Activation of the Ral and phosphatidylinositol 3 ' kinase signaling pathways by the ras-related protein TC21

TC21 is a member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating path ways. We have previously identified the RaE/mitogen-activated protein kinas e pathway as a direct TC21 effector pathway required for TC21-induced trans formation (M, Rosario, H. F, Paterson, and C, J, Marshall, EMBO J, 18:1270- 1279, 1999), In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the phosphat idylinositol 3 ' kinase (PI-3K) and Ral signaling pathways. Expression of c onstitutively active TC21 leads to the activation of Ral A and the PI-3K-de pendent activation of Akt/protein kinase B, Strong activation of the PI-3WA kt pathway is seen even with very low levels of TC21 expression, suggesting : that TC21 may be a key small GTPase-regulator of PI-3K. TC21-induced alte rations in cellular morphology in NIH 3T3 and PC12 cells are also PI-3K dep endent. On the other hand, activation of the Ral pathway by TC21 is require d for TC21-stimulated DNA synthesis but not transformed morphology, We show that inhibition of Ral signaling blocks DNA synthesis in human tumor cell lines containing activating mutations in TC21, demonstrating for the first time that this pathway is required for the proliferation of human tumor cel ls. Finally, we provide mechanisms for the activation of these pathways, na mely, the direct in vivo interaction of TC21 with guanine nucleotide exchan ge factors for Ral, resulting in their translocation to the plasma membrane , and the direct interaction of TC21 with PI-3K. In both cases, the effecto r domain region of TC21 is required since point mutations in this region ca n interfere with activation of downstream signaling.