Secondary literature sources for RBD

The following references were automatically generated.

Zeng J et al.
Design of inhibitors of Ras--Raf interaction using a computational combinatorial algorithm.
Protein Eng. 2001; 14: 39-45
Display abstract
Drugs that inhibit important protein-protein interactions are hard to find either by screening or rational design, at least so far. Most drugs on the market that target proteins today are therefore aimed at well-defined binding pockets in proteins. While computer-aided design is widely used to facilitate the drug discovery process for binding pockets, its application to the design of inhibitors that target the protein surface initially seems to be limited because of the increased complexity of the task. Previously, we had started to develop a computational combinatorial design approach based on the well-known 'multiple copy simultaneous search' (MCSS) procedure to tackle this problem. In order to identify sequence patterns of potential inhibitor peptides, a three-step procedure is employed: first, using MCSS, the locations of specific functional groups on the protein surface are identified; second, after constructing the peptide main chain based on the location of favorite locations of N-methylacetamide groups, functional groups corresponding to amino acid side chains are selected and connected to the main chain C(alpha) atoms; finally, the peptides generated in the second step are aligned and probabilities of amino acids at each position are calculated from the alignment scheme. Sequence patterns of potential inhibitors are determined based on the propensities of amino acids at each C(alpha) position. Here we report the optimization of inhibitor peptides using the sequence patterns determined by our method. Several short peptides derived from our prediction inhibit the Ras--Raf association in vitro in ELISA competition assays, radioassays and biosensor-based assays, demonstrating the feasibility of our approach. Consequently, our method provides an important step towards the development of novel anti-Ras agents and the structure-based design of inhibitors of protein--protein interactions.

Cliffe A
Ras vs Rap.
Trends Cell Biol. 2001; 11: 400-400

Fridman M, Walker F, Catimel B, Domagala T, Nice E, Burgess A
c-Raf-1 RBD associates with a subset of active v-H-Ras.
Biochemistry. 2000; 39: 15603-11
Display abstract
Mutational analysis of the cRaf-1 Ras binding domain (RBD) identified several point mutants with elevated Ras binding. Detailed examination of the binding kinetics of one mutant (A85K) suggests that it associates with a greater range of isomeric conformers of v-H-Ras than wt-RBD. At limiting v-H-Ras concentrations, saturation binding to A85K-RBD is higher than to wt-RBD. Notably, in assay systems where the RBD concentration is limiting, no difference exists between wt-RBD and A85K-RBD saturation levels in the presence of a sufficiently large molar excess of Ras. The inability of wt-RBD to saturate all bindable Ras/GTP (defined by its binding to A85K-RBD) suggests that Ras/GTP exists as several isoforms and that only a minority of these isoforms are capable of associating with wt-RBD. These findings provide the first experimental evidence in support of functionally distinct Ras/GTP isoforms. We also describe a novel analysis of such isoforms.

Okada T, Hu CD, Jin TG, Kariya K, Yamawaki-Kataoka Y, Kataoka T
The strength of interaction at the Raf cysteine-rich domain is a critical determinant of response of Raf to Ras family small GTPases.
Mol Cell Biol. 1999; 19: 6057-64
Display abstract
To be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-Raf, which clearly indicated that the nature of CRD determines the specificity of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.

Hu CD, Kariya K, Okada T, Qi X, Song C, Kataoka T
Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation.
J Biol Chem. 1999; 274: 48-51
Display abstract
Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152-184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.

Terada T et al.
Nuclear magnetic resonance and molecular dynamics studies on the interactions of the Ras-binding domain of Raf-1 with wild-type and mutant Ras proteins.
J Mol Biol. 1999; 286: 219-32
Display abstract
The Ras protein and its homolog, Rap1A, have an identical "effector region" (residues 32-40) preceded by Asp30-Glu31 and Glu30-Lys31, respectively. In the complex of the "Ras-like" E30D/K31E mutant Rap1A with the Ras-binding domain (RBD), residues 51-131 of Raf-1, Glu31 in Rap1A forms a tight salt bridge with Lys84 in Raf-1. However, we have recently found that Raf-1 RBD binding of Ras is indeed reduced by the E31K mutation, but is not affected by the E31A mutation. Here, the "Rap1A-like" D30E/E31K mutant of Ras was prepared and shown to bind the Raf-1 RBD less strongly than wild-type Ras, but slightly more tightly than the E31K mutant. The backbone 1H, 13C, and 15N magnetic resonances of the Raf-1 RBD were assigned in complexes with the wild-type and D30E/E31K mutant Ras proteins in the guanosine 5'-O-(beta,gamma-imidotriphosphate)-bound form. The Lys84 residue in the Raf-1 RBD exhibited a large change in chemical shift upon binding wild-type Ras, suggesting that Lys84 interacts with wild-type Ras. The D30E/E31K mutant of Ras caused nearly the same perturbations in Raf-1 chemical shifts, including that of Lys84. We hypothesized that Glu31 in Ras may not be the major salt bridge partner of Lys84 in Raf-1. A molecular dynamics simulation of a model structure of the Raf-1 RBD.Ras.GTP complex suggested that Lys84 in Raf-1 might instead form a tight salt bridge with Asp33 in Ras. Consistent with this, the D33A mutation in Ras greatly reduced its Raf-I RBD binding activity. We conclude that the major salt bridge partner of Lys84 in Raf-1 may be Asp33 in Ras.

Kimoto M, Sakamoto K, Shirouzu M, Hirao I, Yokoyama S
RNA aptamers that specifically bind to the Ras-binding domain of Raf-1.
FEBS Lett. 1998; 441: 322-6
Display abstract
RNA aptamers that bind to the Ras-binding domain (RBD) of a proto-oncogene product, Raf-1, were isolated from a pool of random sequences using a glutathione S-transferase-fused RBD (GST-RBD). The RNA molecules bind to the GST-RBD, but not to GST, with dissociation constants of about 300 nM. In contrast, these RNA aptamers do not bind to the Ras-binding domain of the RGL protein, which is also known to be activated by Ras. The aptamers actually compete with Ras for binding to the Raf-1 RBD. The anti-Raf-1 aptamers may be used to specifically inhibit the Ras-Raf interaction in the complicated signaling network in mammalian cells.

Sydor JR, Engelhard M, Wittinghofer A, Goody RS, Herrmann C
Transient kinetic studies on the interaction of Ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex.
Biochemistry. 1998; 37: 14292-9
Display abstract
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring at several hundred per second. The reversal of this step determines the rate constant for dissociation, which is on the order of 10 s-1. The lifetime of the complex is therefore on the order of 50-100 ms, which is much shorter than the lifetime of GTP at the active site of H-Ras as determined by the intrinsic GTPase reaction. This suggests that multiple interactions of a single activated Ras molecule and Raf can occur, the number being limited by the competing interaction with GAP. The GDP complex of H-Ras binds more than 2 orders of magnitude more weakly than the GTP-analogue complex, mainly due to a significant weakening of the initial binding equilibrium reaction in the GDP state, thereby avoiding even short-lived recruitment of Raf to the plasma membrane by the inactive Ras form.

