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...

Activated Ras but not Raf can transform RIE-1 and other epithelial cells, indicating the critical importance of Raf-independent effector function in Ras transformation of epithelial cells. To elucidate the nature of these Raf-independent activities, we utilized representational difference analysis to identify genes aberrantly expressed by Ras through Raf-independent mechanisms in RIE-1 cells. We identified a total of 22 genes, both known and novel, whose expression was either activated (10) or abolished (12) by Ras but not Raf. The genes up-regulated encode proteins involved in protein or DNA synthesis, regulation of protease activity, or ligand binding, whereas those genes down-regulated encode actin cytoskeletal-, extracellular matrix-, and gap junction-associated proteins, and transmembrane receptor-or cytokine-like proteins. These results suggest that a key function of Raf-independent signaling involves deregulation of gene expression. We further characterized transgelin as a gene whose expression was abolished by Ras. Transgelin was identified previously as a protein whose expression was lost in virally transformed cell lines. We show that this loss is regulated at the level of gene expression and that both Raf-dependent and Rafindependent pathways are required to cause Ras downregulation of transgelin in RIE-1 cells, whereas Raf alone is sufficient to cause its loss in NIH 3T3 fibroblasts. We also found that Ras-dependent and Ras-independent mechanisms can cause the down-regulation of transgelin in human breast and colon carcinoma cells lines and patient-derived tumor samples. We conclude that loss of transgelin gene expression may be an important early event in tumor progression and a diagnostic marker for breast and colon cancer development.

M-Ras is a Ras-related protein that shares ϳ55% identity with K-Ras and TC21. The M-Ras message was widely expressed but was most predominant in ovary and brain. Similarly to Ha-Ras, expression of mutationally activated M-Ras in NIH 3T3 mouse fibroblasts or C2 myoblasts resulted in cellular transformation or inhibition of differentiation, respectively. M-Ras only weakly activated extracellular signal-regulated kinase 2 (ERK2), but it cooperated with Raf, Rac, and Rho to induce transforming foci in NIH 3T3 cells, suggesting that M-Ras signaled via alternate pathways to these effectors. Although the mitogen-activated protein kinase/ ERK kinase inhibitor, PD98059, blocked M-Ras-induced transformation, M-Ras was more effective than an activated mitogen-activated protein kinase/ERK kinase mutant at inducing focus formation. These data indicate that multiple pathways must contribute to M-Ras-induced transformation. M-Ras interacted poorly in a yeast two-hybrid assay with multiple Ras effectors, including c-Raf-1, A-Raf, B-Raf, phosphoinositol-3 kinase ␦, RalGDS, and Rin1. Although M-Ras coimmunoprecipitated with AF6, a putative regulator of cell junction formation, overexpression of AF6 did not contribute to fibroblast transformation, suggesting the possibility of novel effector proteins. The M-Ras GTP/GDP cycle was sensitive to the Ras GEFs, Sos1, and GRF1 and to p120 Ras GAP. Together, these findings suggest that while M-Ras is regulated by similar upstream stimuli to Ha-Ras, novel targets may be responsible for its effects on cellular transformation and differentiation.The mammalian Ras superfamily is made up of over 60 GTPases that serve as molecular switches to regulate a diverse array of cellular functions. These include intracellular signal transduction for cell growth and differentiation (Ras subfamily), regulation of the actin cytoskeleton (Rho subfamily), membrane trafficking (Rab subfamily), and nuclear transport (Ran) (1-4). The Ras subfamily consists of Ha-, Ki-, and N-Ras; Krev-1/Rap1A and -1B; Rap2A and -2B; R-Ras; TC21(R-Ras2); Ral A and B; Rheb; Dex-Ras; Rin; and Rit that share several common features outside of the core GTP-binding domain (2).The classic/prototypic Ras proteins, Ha-, Ki-, and N-Ras, transduce signals for growth and differentiation from ligand-bound receptors to the nuclear transcriptional machinery and to the cytoskeleton (2, 3, 5, 6). These proteins can be constitutively activated by point mutation, contributing to the development of a broad spectrum of human malignancies (7). The introduction of equivalent activating mutations into the closely related TC21 and R-Ras proteins also results in transformation in tissue culture models (8, 9), and TC21 mutants have been identified in human tumor cell lines (10, 11). R-Ras has also been associated with apoptosis and integrin activation (12, 13). Overexpression of Rap1A/Krev-1 can induce transformation in some cells (14) but typically has been found to counter Rasinduced activities, due to competitive binding to Ras effectors (15,16). Rheb...

No abstract

Pleckstrin homology domains are structurally conserved functional domains that can undergo both protein/protein and protein/lipid interactions. Pleckstrin homology domains can mediate inter-and intra-molecular binding events to regulate enzyme activity. They occur in numerous proteins including many that interact with Ras superfamily members, such as p120 GAP. The pleckstrin homology domain of p120 GAP is located in the NH 2 -terminal, noncatalytic region of p120 GAP. Overexpression of the noncatalytic domains of p120 GAP may modulate Ras signal transduction pathways. Here, we demonstrate that expression of the isolated pleckstrin homology domain of p120 GAP specifically inhibits Ras-mediated signaling and transformation but not normal cellular growth. Furthermore, we show that the pleckstrin homology domain binds the catalytic domain of p120 GAP and interferes with the Ras/GAP interaction. Thus, we suggest that the pleckstrin homology domain of p120 GAP may specifically regulate the interaction of Ras with p120 GAP via competitive intramolecular binding.

Oncogenic Ras and activated forms of the Ras-related protein TC21/R-Ras2 share similar abilities to alter cell proliferation. However, in contrast to Ras, we found previously that TC21 fails to activate the Raf-1 serine/ threonine kinase. Thus, TC21 must utilize non-Raf effectors to regulate cell function. In this study, we determined that TC21 interacts strongly with some (RalGDS, RGL, RGL2/Rlf, AF6, and the phosphatidylinositol 3-kinase (PI3K) catalytic subunit p110␦), and weakly with other Ras⅐GTP-binding proteins. In addition, library screening identified novel TC21-interacting proteins. We also determined that TC21, similar to Ras, mediates activation of phospholipase C⑀. We then examined if RalGDS, a RalA guanine nucleotide exchange factor, or PI3K are effectors for TC21-mediated signaling and cell proliferation in murine fibroblasts. We found that overexpression of fulllength RalGDS reduced the focus forming activity of activated TC21. Furthermore, expression of activated Ras, but not TC21, enhanced GTP loading on RalA. In fact, TC21 attenuated insulin-stimulated RalA⅐GTP formation. In contrast, like Ras, expression of activated TC21 resulted in membrane translocation and an increase in the PI3K-dependent phosphorylation of Akt, and inhibition of PI3K activity interfered with TC21 focus formation. Finally, unlike Ras, TC21 did not activate the Rac small GTPase, indicating that Ras may not activate Rac by PI3K. Taken together, these results suggest that PI3K, but not RalGDS, is an important mediator of cell proliferation by TC21.