HTLV-1 TAX1 AS AN EXTRACELLULAR CYTOKINE

HTLV-1 TAX1 作为细胞外细胞因子

• 批准号: 2468441
• 负责人: J BRADY
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2468441
• 关键词: chimeric proteins; cytokine; enzyme activity; gel mobility shift assay; gene expression; genetic regulation; glutathione transferase; human T cell lymphotropic virus type 1; lymphocyte; nuclear factor kappa beta; protein kinase C; protein structure function; tissue /cell culture; transcription factor; virus genetics; virus protein

Human T-cell lymphotropic virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL), and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). HTLV-I Tax1 is released from infected lymphocytes and functions as an extracellular cytokine to stimulate gene expression in, and proliferation of, uninfected lymphocytes. Human T-cell lymphotropic virus type I (HTLV-I) Tax1 induces the activation and nuclear localization of the cellular transcription factor, NF-kappaB. Treatment of cells with calphostin C, a protein kinase C (PKC) inhibitor, blocked induction of NF-kappaB DNA binding activity in HTLV-I-transformed C81 cells and Tax1-stimulated murine pre-B cells, suggesting that PKC was an important intermediate in the NF-kappaB induction pathway. It was further demonstrated that Tax1 associates with, and activates, PKC. PKC was coimmunoprecipitated with anti-Tax1 sera from Tax1-expressing MT4 extracts and Jurkat extracts in the presence of exogenous Tax1 protein. In addition, Glutathione-S-transferase (GST)-Tax1 protein bound specifically to the alpha, delta and eta PKC isoenzymes synthesized in rabbit reticulocyte lysates. The addition of Tax1 to in vitro kinase reactions leads to the phosphorylation of Tax1 and an eighteen-fold increase in the autophosphorylation of PKC. Transfection of Jurkat cells with wild-type Tax1 stimulated membrane translocation of PKC. In contrast, Tax1 mutant M22, which fails to stimulate NF-kappaB-dependant transcription, failed to stimulate membrane translocation of PKC. Tax1 did not directly increase PKC phosphorylation of IkappaBalpha. Our results are consistent with a model in which Tax1 interacts with PKC and stimulates membrane translocation and triggering of the PKC pathway. Subsequent steps in the PKC cascade likely stimulate phosphorylation of IkappaBalpha. NF-kappaB regulates expression of several viral and cellular genes including the HIV LTR, MHC class I and IL-2Ralpha cytokine genes. It has been demonstrated that Rb stimulates binding of the NF-kappaB p50 homodimer. Addition of Rb protein to an in vitro gel shift binding assay stimulated p50 binding greater than ten-fold. Interestingly, by analyzing NF-kappaB-dependent transcription activity in vitro, we demonstrate that Rb suppresses transcriptional activity of p50. Chymotrypsin analysis suggests that Rb induces a conformational change in the NF-kappaB-DNA complex resulting in binding of a transcriptionally inactive complex. Finally, we demonstrate by coimmunoprecipitation analysis that the Rb-p50 complex is present in Jurkat cell extracts. Our results suggest that Rb may play an important role in regulation of NF-kappaB transcriptional activity.

人类T细胞嗜淋巴细胞病毒I型（HTLV-I）与两种 人类疾病、成人T细胞白血病（ATL）和热带痉挛性 轻瘫/HTLV-I相关脊髓病（TSP/HAM）。 HTLV-I Tax 1是 从受感染的淋巴细胞中释放出来， 细胞因子，刺激基因表达和增殖， 未感染的淋巴细胞 人嗜T淋巴细胞病毒I型 （HTLV-I）Tax 1诱导细胞核内的活化和核定位。 细胞转录因子NF-κ B。 处理细胞 calphostin C是一种蛋白激酶C（PKC）抑制剂， 在HTLV-I转化的C81细胞中的NF-κ B DNA结合活性和 Tax 1刺激的小鼠前B细胞，表明PKC是一种免疫抑制剂。 NF-κ B诱导途径中的重要中间体。 这是 进一步证明Tax 1与PKC相关并激活PKC。 PKC与来自Tax 1表达细胞的抗Tax 1血清共免疫沉淀， MT4提取物和Jurkat提取物在外源性Tax 1存在下 蛋白 此外，谷胱甘肽-S-转移酶（GST）-Tax 1蛋白 与α、δ和eta PKC同工酶特异性结合 在兔网织红细胞裂解物中合成。 增加Tax 1至 体外激酶反应导致Tax 1的磷酸化， PKC自身磷酸化增加了18倍。 转染 具有野生型Tax 1刺激的膜易位的Jurkat细胞 的PKC。 相比之下，Tax 1突变体M22，它不能刺激 NF-κ B依赖性转录，未能刺激膜 PKC易位。 Tax 1不直接增加PKC I κ B α的磷酸化。 我们的研究结果与 Tax 1与PKC相互作用并刺激膜的模型 易位和触发PKC途径。 中的后续步骤 PKC级联可能刺激I κ B α磷酸化。 NF-κ B调节几种病毒和细胞基因的表达 包括HIV LTR、MHC I类和IL-2 R α细胞因子基因。 它 已经证明Rb刺激NF-κ B p50的结合 同二聚体。 添加Rb蛋白到体外凝胶位移结合 测定刺激p50结合大于10倍。 有趣的是， 在体外分析NF-κ B依赖的转录活性，我们 表明Rb抑制p50的转录活性。 糜蛋白酶分析表明，Rb诱导构象变化 在NF-κ B-DNA复合物中， 转录不活跃的复合物。 最后，我们证明了 免疫共沉淀分析表明Rb-p50复合物存在于 Jurkat细胞提取物。 我们的研究结果表明Rb可能在 在调节NF-κ B转录活性中的重要作用。