Hemmings BA
PtdIns(3,4,5)P3 gets its message across.
Science. 1997; 277: 534-534

Pritchard C, McMahon M
Raf revealed in life-or-death decisions.
Nat Genet. 1997; 16: 214-5

Akashi S, Shirouzu M, Terada T, Ito Y, Yokoyama S, Takio K
Characterization of the structural difference between active and inactive forms of the Ras protein by chemical modification followed by mass spectrometric peptide mapping.
Anal Biochem. 1997; 248: 15-25
Display abstract
Ras is one of the guanosine triphosphate (GTP) binding proteins that plays a significant role in signaling events of cell growth and differentiation. It can exist in two states: guanosine diphosphate (GDP)-bound from (Ras.GDP; inactive) and GTP-bound form (Ras.GTP; active). This paper discusses the difference in tertiary structure between the active and inactive forms using the combination of chemical modification and mass spectrometry. This difference can be clearly recognized in the presence of a target protein. Raf-1 RBD (Raf-1 Ras-binding domain), as differing glycinamidation of carboxyl groups. It was possible to observe the difference between these two states using several hundred picomoles of sample. While it is true that it is difficult to obtain the whole picture of a protein by the combination of chemical modification and mass spectrometry, it is a promising approach for the characterization of surface structure using very small amounts of sample.

Monia BP
First- and second-generation antisense inhibitors targeted to human c-raf kinase: in vitro and in vivo studies.
Anticancer Drug Des. 1997; 12: 327-39

Chang HC, Rubin GM
14-3-3 epsilon positively regulates Ras-mediated signaling in Drosophila.
Genes Dev. 1997; 11: 1132-9
Display abstract
We have isolated mutations in the gene encoding a Drosophila 14-3-3 epsilon protein as suppressors of the rough eye phenotype caused by the ectopic expression of RAS1(V12). Using a simple loss-of-function 14-3-3 epsilon mutation, we show that 14-3-3 epsilon acts to increase the efficiency of RAS1 signaling. The 14-3-3 epsilon protein appears to function in multiple RTK pathways, suggesting that it is a general component of RAS1 signaling cascade. Sequence analysis of three dominant-negative alleles defines two regions of 14-3-3 epsilon that participate in RAS1 signaling. We also present evidence that 14-3-3 epsilon and 14-3-3 zeta, two 14-3-3 protein family members, are partially redundant for RAS1 signaling in photoreceptor formation and in animal viability. Our genetic data suggest that 14-3-3 epsilon functions downstream of or parallel to RAF, but upstream of nuclear factors in RAS1 signaling.

Troppmair J, Rapp UR
Apoptosis regulation by Raf, Bcl-2, and R-Ras.
Recent Results Cancer Res. 1997; 143: 245-9

de Rooij J, Bos JL
Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras.
Oncogene. 1997; 14: 623-5
Display abstract
Ras is a small GTPase that cycles between an inactive GDP-bound and an active GTP-bound form. A large variety of ligands that stimulate cell surface receptors induce the activation of Ras. Thus far, this activation could only be measured by the increase of GTP bound to Ras, which was precipitated from radio-labelled cell extract. We have used the minimal Ras-binding domain (RBD) of Raf1 (aa 51-131) to identify in vivo activated Ras. This novel method is based on the observation that RBD binds RasGTP in vitro with a Kd of 20 nM whereas the affinity between RBD and RasGDP is three orders of magnitude lower. Here we show that the Gst-RBD fusion protein precipitates transfected RasL61 (RasGTP) but not RasN17 (RasGDP) from cell lysates. In addition, we demonstrate for two different cell lines that the increase in RasGTP is reflected by an increase in Ras bound to Gst-RBD. From these results we conclude that the minimal Ras-binding domain of Raf1 is an excellent activation specific-probe for Ras.

Stokoe D, McCormick F
Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro.
EMBO J. 1997; 16: 2384-96
Display abstract
The c-Raf-1 protein kinase plays a critical role in intracellular signaling downstream from many tyrosine kinase and G-protein-linked receptors. c-Raf-1 binds to the proto-oncogene Ras in a GTP-dependent manner, but the exact mechanism of activation of c-Raf-1 by Ras is still unclear. We have established a system to study the activation of c-Raf-1 in vitro. This involves mixing membranes from cells expressing oncogenic H-RasG12V, with cytosol from cells expressing epitope-tagged full-length wild-type c-Raf-1. This results in a fraction of the c-Raf-1 binding to the membranes and a concomitant 10- to 20-fold increase in specific activity. Ras was the only component in these membranes required for activation, as purified recombinant farnesylated K-Ras.GTP, but not non-farnesylated K-Ras.GTP or farnesylated K-Ras.GDP, was able to activate c-Raf-1 to the same degree as intact H-RasG12V membranes. The most potent activation occurred under conditions in which phosphorylation was prohibited. Under phosphorylation-permissive conditions, activation of c-Raf-1 by Ras was substantially inhibited. Consistent with the results from other groups, we find that the activation of c-Raf-1 by Src in vivo occurs concomitant with tyrosine phosphorylation on c-Raf-1, and in vitro, activation of c-Raf-1 by Src requires the presence of ATP. Therefore we propose that activation of c-Raf-1 by Ras or by Src occurs through different mechanisms.

Verma IM, Stevenson J
IkappaB kinase: beginning, not the end.
Proc Natl Acad Sci U S A. 1997; 94: 11758-60

Gorman C, Skinner RH, Skelly JV, Neidle S, Lowe PN
Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf.
J Biol Chem. 1996; 271: 6713-9
Display abstract
Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected. This difference was probably due to the presence of a high percentage of inactive Raf protein. Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion. With this protein, binding with Ras could readily be measured under equilibrium conditions. The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras. The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar. The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell. In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant.

Rommel C et al.
Activated Ras displaces 14-3-3 protein from the amino terminus of c-Raf-1.
Oncogene. 1996; 12: 609-19
Display abstract
The serine/threonine protein kinase c-Raf-1 interacts with a number of cellular proteins including 14-3-3 isoforms which may be regulators or substrates of c-Raf-1 in signal transduction pathways. In vivo and in vitro binding analyses of c-Raf-1 and mutant proteins with 14-3-3 zeta indicate bivalent binding of 14-3-3 zeta to the amino terminus as well as to the carboxy terminus of c-Raf-1. Although 14-3-3 zeta and Ras use different binding regions on the amino terminal regulatory domain of c-Raf-1 (c-Raf-NT), 14-3-3 zeta is displaced from the amino terminus upon binding of activated Ras. In contrast, if c-Raf-1 full length is analysed instead of the separately expressed c-Raf-NT, binding of 14-3-3 zeta is only slightly effected by co-expression of activated Ras. This is explained by a second binding site of 14-3-3 zeta at the carboxy terminus of c-Raf-1. The mutant c-Raf-NT (S259A) cannot bind 14-3-3 zeta, suggesting a regulatory role of this in vivo phosphorylation site. However, c-Raf-NT phosphorylated or unphosphorylated at S259, is able to bind 14-3-3 zeta. Even though 14-3-3 zeta can be phosphorylated in vivo, only the unphosphorylated form binds to the amino terminus of c-Raf-1. The data presented indicate, that 14-3-3 zeta binds to c-Raf-1 in a bivalent fashion in unstimulated cells. 14-3-3 zeta is displaced from the amino terminus but not from the carboxy terminus of c-Raf-1 by binding of activated Ras to c-Raf-1.

