and with a similar sequence fails to bind. FIG. 2. Sf9 cell-expressed Raf-1 binds to Ras but not to Rac-1 in a GTP-dependent fashion. Clarified lysates from 1.25.
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1995, p. 398–406 0270-7306/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 15, No. 1
Raf-1 N-Terminal Sequences Necessary for Ras-Raf Interaction and Signal Transduction KEVIN PUMIGLIA,1,2 YU-HUA CHOW,1 JOHN FABIAN,3 DEBORAH MORRISON,3 STUART DECKER,2 AND RICHARD JOVE1* Department of Microbiology and Immunology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 481091; Parke-Davis Pharmaceutical Research Division, Ann Arbor, Michigan 481062; and Molecular Mechanisms of Carcinogenesis Laboratory, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 217023 Received 17 August 1994/Returned for modification 16 September 1994/Accepted 13 October 1994
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. Together, these results provide evidence that the direct physical interaction of Ras with Raf-1 amino acids 53 to 132 is required for activation of the Raf-1 kinase and signaling activities by Ras but not by Src. Furthermore, the adjacent zinc finger motif in Raf-1 is not essential either for interaction with Ras or for activation of the Raf-1 kinase. is rich in cysteine residues and coordinates 2 mol of zinc (14). CR2 is a short sequence in the N-terminal half that is rich in serine and threonine residues and contains several identified sites of phosphorylation (28). CR3 contains the protein-kinase domain and spans most of the C-terminal half of the protein. The N-terminal portion of the molecule containing CR1 and CR2 is considered to be critical for normal regulation of Raf-1 activity, and mutations in this region activate the oncogenic potential of Raf-1 (18, 21, 37). In the case of oncogenic v-Raf encoded in murine sarcoma virus 3611, viral Gag sequences are fused to the C-terminal half of Raf-1, resulting in truncation of N-terminal regulatory sequences (19). Genetic evidence places the regulation of Raf downstream from Ras in eye development in D. melanogaster (8) and vulval differentiation in C. elegans (17). Meiotic maturation of Xenopus oocytes induced by the activated Tpr-Met tyrosine kinase or activated Ras mutants is blocked by expression of a kinasedefective mutant of Raf-1 (11). Raf-1 also acts downstream of Ras in mammalian cells, as evidenced by experiments with neutralizing anti-Ras antibodies (35), dominant negative Raf-1 constructs (3, 22, 46), and antisense RNA constructs (22). Recently, the connection between Ras and Raf-1 has been clarified by the observation that these two proteins physically couple (27). Studies with the yeast two-hybrid system (40, 41, 49), as well as with bacterially expressed purified proteins (42, 49), have demonstrated that Ras and Raf-1 proteins can interact directly. Furthermore, Raf-1 can be detected in anti-Ras immunoprecipitates from stimulated T cells and fibroblasts (13, 16). These data imply that Ras may activate Raf-1, either directly or indirectly, through physical interaction.
Raf-1 is a 74-kDa cytoplasmic serine/threonine protein kinase implicated in the transduction of signals from cell surface receptors to the nucleus (19, 34). Much evidence suggests that Raf-1 is poised at a key relay point in a kinase cascade that ultimately regulates cell proliferation, differentiation, and development (5, 9). Activated Raf-1 phosphorylates mitogenactivated protein kinase kinase (also known as MEK or MKK), activating its kinase activity toward mitogen-activated protein kinases (MAP kinases; also known as extracellular signal-related kinases [ERKs]) (6). Phosphorylated MAP kinases are in turn activated and phosphorylate a number of diverse substrates believed to be involved in regulation of gene expression and DNA synthesis (1). While expression of Raf-1 is ubiquitous, two other mammalian forms of Raf, A-Raf (2) and B-raf (2, 20), show more tissue-specific distribution. A-Raf expression is localized primarily in reproductive tissues, and B-raf expression is detected mainly in the brain (39). Raf homologs also have been identified in a number of nonmammalian organisms, including Xenopus laevis, Caenorhabditis elegans, and Drosophila melanogaster (17, 24, 29). Sequence analysis of the Raf isoforms and homologs reveals three distinct regions of conservation, termed conserved region 1 (CR1), CR2, and CR3 (19). CR1 is the most N-terminal of the conserved regions, corresponding to approximately amino acids 53 to 200. This region contains a zinc finger motif, which
