この手法にヒトAPC/Cのそれぞれのサブユニットに対するRNAi法によるノックダウン実験を組み合わせることにより，特定のサブユニットが制御タンパク質の分解に対しどのように影響をあたえるかを解析した．\r\n\r\n1．APC3とAPC11は前中期と中期における制御タンパク質の分解に必要である\r\n　APC11はユビキチン付加反応に必須であり，APC3は活性化タンパク質と直接に結合することが示唆されている6)．これらのサブユニットをRNAi法によりノックダウンすることによってAPC/Cの活性が低下することが予想される．はじめに対照実験として，APC/Cの活性が低下したときサイクリンAおよびサイクリンB1の分解がどのように影響をうけるかを解析した．予想されたとおり，APC11とAPC3の対するノックダウン実験ではサイクリンAとサイクリンB1はともに分解が非常に遅延した．興味深いことに，APC/Cの活性を低下させた状況では，通常は前中期からはじまるサイクリンAの分解がほとんどの細胞において中期からはじまっていた．くわえて，薬剤により微小管の重合を阻害しスピンドルチェックポイント機構の活性を持続させて細胞周期を前中期で停止させると，サイクリンAは分解されず安定化していた．\r\n\r\n2．APC3は前中期におけるAPC/CとCdc20との結合に必要ない\r\n　蛍光顕微鏡による観察からAPC3は制御タンパク質の分解に必要であることがわかった．この結果は，酵母やショウジョウバエのAPC3の変異体の結果と一致した．つぎに免疫沈降実験を行い，APC3をRNAi法によりノックダウンした状態において，APC/CとCdc20との相互作用，また，制御タンパク質との結合がどのように影響をうけるかを解析した．APC3をノックダウンしたうえで前中期もしくは中期の細胞から調製した細胞抽出液を用いてAPC/Cを免疫沈降した．この状況において，APC/CはサイクリンAおよびサイクリンB1との結合が非常に弱まっていた．この結果は，APC3は制御タンパク質の認識に必要なことを示唆していた．興味深い結果として，APC3をノックダウンした前中期の細胞抽出液においてAPC/Cを免疫沈降したところCdc20がスピンドルチェックポイント機構に機能するタンパク質であるBubR1とともに結合していた．しかし，スピンドルチェックポイント機構が不活性化している前中期の細胞抽出液から免疫沈降した場合，APC/CとCdc20との結合は低下していた．これまでの知見ではin vitroにおいてAPC3はCdc20と直接に結合することが知られていたが6)，スピンドルチェックポイント機構の活性化している前中期においてはCdc20がAPC/Cと相互作用するにあたってAPC3は必要のないことが示唆された．さらにこの結果を支持する結果として，昆虫細胞で発現および精製したヒトCdc20はAPC/Cと結合するが，その結合にはAPC3が必要であった．中期の結果と同様に，スピンドルチェックポイント機構に機能するタンパク質と結合していないCdc20はAPC/Cと相互作用するためAPC3を必要とすることが示された．\r\n\r\n3．APC10はサイクリンB1の分解には必要だがサイクリンAの分解には必要ない\r\n　つぎに，APC/Cの制御タンパク質の認識に必要と考えられているAPC10の解析を行った．出芽酵母および分裂酵母のAPC10の変異体においては体細胞分裂期にサイクリンB1が安定化されることが知られている．APC10をRNAi法によりノックダウンした状況において制御タンパク質の分解を解析したところ，興味深いことに，サイクリンB1の分解は影響をうけ分解速度は低下していたが，サイクリンAの分解は影響をうけず分解の時期および速度ともまったく変化がなかった．この結果は，APC10はサイクリンAの分解には必要ないことを示唆していた．APC10をノックダウンした細胞抽出液においてAPC/Cを免疫沈降したところ，サイクリンAの相互作用は影響をうけないが，サイクリンB1とCdc20との結合は弱まっていた．これらの結果はさきの実験結果とも一致しており，前中期に分解される制御タンパク質であるサイクリンAはAPC10に依存せずAPC/Cに認識されるが，APC/Cが中期に分解される制御タンパク質であるサイクリンB1を認識するにはAPC10を必要とすることが明らかになった．\r\n　最近，筆者らの研究室において，サイクリンAはCksと複合体を形成し，このCksがAPC/Cのリン酸化修飾を認識することで，サイクリンAはCdc20に依存せずにAPC/Cと相互作用することが明らかになった7)．さらに，APC3に対するノックダウン実験においてはAPC/CとサイクリンAとの結合が低下することから，CksはAPC3のリン酸化修飾を認識していると推測できた．\r\n\r\n4．