Miyake M, Mizutani S, Koide H, Kaziro Y
Unfarnesylated transforming Ras mutant inhibits the Ras-signaling pathway by forming a stable Ras.Raf complex in the cytosol.
FEBS Lett. 1996; 378: 15-8
Display abstract
Farnesyltransferase inhibitors cause the growth arrest of ras-transformed cells, but not that of normal cells. To elucidate the mechanism of this differential effect, we examined the effect of accumulation of unfarnesylated Ras in the cytosol by using RasG12V,C186S and RasC186S, which mimic unfarnesylated form of the oncogenic and the normal Ras, respectively. We found that RasG12,C186S inhibited activation and membrane translocation of Raf by forming a stable complex with Raf in the cytosol. In contrast, RasC186S showed inhibitory effect on neither Raf activation nor Raf translocation. These results indicate that unfarnesylated oncogenic Ras interacts with Raf in the cytosol and inhibits its membrane translocation, a crucial step for the Raf activation, while unfarnesylated normal Ras does not.

Dent P et al.
Inactivation of raf-1 by a protein-tyrosine phosphatase stimulated by GTP and reconstituted by Galphai/o subunits.
J Biol Chem. 1996; 271: 3119-23
Display abstract
A membrane-associated form of Raf-1 in v-Ras transformed NIH 3T3 cells can be inactivated by protein phosphatases regulated by GTP. Herein, a distinct protein-tyrosine phosphatase (PTPase) in membrane preparations from v-Ras transformed NIH 3T3 cells was found to be activated by guanyl-5'-yl imidodiphosphate (GMPPNP) and was identified as an effector for pertussis toxin (PTx)-sensitive G-protein alpha subunits. PTPase activation was blocked by prior treatment of cells with PTx. PTPase activation by GTP, but not GMPPNP, was transient. A GMPPNP-stimulated PTPase (PTPase-G) co-purified with Galphai/o subunits during Superose 6 and Mono Q chromatography. PTPase-G activity in Superose 6 fractions from GDP-treated membranes was reconstituted by activated Galphai/o, but not G beta gamma, subunits. PTPase-G may contribute to GMPPNP-stimulated inactivation of Raf-1 in v-Ras cell membranes because Raf-1 inactivation was PTx-sensitive and PTPase-G inactivated exogenous Raf-1.

Joneson T, White MA, Wigler MH, Bar-Sagi D
Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS.
Science. 1996; 271: 810-2
Display abstract
The RAS guanine nucleotide binding proteins activate multiple signaling events that regulate cell growth and differentiation. In quiescent fibroblasts, ectopic expression of activated H-RAS (H-RASV12, where V12 indicates valine-12) induces membrane ruffling, mitogen-activated protein (MAP) kinase activation, and stimulation of DNA synthesis. A mutant of activated H-RAS, H-RASV12C40 (where C40 indicates cysteine-40), was identified that was defective for MAP kinase activation and stimulation of DNA synthesis, but retained the ability to induce membrane ruffling. Another mutant of activated H-RAS, H-RASV12S35 (where S35 indicates serine-35), which activates MAP kinase, was defective for stimulation of membrane ruffling and induction of DNA synthesis. Expression of both mutants resulted in a stimulation of DNA synthesis that was comparable to that induced by H-RASV12. These results indicate that membrane ruffling and activation of MAP kinase represent distinct RAS effector pathways and that input from both pathways is required for the mitogenic activity of RAS.

Mischak H et al.
Negative regulation of Raf-1 by phosphorylation of serine 621.
Mol Cell Biol. 1996; 16: 5409-18
Display abstract
The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

Hiwasa T, Sawada T, Sakiyama S
Synergistic induction of anchorage-independent growth of NIH3T3 mouse fibroblasts by cysteine proteinase inhibitors and a tumor promoter.
J Biol Chem. 1996; 271: 9181-4
Display abstract
We have previously reported that Ras protein is a potent cysteine proteinase inhibitor. In order to examine whether the cysteine proteinase-inhibitory activity of Ras is involved in carcinogenesis, the effects of the following probes were investigated. Cystatin alpha is a cysteine proteinase-specific inhibitor and has some amino acid sequence homology with Ras. Ras has a CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) at the carboxyl terminus, which is indispensable for the biological activity. Thus, cystatin alpha carrying a CAAX motif (cystatin alpha-CVLS) was examined. A v-Ha-Ras deletion mutant, Ras delta 42-49, has undetectable GTP binding activity, yet it retains a similar protease inhibitory activity to that of wild-type v-Ras. These genes were inserted into a eukaryotic inducible expression vector and transfected into NIH3T3 cells. The expression was effectively induced by treatment with a glucocorticoid hormone, dexamethasone. The expression of cystatin alpha-CVLS or Ras delta 42-49 alone induced neither transformation nor morphological changes. However, when their expression was induced in the presence of a tumor-promoting phorbol ester, a remarkable increase in the anchorage-independent growth was observed in cystatin alpha-CVLS- and Ras delta 42-49-transfected clones. These results suggest that cysteine proteinase inhibitors and a tumor promoter synergistically transformed NIH3T3 cells. It is thus possible that the cysteine proteinase-inhibitory activity of Ras might play a key role in the early stage of carcinogenesis.

Xu RH et al.
Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.
Proc Natl Acad Sci U S A. 1996; 93: 834-8
Display abstract
Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway.

Allard JD, Chang HC, Herbst R, McNeill H, Simon MA
The SH2-containing tyrosine phosphatase corkscrew is required during signaling by sevenless, Ras1 and Raf.
Development. 1996; 122: 1137-46
Display abstract
The sevenless gene encodes a receptor tyrosine kinase which is required for the development of the R7 photoreceptor cell in each ommatidium of the Drosophila eye. We have previously used a sensitized genetic screen to identify mutations, designated Enhancers of sevenless (E(sev)), which affect genes that encode components of the sevenless signaling pathway. Here, we report that one of these mutations, E(sev)1Ae0P is a dominantly inhibiting allele of corkscrew, which encodes an SH2 domain-containing protein tyrosine phosphatase (Perkins et al., 1992). We show that corkscrew function is essential for sevenless signaling and that expression of a membrane-targeted form of corkscrew can drive R7 photoreceptor development in the absence of sevenless function. Furthermore, we have used the dominantly inhibiting corkscrew allele to examine the role of corkscrew during signaling by activated forms of Ras1 and Raf. Our analysis indicates that corkscrew function is still required during signaling by activated forms Ras1 and Raf proteins. These results define a function for corkscrew that is either downstream of Ras1 activation or in a parallel pathway that acts with activated Ras1/Raf to specify R7 photoreceptor development.

Shinkai M et al.
Difference in the mechanism of interaction of Raf-1 and B-Raf with H-Ras.
Biochem Biophys Res Commun. 1996; 223: 729-34
Display abstract
Ras is known to possess multiple cellular targets including Raf-1. Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms. B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays. Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1. On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK. These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf.

Kolch W et al.
Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding.
Oncogene. 1996; 13: 1305-14
Display abstract
Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.

Luo Z, Tzivion G, Belshaw PJ, Vavvas D, Marshall M, Avruch J
Oligomerization activates c-Raf-1 through a Ras-dependent mechanism.
Nature. 1996; 383: 181-5
Display abstract
The c-Raf-1 proto-oncoprotein is a Ras-GTP-regulated protein kinase that associates in situ with 14-3-3 proteins, which are naturally dimeric. In COS cells, recombinant Raf is found in oligomeric assemblies. To examine whether induced oligomerization can alter Raf kinase activity, sequences encoding the FK506-binding protein FKBP12 were fused to the amino terminus of c-Raf-1, introducing a binding site for FK506. Oligomerization of recombinant FKBP-Raf in situ, induced by the addition of the dimeric FK506 derivative FK1012A, activated Raf kinase activity at least half as well as epidermal growth factor (EGF). As with EGF, activation of FKBP-Raf by FK1012A is entirely Ras-GTP dependent. Thus oligomerization of Raf per se promotes Raf activation through a Ras-dependent mechanism.

