ASB11在BIK泛素化與細胞凋亡調控中所扮演的角色

Abstract

BIK (Bcl-2-interacting killer)屬Bcl-2蛋白質家族中之BH3-only成員，且因其促細胞凋亡之性質而被認為是腫瘤抑制基因。然而，細胞內BIK蛋白表現量之調控機制至今尚未被完全了解。基於過表現ASB11 (ankyrin and SOCS box protein 11)可促進BIK泛素化之事實，本實驗室先前已鑑別出ASB11蛋白可能作為負責介導BIK泛素化之受質辨識單元(substrate recognition subunit, SRS)，參與在Cullin5 E3接合酶複合體(Cul5 E3 ligases complex)所催化之泛素化反應中。而在本次研究中，我們藉Co-IP以及in vitro binding assay驗證了ASB11與BIK之間所存在的直接性結合，並於in vitro ubiquitination assay中證明重組之ElonginBC-Cul5-ROC2-ASB11複合體具有催化BIK泛素化之酵素活性。進一步的實驗則顯示，過表現ASB11將會導致293T細胞株內之BIK蛋白含量下降；且此現象可藉由施以蛋白酶體抑制劑MG132所排除，指出ASB11可促使BIK蛋白經泛素-蛋白酶體系統(ubiquitin-proteasome system, UPS)被降解。與此相應，過表現ASB11亦可部分性地抑制由DNA損害藥物(DNA damage agents)處理所引發之細胞凋亡，顯示ASB11具有促進細胞存活之能力。另一方面，HCT116細胞株中ASB11之mRNA表現量被發現會在經過cisplatin, doxorubicin以及5-fu等DNA損害藥物之處理後經由p53路徑被負調控，顯示ASB11可能參與在DNA損害反應(DNA damage response)之中。此外，tunicamycin及thapsigargin等內質網壓力誘導物(ER-stress inducers)之處理則被發現可以透過XBP1路徑提高293T細胞株中ASB11之mRNA表現量，並且降低BIK蛋白之表現量；此結果暗示ASB11可能參與在不正常蛋白質摺疊反應(unfolded protein response, UPR)的適應性反應(adaptive response)之中。綜合以上結果，本研究發現了Cul5-ASB11所介導之BIK泛素化機制；並且指出此機制在抑制DNA損害藥物所引發之細胞凋亡，以及在針對內質網壓力之適應性反應中所扮演的角色。

BIK (Bcl-2-interacting killer) is a BH3-only Bcl-2 family protein which has been considered as a tumor suppressor based on its pro-apoptotic function. However, the mechanism that regulates BIK protein level has not been completely understood. Previous studies in our laboratory identified ASB11 (ankyrin and SOCS box protein 11) as a candidate of the substrate recognition subunit (SRS) that recruits BIK to Cullin5 (Cul5) E3 ligases complex. In this study, we first examined the direct relationship between BIK and ASB11. Through co-IP and in vitro binding assay, the interaction between ASB11 and BIK was demonstrated. Furthermore, in vitro ubiquitination assay revealed the capacity of reconstituted ElonginBC-Cul5-ROC2-ASB11 complex to promote BIK polyubiquitination. Accordingly, overexpression of ASB11 in 293T cells decreases BIK protein abundance. Together with the finding that MG132 treatment blocks BIK downregulation induced by ASB11 overexpression, our data indicate that ASB11 promotes BIK degradation through ubiquitin-proteasome system (UPS). Consistent with the downregulation of pro-apoptotic BIK, ASB11 elicits a pro-survival effect to inhibit apoptosis induced by DNA damage agents. Furthermore, we show that DNA damage agents induced downregulation of ASB11 mRNA in a p53-dependent manner. On the contrary, the ER-stress inducers tunicamycin and thapsigargin unregulated ASB11 mRNA expression and reduced BIK protein abundance in 293T cells through a XBP1 dependent pathway. Together, this study identifies a BIK degradation pathway mediated by Cul5-ASB11 ubiquitin ligase complex and implies a role of this pathway in both antagonizing p53-dependent apoptosis in response to DNA damage agents and the adaptive response to ER stress.