Senescence is a form of stable proliferative arrest historically associated with the exhaustion of replicative potential of cells. Activated oncogenes, such as ras, can induce senescence prematurely in young cells. Recent studies demonstrate that like apoptosis, oncogene-induced senescence is a bona fide tumor suppressing mechanism in vivo, which needs to be compromised during cancer development. However, the signaling pathways responsible for this important anti-tumorigenic response are poorly understood. Studies from our lab indicate that the p38 MAPK and its downstream substrate kinase PRAK play a key role in oncogenic ras-induced senescence and tumor suppression both in vitro and in vivo, and that PRAK is likely to be a tumor suppressor protein. Studies from the last funding period demonstrated that a multifunctional acetyltransferase Tip60 is also essential for oncogenic ras-induced senescence. Further analyses revealed a novel posttranslational modification cascade involving p38, Tip60 and PRAK, which plays an essential role in oncogenic ras-induced senescence. Upon activation by ras, p38 induces the acetyltransferase activity of Tip60 through phosphorylation of Thr158; activated Tip60, which directly interacts with PRAK, in turn induces the protein kinase activity of PRAK through acetylation of K364 in a manner that depends on phosphorylation of both Tip60 and PRAK by p38. These posttranslational modifications are critical for the pro-senescent function of Tip60 and PRAK, respectively. In the current renewal application, we propose to investigate the mechanism by which Tip60-mediated acetylation induces the activity and function of PRAK (Aims 1 and 2), and to determine the role of this novel posttranslational modification cascade in senescence induction and tumor suppression in a murine cancer model (Aim 3). In Aim 1, we will test a hypothesis that Tip60-mediated acetylation induces PRAK activity and function by enhancing p38 docking and/or by modulating its subcellular localization, 2 mechanisms that may not be mutually exclusive. In Aim 2, we will examine a possibility that Tip60-mediated acetylation disrupts the autoinhibitory conformation in the PRAK protein, leading to activation of PRAK, using biochemical and structural biology approaches. Finally in Aim 3, we will investigate the role of the p38-Tip60-PRAK cascade during senescence induction in vivo using the DMBA-induced skin carcinogenesis mouse model. These studies will likely reveal novel molecular mechanisms that regulate the activity and function of PRAK and other similar protein kinases, and provide new insights into signaling pathways mediating senescence and tumor suppression. Investigation of the p38-Tip60-PRAK cascade in vivo may lead to the development of novel cancer therapies targeting components of the senescence pathway.