Ohtsuka T, Shimizu K, Yamamori B, Kuroda S, Takai Y
Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein.
J Biol Chem. 1996; 271: 1258-61
Display abstract
Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.

Farrar MA, Alberol-Ila, Perlmutter RM
Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization.
Nature. 1996; 383: 178-81
Display abstract
The Raf-1 serine/threonine kinase is a key component of the MAP kinase cascade, regulating both proliferation and commitment to cell fate. Raf activation is stimulated following its translocation to the plasma membrane, a process that ordinarily requires interaction with the membrane-localized GTPase, Ras-GTP. To investigate the mechanisms underlying Raf activation, we have developed a coumermycin-induced chemical dimerization method. We find that dimerization is by itself sufficient, in the absence of any membrane components, both to activate a modified Raf protein and to stimulate the MAP kinase cascade appropriately. As Ras-GTP-induced membrane localization increases the effective intracellular Raf concentration, our results indicate that homotypic oligomerization may ordinarily act to promote Raf activation in vivo.

Marshall CJ
Cell signalling. Raf gets it together.
Nature. 1996; 383: 127-8

Nishida Y et al.
The Raf/MAP kinase cascade in cell cycle regulation and differentiation in Drosophila.
Cell Struct Funct. 1996; 21: 437-44

Zhong Y
Genetic dissection of signal transduction mechanisms underlying PACAP-like neuropeptide transmission in Drosophila: synergy of cAMP and Ras/Raf pathways.
Ann N Y Acad Sci. 1996; 805: 67-79

Zhang Y, Saez R, Leal MA, Chan AM
Synergism between two growth regulatory pathways: cooperative transformation of NIH3T3 cells by G alpha 12 and c-raf-1.
Oncogene. 1996; 12: 2377-83
Display abstract
The alpha-subunit of the heterotrimeric G-protein, G12, has been shown to induce cellular transformation when overexpressed or oncogenically activated in rodent fibroblasts. To investigate the interaction between Galpha12 transforming pathway and the Ras-Raf-MAPK pathway, we examined the ability of mitogenic signaling molecules in cooperating with Galpha12 in transforming NIH3T3 fibroblasts. We observed a striking cooperative effect on focus-forming ability when Galpha12 and c-raf-1 cDNAs were co-transfected into NIH3T3 cells. NIH3T3 cells coexpressing both Galpha12 and c-raf-1 resulted in the constitutive activation of the mitogenic-activated protein kinase (MAPK). In addition, the levels of GTP-bound Ras were elevated in Galpha12 transformed NIH3T3 cells. Our results provide a model for studying the effects of simultaneous activation of two distinct growth regulatory pathways in cellular transformation.

Kahl SD, Gygi T, Eichelberger LE, Manetta JV
A multiple-approach scintillation proximity assay to measure the association between Ras and Raf.
Anal Biochem. 1996; 243: 282-3

Lee JE, Beck TW, Wojnowski L, Rapp UR
Regulation of A-raf expression.
Oncogene. 1996; 12: 1669-77
Display abstract
The Raf family proto-oncogenes encode cytoplasmic protein serine/threonine kinases which play a critical role in cell growth and development. A-raf shares several functional properties with Raf-1 including transforming activity, stimulation of the Raf/MAPK pathway and the ability of dominant negative versions to functionally block Ras signalling. A-raf transcripts are predominantly expressed in the mouse urogenital tissues. Interestingly, the human A-raf promoter region contains three potential glucocorticoid response elements GRE-1, GRE-2 and GRE-3, at positions -17, -34 and -168 respectively from the transcriptional start site. DNA sequence analysis of the mouse A-raf promoter region demonstrated that GRE-1 and -2 were conserved evolutionarily. To determine whether the human A-raf GREs represent functional motifs, an expression vector for the glucocorticoid receptor was cotransfected with A-raf promoter/reporter constructs into HeLa cells. A fivefold dexamethasone-dependent induction of A-raf promoter activity was observed using constructs containing all three GRE motifs whereas point mutations in the GREs either diminished or abolished dexamethasone induction. Electrophoretic mobility shift assays (EMSAs) using purified glucocorticoid receptor DNA binding domain (DBD) demonstrated that both GRE-2 and -3 motifs interact with DBD and oligonucleotide competition experiments established that these have different affinities for DBD. Using nuclear extracts from human and rodent cell lines in EMSAs, a specific protein-DNA complex was observed with GRE-1 which displayed binding properties unlike that of glucocorticoid receptor. These results demonstrate that the A-raf promoter is regulated in part by members of the glucocorticoid family of steroid hormone receptors and suggest a model for the regulation of A-raf expression in urogenital tissues.

Drugan JK et al.
Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation.
J Biol Chem. 1996; 271: 233-7
Display abstract
Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).

Pumiglia K, Chow YH, Fabian J, Morrison D, Decker S, Jove R
Raf-1 N-terminal sequences necessary for Ras-Raf interaction and signal transduction.
Mol Cell Biol. 1995; 15: 398-406
Display abstract
Raf-1 is a serine/threonine protein kinase that transduces signals from cell surface receptors to the nucleus. Interaction of Ras with a regulatory domain in the N-terminal half of Raf-1 is postulated to regulate Raf-1 protein kinase and signaling activities. To better understand molecular interactions of Ras with Raf-1 and regulation of the Raf-1 kinase, a panel of Raf-1 N-terminal mutants expressed in the baculovirus-insect cell system was used for mapping the precise region necessary for Ras interaction in the context of full-length, functional Raf-1 kinase. An 80-amino-acid sequence in Raf-1 between positions 53 and 132 was found to confer the ability to bind Ras protein in vitro and in infected insect cells. Deletion of residues 53 to 132 abolished Raf-1 kinase activation by Ras in insect cells, indicating that activation of the Raf-1 kinase by Ras requires the capacity to physically interact with Ras. By contrast, deletion of this Ras-binding site did not diminish activation of Raf-1 kinase by Src, implying that Src and Ras can activate Raf-1 through independent mechanisms. Significantly, Raf-1 mutants lacking the entire zinc finger motif or containing substitutions of two critical cysteine residues in the zinc finger retained the ability to bind Ras and to be activated by this interaction. Consistent with results obtained in the baculovirus-insect cell system, deletion of residues 53 to 132 but not mutations in the zinc finger motif abrogated the ability of kinase-inactive, dominant negative Raf-1 to block Ras-mediated signaling in Xenopus oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Finney R, Herrera D
Ras-Raf complexes: analyses of complexes formed in vivo.
Methods Enzymol. 1995; 255: 310-23

Zhang XF, Marshall MS, Avruch J
Ras-Raf complexes in vitro.
Methods Enzymol. 1995; 255: 323-31

Vojtek AB, Hollenberg SM
Ras-Raf interaction: two-hybrid analysis.
Methods Enzymol. 1995; 255: 331-42
Display abstract
The identification of proteins mediating Ras effects, such as the serine/threonine kinases of the Raf family, has advanced our understanding of how signals are transmitted from the extracellular milieu to the nucleus. The modified two-hybrid system has proved to be a powerful tool for identifying specific protein interactions, such as those between Ras and Raf. We hope that the insight gained from the Ras screen, as well as insights from other two hybrid screens, will prove valuable in the application of this system to other enigmatic questions in biology.

