Critical function of the Ras-associating domain as a primary Ras-binding site for regulation of Saccharomyces cerevisiae adenylyl cyclase.

Mammalian candidate effectors of the small GTPase Ras, such as RalGDS, afadin/AF-6, Rin1, and phospholipase Cepsilon, have been shown to share structurally conserved modules termed Ras-associating (RA) domains at their Ras-binding sites. The Ras-binding domains of Raf-1 and phosphoinositide 3-kinase gamma (other Ras effectors) also share a similar tertiary structure with the RA domains. On the other hand, the primary Ras-binding site of Saccharomyces cerevisiae adenylyl cyclase, the best characterized Ras effector, has been mapped by mutational studies to the leucine-rich repeats (LRR) domain (amino acids 674-1300), whose structure apparently bears no resemblance to the RA domains. By a computer algorithm-based search we have unexpectedly found an RA domain in the N-terminal 81 amino acid residues (676) of the LRR domain. The purified RA-domain polypeptide exhibits an ability to bind directly to Ras in a GTP-dependent manner and to competitively inhibit Ras-dependent activation of adenylyl cyclase in vitro, with an affinity comparable with that of the whole LRR domain. The specificity of binding of the RA domain to various Ras effector region mutants is indistinguishable from that of the full-length adenylyl cyclase. The activated RAS2 (RAS2(Val-19))-dependent heat shock sensitivity of yeast cells is suppressed by overexpression of the RA domain polypeptide. Further, mutations of the RA domain abolish its Ras binding activity, and adenylyl cyclase molecules carrying these mutations are rendered unactivatable by Ras in vitro. This RA domain bears highest similarity to the Ras-binding domain of Raf-1 based on comparison of its primary and predicted secondary structures with those of other Ras effectors. These results indicate that the RA domain is a primary Ras-binding site for activation of adenylyl cyclase, implicating RA domains as universal modules for interaction of effectors with Ras, conserved from yeast to mammals.     

RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.     

Human Sin1 contains Ras-binding and pleckstrin homology domains and suppresses Ras signalling

Human Sin1 (SAPK-interacting protein 1) is a member of a conserved family of orthologous proteins that have an essential role in signal transduction in yeast and Dictyostelium. This study demonstrates that most Sin1 orthologues contain both a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain. These domains are functional in the human Sin1 protein, with the PH domain involved in lipid and membrane binding by Sin1, and the RBD able to bind activated H-and K-Ras. Sin1 and Ras co-immunoprecipitated and co-localised, showing that these proteins associate with each other in vivo. Overexpression of Sin1 inhibited the activation of ERK, Akt and JNK signalling pathways by Ras. In contrast, siRNA knockdown of endogenous Sin1 protein expression in HEK293 cells enhanced the activation of ERK1/2 by Ras. These data suggest that Sin1 is a mammalian Ras-inhibitor.     

The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.     

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.     

RA domain-mediated interaction of Cdc35 with Ras1 is essential for increasing cellular cAMP level for Candida albicans hyphal development

Many Ras GTPases activate their effectors through binding at a conserved Ras association (RA) domain. An example is the activation of the budding yeast adenylate cyclase Cyr1 by Ras1 and Ras2. Candida albicans Ras1 is speculated to similarly activate Cdc35, the orthologue of Cyr1, for hyphal development. Here, we have investigated whether the RA domain mediates Ras1-Cdc35 interaction and how this interaction regulates cAMP levels and morphogenesis. Yeast two-hybrid assays suggested that Ras1 interacts only with the RA but not any other identifiable domains of Cdc35. The Ras1-RA interaction was further confirmed by in vitro binding assays of purified RA domain and Ras1 and by co-immunoprecipitation of Ras1 and Cdc35 from cell lysates. Substituting Ala for the conserved residue K(338) or L(349) in the RA domain or deleting the RA domain abolished the Ras1-RA or Ras1-Cdc35 interactions. cdc35 mutants with the RA domain deleted or carrying K388A or L349A mutation exhibited rather normal yeast growth but were completely defective in hyphal morphogenesis. Further, the mutants contained nearly wild-type levels of cAMP during yeast growth but were unable to increase it upon hyphal induction. These results suggest an essential role for the RA-mediated Ras1-Cdc35 interaction in raising cellular cAMP levels for hyphal morphogenesis.     

Direct activation of phospholipase C-epsilon by Rho

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.     

G-protein binding features and regulation of the RalGDS family member, RGL2

RGL2 [RalGDS (Ral guanine nucleotide dissociation stimulator)-like 2] is a member of the RalGDS family that we have previously isolated and characterized as a potential effector for Ras and the Ras analogue Rap1b. The protein shares 89% sequence identity with its mouse orthologue Rlf (RalGDS-like factor). In the present study we further characterized the G-protein-binding features of RGL2 and also demonstrated that RGL2 has guanine-nucleotide-exchange activity toward the small GTPase RalA. We found that RGL2/Rlf properties are well conserved between human and mouse species. Both RGL2 and Rlf have a putative PKA (protein kinase A) phosphorylation site at the C-terminal of the domain that regulates the interaction with small GTPases. We demonstrated that RGL2 is phosphorylated by PKA and phosphorylation reduces the ability of RGL2 to bind H-Ras. As RGL2 and Rlf are unique in the RalGDS family in having a PKA site in the Ras-binding domain, the results of the present study indicate that Ras may distinguish between the different RalGDS family members by their phosphorylation by PKA.     