* Corresponding author. Mailing address: Department of Microbiology and Immunology, 6606 Medical Science II, University of Michigan Medical School, Ann Arbor, MI 48109-0620. 398
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Raf-1 BINDING TO Ras
Binding of Raf-1 to Ras is dependent on the guanine nucleotide-bound state of Ras (27, 40–42, 49) and can be disrupted by mutations in the effector loop of Ras (amino acids 32 to 40); these mutations also perturb biological activity (41, 42, 49). Mutation of cysteine 168 in the zinc finger motif of Raf-1 has been reported to inhibit the Raf-Ras interaction (49). Similarly, a glutathione-S-transferase (GST)-Raf fusion protein containing the cysteine-rich region of CR1 (amino acids 128 to 196) binds to Ras (14). These findings have been interpreted to implicate the zinc finger region of the Raf-1 protein as the critical regulatory region for binding to Ras. By contrast, using the yeast two-hybrid system, other investigators (41) found several clones encoding Raf-1 sequences which were truncated prior to the zinc finger region but which interacted with Ras. From these last studies, it appears that the zinc finger region is not necessary for Ras-Raf interaction. Resolution of these conflicting findings is complicated by the fact that most of the previous experiments were done with Raf-1 fragments rather than full-length proteins. In addition, most were done with GST-Raf fragments expressed in bacteria, conditions which may not favor optimal protein conformation. To better understand the requirements for Raf-1 binding to Ras in the context of full-length functional Raf-1 kinase, we expressed in animal cells a panel of unique deletion mutants spanning the entire N-terminal portion of Raf-1. Earlier studies demonstrated the utility of the baculovirus-Sf9 insect cell system for analysis of signal transduction through the Raf-1 kinase (10, 25, 45). Using this baculovirus system, we analyzed our Raf-1 mutants for the ability to bind Ras both in vitro and in vivo. Results of our experiments demonstrate that the binding of Raf-1 to Ras requires the N-terminal portion of CR1 (residues 53 to 132). In contrast, deletion of the entire zinc finger region or targeted disruption of the zinc finger by point mutation did not abolish the ability of Raf-1 to bind Ras. Experiments involving coinfection with viruses coding for Raf-1 and its mutants together with the activators v-Ras or c-Src revealed that physical interaction of Ras and Raf-1 is necessary for activation of Raf-1 by Ras but not by Src. Furthermore, deletion of amino acids 53 to 132 (necessary for Ras binding) in a kinase-defective, dominant negative Raf-1 construct resulted in abolition of the dominant negative characteristics in developing Xenopus oocytes. Our data suggest that the N-terminal portion of CR1 is critical for binding of fulllength Raf-1 to Ras and that the physical interaction of these two proteins is essential for biologic responses downstream of Ras, but not necessarily of Src, which use the Raf-1 signaling pathway. MATERIALS AND METHODS Construction of Raf-1 mutants and vectors. A unique series of Raf-1 mutants with N-terminal deletions was generated by combining selected pairs of XhoI linker-insertion mutations in a human c-raf-1 cDNA clone (Fig. 1). Details on the construction and complete characterization of these Raf-1 mutants will be presented elsewhere (3a). The 22W cDNA was generously provided by G. M. Cooper, Harvard University, and has been described previously (37). Raf-NTE was constructed by cleaving a c-raf-1 linker insertion mutant with XhoI, filling in overhangs with the Klenow fragment of DNA polymerase I, and then digesting with BamHI. This N-terminal coding fragment of c-raf-1 was blunt-end ligated to the C-terminal coding fragment at the EcoRV site, creating a construct with a deletion of the entire C-terminal portion of Raf-1 (amino acids 290 to 633) except for the epitope for the polyclonal anti-Raf serum. All constructs were subcloned into the pBlueBacIII (Invitrogen) baculovirus transfer vector. Purified recombinant transfer vectors were transfected with wild-type Autographa californica nuclear polyhedrosis virus DNA into Sf9 insect cells. Recombinant viruses were plaque purified and their titers were determined by standard procedures (31). CRM-Raf, which contains substitutions of cysteines 165 and 168 to serine residues, was generated by site-directed mutagenesis with the full-length wildtype cRaf-1 cDNA clone and a custom oligonucleotide primer to introduce the desired base changes.