Pumiglia KM, LeVine H, Haske T, Habib T, Jove R, Decker SJ
A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase.
J Biol Chem. 1995; 270: 14251-4
Display abstract
Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.

Aitken A, Howell S, Jones D, Madrazo J, Patel Y
14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF.
J Biol Chem. 1995; 270: 5706-9
Display abstract
The 14-3-3 protein family has received considerable attention recently in the literature, because of the finding that beta and zeta isoforms interact with and activate Raf. We had previously shown that these 14-3-3 isoforms also exist as phosphorylated forms in mammalian and avian brain. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly 2-fold. In this report we show by a combination of electrospray mass spectrometry and protein microsequencing that alpha and delta are in vivo post-translationally modified forms of beta and zeta, respectively, and the site of phosphorylation, serine 185, is in a consensus sequence motif for proline-directed kinases.

Marshall M
Interactions between Ras and Raf: key regulatory proteins in cellular transformation.
Mol Reprod Dev. 1995; 42: 493-9
Display abstract
Ras proteins function during cell growth and development as essential, plasma membrane-bound signaling proteins. Current evidence suggests that Ras is part of a signal transduction chain extending from extracellular signals to transcriptional regulation in the nucleus. Growth factor and cytokine activation of many tyrosine kinase and kinase-linked receptors recruits many proteins to the plasma membrane including Ras-specific guanine nucleotide releasing proteins (GNRP). Under the influence of a GNRP, Ras proteins bind GTP, resulting in activation of the Ras signal. The GTP-bound form of Ras is capable of interacting directly with RasGAP, neurofibromin, and the Raf kinases. Although believed to be endowed with some signaling capacity, RasGAP and neurofibromin act primarily to negatively regulate Ras. Based upon genetic and biochemical studies in a variety of diverse organisms, the Raf kinases are considered the primary targets of Ras signaling. Activation of the Raf kinases is the first step in a cascade of multiple protein kinases, including Mek, Erk1, and Erk2. We are attempting to understand structurally how activated Ras proteins interact specifically with Raf kinases to induce the downstream signals necessary for cell division. Using mutagenesis, peptide epitope scanning, and in vitro reconstitution of protein interactions, we have identified specific sites of association between the Ras-GTP and c-Raf-1 proteins. The interaction between these contact points is essential for the plasma membrane localization of Raf, which ultimately leads to kinase activation. The formation of this protein complex is negatively regulated by protein kinase A (PKA) through phosphorylation of the c-Raf-1 N-terminus. Phosphorylation of c-Raf-1 serine 43 is believed to cause an N-terminal cap structure to cover the Ras docking site.

Morrison DK
Mechanisms regulating Raf-1 activity in signal transduction pathways.
Mol Reprod Dev. 1995; 42: 507-14
Display abstract
Raf-1 is a key protein involved in the transmission of developmental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Biochemical and genetic studies have demonstrated that Raf-1 functions downstream of activated tyrosine kinases and Ras and upstream of mitogen-activated protein kinase (MAPK) and MAPK kinase (MKK or MEK) in many signaling pathways. A major objective of our laboratory has been to determine how Raf-1 becomes activated in response to signaling events. Using mammalian, baculovirus, and Xenopus systems, we have examined the roles that phosphorylation and protein-protein interactions play in regulating the biological and biochemical activity of Raf-1. Our studies have provided evidence that the activity of Raf-1 can be modulated by both Ras-dependent and Ras-independent pathways. Recently, we reported that Arg89 of Raf-1 is a residue required for the association of Raf-1 and Ras. Mutation of this residue disrupted interaction with Ras and prevented Ras-mediated, but not protein kinase C-or tyrosine kinase-mediated, enzymatic activation of Raf-1 in the baculovirus expression system. Further analysis of this mutant demonstrated that kinase-defective Raf-1 proteins interfere with the propagation of proliferative and developmental signals by binding to Ras and blocking Ras function. Our findings have also shown that phosphorylation events play a role in regulating Raf-1. We have identified sites of in vivo phosphorylation that positively and negatively alter the biological and enzymatic activity of Raf-1. In addition, we have found that some of these phosphorylation sites are involved in mediating the interaction of Raf-1 with potential activators (Fyn and Src) and with other cellular proteins (14-3-3). Results from our work suggest that Raf-1 is regulated at multiple levels by several distinct mechanisms.

Suen KL, Bustelo XR, Barbacid M
Lack of evidence for the activation of the Ras/Raf mitogenic pathway by 14-3-3 proteins in mammalian cells.
Oncogene. 1995; 11: 825-31
Display abstract
We have isolated cDNA clones encoding the rodent 14-3-3 zeta and epsilon isoforms by screening bacteriophage expression libraries with a probe derived from the carboxy-terminus of the Vav oncoprotein. These isoforms, however, did not recognize the full length Vav protein under physiological conditions. In agreement with previous studies (see D Morrison, Science 266, 56-57, 1994), these 14-3-3 proteins bound very efficiently to Raf. The interaction between 14-3-3 zeta and Raf involves the central region of 14-3-3 zeta which includes a motif related to annexins. 14-3-3 zeta binds to Raf independently of Ras and forms stable ternary complexes with these two molecules. In contrast to published reports, we have observed that the catalytic activity of Raf was not activated in Raf/14-3-3 zeta immunocomplexes. Likewise, purified preparations of 14-3-3 zeta had no effect on the kinase activity of Raf immunoprecipitates. In addition, Ras activated Raf regardless of whether it was bound or not to 14-3-3 zeta. Finally, overexpression of 14-3-3 zeta cDNA clones in NIH3T3 cells did not result in detectable morphologic transformation even when co-transfected with plasmids encoding Raf and/or Ras proteins. These observations argue against a critical regulatory role of the 14-3-3 proteins in the Raf mitogenic pathway.

Morrison DK
Activation of Raf-1 by Ras in intact cells.
Methods Enzymol. 1995; 255: 301-10

Ito A, Satoh T, Kaziro Y, Itoh H
G protein beta gamma subunit activates Ras, Raf, and MAP kinase in HEK 293 cells.
FEBS Lett. 1995; 368: 183-7
Display abstract
Using transient transfection of HEK 293 cells, we have studied the activation of Ras, c-Raf, and MAP kinase by G protein-coupled receptors, activated G protein alpha subunit (G alpha), and beta gamma subunits (G beta gamma). The expression of constitutively activated Gs alpha, Gi alpha, and G11 alpha did not have any effect on MAP kinase phosphorylation. In contrast, overexpression of G beta gamma could stimulate the phosphorylation of MAP kinase and enhance the MEK kinase activity of c-Raf. Coexpression of dominant negative Ras inhibited G beta gamma-induced phosphorylation of MAP kinase. Furthermore, the GTP-bound form of Ras was increased by overexpression of G beta gamma. These results strongly suggest that the G beta gamma may play an important role in signaling from G protein-coupled receptors to the MAP kinase pathway, and the activation of Ras and c-Raf may be involved in this signaling cascade in HEK 293 cells.

