Greenfeld Hannah, Takasaki Kaoru, Walsh Michael J, Ersing Ina, Bernhardt Katharina, Ma Yijie, Fu Bishi, Ashbaugh Camille W, Cabo Jackson, Mollo Sarah B, Zhou Hufeng, Li Shitao, Gewurz Benjamin E
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS Pathog. 2015 May 21;11(5):e1004890. doi: 10.1371/journal.ppat.1004890. eCollection 2015 May.
The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.
爱泼斯坦-巴尔病毒（EBV）编码的癌蛋白潜伏膜蛋白1（LMP1）通过两个C末端结构域发出信号，以驱动细胞生长、存活和转化。LMP1膜近端的TES1/CTAR1结构域募集肿瘤坏死因子受体相关因子（TRAFs）以激活丝裂原活化蛋白激酶（MAP激酶）、非经典和经典核因子κB（NF-κB）信号通路，并且对于EBV介导的B细胞转化至关重要。TRAF1是TES1诱导程度最高的靶基因之一，并且在EBV相关的淋巴增殖性疾病中大量表达。我们发现，TRAF1的表达增强了LMP1 TES1结构域介导的p38、JNK、ERK和经典NF-κB信号通路的激活，但不影响非经典NF-κB信号通路的活性。为了深入了解TRAF1如何放大LMP1 TES1 MAP激酶和经典NF-κB信号通路，我们对从未诱导或诱导LMP1 TES1信号的细胞中免疫纯化的TRAF1复合物进行了蛋白质组学分析。出乎意料的是，我们发现LMP1 TES1结构域信号传导诱导了TRAF1与线性泛素链组装复合物（LUBAC）之间的关联，并刺激了线性（M1）连接的多聚泛素链附着到TRAF1复合物上。从EBV转化的淋巴母细胞样B细胞系（LCLs）中分离出的LMP1或TRAF1复合物被M1连接的多聚泛素链高度修饰。在表达LMP1的细胞中，M1泛素结合蛋白IKK-γ/NEMO、A20和ABIN1均与TRAF1相关联。TRAF2而非细胞凋亡抑制蛋白1（cIAP1）或细胞凋亡抑制蛋白2（cIAP2）泛素连接酶在LUBAC募集和M1链附着到TRAF1复合物中起关键作用，这表明TRAF1:TRAF2异源三聚体参与LMP1 TES1依赖性LUBAC激活。TRAF1或LUBAC泛素E3连接酶亚基HOIP的缺失均显著损害LCL的生长。同样，从LCLs中纯化的LMP1或TRAF1复合物被赖氨酸63（K63）连接的多聚泛素链修饰。LMP1 TES1信号传导诱导K63-多聚泛素链附着到TRAF1复合物上，并且TRAF2被确定为K63-泛素链的靶标。M1和K63连接的多聚泛素链在LMP1复合物上的共定位可能有助于下游经典NF-κB信号通路的激活。我们的结果突出了LUBAC作为EBV相关淋巴增殖性疾病中一种新的潜在治疗靶点。
