DataSheet6_Endogenous Bok is stable at the endoplasmic reticulum membrane and does not mediate proteasome inhibitor-induced apoptosis.PDF

Controversy surrounds the cellular role of the Bcl-2 family protein Bok. On one hand, it has been shown that all endogenous Bok is bound to inositol 1,4,5-trisphosphate receptors (IP3Rs), while other data suggest that Bok can act as a pro-apoptotic mitochondrial outer membrane permeabilization mediator, apparently kept at very low and non-apoptotic levels by efficient proteasome-mediated degradation. Here we show that 1) endogenous Bok is expressed at readily-detectable levels in key cultured cells (e.g., mouse embryonic fibroblasts and HCT116 cells) and is not constitutively degraded by the proteasome, 2) proteasome inhibitor-induced apoptosis is not mediated by Bok, 3) endogenous Bok expression level is critically dependent on the presence of IP3Rs, 4) endogenous Bok is rapidly degraded by the ubiquitin-proteasome pathway in the absence of IP3Rs at the endoplasmic reticulum membrane, and 5) charged residues in the transmembrane region of Bok affect its stability, ability to interact with Mcl-1, and pro-apoptotic activity when over-expressed. Overall, these data indicate that endogenous Bok levels are not governed by proteasomal activity (except when IP3Rs are deleted) and that while endogenous Bok plays little or no role in apoptotic signaling, exogenous Bok can mediate apoptosis in a manner dependent on its transmembrane domain.

关于B细胞淋巴瘤因子2（Bcl-2）家族蛋白Bok的细胞功能，学界尚存争议。一方面，已有研究表明所有内源性Bok均结合于肌醇1,4,5-三磷酸受体（IP3Rs）；另一方面，另有数据显示Bok可作为促凋亡线粒体外膜通透介导因子，其水平似乎通过高效的蛋白酶体介导降解维持在极低且非凋亡状态。本研究证实：1）内源性Bok在关键培养细胞（如小鼠胚胎成纤维细胞与HCT116细胞）中可被稳定检测到表达，且并非通过蛋白酶体发生持续性降解；2）蛋白酶体抑制剂诱导的细胞凋亡并非由Bok介导；3）内源性Bok的表达水平严格依赖于IP3Rs的存在；4）在IP3Rs缺失的情况下，内源性Bok会在内质网膜上通过泛素-蛋白酶体通路快速降解；5）Bok跨膜区的带电残基会影响其稳定性、与髓系细胞白血病因子1（Mcl-1）的相互作用能力，以及过表达时的促凋亡活性。综上，本研究数据表明，内源性Bok的水平并不受蛋白酶体活性调控（IP3Rs缺失的情况除外）；尽管内源性Bok在细胞凋亡信号通路中几乎或完全不发挥作用，但外源性Bok可通过依赖其跨膜结构域的方式介导细胞凋亡。