Zhong Y
Mediation of PACAP-like neuropeptide transmission by coactivation of Ras/Raf and cAMP signal transduction pathways in Drosophila.
Nature. 1995; 375: 588-92
Display abstract
Much work on the signal transduction mechanisms underlying neurotransmission has been directed towards studying the roles of the cyclic AMP and phosphoinositide pathways. Upon ligand binding, the transmitter receptors interact with heterotrimeric G proteins, allowing G alpha and G beta gamma subunits to disengage. The free G alpha then modulates the activity of adenylyl cyclase and phospholipase C. It has been suggested that the G beta gamma complex which is activated through muscarinic or neuropeptide receptors can stimulate mitogen-activated protein kinase (MAPK) via activation of the small guanine-nucleotide-binding protein Ras. Sequential activation of the intermediates in the Ras/Raf serine-threonine protein kinase/MAPK kinase/MAPK/transcription factor pathway has emerged as a central mechanism for controlling cell proliferation and differentiation in yeast, worms, fruitflies and mammals. Here we show, by analysis of Drosophila mutants, that synaptic current and modulation of K+ current, triggered by a pituitary adenylyl cyclase-activating polypeptide-like neuropeptide, are mediated by coactivation of the Ras/Raf and Rutabaga-adenylyl cyclase pathways. Thus the Ras/Raf pathway also appears to be essential for G-protein-coupled neurotransmission.

Michaud NR, Fabian JR, Mathes KD, Morrison DK
14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3- and Ras-independent manner.
Mol Cell Biol. 1995; 15: 3390-7
Display abstract
Recent reports have demonstrated the in vivo association of Raf-1 with members of the 14-3-3 protein family. To address the significance of the Raf-1-14-3-3 interaction, we investigated the enzymatic activity and biological function of Raf-1 in the presence and absence of associated 14-3-3. The interaction between these two molecules was disrupted in vivo and in vitro with a combination of molecular and biochemical techniques. Biochemical studies demonstrated that the enzymatic activities of Raf-1 were equivalent in the presence and absence of 14-3-3. Furthermore, mixing of purified Raf-1 and 14-3-3 in vitro was not sufficient to activate Raf-1. With a molecular approach, Cys-165 and Cys-168 as well as Ser-259 were identified as residues of Raf-1 required for the interaction with 14-3-3. Cys-165 and Cys-168 are located within the conserved cysteine-rich region of the CR1 domain, and Ser-259 is a conserved site of serine phosphorylation found within the CR2 domain. Mutation of either Cys-165 and Cys-168 or Ser-259 prevented the stable interaction of Raf-1 with 14-3-3 in vivo. Consistent with the model in which a site of serine phosphorylation is involved in the Raf-1-14-3-3 interaction, dephosphorylated Raf-1 was unable to associate with 14-3-3 in vitro. Phosphorylation may represent a general mechanism mediating 14-3-3 binding, because dephosphorylation of the Bcr kinase (known to interact with 14-3-3) also eliminated its association with 14-3-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Burgering BM, Bos JL
Regulation of Ras-mediated signalling: more than one way to skin a cat.
Trends Biochem Sci. 1995; 20: 18-22
Display abstract
A powerful combination of genetics and biochemistry has provided details of how Ras-directed signalling interacts with and is regulated by other cellular signalling pathways. This might ultimately lead to the control of deregulated signalling by oncogenic Ras. Recently, progress has been made in understanding the regulation of Ras-mediated activation of the Raf-1-ERK2 kinase cascade through crosstalk with protein kinase C and cyclic-AMP-dependent protein kinase.

Brtva TR et al.
Two distinct Raf domains mediate interaction with Ras.
J Biol Chem. 1995; 270: 9809-12
Display abstract
A key event for Ras transformation involves the direct physical association between Ras and the Raf-1 kinase. This interaction promotes both Raf translocation to the plasma membrane and activation of Raf kinase activity. Although substantial experimental evidence has demonstrated that Raf residues 51-131 alone are sufficient for Ras binding, conflicting observations have suggested that the Raf cysteine-rich domain (residues 139-184) may also be important for interaction with Ras. To clarify the role of the Raf cysteine-rich domain in Ras-Raf binding, we have compared the ability of two distinct Raf fragments to interact with Ras using both in vitro Ras binding and in vivo Ras inhibition assays. First, we determined that both Raf sequences 2-140 and 139-186 (designated Raf-Cys) showed preferential binding to active, GTP-bound Ras in vitro. Second, we observed that Raf-Cys antagonized oncogenic Ras(Q61L)-mediated transactivation of Ras-responsive elements and focus-forming activity in NIH 3T3 cells and insulin-induced germinal vesicle breakdown in Xenopus laevis oocytes in vivo. This inhibitory activity suggests that Raf-Cys can interact with Ras in vivo. Taken together, these results suggest that Ras interaction with two distinct domains of Raf-1 may be important in Ras-mediated activation of Raf kinase activity.

Fridman M, Tikoo A, Varga M, Murphy A, Nur-E-Kamal MS, Maruta H
The minimal fragments of c-Raf-1 and NF1 that can suppress v-Ha-Ras-induced malignant phenotype.
J Biol Chem. 1994; 269: 30105-8
Display abstract
v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton. The kinase activity of c-Raf-1 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-induced malignancy. In this paper we demonstrate for the first time that the 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-binding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% reduction in the Ras binding of Raf81. The resultant 75 amino acid fragment (Raf75, residues 57-131) consists of a single alpha-helix, five anti-paralleled beta-sheets and five loops. We have found that a further deletion of either the first beta-sheet/loop or the last two beta-sheets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) does not abolish its ability to suppress the Ras-induced malignancy.

Freed E, Symons M, Macdonald SG, McCormick F, Ruggieri R
Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation.
Science. 1994; 265: 1713-6
Display abstract
To identify proteins that may participate in the activation of the protein kinase Raf, proteins that interact with Raf were selected in a two-hybrid screen. Two members of the 14-3-3 protein family were isolated that interacted with both the amino terminal regulatory regions of Raf and the kinase domain of Raf, but did not compete with the guanine nucleotide-binding protein Ras for binding to Raf. 14-3-3 proteins associated with Raf in mammalian cells and accompanied Raf to the membrane in the presence of activated Ras. In yeast cells expressing Raf and MEK, mammalian 14-3-3 beta or 14-3-3 zeta activated Raf to a similar extent as did expression of Ras. Therefore, 14-3-3 proteins may participate in or be required for the regulation of Raf function. These findings suggest a role for 14-3-3 proteins in Raf-mediated signal transduction.

Schramm K, Niehof M, Radziwill G, Rommel C, Moelling K
Phosphorylation of c-Raf-1 by protein kinase A interferes with activation.
Biochem Biophys Res Commun. 1994; 201: 740-7
Display abstract
c-Raf-1 is a serine/threonine-specific protein kinase which is regulated by phosphorylation. A putative c-AMP dependent protein kinase PKA phosphorylation site with the consensus sequence RRXS, Ser43, and a predominant phosphorylation site of c-Raf-1, Ser259, can be phosphorylated by PKA in vitro as shown by comparison of phosphopeptide maps of recombinant wild-type c-Raf-1 and the corresponding mutants. In vivo stimulation of the PKA pathway by treatment of A431 cells with Forskolin results in increase of phosphorylation in Ser43. Forskolin reduces the upshift of c-Raf-1 induced by EGF-treatment. It inhibits the EGF-activation of the c-Raf-1 protein kinase activity tested in vitro with a peptide substrate.

