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.
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