Synergistic promotion of c-Src activation and cell migration by Cas and AND-34/BCAR3

The adapter molecule p130Cas (Cas) plays a role in cellular processes such as proliferation, survival, cell adhesion, and migration. The ability of Cas to promote migration has been shown to be dependent upon its carboxyl terminus, which contains a bipartite binding site for the protein tyrosine kinase c-Src (Src). The association between Src and Cas enhances Src kinase activity, and like Cas, Src plays an important role in cell proliferation and migration. In this study, we show that Src and Cas function cooperatively to promote cell migration in a manner that depends upon kinase-active Src. Another carboxyl-terminal binding partner of Cas, AND-34/BCAR3 (AND-34), functions synergistically with Cas to enhance Src activation and cell migration. The carboxyl-terminal guanine nucleotide exchange factor domain of AND-34, as well as the activity of its putative target Rap1, contribute to these events. A mechanism through which AND-34 may regulate Cas-dependent cell migration is suggested by the finding that Cas becomes redistributed from focal adhesions to lamellipodia located at the leading edge of AND-34 overexpressing cells. These data thus provide insight into how Cas and AND-34 may function together to stimulate Src signaling pathways and promote cell migration.     

AND-34/BCAR3 regulates adhesion-dependent p130Cas serine phosphorylation and breast cancer cell growth pattern

NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS-PAGE while NSP1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.     

AND-34 activates phosphatidylinositol 3-kinase and induces anti-estrogen resistance in a SH2 and GDP exchange factor-like domain-dependent manner

AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor-like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser(473) phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERalpha levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERalpha-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 micromol/L LY294002 or 10 micromol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.     

Identification and characterization of RA-GEF-2, a Rap guanine nucleotide exchange factor that serves as a downstream target of M-Ras

The Ras family small GTPase Rap is regulated by an array of specific guanine nucleotide exchange factors (GEFs) in response to upstream stimuli. RA-GEF-1 was identified as a novel Rap GEF, which possesses a Ras/Rap1-associating (RA) domain. Here we report a protein closely related to RA-GEF-1, named RA-GEF-2. Like RA-GEF-1, a putative cyclic nucleotide monophosphate-binding domain, a Ras exchanger motif, a PSD-95/DlgA/ZO-1 domain, and an RA domain in addition to the GEF catalytic domain are found in RA-GEF-2. However, RA-GEF-2 displays a different tissue distribution profile from that of RA-GEF-1. RA-GEF-2 stimulates guanine nucleotide exchange of both Rap1 and Rap2, but not Ha-Ras. The RA domain of RA-GEF-2 binds to M-Ras in a GTP-dependent manner, but not to other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, and Rheb, in contrast to the RA domain of RA-GEF-1, which specifically binds to Rap1. In accordance with this, RA-GEF-2 colocalizes with activated M-Ras in the plasma membrane in COS-7 cells, suggesting a role of RA-GEF-2 in the regulation of Rap1 and Rap2, particularly in the plasma membrane. In fact, an increase in the level of the GTP-bound form of plasma membrane-located Rap1 was observed when coexpressed with RA-GEF-2 and activated M-Ras. Thus, RA-GEF-2 acts as a GEF for Rap1 and Rap2 downstream of M-Ras in the plasma membrane, whereas RA-GEF-1 exerts Rap GEF function in perinuclear compartments including the Golgi apparatus.     

CalDAG-GEFIII activation of Ras, R-ras, and Rap1

We characterized a novel guanine nucleotide exchange factor (GEF) for Ras family G proteins that is highly homologous to CalDAG-GEFI, a GEF for Rap1 and R-Ras, and to RasGRP/CalDAG-GEFII, a GEF for Ras and R-Ras. This novel GEF, referred to as CalDAG-GEFIII, increased the GTP/GDP ratio of Ha-Ras, R-Ras, and Rap1 in 293T cells. CalDAG-GEFIII promoted the guanine nucleotide exchange of Ha-Ras, R-Ras, and Rap1 in vitro also, indicating that CalDAG-GEFIII exhibited the widest substrate specificity among the known GEFs for Ras family G proteins. Expression of CalDAG-GEFIII was detected in the glial cells of the brain and the glomerular mesangial cells of the kidney by in situ hybridization. CalDAG-GEFIII activated ERK/MAPK most efficiently, followed by CalDAG-GEFII and CalDAG-GEFI in 293T cells. JNK activation was most prominent in cells expressing CalDAG-GEFII, followed by CalDAG-GEFIII and CalDAG-GEFI. Expression of CalDAG-GEFIII induced neuronal differentiation of PC12 cells and anchorage-independent growth of Rat1A cells less efficiently than did CalDAG-GEFII. Thus, co-activation of Rap1 by CalDAG-GEFIII apparently attenuated Ras-MAPK-dependent neuronal differentiation and cellular transformation. Altogether, CalDAG-GEFIII activated a broad range of Ras family G proteins and exhibited a biological activity different from that of either CalDAG-GEFI or CalDAG-GEFII.     