Saez R, Chan AM, Miki T, Aaronson SA
Oncogenic activation of human R-ras by point mutations analogous to those of prototype H-ras oncogenes.
Oncogene. 1994; 9: 2977-82
Display abstract
R-ras, K-rev-1/rap and TC21, are more closely related to prototype H-ras than any other known members of the ras superfamily. We recently isolated a mutationally activated TC21 oncogene from a human ovarian carcinoma cell line. Based upon these observations, we sought to re-examine the transforming potential of R-ras, which was reported earlier to lack transforming capacity. Mutations were introduced into the R-ras gene at codons 38 or 87, analogous to positions 12 and 61, respectively, responsible for H-ras oncogene activation. While both mutations resulted in acquisition of R-ras transforming capacity for NIH3T3 cells the position 61 was shown to be more active. Transfectants expressing either R-ras mutant formed colonies in soft agar and were tumorigenic in vivo. As has been reported for H-ras, R-ras cooperated with c-raf-1 in inducing transformation of NIH3T3 cells. These results imply interactions in R-ras and c-raf-1 signaling pathways. We observed R-ras transcripts of 4.6 and 1.2 kb ubiquitously expressed in each of a variety of tissues examined. All these findings raise the possibility that R-ras, like prototype ras genes, may be mutationally activated as an oncogene in some human malignancies.

Shirouzu M et al.
Mutations that abolish the ability of Ha-Ras to associate with Raf-1.
Oncogene. 1994; 9: 2153-7
Display abstract
Recent studies have revealed that Ras can associate physically with Raf. In the present study, we tested 34 mutants of Ha-Ras carrying substitution(s) in the region of residues 23-71 for their ability to associate with Raf-1. Mouse Ba/F3 cell lysates were incubated with each mutant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S)-bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an anti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A and A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1. All the other mutants could associate with Raf-1 with nearly the same efficiency as that of wild-type Ras. Thus, the Raf-I-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (residues 32-40) and in the region (residues 56-59) flanking the N-terminal of Switch II. The abilities to bind Raf-1 and to induce neurite outgrowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurite-inducing or transforming activities, abolishes the ability to bind Raf-1. In contrast, mutations Y32F, K42A and L53A, which impair the neurite-inducing activity of Ras, have no effect on the Ras.Raf-1 association. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R41A, V44A, L56A and T58A, which exhibit full neurite-inducing activity, and also for mutant V45E, which has no activity of neurite induction.

Avruch J, Zhang XF, Kyriakis JM
Raf meets Ras: completing the framework of a signal transduction pathway.
Trends Biochem Sci. 1994; 19: 279-83
Display abstract
The Ras oncoprotein, a GTP-activated molecular switch, interacts directly with the Raf oncoprotein to recruit the MAP kinases and their subordinates. In this way, a mitogenic signal initiated by tyrosine kinases is converted by Ras into a wave of regulatory phosphorylation on serine and threonine residues that, depending on its intensity and duration, and the variety of substrates available, results in cell differentiation or cell division.

Miwa W, Yasuda J, Yashima K, Makino R, Sekiya T
Absence of activating mutations of the RAF1 protooncogene in human lung cancer.
Biol Chem Hoppe Seyler. 1994; 375: 705-9
Display abstract
Recently, the RAF protein has been demonstrated to be a direct effector of RAS protein in a RAS-mediated signal transduction pathway. Activations of the RAF1 gene by small mutations, such as point mutations in the kinase domain and a tetrapeptide insertion into conserved region 2, have been suggested from analyses of chemically induced lung cancers in mice and by site-directed mutagenesis. We investigated the presence of small mutations of the RAF1 gene in human lung carcinomas, especially in those not carrying the mutated RAS gene, expecting that aberrations of the RAF1 gene might play a role complementary to RAS gene mutations in tumorigenesis. Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products of DNA samples from 140 patients revealed no tumor specific mutations of the RAF1 gene in any of these specimens. This result suggests that mutations of the RAF1 gene are not involved in tumorigenesis in human lung.

Ghosh S, Bell RM
Identification of discrete segments of human Raf-1 kinase critical for high affinity binding to Ha-Ras.
J Biol Chem. 1994; 269: 30785-8
Display abstract
A critical event in a signal transduction pathway downstream of receptor tyrosine kinases is the physical association of GTP-liganded Ras with the serine/threonine kinase, Raf-1. The binding of Raf-1 to Ras results in translocation of the kinase to the plasma membrane and facilitates its activation by an unknown mechanism. A deletion mutagenesis approach was employed to elucidate critical sequences in Raf-1 necessary for binding to Ras and to resolve seemingly contradictory data in the literature. While an N-terminal fragment consisting of residues 2-130 of Raf-1 was able to bind Ras, residues 131-147 were found to be critically important for conferring high affinity binding to Ras. Surprisingly, a second domain between residues 52-64 was an essential element for Raf-Ras interaction, although it did not appear to form an independent binding site for Ras. These findings may prove useful for the design of peptides or peptidomimetic drugs for the modulation of Raf-Ras interaction in neoplastic disorders.

Lovric J, Bischof O, Moelling K
Cell cycle-dependent association of Gag-Mil and hsp90.
FEBS Lett. 1994; 343: 15-21
Display abstract
Immunoprecipitated p100Gag-Mil protein kinase from MH2-transformed quail embryo fibroblasts is associated with an 89 kDa protein. The molar ratio between p89 and Gag-Mil in the immunocomplex is 0.72, indicating that the majority of Gag-Mil is complexed with p89. During mitosis part of Gag-Mil is shifted to a form with reduced electrophoretic mobility, p102Gag-Mil. Appearance of p102Gag-Mil leads to a reduced association with p89 indicating that p102 is not associated with p89. Microsequencing of p89 isolated from immunoprecipitates of Gag-Mil identified the protein as the quail homologue of chicken hsp90. Our results show that p100Gag-Mil is associated with hsp90 with a high stoichiometry and that upshifted p102Gag-Mil is released from the complex with hsp90.

Beimling P, Niehof M, Radziwill G, Moelling K
The aminoterminus of c-Raf-1 binds a protein kinase phosphorylating Ser259.
Biochem Biophys Res Commun. 1994; 204: 841-8
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The kinase negative aminoterminal domain of c-Raf-1 expressed as glutathione S-transferase fusion protein was phosphorylated in vitro after treatment with lysates from A431 cells and subsequent in vitro protein kinase assay. This phosphorylation was independent of stimulation of the cells with EGF; it occurred exclusively on serine and was mapped to Ser259. The identical site of c-Raf-1 was phosphorylated in A431 cells by metabolic labelling in vivo. The kinase binding domain was mapped by various GST-Raf deletion mutants to c-Raf-1 aminoacid residues 181 to 255.

Daum G, Eisenmann-Tappe I, Fries HW, Troppmair J, Rapp UR
The ins and outs of Raf kinases.
Trends Biochem Sci. 1994; 19: 474-80
Display abstract
Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.

Yamamoto D
Signaling mechanisms in induction of the R7 photoreceptor in the developing Drosophila retina.
Bioessays. 1994; 16: 237-44
Display abstract
The Drosophila compound eye is an excellent experimental system for analysing fate induction of identifiable single cells. Each ommatidium, a unit eye, contains eight photoreceptors (R1-R8), and the differentiation of these photoreceptors occurs in the larval eye imaginal disc in discrete steps: first R8 is determined, then R2/R5, R3/R4, R1/R6 and finally R7. Induction of R7, in particular, has been extensively studied at the molecular level. The R8 photoreceptor presents on its surface a ligand, Bride of Sevenless, that binds and activates Sevenless receptor tyrosine kinase in the R7 precursor. Autophosphorylated Sevenless initiates a Ras1-mediated cascade, which eventually activates transcription factors in the nucleus via Raf1 and MAP kinases, resulting in R7 development. However, recent studies indicate that Sevenless (Sev) functions just to neuralize the cell and has no role in R7 fate determination per se. It appears that the R7 fate may represent the lowest rung of a 'neuronal ground state', which is attained without any specific inductive cue. It is plausible that the R7 precursor is actively prevented from taking on the neuronal fate and this inhibition is removed by activation of Sev.