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FIG. 1. Structures of Raf-1 mutant proteins. A series of deletions or point mutations were introduced into the human Raf-1 protein. Amino acid positions of the deletions and point mutations are indicated on the right and represented schematically by the solid boxes (deletions) and asterisks (point mutations). Raf isoforms are conserved in the regions outlined and denoted CRI, CRII, and CRIII. The C-terminal epitope for the anti-Raf antibodies is represented by the sawtooth pattern. The cysteine-rich region, containing a zinc finger similar to that found in protein kinase C, is shown by the diagonal lines. All constructs were subcloned into baculovirus transfer vectors (as described in Materials and Methods), and high-titer, purified baculovirus stocks were used to express proteins in Sf9 cells. WT, wild type.
Protein expression and cell lysis. Expression of recombinant proteins in Sf9 cells was routinely done at a multiplicity of infection of 10, unless otherwise noted. Infected cells were harvested at 48 to 50 h postinfection, washed in phosphate-buffered saline, and lysed in MILY (modified insect lysis) buffer (20 mM Tris [pH 7.4], 100 mM KCl, 1 mM MgCl2, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsufonyl fluoride, 1 mM leupeptin, 1 mM antipain, 0.1 mM aprotinin, 5 mM sodium orthovanadate). Lysates were clarified by centrifugation at 12,000 3 g for 15 min prior to use in experiments. In some cases, clarified lysates were stored at 2808C without loss of activity. Immunoblotting and antibodies. Rabbit polyclonal anti-Raf antibodies were generated against a synthetic peptide corresponding to the C-terminal 12 amino acids of Raf-1 coupled to purified human immunoglobulin G. This antiserum, which will be described elsewhere (3a), is specific for Raf-1 as determined by Western blotting and competition with the synthetic peptide antigen. For immunoblotting, proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to nitrocellulose membranes electrophoretically. Membranes were blocked with 5% nonfat milk proteins and probed with either polyclonal anti-Raf or monoclonal anti-Ras (LA-069; National Cancer Institute Repository) antibodies at a dilution of 1:5,000 or a monoclonal anti-Src antibody (MAb 327; a gift from J. S. Brugge, ARIAD Pharmaceuticals) at 1:1,000. Bound antibodies were detected by the appropriate horseradish peroxidase-conjugated secondary antibodies followed by enhanced chemiluminescence (ECL; Amersham) reagents. In some experiments, quantitative analysis was performed with an AMBIS 4000 optical imaging system, which measures the band volume as defined by height 3 width 3 density. This method was determined to be linear within the linear range of film. GST-Ras construction and purification. A HindIII fragment of plasmid pBW1631 (kindly provided by D. Lowy, National Institutes of Health) encoding c-H-Ras (48) was excised and cloned into the HindIII site of the GST fusion vector pGEX-KG (a gift of K.-L. Guan and J. Dixon, University of Michigan) (15). Expression of GST-Ras fusion protein was induced with 1 mM isopropylb-D-thiogalactopyranoside (IPTG), and cells were allowed to grow for 3 h postinduction. Bacteria were lysed, and GST-Ras was purified on glutathione-agarose as previously described (15), except that 100 mM GTP was present at all steps of the purification. Protein immobilized on beads was kept in storage buffer (50 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid [HEPES; pH 7.4], 150 mM NaCl, 10% glycerol, 10 mM GTP, 0.1 mM dithiothreitol, 5 mM benzamidine) and was stable for at least 2 weeks under these conditions. The purified protein migrated as a single band on Coomassie blue-stained polyacrylamide gels. This GST-Ras fusion protein was functionally active as assessed by the ability to specifically bind [32P]GTP in a filter-binding assay. Furthermore, the addition of Sf9 cell-expressed Ras GTPase-activating protein (31) resulted in a marked increase in hydrolytic capacity toward GTP, demonstrating preservation of effector domain function in the fusion protein. GST–Rac-1 was a kind gift of R. Herrera, Parke-Davis Pharmaceutical Research Division. In vitro binding assays. Purified GST-Ras or GST alone (1 mg) immobilized on glutathione agarose was added to each tube. Beads were washed once with 10
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FIG. 2. Sf9 cell-expressed Raf-1 binds to Ras but not to Rac-1 in a GTP-dependent fashion. Clarified lysates from 1.25 3 105 Sf9 cells expressing full-length Raf-1 were incubated with purified GST-Ras (A and B) or GST–Rac-1 (B) coupled to glutathione-agarose beads. Coupled beads were preloaded with either GDP, GTP, or GTPgS, as indicated. Beads were washed, and proteins (2.5 3 104 cell equivalents per lane) were electrophoresed on 12% polyacrylamide–SDS gels, transferred to nitrocellulose, and detected by simultaneous immunoblotting with anti-Ras and anti-Raf antibodies (A) or anti-Raf antibodies alone (B). Whole-cell lysate (WCL) (one-half equivalent) (lane 4 in panel A and lane 5 in panel B) and 100 ng of purified GST-Ras (lane 4 in panel A) were included to facilitate the identification of proteins and qualitatively assess the binding. As a control, GST polypeptide alone (GST) coupled to glutathione-agarose beads was incubated with cell lysates.