APC8は前中期においてAPC/CとCdc20との相互作用またサイクリンAの分解に必要である\r\n　APC3に対するノックダウン実験から前中期におけるAPC/CとCdc20との結合にはAPC3は必要ないことが明らかになったが，ほかのどのサブユニットがAPC/CとCdc20との結合に必要であるかは不明であった．さらなる探索を行った結果，APC6またはAPC8をRNAi法によりノックダウンした状況では，前中期においてAPC/CとCdc20とのあいだに相互作用は検出されなかった．しかし，このときAPC/Cの構造も部分的に崩壊していることがわかり，APC6もしくはAPC8がAPC/CとCdc20との相互作用に必要であることは示唆されたが，APC6またはAPC8がCdc20と直接に結合しているとはいえなかった．そこで，APC6もしくはAPC8に変異を導入してAPC/Cの構造を壊すことなくAPC/CとCdc20との結合を低下させる試みを行った．同じ時期にほかのグループにより，出芽酵母においてAPC/CとCdh1との相互作用に必要な部位がAPC3およびAPC8に変異を導入することによって同定された8)．この情報にもとづいて，ヒトAPC8においても保存されたアミノ酸残基に同様な変異を導入したところ，前中期においてAPC/Cの構造に影響を及ぼすことなくCdc20とAPC/Cとの相互作用を低下させることができた．\r\n　APC8への変異の導入によりAPC/CとCdc20との結合を前中期に阻害することができたことから，内在性のAPC8をRNAi法によりノックダウンしたうえでAPC8の変異体を発現させる相補実験において，サイクリンAの分解がどのような影響をうけるかを観察した．野生型のAPC8を相補したときにはサイクリンAは通常どおり前中期の開始とともに分解されたが，APC8の変異体を相補したときにはサイクリンAの分解は遅延し中期から分解がはじまった．さらに，薬剤によりスピンドルチェックポイント機構を活性化させ細胞周期を前中期で停止させると，APC8の変異体を相補したときサイクリンAは非常に安定化した．これらの結果から，APC8は前中期においてAPC/CとCdc20との相互作用に必要であり，さらに，その相互作用は前中期におけるサイクリンAの分解に必要であることが示唆された．\r\n\r\n5．APC8は中期におけるAPC/CとCdc20との結合にも必要である\r\n　前中期においてAPC/CがCdc20と結合するためにはAPC8が重要なことが明らかになったことから，つぎに，中期におけるAPC8の役割を解析した．その結果，中期においてもAPC8はAPC/CとCdc20との結合に必要であった．この結果と一致して，内在性のAPC8をRNAi法によりノックダウンしたうえでAPC8の変異体を相補したときにはサイクリンB1の分解が阻害されていた．さらに，in vitroでのAPC/Cによるユビキチン付加実験においても，APC8を変異体で置き換えたAPC/Cは内在性のAPC8をもつAPC/Cと比較して弱いユビキチン付加活性を示したことから，APC8はCdc20と結合し，その相互作用はAPC/Cの活性化にも重要な役割をはたしていることがわかった．\r\n\r\nおわりに\r\n　この研究は，APC/CとCdc20との相互作用に関してより詳細な知見をあたえた．以前から，APC/CとCdc20はスピンドルチェックポイント機構を介し前中期と中期では質的に異なる相互作用をしていることは考えられていたが，今回，その相互作用には異なるサブユニットを要求していることが発見された（図2）．前中期において，APC/CとCdc20との相互作用にはAPC3は必要とされずAPC8だけが必要とされ，中期においてはAPC3とAPC8の両方が必要とされる．出芽酵母においてAPC/CともうひとつのAPC/C活性化タンパク質であるCdh1との相互作用にはAPC3とAPC8の両方が要求されることが報告されており8)，これは，筆者らのヒト細胞における実験結果と一致する．最新の研究からAPC/Cの詳細な立体構造がかなり明らかになり9)，Cdh1はAPC/Cの複数のサブユニットと近接していて，これはAPC/CとCdc20との相互作用においても同様であることが推測される．in vitroの実験ではCdc20およびCdh1のC末端にあるイソロイシン-アルギニンモチーフがAPC3と直接に結合することが知られており，in vivoにおいてもCdc20はこのモチーフを介してAPC3と結合しているものと推測される．しかし，APC8はCdc20のどのモチーフと結合しているかは未解明であり，現在，APC8とCdc20との相互作用についてさらなる解析を行っている．また，スピンドルチェックポイント機構がどのようにCdc20とAPC/Cとの相互作用を制御しているかという重要な疑問も未解明であり，その関係についても解析を進めている．