Kaneko Y, Tanzawa H, Sato K
The proto-oncogene C-raf-1 is highly expressed only in the hypertrophic zone of the growth plate.
Calcif Tissue Int. 1994; 54: 426-30
Display abstract
Proto-oncogene c-raf-1, the cellular homologue of the acutely transforming oncogene v-raf, has a central role in the signal transduction pathways. The growth plate, due to its non-overlapping zones of chondrocyte maturation, provides a physiological in situ model for investigating the role of c-raf-1 in proliferation and differentiation of chondrocytes. In this study, Northern blotting was first performed to examine the expression of mRNA for c-raf-1 in the embryonic chick tibial growth plate. It revealed that the normal levels of c-raf-1 mRNA were associated with the whole growth plate. We then investigated the localization of c-raf-1 mRNA and c-raf-1 protein in the growth plate by in situ hybridization and immunohistochemistry in order to determine whether c-raf-1 is involved in chondrocyte maturation. Our results showed that c-raf-1 mRNA and c-raf-1 protein were detected only in the hypertrophic zone. The data suggest involvement of this proto-oncogene in chondrocyte differentiation and/or hypertrophy rather than in proliferation.

Mihaly A, Rapp UR
Neuronal expression of the RAF protooncogene in the spinal cord of adult guinea pigs.
Acta Histochem. 1993; 94: 189-96
Display abstract
The cellular raf oncogenes encode cytoplasmic serine/threonine specific protein kinases. Since there are some indications that they are involved in neuronal plasticity, we investigated the localization of raf protein kinases immunohistochemically. The spinal cord of adult guinea pigs was fixed by means of transcardial perfusion, transverse sections were cut from each segment and stained with monoclonal and polyclonal antibodies, raised against synthetic raf peptide and recombinant raf protein, respectively. The motor neurons of the ventral horn and the large- and medium sized neurons of laminae I, IV, V, VI, VII and VII were stained in every segment of the spinal cord. The widespread occurrence of raf kinases in the spinal cord and the literature data indicating their participation in growth factor mediated processes, raise the possibility that the raf protooncogenes play a fundamental role in the regulation of transmembrane signalling of these neurons.

Murakoshi T, Hamasaki H, Ikawa Y, Urayama O
Expression of Krev-1 and c-H-ras proto-oncogenes in the rat spinal cord and the effects of noxious stimulation.
Regul Pept. 1993; 46: 370-2

Moodie SA, Willumsen BM, Weber MJ, Wolfman A
Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase.
Science. 1993; 260: 1658-61
Display abstract
The guanosine triphosphate (GTP)-binding protein Ras functions in regulating growth and differentiation; however, little is known about the protein interactions that bring about its biological activity. Wild-type Ras or mutant forms of Ras were covalently attached to an insoluble matrix and then used to examine the interaction of signaling proteins with Ras. Forms of Ras activated either by mutation (Gly12Val) or by binding of the GTP analog, guanylyl-imidodiphosphate (GMP-PNP) interacted specifically with Raf-1 whereas an effector domain mutant, Ile36Ala, failed to interact with Raf-1. Mitogen-activated protein kinase (MAP kinase) activity was only associated with activated forms of Ras. The specific interaction of activated Ras with active MAP kinase kinase (MAPKK) was confirmed by direct assays. Thus the forming of complexes containing MAPKK activity and Raf-1 protein are dependent upon the activity of Ras.

Calogeraki I, Barnier JV, Eychene A, Felder MP, Calothy G, Marx M
Genomic organization and nucleotide sequence of the coding region of the chicken c-Rmil(B-raf-1) proto-oncogene.
Biochem Biophys Res Commun. 1993; 193: 1324-31
Display abstract
c-Rmil is the cellular allele of the v-Rmil oncogene, transduced during in vitro passaging of Rous associated virus type 1 in chicken embryonic neuroretina (NR) cells. The c-Rmil proto-oncogene is the avian homolog of the mammalian B-raf gene and belongs to the mil/raf oncogene family of serine/threonine protein kinases. We recently reported that the avian c-Rmil gene encodes two proteins of 94 and 95 kDa, resulting from an alternative splicing mechanism. We describe here the exon-intron organization of the coding region of the chicken c-Rmil locus. We show that c-Rmil proteins are encoded by 19 exons lying within about 100 kbp of genomic DNA. Comparison of the organization of this gene with those of the other mil/raf genes shows strong similarities within three conserved domains previously identified in mil/raf protein kinases. However, c-Rmil contains two additional 5' coding exons that are not present in the other mil/raf genes.

Clark GJ, Quilliam LA, Hisaka MM, Der CJ
Differential antagonism of Ras biological activity by catalytic and Src homology domains of Ras GTPase activation protein.
Proc Natl Acad Sci U S A. 1993; 90: 4887-91
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Ras p120 GTPase activation protein (GAP), a cytosolic protein, is a negative mediator and potential downstream effector of Ras function. Since membrane association is critical for Ras function, we introduced the Ras membrane-targeting signal (a 19-residue peptide ending in CAAX, where C = cysteine, A = aliphatic amino acid, and X = any amino acid) onto the GAP N-terminal Src homology 2 and 3 and the C-terminal catalytic domains (designated nGAP/CAAX and cGAP/CAAX, respectively) to determine the role of membrane association in GAP function. cGAP/CAAX and full-length GAP/CAAX, but not GAP or nGAP/CAAX, exhibited potent growth inhibitory activity. Whereas both oncogenic and normal Ras activity were inhibited by cGAP/CAAX, nGAP/CAAX, despite lacking the Ras binding domain, inhibited the activity of oncogenic Ras without affecting the action of normal Ras. Altogether, these results demonstrate that membrane association potentiates GAP catalytic activity, support an effector function for GAP, and suggest that normal and oncogenic Ras possess different downstream interactions.

Winer MA, Wadewitz AG, Wolgemuth DJ
Members of the raf gene family exhibit segment-specific patterns of expression in mouse epididymis.
Mol Reprod Dev. 1993; 35: 16-23
Display abstract
The proto-oncogene c-raf-1 and the related genes A-raf and B-raf encode serine/threonine protein kinases thought to be involved in regulating gene expression by acting as part of second-messenger signaling pathways within the cell. Among the tissues in which A-raf and c-raf-1 have been shown to be expressed was mouse epididymis. The present studies were undertaken to determine if the raf family genes exhibited specificity in their pattern of expression that might be indicative of specific function in the epididymis. Northern and in situ hybridization analyses demonstrated that c-raf-1 mRNA was expressed as a 3.1 kb transcript at uniform levels throughout the length of the epididymis in all types of epididymal epithelial cells. Neither the germ cell-specific testicular transcripts nor the somatic transcripts of B-raf were detected by either Northern or in situ hybridization analysis in any region of the epididymis. A-raf, expressed as two transcripts of 2.6 and 4.3 kb, was the only gene examined which exhibited a segment-specific pattern of expression, being highest in the principal epithelial cells of the proximal caput epididymis and decreasing progressively in more distal regions of the tubule. These studies indicate that each raf gene exhibits a characteristic pattern of expression in the epididymis; A-raf in particular may play a unique regulatory role in the regionalized functions of the epididymis.