Immunohistochemical study of the BCAR1/p130Cas protein in non-malignant and malignant human breast tissue

BCAR1/p130Cas is a docking protein involved in intracellular signaling pathways and in vitro resistance of estrogen-dependent breast cancer cells to antiestrogens. The BCAR1/p130Cas protein level in primary breast cancer cytosols was found to correlate with rapid recurrence of disease. A high BCAR1/p130Cas level was also associated with a higher likelihood of resistance to first-line tamoxifen treatment in patients with advanced breast cancer. Using antibodies raised against the rat p130Cas protein, we determined by immunohistochemical methods the BCAR1/p130Cas localization in primary breast carcinomas, in tumors of stromal origin, and in non-neoplastic breast tissues. The BCAR1/p130Cas protein was detected in the cytoplasm of non-malignant and neoplastic epithelial cells and in the vascular compartment of all tissue sections analyzed. Immunohistochemistry demonstrated variable intensity of BCAR1/p130Cas staining and variation in the proportion of BCAR1/p130Cas-positive epithelial tumor cells for the different breast carcinomas. Double immunohistochemical staining for BCAR1/p130Cas and estrogen receptor confirmed coexpression in non-malignant luminal epithelial cells and malignant breast tumor cells. The stromal cells in non-malignant tissues and tumor tissues as well as breast tumors of mesodermal origin did not stain for BCAR1/p130Cas. This immunohistochemical study demonstrates a variable expression of BCAR1/p130Cas in malignant and non-malignant breast epithelial cells, which may be of benefit for diagnostic purposes.     

Breast Cancer Anti-estrogen Resistance 3 (BCAR3) Protein Augments Binding of the c-Src SH3 Domain to Crk-associated Substrate (p130cas)

The focal adhesion adapter protein p130(cas) regulates adhesion and growth factor-related signaling, in part through Src-mediated tyrosine phosphorylation of p130(cas). AND-34/BCAR3, one of three NSP family members, binds the p130(cas) carboxyl terminus, adjacent to a bipartite p130(cas) Src-binding domain (SBD) and induces anti-estrogen resistance in breast cancer cell lines as well as phosphorylation of p130(cas). Only a subset of the signaling properties of BCAR3, specifically augmented motility, are dependent upon formation of the BCAR3-p130(cas) complex. Using GST pull-down and immunoprecipitation studies, we show that among NSP family members, only BCAR3 augments the ability of p130(cas) to bind the Src SH3 domain through an RPLPSPP motif in the p130(cas) SBD. Although our prior work identified phosphorylation of the serine within the p130(cas) RPLPSPP motif, mutation of this residue to alanine or glutamic acid did not alter BCAR3-induced Src SH3 domain binding to p130(cas). The ability of BCAR3 to augment Src SH3 binding requires formation of a BCAR3-p130(cas) complex because mutations that reduce association between these two proteins block augmentation of Src SH3 domain binding. Similarly, in MCF-7 cells, BCAR3-induced tyrosine phosphorylation of the p130(cas) substrate domain, previously shown to be Src-dependent, was reduced by an R743A mutation that blocks BCAR3 association with p130(cas). Immunofluorescence studies demonstrate that BCAR3 expression alters the intracellular location of both p130(cas) and Src and that all three proteins co-localize. Our work suggests that BCAR3 expression may regulate Src signaling in a BCAR3-p130(cas) complex-dependent fashion by altering the ability of the Src SH3 domain to bind the p130(cas) SBD.     