\r\n\r\n〈文 献〉\r\n 1) Pines, J. \u0026 Rieder, C. L.: Re-staging mitosis: a contemporary view of mitosis. Nat. Cell Biol., 3, E3-E6 (2001)\r\n 2) Peters, J. M.: The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat. Rev. Mol. Cell Biol., 7, 644-656 (2006)\r\n 3) Pines, J.: Mitosis: a matter of getting rid of the right protein at the right time. Trends Cell Biol., 16, 55-64 (2006)\r\n 4) Thornton, B. R., Ng, T. M., Matyskiela, M. E. et al.: An architectural map of the anaphase promoting complex. Genes Dev., 20, 449-460 (2006)\r\n 5) Yu, H.: Cdc20: a WD40 activator for a cell cycle degradation machine. Mol. Cell, 27, 3-16 (2007)\r\n 6) Vodermaier, H. C., Gieffers, C., Maurer-Stroh, S. et al.: TPR subunits of the anaphase promoting complex mediate binding to the activator protein CDH1. Curr. Biol., 12, 1459-1468 (2003)\r\n 7) Di Fiore, B., Pines, J.: How cyclin A destruction escapes the spindle assembly checkpoint. J. Cell Biol., 190, 501-509 (2010)\r\n 8) Matyskiela, M. E., Morgan, D. O.: Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism. Mol. Cell, 32, 68-80 (2009)\r\n 9) Schreiber, A., Stengel, F., Zhang, Z. et al.: Structural basis for the subunit assembly of the anaphase promoting complex. Nature, 470, 227-232 (2011)\r\n\r\n〈著者プロフィール〉\r\n伊澤 大介（Daisuke Izawa）\r\n略歴：2006年 東京大学大学院理学系研究科博士課程 修了，同年より英国Cancer Research UK Gurdon Institute研究員．\r\n研究テーマ：ヒト培養細胞を用いた体細胞分裂期におけるAPC/Cの機能解析．\r\n抱負：体細胞分裂期だけでなく，間期や停止期においても，APC/Cを介するどのような細胞周期の制御機構があるのかを追求していきたい．\r\n\r\n〈図説明〉\r\n図1　APC/Cは体細胞分裂期においてさまざまな制御タンパク質を時期に特異的に分解する\r\nAPC/Cとその活性化タンパク質Cdc20は前中期および中期において制御タンパク質の分解を促進し，APC/Cとそのもうひとつの活性化タンパク質であるCdh1は後期からG1期にかけて活性化しさまざまな制御タンパク質の分解を促進する．前中期にはスピンドルチェックポイント機構が活性化してCdc20の活性を制限する．\r\n図2　APC/CとCdc20との相互作用には異なるサブユニットが必要である\r\n（a）スピンドルチェックポイント機構の活性化している前中期において，APC/CはAPC8を介してCdc20と相互作用し，APC3は分解されるべき制御タンパク質を認識する．スピンドルチェックポイント機構に機能するタンパク質（SAC）はCdc20と結合し，Cdc20とAPC3との相互作用を阻害しているものと推測される．\r\n（b）スピンドルチェックポイント機構の不活性化している中期において，APC/CはAPC3とAPC8の両方を介してCdc20と相互作用し，APC10とCdc20とが協調して分解されるべき制御タンパク質を認識する．\r\nIR：イソロイシン-アルギニンモチーフ．"}