Association of yeast adenylyl cyclase with cyclase-associated protein CAP forms a second Ras-binding site which mediates its Ras-dependent activation

Posttranslational modification, in particular farnesylation, of Ras is crucial for activation of Saccharomyces cerevisiae adenylyl cyclase (CYR1). Based on the previous observation that association of CYR1 with cyclase-associated protein (CAP) is essential for its activation by posttranslationally modified Ras, we postulated that the associated CAP might contribute to the formation of a Ras-binding site of CYR1, which mediates CYR1 activation, other than the primary Ras-binding site, the leucine-rich repeat domain. Here, we observed a posttranslational modification-dependent association of Ras with a complex between CAP and CYR1 C-terminal region. When CAP mutants defective in Ras signaling but retaining the CYR1-binding activity were isolated by screening of a pool of randomly mutagenized CAP, CYR1 complexed with two of the obtained three mutants failed to be activated efficiently by modified Ras and exhibited a severely impaired ability to bind Ras, providing a genetic evidence for the importance of the physical association with Ras at the second Ras-binding site. On the other hand, CYR1, complexed with the other CAP mutant, failed to be activated by Ras but exhibited a greatly enhanced binding to Ras. Conversely, a Ras mutant E31K, which exhibits a greatly enhanced binding to the CYR1-CAP complex, failed to activate CYR1 efficiently. Thus, the strength of interaction at the second Ras-binding site appears to be a critical determinant of CYR1 regulation by Ras: too-weak and too-strong interactions are both detrimental to CYR1 activation. These results, taken together with those obtained with mammalian Raf, suggest the importance of the second Ras-binding site in effector regulation.     

Chemoattractant-induced Ras activation during Dictyostelium aggregation

Ras proteins are highly conserved molecular switches that regulate cellular response to external stimuli. Dictyostelium discoideum contains an extensive family of Ras proteins that function in regulation of mitosis, cytoskeletal function and motility, and the onset of development. Little is known about the events that lead to the activation of Ras proteins in Dictyostelium, primarily owing to a lack of a biochemical assay to measure the levels of activated Ras. We have adapted an assay, used successfully to measure activated Ras in mammalian cells, to monitor activation of two Dictyostelium Ras proteins, RasC and RasG. We have found that the Ras-binding domain (RBD) of mammalian Raf1 was capable of binding to the activated form of RasG, but not to the activated form of RasC; however, the RBD of Schizosaccharomyces pombe Byr2 was capable of binding preferentially to the activated forms of both RasC and RasG. Using this assay, we discovered that RasC and RasG showed a rapid and transient activation when aggregation-competent cells were stimulated with the chemoattractant cAMP, and this activation did not occur in a number of cAMP signalling mutants. These data provide further evidence of a role for both RasC and RasG in the early development of Dictyostelium.     

Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity

nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.     

Effector recognition by the small GTP-binding proteins Ras and Ral.

The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.     

Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.     

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.     

The tumor suppressor RASSF1A is a novel effector of small G protein Rap1A

Rap1A is a small G protein implicated in a spectrum of biological processes such as cell proliferation, adhesion, differentiation, and embryogenesis. The downstream effectors through which Rap1A mediates its diverse effects are largely unknown. Here we show that Rap1A, but not the related small G proteins Rap2 or Ras, binds the tumor suppressor Ras association domain family 1A (RASSF1A) in a manner that is regulated by phosphorylation of RASSF1A. Interaction with Rap1A is shown to influence the effect of RASSF1A on microtubule behavior.     

Conservation and divergence of Grb7 family of Ras-binding domains

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTR-CNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%–70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.     

Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor

Members of the plexin family are unique transmembrane receptors in that they interact directly with Rho family small GTPases; moreover, they contain a GTPase-activating protein (GAP) domain for R-Ras, which is crucial for plexin-mediated regulation of cell motility. However, the functional role and structural basis of the interactions between the different intracellular domains of plexins remained unclear. Here we present the 2.4 Å crystal structure of the complete intracellular region of human plexin-B1. The structure is monomeric and reveals that the GAP domain is folded into one structure from two segments, separated by the Rho GTPase binding domain (RBD). The RBD is not dimerized, as observed previously. Instead, binding of a conserved loop region appears to compete with dimerization and anchors the RBD to the GAP domain. Cell-based assays on mutant proteins confirm the functional importance of this coupling loop. Molecular modeling based on structural homology to p120^GAP·H-Ras suggests that Ras GTPases can bind to the plexin GAP region. Experimentally, we show that the monomeric intracellular plexin-B1 binds R-Ras but not H-Ras. These findings suggest that the monomeric form of the intracellular region is primed for GAP activity and extend a model for plexin activation.     

In vivo interaction of AF-6 with activated Ras and ZO-1.

AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c-fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.