volumes of loading buffer (50 mM HEPES, 5 mM EDTA [pH 7.4]), and following aspiration, beads were resuspended in 200 ml of loading buffer supplemented with 1 mM dithiothreitol, 75 mg of bovine serum albumin (BSA) per ml, and 1 mM of the appropriate guanine nucleotide. Samples were loaded with nucleotide by incubation at 308C for 15 min, and loading reactions were stopped by the addition of MgCl2 to a final concentration of 20 mM. Loading buffer was then replaced with 150 ml of binding buffer (100 mM KCl, 6.33 mM MgCl2, 20 mM Tris, 1.0 mg of BSA per ml, 25 mM ZnCl2, 0.5 mM of appropriate nucleotide). Sf9 cell lysates containing expressed Raf proteins were added in combination with MILY buffer to achieve a final volume of 200 ml. Differential expression and solubilization of Raf proteins were corrected for by adjusting the ratio of lysate and MILY buffer. Incubations were routinely carried out for 1 h at 48C and were followed by three washes, 1 ml each, with wash buffer (100 mM KCl, 6.33 mM MgCl2, 20 mM Tris, 0.25% Triton X-100). Wash buffer was completely aspirated and replaced with 40 ml of SDS sample buffer, and the mixture was boiled for 3 min. Following SDS-PAGE, samples were analyzed by Western immunoblotting for the presence of bound Raf proteins. Coimmunoprecipitation of Ras and Raf. Sf9 cells were infected with a combination of v-Ras (45) (kindly provided by N. Williams and T. Roberts, DanaFarber Cancer Institute) and the indicated Raf baculoviruses at a multiplicity of infection of 10 for each. At 48 h postinfection, cells were lysed in MILY buffer and clarified. Clarified extracts (200 ml) were incubated with 3 ml of the anti-Ras monoclonal antibody LA-069 for 1 h, and then immune complexes were collected with 25 ml of immobilized anti-mouse immunoglobulin G (Calbiochem) for 40 min. Immunoprecipitates were washed three times with MILY buffer before being resuspended in 40 ml of SDS sample buffer. Samples were analyzed by immunoblot with anti-Ras antibodies to ensure that equivalent amounts of Ras were precipitated and with anti-Raf antibodies to determine the Raf forms which coprecipitated with Ras. Equivalent expression and solubilization of Raf forms was verified by immunoblotting samples of the clarified lysates. Similar results were obtained when immunoprecipitations were done with the rat monoclonal anti-Ras antibody Y13-238 (Oncogene Science). Immune-complex kinase reactions. Sf9 cells were infected with the indicated Raf constructs at a multiplicity of infection of 5, alone or in combination with either v-Ras or c-Src (31) at multiplicities of infection of 20 (to ensure a favorable activator/Raf ratio). Clarified lysates were prepared in MILY buffer. Aliquots (200 ml) of each lysate were incubated with 2 ml of polyclonal anti-Raf antiserum for 1 h at 48C, and then the immune complexes were collected with 25 ml of protein A-Sepharose (Pharmacia). Beads were washed three times with MILY buffer and once with kinase buffer (30 mM HEPES, 7 mM MnCl2, 5 mM MgCl2, 1 mM dithiothreitol [pH 7.4]). They were then resuspended in 27 ml of kinase buffer together with 10 mCi of [32P]ATP (final concentration, 20 mM) and 300 ng of recombinant, purified MKK1 protein (7) (generously provided by P. Dent and T. Sturgill, University of Virginia). Reactions were allowed to proceed at 308C for 20 min and were then stopped by the addition of 12 ml of 43 SDS sample buffer. Samples were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose. Phosphorylation of MKK substrate was analyzed and quantified by direct counting in an AMBIS 4000 radioisotope detector followed by autoradiography with an intensifying screen at 2808C. Nitrocellulose filters from kinase assays were routinely probed with anti-Raf antibodies to ensure equivalent precipitation of Raf proteins in the kinase assays. Oocyte microinjection and analysis. The N-terminal domains of Raf-d2, Rafd3, and CRM-Raf were substituted into kinase-defective Raf-1 containing a serine-to-alanine substitution at position 621 (11) (KD/Raf-1) to generate KD/2, KD/3, and KD/CRM-Raf. These constructs were subcloned into the in vitro transcription vector pSP64T. In vitro transcribed and capped RNA was microinjected into oocytes and then scored for germinal vesicle breakdown in response to c-H-RasV12 (v-Ras) as previously described (11).