BCAR3/AND-34 can signal independent of complex formation with CAS family members or the presence of p130Cas

BCAR3 binds to the carboxy-terminus of p130Cas, a focal adhesion adapter protein. Both BCAR3 and p130Cas have been linked to resistance to anti-estrogens in breast cancer, Rac activation and cell motility. Using R743A BCAR3, a point mutant that has lost the ability to bind p130Cas, we find that BCAR3-p130Cas complex formation is not required for BCAR3-mediated anti-estrogen resistance, Rac activation or discohesion of epithelial breast cancer cells. Complex formation was also not required for BCAR3-induced lamellipodia formation in BALB/c-3T3 fibroblasts but was required for optimal BCAR3-induced motility. Although both wildtype and R743A BCAR3 induced phosphorylation of p130Cas and the related adapter protein HEF1/NEDD9, chimeric NSP3:BCAR3 experiments demonstrate that such phosphorylation does not correlate with BCAR3-induced anti-estrogen resistance or lamellipodia formation. Wildtype but not R743A BCAR3 induced lamellipodia formation and augmented cell motility in p130Cas^−/− murine embryonic fibroblasts (MEFs), suggesting that while p130Cas itself is not strictly required for these endpoints, complex formation with other CAS family members is, at least in cells lacking p130Cas. Overall, our work suggests that many, but not all, BCAR3-mediated signaling events in epithelial and mesenchymal cells are independent of p130Cas association. These studies also indicate that disruption of the BCAR3-p130Cas complex is unlikely to reverse BCAR3-mediated anti-estrogen resistance.     

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas–Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.     

Structural Insights into the Association between BCAR3 and Cas Family Members, an Atypical Complex Implicated in Anti-Oestrogen Resistance

The association between novel Src homology 2-containing protein (NSP) and Crk-associated substrate (Cas) family members contributes to integrin and receptor tyrosine kinase signalling and is involved in conferring anti-oestrogen resistance to human breast carcinomas. The precise role of this association in tumorigenesis remains controversial, and the molecular basis for the complex NSP and Cas protein form is unknown. Here we present a pluridisciplinary approach, including small-angle X-ray scattering, that provides first insights into the structure of the complex formed between breast cancer anti-oestrogen resistance 3 (BCAR3, an NSP family member) and human enhancer of filamentation 1 (HEF1, also named NEDD9 or Cas-L, a Cas family protein). Our analysis corroborates a four-helix bundle structure for the NSP-binding domain of HEF1 and a Cdc25-like guanine nucleotide exchange factor (GEF) fold for the Cas-binding domain of BCAR3. Using residues located on helix 2 of the four-helix bundle, HEF1 binds very tightly to a site on BCAR3 that is remote from the putative guanosine triphosphatase binding site of the GEF domain, but similar to a site implicated in allosteric regulation of the homologous SOS (Son of Sevenless) GEF domain. Thus, the association between NSP and Cas proteins might not only create a very stable link between these molecules, co-localising their cellular functions, but also modulate the function of the NSP GEF domains. Such modulation may explain, at least in part, the controversial results published for NSP GEF function.     

R-Ras promotes focal adhesion formation through focal adhesion kinase and p130(Cas) by a novel mechanism that differs from integrins

R-Ras regulates integrin function, but its effects on integrin signaling pathways have not been well described. We demonstrate that activation of R-Ras promoted focal adhesion formation and altered localization of the alpha2beta1 integrin from cell-cell to cell-matrix adhesions in breast epithelial cells. Constitutively activated R-Ras(38V) dramatically enhanced focal adhesion kinase (FAK) and p130(Cas) phosphorylation upon collagen stimulation or clustering of the alpha2beta1 integrin, even in the absence of increased ligand binding. Signaling events downstream of R-Ras differed from integrins and K-Ras, since pharmacological inhibition of Src or disruption of actin inhibited integrin-mediated FAK and p130(Cas) phosphorylation, focal adhesion formation, and migration in control and K-Ras(12V)-expressing cells but had minimal effect in cells expressing R-Ras(38V). Therefore, signaling from R-Ras to FAK and p130(Cas) has a component that is Src independent and not through classic integrin signaling pathways and a component that is Src dependent. R-Ras effector domain mutants and pharmacological inhibition suggest a partial role for phosphatidylinositol 3-kinase (PI3K), but not Raf, in R-Ras signaling to FAK and p130(Cas). However, PI3K cannot account for the Src-independent pathway, since simultaneous inhibition of both PI3K and Src did not completely block effects of R-Ras on FAK phosphorylation. Our results suggest that R-Ras promotes focal adhesion formation by signaling to FAK and p130(Cas) through a novel mechanism that differs from but synergizes with the alpha2beta1 integrin.     

