摘要
DAPK 是一個具有多個功能區域的絲/酥胺酸(serine/threonine)蛋白質激脢，具有抑癌功能，同時也會參與各式的細胞凋亡系統。DAPK 的分子作用機制目前仍不太清楚，其在T 細胞中所扮演的角色也從未被探討過。我們發現DAPK 在T 細胞活化時亦會被活化，然後DAPK 會被分解。我們在T 細胞株和正常的小鼠T 細胞轉殖基因表現活化的DAPK 和會抑制DAPK 活性的變異體，或是利用siRNA 降低DAPK 的表現，發現DAPK 會負向調控T 細胞的活化。DAPK 會影響T 細胞活化後之增殖反應以及IL-2 的產生。同時也發現DAPK 的作用目標之ㄧ是TCR 刺激後引發的NF-κB 活化。在DAPK 被抑制或調降的T 細胞中，PKCθ磷酸化會增加，而進入核中之NF-κBp65 也提升。相對的，IL-1β和TNF-α刺激所引發的NF-κB活化並不受DAPK 影響。我們更進一步發現在T 細胞活化時，DAPK 只會選擇性地影響PKCθ、Bcl-10 和 IKK 等與NF-κB 活化相關的訊息分子進入脂質筏，但對於其他位於脂質筏的分子，例如LAT 和Lck，則沒有影響。 我們接著探討DAPK 是否藉著抑制T 細胞活化而參與T 細胞耐受性之產生。以A23187 增加鈣離子濃度在體外誘導T 細胞去活化，或以靜脈注射CD3 抗體到小鼠體內直接誘導T 細胞去活化的方式，都可以發現在去活化的T 細胞，DAPKmRNA 的表現增加。DAPK 的表現量與T 細胞去活化的程度成正比，顯示DAPK可能在T 細胞耐受度上扮演角色。此外，我們也發現表現活化的DAPK 會抑制小鼠誘發產生實驗性自身免疫性腦脊髓炎。我們的結果顯示了DAPK 是個新的T 細胞調控分子，會主導T 細胞活化以及TCR 刺激引發的NF-κB 活化。同時，DAPK亦可調節T 細胞去活化而維持活體免疫耐受度以避免自體免疫之產生。
關鍵字
T細胞活化並列摘要
Death-associated protein kinase (DAP-kinase, DAPK) is a unique multi-domain kinase acting both as a tumor suppressor and an apoptosis inducer. The molecular mechanism underlying the effector function of DAPK is not fully understood, while the role of DAPK in T lymphocyte activation is mostly unknown. DAPK was activated after TCR stimulation, followed by DAPK degradation. Through the expression of a dominant negative form and a constitutively active form of DAPK in T cell lines and in transgenic mice, we found that DAPK negatively regulated T cell activation. DAPK markedly affected T cell proliferation and IL-2 production. We identified TCR-induced NF-κB activation as a target of DAPK. There was an increased PKCθ phosphorylation and an enhanced NF-κBp65 nuclear translocation in T cells with DAPK inhibited or down-regulated. In contrast, IL-1β- and TNF-α-triggered NF-κB activation was not affected by DAPK. We further found that DAPK selectively modulated the TCR-induced translocation of PKCθ, Bcl-10, and IKK into membrane rafts. Notably, the effect of DAPK on the raft entry was specific for NF-κB pathway, as other raft-associated molecules, such as LAT, were not affected. We further examined the possibility that, by negatively regulating T cell activation, DAPK contributes to the generation of T cell tolerance. DAPK mRNA expression was up-regulated when T cell anergy was induced either in vitro by calcium ionophore, or in vivo by intravenous administration of anti-CD3 antibody. There was a correlation between the amount of DAPK expression and the extent of T cell anergy, suggesting a role of DAPK in T cell tolerance induction. In addition, active DAPK transgene inhibited the induction and progression of autoimmune experimental autoimmune encephalomyelitis (EAE). Our results demonstrate that DAPK is a novel T cell regulator on TCR-activated NF-κB and T cell activation, and DAPK may contribute to maintenance of immune tolerance and avoidance of autoimmunity by promoting T cell anergy.