接合酶酵素KLHL20-Cul3-Roc1對抑癌蛋白DAPK及PML調控機制之探討

Abstract

死亡相關蛋白激脢(DAPK)是一會受攜鈣素(calmodulin)所調控的serine/thronine死亡相關蛋白激脢。一開始DAPK被發現的原因是因為其會參與interferon所調控的細胞死亡。但至今，interferon如何活化DAPK，其間的機制仍不清楚。在本篇論文中，我們找到了KLHL20。它是一個含有BTB-Kelch功能區塊的蛋白。我們發現它會藉由與DAPK結合加速DAPK的本身的蛋白質降解。我們也找出其間的調控機制，那是KLHL20利用它的Kelch repeat與DAPK結合，再利用它的BTB功能區塊與Cullin3結合。而這樣一個由KLHL20-Cul3-ROC1組合而成的E3連接脢複合體我們也證實的確會促進DAPK的泛素化作用。我們更發現在interferon的刺激下，這樣一個訊號會減弱KLHL20對DAPK的泛素化作用。由於干擾素引發PML及PML nuclear body生成，KLHL20藉由與PML結合作用進入PML nuclear body，與DAPK分離，進而抑制DAPK蛋白降解作用。而此干擾素促進之DAPK穩定作用參與了干擾素引發之細胞凋亡及細胞自噬。此外，本論文以骨髓癌細胞模式，證實了干擾素抑制KLHL20對DAPK降解作用為骨髓癌細胞對干擾素敏感性之一項決定因子，因此對骨髓癌之治療策略上具重要參考價值。 除了死亡相關蛋白激脢，我們也發現KLHL20會在缺氧的情況下造成PML之反向調控。腫瘤缺氧與病情進展和治療失效有關，但缺氧的訊息機制並未完全明朗。在此，我們發現缺氧可透過HIF-1所誘發的KLHL20轉錄活化作用，由KLHL20扮演Cul3泛素接合酶之受質轉接蛋白，觸發泛素所媒介的PML腫瘤抑制蛋白之降解。然而，PML必需被CDK1/2於其S518位置進行磷酸化，才會被送至Cul3-KLHL20接合酶。我們證實由HIF-1所誘發、KLHL20所媒介的PML降解，以反饋機制充分地在缺氧情況下誘發HIF-1，進而強化缺氧所引起的代謝重整及上皮間質轉化現象。我們的研究找到一條涉及KLKL20、CDK1/2以及PML的HIF-1自我調控迴路，並提出此迴路對腫瘤進程之影響。

Death-associated protein kinase (DAPK) was identified as a mediator of interferon (IFN)-induced cell death. How IFN controls DAPK activation remains largely unknown. Here we identify the BTB-Kelch protein KLHL20 as a negative regulator of DAPK. KLHL20 binds DAPK and Cullin 3 (Cul3) via its Kelch-repeat domain and BTB domain, respectively. The KLHL20-Cul3-ROC1 E3 ligase complex promotes DAPK polyubiquitination, thereby inducing the proteasomal degradation of DAPK. Accordingly, depletion of KLHL20 diminishes DAPK ubiquitination and degradation. The KLHL20-mediated DAPK ubiquitination is suppressed in cells receiving interferon (IFN) –α or IFN-γ, which induces an enrichment/sequestration of KLHL20 in the PML nuclear bodies, thereby separating KLHL20 from DAPK. Consequently, IFN triggers the stabilization of DAPK. This mechanism of DAPK stabilization is crucial for determining IFN responsiveness of tumor cells and contributes to IFN-induced autophagy. This study identifies KLHL20-Cul3-ROC1 as an E3 ligase for DAPK ubiquitination and reveals a regulatory mechanism of DAPK, through blocking its accessibility to this E3 ligase, in IFN-induced apoptotic and autophagic death. Our findings may be relevant to the problem of IFN resistance in cancer therapy. Apart from DAPK, we also identify KLHL20 as a negative regulator of PML, and this regulation is controlled by hypoxia. Tumor hypoxia is associated with disease progression and treatment failure, but the hypoxia signaling mechanism is not fully understood. Here, we show that hypoxia triggers ubiquitin-dependent proteolysis of PML tumor suppressor through HIF-1-induced transactivation of KLHL20. Targeting PML to the Cul3-KLHL20 ligase, however, requires PML phosphorylation by CDK1/2 at S518. We present evidence indicating that this HIF-1-induced, KLHL20-mediated PML destruction participates in a feedback mechanism to maximize HIF-1 induction by hypoxia, thereby potentiating hypoxia-induced metabolic reprogramming and epithelial-mesenchymal transition. Our study identifies a HIF-1 autoregulatory loop involving KLHL20, CDK1/2 and PML and suggests a contribution of this loop to tumor progression.