Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells

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Abstract
TC21 is a member of the Ras superfamily of small GTP‐binding proteins and, like Ras, has been implicated in the regulation of growth‐stimulating pathways. Point mutations introduced into TC21 based on equivalent H‐Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf‐1 and B‐Raf has been implicated in signalling pathways leading to transformation by H‐Ras, it has been argued that TC21 does not activate Raf‐1 or B‐Raf. Since the Raf‐signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf‐1 and B‐Raf are constitutively active in TC21‐transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf‐1 and B‐Raf (but not with A‐Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf‐1 is crucial for transformation.