Regulation of the Tp12 Kinase

在逆转录病毒诱导的啮齿动物淋巴瘤和乳腺癌中，编码TPL-2激酶的原癌基因被前病毒整合激活。Tpl2在多种类型的细胞中过表达可激活ERK、JNK、p38MAPK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，表达构成活性形式的TPL-2的转基因小鼠会患上胸腺淋巴瘤。最后，Tpl2有助于 LMP1和MyrAkt1诱导的致癌信号转导。我们用Tpl2基因敲除小鼠的研究表明，Tpl2在Toll样受体和死亡受体信号的转导中起着必须的作用。在巨噬细胞中Tpl2的消融只干扰激活ERK的信号，而在小鼠胚胎成纤维细胞中消融Tpl2则干扰激活ERK、JNK和NF-KB的信号。由于这些信号缺陷，TPL-2基因敲除小鼠 抵抗内毒素/D-氨基半乳糖和大剂量内毒素诱导的休克，以及肿瘤坏死因子-α诱导的关节炎和炎症性肠病。在未受刺激的巨噬细胞中，Tpl2按化学计量与核因子-kB1结合。内毒素刺激促进这两种蛋白的解离。未结合的Tpl2是活性的，但不稳定，通过蛋白酶体快速降解。拟议的实验将解决调控Tpl2基因的分子机制。 对内毒素或肿瘤坏死因子-α信号的反应。这些实验将基于我们最近的几个观察结果：a)Tpl2在Thr290处被磷酸化，以响应内毒素。磷酸化促进内毒素诱导的Tpl2从核因子-kB1解离并激活Tpl2；b)而野生型Tpl2经历泛素化，激酶失活 TPL2则不能。蛋白酶体抑制剂增加了Tpl2泛素化的化学计量比，并增强了Tpl2激活的信号通路。最后，Tpl2结合了E3泛素连接酶TRAF6和TRAF2，是TRAF6和TRAF2诱导的信号转导所必需的。这些数据结合在一起，表明泛素化可能有助于外部信号对Tpl2的激活。基于这一信息，我们设计了利用分子遗传学、组织培养技术来研究内毒素和肿瘤坏死因子-α激活Tpl2的机制的实验。

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.