The broad, long-term objective of this proposal is to explore the interactions of the Rac exchange factor Tiam1 with three different scaffold protein complexes. Both Tiam1 and Rac have multiple roles in cell signaling, and both have been implicated in malignant cell behaviors, including altered cell motility, invasion, apoptosis and survival. The factors regulating Tiam1 and Rac signaling specificity and the resulting effects on cellular behavior have not yet been well characterized. This proposal tests the hypothesis that by interacting with a specific scaffold protein, Tiam1 brings activated Rac into a complex with a particular downstream effector pathway, thereby contributing to Rac signaling specificity and Rac-mediated effects on cellular function. The first part of the project investigates in detail the factors that may contribute to regulating Tiam1 interaction with each scaffold protein complex, including calcium signals, phosphorylation, Ras-mediated signals, ligand stimulation, and extra-cellular matrix. Methods used here include transfection, immunoprecipitation, immunofluorescence, cell lines with inducible protein expression, and RNA interference. The results of these experiments will form the basis for future study of the specific upstream pathways promoting these associations. The second part of the project investigates the effect of directed Tiam1/Rac signaling through each scaffold protein complex on cellular migration/invasion and survival/apoptosis. This will entail additional methods including mutational analysis with reverse two hybrid and differential interaction trap techniques in yeast and engineering of stable DNA-based RNAi knock-out lines using a retroviral vector, along with transwell migration and invasion assays and apoptosis assays. This work will develop a powerful set of reagents for future study of how Tiam1 interactions with particular scaffold protein complexes affect signaling pathways downstream of Rac. Taken together, the results of this work should provide better understanding of how scaffold protein complexes direct Tiam1 and Rac signaling specificity. Determining how specific Tiam1-scaffold interactions can influence Rac-mediated cellular behavior will provide important information as to the regulation of signals governing normal and malignant cell behavior. Ultimately this may allow development of therapeutic reagents targeted specifically against Rac-mediated cancer cell pathophysiology.
| Liu, Jiewei; Xu, Kun; Chase, Maya et al. (2012) Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells. J Cell Sci 125:376-86 |
| Xu, K; Rajagopal, S; Klebba, I et al. (2010) The role of fibroblast Tiam1 in tumor cell invasion and metastasis. Oncogene 29:6533-42 |
| Rajagopal, Soumitra; Ji, Yuxin; Xu, Kun et al. (2010) Scaffold proteins IRSp53 and spinophilin regulate localized Rac activation by T-lymphocyte invasion and metastasis protein 1 (TIAM1). J Biol Chem 285:18060-71 |
| Connolly, Beth A; Rice, Jared; Feig, Larry A et al. (2005) Tiam1-IRSp53 complex formation directs specificity of rac-mediated actin cytoskeleton regulation. Mol Cell Biol 25:4602-14 |
