Oncogenic Ras Activation of Raf/Mitogen-Activated Protein Kinase-Independent Pathways Is Sufficient To Cause Tumorigenic Transformation

Abstract

Substantial evidence supports a critical role for the activation of the Raf-1/MEK/mitogen-activated protein kinase pathway in oncogenic Ras-mediated transformation. For example, dominant negative mutants of Raf-1, MEK, and mitogen-activated protein kinase all inhibit Ras transformation. Furthermore, the observation that plasma membrane-localized Raf-1 exhibits the same transforming potency as oncogenic Ras suggests that Raf-1 activation alone is sufficient to mediate full Ras transforming activity. However, the recent identification of other candidate Ras effectors (e.g., RalGDS and phosphatidylinositol-3 kinase) suggests that activation of other downstream effector-mediated signaling pathways may also mediate Ras transforming activity. In support of this, two H-Ras effector domain mutants, H-Ras(12V, 37G) and H-Ras(12V, 40C), which are defective for Raf binding and activation, induced potent tumorigenic transformation of some strains of NIH 3T3 fibroblasts. These Raf-binding defective mutants of H-Ras induced a transformed morphology that was indistinguishable from that induced by activated members of Rho family proteins. Furthermore, the transforming activities of both of these mutants were synergistically enhanced by activated Raf-1 and inhibited by the dominant negative RhoA (19N) mutant, indicating that Ras may cause transformation that occurs via coordinate activation of Raf-dependent and -independent pathways that involves Rho family proteins. Finally, cotransfection of H-Ras(12V, 37G) and H-Ras(12V, 40C) resulted in synergistic cooperation of their focus-forming activities, indicating that Ras activates at least two Raf-independent, Ras effector-mediated signaling events.Ras proteins act as molecular switches that cycle between active GTP-and inactive GDP-bound forms (reviewed in references 6 and 9) and function as essential components of signal transduction pathways that regulate cell growth and differentiation (reviewed in references 21, 36, and 51). Upon activation by ligand-stimulated receptors, activated Ras proteins transmit their signals to a cascade of serine/threonine kinases (reviewed in references 59, 64, 68, and 86). Activated Ras complexes with and promotes activation of the Raf-1 serine/threonine kinase (50,76,78,79,87). Raf-1 in turn activates mitogen-activated protein kinase (MAPK) kinases (MEK1 and MEK2), which in turn activate p42 and p44 MAPKs/extracellular signal-regulated kinases (ERKs) (16,34,41,42,61,88). Activated MAPKs then translocate into the nucleus, where they phosphorylate and activate nuclear transcription factors such as Elk-1 (30

Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation

“…The ability of RasC40 to confer TSH-independent proliferation supports earlier studies where RasC40 enhanced the proliferation of NIH3T3 ®broblasts in low serum (Khosravi-Far et al, 1996). In contrast, RasC40 failed to stimulate cell cycle progression in REF52 ®broblasts (Joneson et al, 1996) or WT19 myeloid cells (Matsuguchi and Kraft, 1998), suggesting that its mitogenic e ects are cell type-dependent.…”

Ras, but not Src, transformation of RIE-1 epithelial cells is dependent on activation of the mitogen-activated protein kinase cascade

“…Therefore, we wanted to determine if Ras activation of Rafindependent pathways alone was su cient to cause transformation. To address this possibility, we utilized two Ras e ector domain mutants, designated H-Ras (12V/37G) and H-Ras(12V/40C), that are impaired in their ability to bind and activate Raf-1 and to activate p42 and p44 MAPKs (White et al, 1995;Khosravi-Far et al, 1996). However, although these mutants were impaired in their ability to cause morphologic transformation of NIH3T3 cells, they retained the ability to promote their growth in low serum and soft agar, and to cause tumor formation in nude mice (Khosravi-Far et al, 1996).…”

“…However, it should be emphasized that both mutants are likely to retain the ability to activate other e ectors. For example, H-Ras (12V/40C) can still bind AF6 (Khosravi-Far et al, 1996), whereas H-Ras (12V/37G) can still bind Rin1 (Han et al, 1997). Thus, the usefulness of these two mutants would be for determination of whether Raf activation is necessary for Ras transformation.…”

“…First, the recent identi®cation of mutant Ras proteins which failed to bind Raf-1 [designated Ras(12V/37G) and Ras(12V/40C)], yet retained signaling activities that cause tumorigenic transformation of NIH3T3 cells, suggests that Raf-1-independent pathways are also important for promoting full Ras transformation (White et al, 1995;Khosravi-Far et al, 1996). Second, there is evidence that Ras transformation requires the function of speci®c Rho family proteins (RhoA, RhoB, Rac1 and CDC42) for full Ras transforming activity (Qiu et al, 1995a(Qiu et al, ,b, 1997Prendergast et al, 1995;Khosravi-Far et al, 1995).…”

Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol

“…The Ras oncogene also activates the MAP kinase pathway, albeit indirectly, via its down-stream e ector, Raf (reviewed in Avruch et al, 1994;Morrison and Cutler, 1997). The importance of Raf in mediating the oncogenic e ects of Ras is well documented (Leevers et al, 1994;Moodie et al, 1993;Vanaelst et al, 1993;Vojtek et al, 1993;Warne et al, 1993;reviewed in Marshall, 1995b), although other studies suggest that additional, Raf-independent events are also important in Ras-mediated transformation (Khosravi-Far et al, 1996; reviewed in Katz and McCormick, 1997). Besides Ras, other proteins that interact with Raf, and perhaps regulate it, include Mitogen-or extracellular-regulated kinase (MEK1/2); proteins of the 14-3-3 family; molecular chaperones such as HSP90 and Cdc37/p50; and the protein Kinase Suppressor of Ras (KSR) (reviewed in Morrison and Cutler, 1997).…”