A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.

The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.     

BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130^Cas (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.     

The R-Ras GTPase mediates cross talk between estrogen and insulin signaling in breast cancer cells

The signaling cascades activated by insulin and IGF-1 contribute to the control of multiple cellular functions, including glucose metabolism and cell proliferation. In most cases these effects are mediated, at least in part, by insulin receptor substrates (IRS), one of which is insulin receptor substrate 1 (IRS-1). R-Ras is a member of the Ras family of GTPases and is involved in a variety of biological processes, including integrin activation, cell migration, and control of cell proliferation. Here we demonstrate that both R-Ras and BCAR3, a regulator of R-Ras activity that has been implicated in breast cancer, regulate the level of IRS-1 protein in estrogen-dependent MCF-7 and ZR75 breast cancer cells. In particular, expression of a constitutively activated R-Ras mutant, R-Ras38V, or of BCAR3 accelerates the degradation of IRS-1, leading to the impairment of signaling through insulin but not epidermal growth factor receptors. Moreover, knockdown of endogenous R-Ras levels in MCF-7 cells inhibits IRS-1 degradation induced by estrogen signaling blockade but not by long-term insulin treatment. Consistent with these results, both R-Ras38V expression and estrogen signaling blockade lead to the degradation of IRS-1, at least in part, through calpain activity. These findings show that R-Ras activity mediates inhibition of insulin signaling associated with suppression of estrogen action, implicating this GTPase in a growth-inhibitory mechanism associated with antiestrogen treatment of breast cancer.     

Splice variants and expression patterns of SHEP1, BCAR3 and NSP1, a gene family involved in integrin and receptor tyrosine kinase signaling

SHEP1, BCAR3 and NSP1 are the three members of a family of cytoplasmic proteins involved in cell adhesion/migration and antiestrogen resistance. All three proteins contain an SH2 domain and an exchange factor-like domain that binds both Ras GTPases and the scaffolding protein Cas. SHEP1, BCAR3 and NSP1 mRNAs are widely expressed in tissues, and SHEP1 and BCAR3 have multiple splice variants that differ in their 5' untranslated regions and in some cases the beginning of their coding regions. Interestingly, our data suggest that SHEP1 is highly expressed in blood vessels in mouse breast cancer models. In contrast, BCAR3 and NSP1 are more highly expressed than SHEP1 in breast cancer cells. These expression patterns suggest differential roles for the three genes during breast cancer progression in either the vasculature or the tumor cells.     

Distinct mechanisms of alpha 5beta 1 integrin activation by Ha-Ras and R-Ras

To investigate the possible roles of the Ras/Rho family members in the inside-out signals to activate integrins, we examined the ability of Ras/Rho small GTPases to stimulate avidity of alpha(5)beta(1) (VLA-5) to fibronectin in bone marrow-derived mast cells. We found that both Ha-Ras(Val-12) and R-Ras(Val-38) had strong stimulatory effects on adhesion and ligand binding activity of VLA-5 to fibronectin. However, only Ha-Ras(Val-12)-, but not R-Ras(Val-38)-induced adhesion was inhibited by wortmannin, which suggests that Ha-Ras(Val-12) is dependent on phosphatidylinositol (PI) 3-kinase on adhesion whereas R-Ras(Val-38) has another PI 3-kinase independent pathway to induce adhesion. The effector loop mutant Ha-Ras(Val-12)E37G, but not Y40C retained the ability to stimulate adhesion of mast cells to fibronectin. Consistently, PI 3-kinase p110delta, predominantly expressed in mast cells, interacted with Ha-Ras(Val-12) E37G, but not Y40C, which was also correlated with the levels of Akt phosphorylation in mast cells. Furthermore, marked adhesion was induced by a membrane-targeted version of p110delta. These results indicate that Ha-Ras(Val-12) activated VLA-5 through PI 3-kinase p110delta. The mutational effects of the R-Ras effector loop region on adhesion were not correlated with PI 3-kinase activities, consistent with our contention that R-Ras has a distinct pathway to modulate avidity of VLA-5.     

Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation.