未熟 B 細胞の BCR クロスリンクによるアポトーシス誘導をα4 が正に制御することを示 した。さらに解析を行う目的で full-length α4 を恒常的に過剰発現させた WEHI-Bcl-xL 細胞の樹立を試みたが作成できなかった。そこで HEK293T 細胞の内在性α4 を siRNA α4 を用いてノックダウンすることで Bcl-xL Ser62 のリン酸化に対する影響を調べた。α4 は 全般性に発現する分子で WEHI-231 細胞をはじめ、HEK293T 細胞にも内在性のα4 が高発現 している。これまでα4 が JNK の Bcl-xL Ser62 のリン酸化を促進することを示してきたが その効果は高くなかった。これは内在性のα4 存在下の条件で外来的にα4 を過剰発現して も、すでに飽和状態となっていたため大きな変化が認められなかったと考えられた。本研
46
究では内在性のα4 をノックダウンすることで JNK による Bcl-xL のリン酸化が強く抑制さ れることが認められた。
本研究ではα4 はその大部分が細胞質に局在し、一部ミトコンドリアにも発現が確認さ れた。α4 は核に局在するという報告があったが (Du, et al., 2013)、これまでに報告さ れているα4 の結合パートナー PP2A、PP6 などが主に細胞質に存在することから、本研究 で認められたα4 の細胞質への局在が正しいと考えられる。JNK は大部分が細胞質に局在し 一部、核とミトコンドリアにも検出された。JNK は同定当初、細胞質に存在し、c-Jun のリ ン酸化に伴い核へ移行すると報告されていた (Davis, 2000) が、近年の研究ではミトコン ドリアへの局在も報告されていることから本研究の結果と一致する (Zhao, et al., 2009)。
Bcl-xL は抗アポトーシス分子としてミトコンドリア膜の安定性に寄与していることが広 く知られている。しかし細胞内に発現している Bcl-xL の約半分は通常、細胞質に存在して いる (Aranovich, et al., 2012)。Bcl-xL は細胞質、核、ミトコンドリア分画と広範囲に 発現が確認されたが、JNK とα4 の局在結果よりα4 は Bcl-xL Ser62 のリン酸化を細胞質 またはミトコンドリアで調節することでアポトーシス制御に関与していると推測した。
47 [Ⅸ. 結語]
未熟 B 細胞の BCR クロスリンクによるアポトーシスはミトコンドリアを介した系である ことを除き、その全容は明らかとされていなかった。未熟 B 細胞のアポトーシスメカニズ ムの解析は自己免疫疾患の解明の足掛けとなるだけでなく、B 細胞の発癌メカニズム解明 の一端を担う重要なテーマである。
これまでに抗アポトーシス分子 Bcl-xL が未熟 B 細胞のアポトーシスに重要であること は示されていたが、その制御メカニズムは不明のままだった。本研究では Bcl-xL Ser62 のリン酸化に着目し、解析を開始した。すると JNK が Bcl-xL Ser62 をリン酸化することで ユビキチン化を誘導し、分解へと導くことがアポトーシスを引き起こすことを初めて明ら かとした。また Bcl-xL Ser62 を PP6 が脱リン化することでアポトーシス耐性を示すことも 明らかとした。
さらに JNK の Bcl-xL リン酸化を制御する分子として JNK および PP6 とも関連が深いα4 に着目し解析を行った。α4 は JNK による Bcl-xL Ser62 のリン酸化を促進することが分か った。また C-terminal α4 mutant はドミナントネガティブとして機能することが示され た。
これらの反応は各分子の細胞内局在から細胞質で行われている反応であることが示唆さ れた。
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