Thakar Nilay Y, Ovchinnikov Dmitry A, Hastie Marcus L, Kobe Bostjan, Gorman Jeffrey J, Wolvetang Ernst J
Stem Cell Engineering Group, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia.
Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Herston, QLD 4029, Australia.
Mol Biol Cell. 2015 Mar 1;26(5):993-1006. doi: 10.1091/mbc.E14-08-1290. Epub 2015 Jan 7.
CD30 (TNFRSF8), a tumor necrosis factor receptor family protein, and CD30 variant (CD30v), a ligand-independent form encoding only the cytoplasmic signaling domain, are concurrently overexpressed in transformed human embryonic stem cells (hESCs) or hESCs cultured in the presence of ascorbate. CD30 and CD30v are believed to increase hESC survival and proliferation through NFκB activation, but how this occurs is largely unknown. Here we demonstrate that hESCs that endogenously express CD30v and hESCs that artificially overexpress CD30v exhibit increased ERK phosphorylation levels, activation of the canonical NFκB pathway, down-regulation of the noncanonical NFκB pathway, and reduced expression of the full-length CD30 protein. We further find that CD30v, surprisingly, resides predominantly in the nucleus of hESC. We demonstrate that alanine substitution of a single threonine residue at position 61 (T61) in CD30v abrogates CD30v-mediated NFκB activation, CD30v-mediated resistance to apoptosis, and CD30v-enhanced proliferation, as well as restores normal G2/M-checkpoint arrest upon H2O2 treatment while maintaining its unexpected subcellular distribution. Using an affinity purification strategy and LC-MS, we identified TRAF2 as the predominant protein that interacts with WT CD30v but not the T61A-mutant form in hESCs. The identification of Thr-61 as a critical residue for TRAF2 recruitment and canonical NFκB signaling by CD30v reveals the substantial contribution that this molecule makes to overall NFκB activity, cell cycle changes, and survival in hESCs.
CD30（肿瘤坏死因子受体超家族成员8，TNFRSF8）是一种肿瘤坏死因子受体家族蛋白，而CD30变体（CD30v）是一种仅编码细胞质信号结构域的非配体依赖性形式，在转化的人胚胎干细胞（hESC）或在抗坏血酸存在下培养的hESC中同时过表达。据信，CD30和CD30v通过激活NFκB来提高hESC的存活率和增殖能力，但具体机制尚不清楚。在这里，我们证明，内源性表达CD30v的hESC和人工过表达CD30v的hESC表现出ERK磷酸化水平升高、经典NFκB途径激活、非经典NFκB途径下调以及全长CD30蛋白表达降低。我们进一步发现，令人惊讶的是，CD30v主要存在于hESC的细胞核中。我们证明，将CD30v中第61位（T61）的单个苏氨酸残基替换为丙氨酸可消除CD30v介导的NFκB激活、CD30v介导的抗凋亡能力和CD30v增强的增殖能力，同时在H2O2处理后恢复正常的G2/M期检查点停滞，同时保持其意外的亚细胞分布。使用亲和纯化策略和液相色谱-质谱联用技术，我们确定TRAF2是hESC中与野生型CD30v相互作用但不与T61A突变体形式相互作用的主要蛋白。将苏氨酸61鉴定为CD30v招募TRAF2和经典NFκB信号传导的关键残基，揭示了该分子对hESC中整体NFκB活性、细胞周期变化和存活的重大贡献。
