TpI2 Kinase in Macrophage Activation by Microbes

描述（由申请人提供）：我们的长期目标是剖析介导巨噬细胞中炎症介质的微生物诱导的信号通路。这一知识对于合理设计调节抗感染免疫应答以及治疗炎症和感染性休克的策略是重要的。本申请的总体目标是阐明调节蛋白激酶Tp 12的信号传导功能的分子机制。最近的遗传研究已经揭示了Tpl 2在介导由脂多糖（LPS）（来自革兰氏阴性细菌的有效炎症诱导物）引起的巨噬细胞活化中的中心作用。然而，Tpl 2的功能如何被调节仍然是未知的。本补助金申请中提出的研究是基于申请人产生的大量初步数据。我们已经证明，在巨噬细胞中，Tp 12与nfkbl基因产物p105形成潜在复合物。遗传和生物化学证据表明，Tp 12的稳定性和激酶功能都受到其伴侣p105的严格控制。有趣的是，LPS刺激的Tp 12活化与其磷酸化和从p105释放相关。我们进一步观察到LPS还刺激Tp 12的降解，这可以作为负反馈机制来防止这种关键炎症介质的不受控制的活化。基于这些发现，待检验的中心假设是Tp 12的信号传导功能受正性和负性机制调节，这涉及其通过上游信号的修饰和与p105的动态相互作用。为了实现本申请的目的，我们将追求四个具体目标：（1）定义介导Tp 12活化的生物化学机制;（2）研究Tp 12如何被靶向降解;（3）确定pl 05调节Tp 12的稳定性和功能的机制;以及（4）鉴定参与Tp 12活化的上游信号传导分子。在本研究完成时，我们期望已经确定Tp 12的信号传导功能在LPS信号传导级联中是如何被正向和负向调节的。我们还希望有新的信号分子参与这一重要的先天免疫反应。

DESCRIPTION (provided by applicant): Our long-range goal is to dissect the signaling pathways mediating microbial induction of inflammatory mediators in macrophages. This knowledge is important for rational design of strategies to modulate immune responses against infections and to treat inflammation and septic shock. The overall objective of this application is to elucidate the molecular mechanisms regulating the signaling function of a protein kinase, Tpl2. Recent genetic studies have revealed a central role for Tpl2 in mediating macrophage activation by lipopolysaccharide (LPS), a potent inflammatory elicitor from Gram-negative bacteria. However, how the function of Tpl2 is regulated remains unknown. The studies proposed in this grant application are based on a large body of preliminary data generated by the applicants. We have demonstrated that in macrophages, Tpl2 forms a latent complex with the nfkbl gene product p105. Genetic and biochemical evidence suggests that both the stability and kinase function of Tpl2 are tightly controlled by its partner p105. Interestingly, LPS-stimulated Tpl2 activation is correlated with its phosphorylation and release from p105. We have further observed that LPS also stimulates the degradation of Tpl2, which may serve as a negative feedback mechanism to prevent uncontrolled activation of this key inflammatory mediator. Based on these findings, the central hypothesis to be tested is that the signaling function of Tpl2 is regulated by both positive and negative mechanisms, which involve its modification by upstream signals and dynamic interaction with p105. To accomplish the objective of this application, we will pursue four specific aims: (1) define the biochemical mechanisms mediating Tpl2 activation; (2) investigate how Tpl2 is targeted for degradation; (3) determine the mechanisms by which p105 regulates the stability and function of Tpl2; and (4) identify upstream signaling molecules involved in Tpl2 activation. At the completion of this research, we expect to have determined how the signaling function of Tpl2 is positively and negatively regulated in the LPS signaling cascade. We also expect to have isolated novel signaling molecules participating in this important innate immune response.