Regulation of the Tp12 Kinase

Tp12 激酶的调节

• 批准号: 6886332
• 负责人: PHILIP N. TSICHLIS
• 金额: $ 28.97万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-09 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6886332
• 关键词: acid aminoacid ligase; biological signal transduction; cell growth regulation; enzyme activity; gene expression; genetically modified animals; laboratory mouse; lipopolysaccharides; mitogen activated protein kinase; molecular site; nuclear factor kappa beta; phosphorylation; protein kinase; protooncogene; tissue /cell culture; tumor necrosis factor alpha; ubiquitin

The proto-oncogene encoding the Tpl-2 kinase, is activated by provirus integration in retrovirus-induced rodent lymphomas and mammary adenocarcinomas. Overexpression of Tpl2 in a variety of cell types activates ERK, JNK, p38 MAPK, and the transcription factors NF-AT and NF-KB. Moreover, transgenic mice expressing a constitutively-active form of Tpl-2 under the control of a T cell specific promoter develop thymic lymphomas. Finally, Tpl2 contributes to the transduction of oncogenic signals induced by LMP1 and MyrAkt1. Our studies using Tpl2 knockout mice revealed that Tpl2 plays an obligatory role in the transduction of Toll-like receptor and death receptor signals. Ablation of Tpl2 in macrophages interferes only with signals that activate ERK, while ablation of Tpl2 in mouse embryo fibroblasts interferes with signals that activate ERK, JNK and NF-KB. Because of these signaling defects, Tpl-2 knockout mice are resistant to LPS/D-galactosamine and high dose LPS-induced shock as well as to TNF-alpha induced arthritis and inflammatory bowel disease. In unstimulated macrophages, Tpl2 is stoichiometrically bound to NF-KB 1. LPS stimulation promotes the dissociation of the two proteins. Unbound Tpl2 is active but unstable, undergoing rapid degradation via the proteasome. The proposed experiments will address the molecular mechanisms that regulate Tpl2 in response to LPS or TNF-alpha signals. These experiments will be based on several observations we have recently made: a) Tpl2 is phosphorylated at Thr290 in response to LPS. Phosphorylation promotes the LPS-induced dissociation of Tpl2 from NF-KB 1 and the activation of the Tpl2 kinase; b) Whereas wild type Tpl2 undergoes ubiquitination, kinase-inactive Tpl2 does not. Proteasome inhibitors increase the stoichiometry of Tpl2 ubiquitination and enhance signaling pathways activated by Tpl2. Finally, Tpl2 binds the E3 ubiquitin ligases TRAF6 and TRAF2 and is required for the transduction of TRAF6, and perhaps TRAF2-induced signals. These data combined, indicate that ubiquitination may contribute to Tpl2 activation by external signals. Based on this information, we designed experiments that utilize molecular genetics, tissue culture technologies to address the mechanisms of Tpl2 activation by LPS and TNF-alpha.

编码Tpl-2激酶的原癌基因在逆转录病毒诱导的啮齿动物淋巴瘤和乳腺腺癌中被前病毒整合激活。Tp 12在多种细胞类型中的过表达激活ERK、JNK、p38 MAPK以及转录因子NF-AT和NF-KB。此外，在T细胞特异性启动子的控制下表达Tpl-2的组成型活性形式的转基因小鼠发生胸腺淋巴瘤。最后，Tpl 2有助于 LMP 1和MyrAkt 1诱导的致癌信号转导。我们使用Tpl 2敲除小鼠的研究揭示了Tpl 2在Toll样受体和死亡受体信号的转导中起强制性作用。巨噬细胞中Tpl 2的消融仅干扰激活ERK的信号，而小鼠胚胎成纤维细胞中Tpl 2的消融干扰激活ERK、JNK和NF-κ B的信号。由于这些信号传导缺陷，Tpl-2敲除小鼠是 对LPS/D-半乳糖胺和高剂量LPS诱导的休克以及对TNF-α诱导的关节炎和炎性肠病具有抗性。在未刺激的巨噬细胞中，Tpl 2化学计量地结合至NF-κ Bl。LPS刺激促进两种蛋白质的解离。未结合的Tp 12是活性的但不稳定的，通过蛋白酶体经历快速降解。所提出的实验将解决调节Tpl 2的分子机制， 对LPS或TNF-α信号的反应。这些实验将基于我们最近进行的几个观察：a）Tp 12响应于LPS在Thr 290处磷酸化。磷酸化促进LPS诱导的Tp 12从NF-κ B1的解离和Tp 12激酶的活化; B）而野生型Tp 12经历泛素化，激酶失活 TPL 2没有。蛋白酶体抑制剂增加Tp 12泛素化的化学计量并增强由Tp 12激活的信号传导途径。最后，Tpl 2结合E3泛素连接酶TRAF 6和TRAF 2，并且是TRAF 6的转导所需的，并且可能是TRAF 2诱导的信号。结合这些数据，表明泛素化可能有助于Tp 12通过外部信号激活。基于这些信息，我们设计了利用分子遗传学、组织培养技术来解决LPS和TNF-α激活Tp 12的机制的实验。