The ras proto-oncogene is frequently mutated in human tumors and functions to chronically stimulate signal transduction cascades resulting in the synthesis or activation of specific transcription factors, including Ets, c-Myc, c-Jun, and nuclear factor kappa B (NF-kappaB). These Ras-responsive transcription factors are required for transformation, but the mechanisms by which these proteins facilitate oncogenesis have not been fully established. Oncogenic Ras was shown to initiate a p53-independent apoptotic response that was suppressed through the activation of NF-kappaB. These results provide an explanation for the requirement of NF-kappaB for Ras-mediated oncogenesis and provide evidence that Ras-transformed cells are susceptible to apoptosis even if they do not express the p53 tumor-suppressor gene product.

Presently, nothing is known about the function of the Ras-related protein Rheb. Since Rheb shares significant sequence identity with the core effector domains of Ras and KRev-1/Rap1A, it may share functional similarities with these two structurally related, yet functionally distinct, small GTPases. Furthermore, since like Ras, Rheb terminates with a COOH terminus that is likely to signal for farnesylation, it may be a target for the farnesyltransferase inhibitors that block Ras processing and function. To compare Rheb function with those of Ras and KRev-1, we introduced mutations into Rheb that generate constitutively active or dominant negative forms of Ras and Ras-related proteins and were designated Rheb(64L) and Rheb(20N), respectively. Expression of wild type or mutant Rheb did not alter the morphology or growth properties of NIH 3T3 cells. Thus, aberrant Rheb function is distinct from that of Ras and fails to cause cellular transformation. Instead, similar to KRev-1, co-expression of Rheb antagonized oncogenic Ras transformation and signaling. In vitro and in vivo analyses showed that like Ras, Rheb proteins are farnesylated and are sensitive to farnesyltransferase inhibition. Thus, it is possible that Rheb function may be inhibited by farnesyltransferase inhibitors treatment and, consequently, may contribute to the ability of these inhibitors to impair Ras transformation.Mutated forms of the three ras genes (H-, K-, and N-ras) are associated with 30% of all human cancers and encode potent transforming and oncogenic mutant proteins (1). Normal Ras proteins function as GDP/GTP-regulated molecular switches (2). Guanine nucleotide exchange factors (SOS and RasGRF/ CDC25) promote formation of the active, GTP-bound state (2-4), whereas GTPase activating proteins (p120-and NF1-GTPase activating proteins) promote formation of inactive, GDP-bound Ras (5). Mutated Ras proteins contain single amino acid substitutions (at residues 12, 13, or 61) that render the proteins insensitive to GTPase activating protein stimulation and, consequently, persist as constitutively activated proteins. Ras proteins serve as key intermediate relay switches in diverse signaling pathways that control cell growth and differentiation (6 -8). Consequently, mutated Ras proteins cause constitutive, ligand-independent activation of these pathways, thereby promoting to the aberrant growth of tumor cells.Ras proteins are prototypes for a large superfamily of Rasrelated proteins (Ͼ60 mammalian members) that function as GDP/GTP-regulated molecular switches (2, 6, 9). However, despite their strong amino acid sequence identity with Ras proteins (30 -55%), the majority of these small GTPases lack the potent transforming potential of Ras proteins. Exceptions include TC21/R-Ras2 (10, 11), R-Ras (12, 13), RhoA (14 -18), RhoB (19), and Rac1 (17,20), where constitutively activated versions of these Ras-related proteins can cause tumorigenic transformation of NIH 3T3 cells. The transforming activities of TC21 and R-Ras reflect the fact that these two Ras-...

Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139 -184) may be required for Raf-1 activation. The Raf-CRD is located in the NH 2 -terminal negative regulatory domain of Raf-1 and is highly homologous to cysteinerich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH 2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.Substantial genetic, biochemical, and biological evidence supports the critical role of the Raf-1 serine/threonine kinase as a key downstream effector of Ras signaling and transformation (1, 2). Ras interaction with Raf-1 promotes the activation of Raf-1 in vivo, in part by facilitating its translocation from the cytoplasm to the plasma membrane. Activated Raf-1 phosphorylates and activates the mitogen-activated protein kinase kinases (MAPK 1 kinases; also referred to as MEKs), which in turn phosphorylate and activate the p42 and p44 MAPKs. Activated MAPKs translocate to the nucleus where they regulate the activity of transcription factors such as Elk-1 (3).Ras interaction with Raf-1 alone is not sufficient to cause full activation of Raf-1, but rather binding of Ras to Raf-1 initiates other events that lead to full activation. These additional events include tyrosine (4, 5) and serine/threonine (6 -9) phosphorylation, phospholipid binding (10, 11), and interactions with other proteins that include members of the 14-3-3 protein family and 14-3-3 associated proteins (12-17). Hence, full kinase activation involves a complex multistep process that remains to be elucidated fully.An additional complexity of Ras-mediated activation of Raf-1 is that the Ras/Raf-1 interaction is more convoluted than originally believed. We and others have shown recently that Ras interacts with two distinct Ras-binding domains in the NH 2 -terminal regulatory region of 19). The first Rasbinding domain encompasses Raf-1 residues 55-131 (20, 21) and appears to interact with Ras prior to exposure of the second binding site (19). This second binding region is contained within the Raf-1 cysteine-rich domain (residues 139 -184, designated the Raf-CRD; als...