The Ras-like GTPases, RalA and RalB, are key components of the oncogenic Ras signaling network. Recent evidence suggests that RalA and RalB collaborate to support tumorigenic transformation through distinct cell regulatory events. While RalA is apparently required to bypass normal restraints on cell proliferation, RalB is required to bypass normal restraints on cell survival. A direct Ral effector protein, Sec5, is a subunit of the exocyst complex, and is required to mediate RalB-dependent survival signals in transformed cells. Further analysis identified TBK1, a key mediator of the host defense response to viral challenge, as a novel Sec5 interacting protein essential for the capacity of RalB and Sec5 to deflect cell death in transformed cells. RalB activation promotes a direct interaction between Sec5 and TBK1 that results in TBK1 kinase activation via an unknown mechanism. Accordingly, both RalB and Sec5 are required for initiating host defense pathway activation upon virus infection. These observations revealed a novel relationship between molecular components of cell-autonomous innate immune signaling pathways and oncogenic transformation, and identified TBK1 as a potential target for therapeutic intervention in cancer. Here we describe details of methods, including protein complex analysis, protein kinase assays, host defense-response pathway activation, and cell transformation analysis, that can be used to investigate the contribution of the RalB-Sec5-TBK1 signaling cascade to both innate immune signaling and cell transformation.
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