Non-Transcriptional Mechanisms of PEAK1 Action During TGFbeta-Induced EMT

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PROJECT SUMMARY Transforming Growth Factor β (TGFβ) is a secreted protein and the first member of the TGFβ su- perfamily of ligands to be described. However, the molecular mechanisms that govern TGFβ’s ability to switch between its paradoxical growth suppressing and epithelial-mesenchymal transition (EMT) promoting functions remain to be fully elucidated. Since TGFβ elicits pleiotropic functions during normal development, adult tissue homeostasis and pathophysiological processes such as cancer and fibrosis, it is essential that future research efforts focus on producing a complete mechanistic understanding of TGFβ function. To this end, the objective of this proposal is to investigate the cellular and molecular mechanisms by which PEAK1 (pseudopodium-enriched atypical kinase one) regulates TGFβ signaling and mediates TGFβ-induced EMT during disease progression. The proposed work is an extension of our recently published and compelling preliminary data showing that (i) PEAK1 mediates TGFβ-induced EMT, migration, proliferation and cancer metastasis; (ii) PEAK1 localizes to membrane actin structures and regulates Src/Grb2/MAPK signaling in response to TGFβ/fibronectin stimulation; and (iii) inhibition of PEAK1 translation blocks the pathophysiological effects of TGFβ signaling. Thus, the central hypoth- esis of this proposal is that eIF5A-driven PEAK1 translation promotes the assembly of a Src/Grb2/PEAK1 complex in the context of membrane ITGB3 activation to enable TGFβ-induced MAPK signaling, ZEB1 upregulation and EMT. The approach is innovative because it will employ a combina- tion of state-of-the-art cellular, molecular, biochemical, microscopy, proteomic and model organism methods to elucidate the mechanisms of action for the novel eIF5A/PEAK1 translation and Src/Grb2/PEAK1/MAPK cytoskeletal signaling nodes as novel regulators of TGFβ-induced EMT. Furthermore, the proposed research is significant because it will address the following two major challenges and needs within the field of TGFβ research: Specific Aim 1 will characterize translational and post-translational mechanisms of TGFβ-induced EMT; and Specific Aim 2 will identify context- dependent spatiotemporal dynamics for molecular regulators of TGFβ responses. The collective knowledge gained from these mechanistic studies will identify and characterize details of the cellular and molecular contexts in which TGFβ is dysregulated to cause disease, and novel methods for block- ing the negative consequences of TGFβ signaling.