RESULTS Characterization of Raf-Ras interactions in the baculovirus system. We chose to measure in vitro Raf-Ras interactions by using Raf proteins expressed in baculovirus-infected Sf9 insect cells. This allows expression of full-length, kinase-active Raf-1 protein in the absence of endogenous mammalian Raf-1 protein. Moreover, because the Raf-1 kinase activity has been shown to be activated in Sf9 cells by coinfection with baculoviruses encoding Ras and/or Src (10, 25, 45), this system allows analysis of both physical interaction and functional activation. To date, however, the binding of Raf to Ras in infected Sf9 cells has not been fully characterized. To test the ability of the full-length Raf-1 protein to interact with Ras in vitro and to ensure the specificity of this assay, we added Sf9 cell lysates containing Raf-1 to purified GST-Ras immobilized on glutathione-agarose. Following incubation and washing, bound proteins were analyzed by SDS-PAGE and immunoblotting. This experiment involved a simultaneous blot for levels of both Raf and Ras proteins. As shown in Fig. 2A, there is an equal amount of Ras protein present in all experiments. Under the conditions used, Raf-1 bound only to GST-Ras that had been preloaded with GTP, showing no interaction with GDP-bound Ras or with GST protein alone. Furthermore, Sf9 cell lysates infected with wild-type baculovirus (A. californica nuclear polyhedrosis virus) yielded no bound Raf proteins, and there was no detectable Raf protein in insect cells (data not shown). Thus, this system replicates the interaction characteristics previously reported for Raf-Ras binding (27, 40–42, 49) and serves as a valid model to analyze in vitro binding of Raf to Ras. Previous studies described Raf-1 binding to the Ras family member Rap1b, which is conserved in the effector domain (amino acids 30 to 40) believed to be critical for Ras-Raf interaction (41, 42). However, the ability to bind other Ras family members that are less highly conserved has not been tested. To explore this possibility, we used a GST–Rac-1 construct. Rac-1 is a low-molecular-weight GTP-binding protein of the Ras family and is believed to be involved in the regulation of cytoskeleton reorganization (32, 33). This protein shows relatively good homology to Ras in the effector domain (50% identical and 70% similar). Despite this homology, Rac-1 had no capacity to bind Raf-1, even when nucleotide loading was performed with the nonhydrolyzable GTP analog GTPgS to compensate for the high intrinsic GTPase activity of Rac-1 (Fig. 2B). Thus, interaction with Raf-1 seems to be highly specific for Ras, because a protein from the same superfamily and with a similar sequence fails to bind.
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FIG. 3. Raf-1 and NTE show similar binding to GST-Ras. (A) Clarified lysates from 0.5 3 105 (lanes 2 and 7) 1.25 3 105 (lanes 3 and 8), or 2.5 3 105 (lanes 4 and 9) Sf9 cells expressing either Raf-1 or NTE were incubated with purified, GTP-loaded GST-Ras. Bound proteins were detected by immunoblotting (two-fifths of the total sample) with anti-Raf antibodies; specificity of binding was monitored by measuring binding to GST alone (lanes 5 and 10). Wholecell lysate (WL; lanes 1 and 6) from 1.25 3 104 cells was analyzed to determine the relative expression of the two proteins. (B) Quantification of Raf binding performed by densitometric analysis of immunoblots. Values have been adjusted to reflect relative differences in Raf expression (1.6-fold). Lysate from 1.25 3 105 cells is equal to 25 ml.
Full-length Raf-1 and an N-terminal fragment show comparable binding to Ras. Earlier studies (41) showed that Ras had a marked preference (27-fold) for an N-terminal fragment of Raf over the full-length Raf-1 protein in quantitative b-galactosidase assays. This raises the possibility that full-length Raf-1 must undergo an ‘‘activation’’ event to unmask the Ras-binding site. Alternatively, because the yeast two-hybrid system requires the physical interaction of two fusion proteins and the subsequent transactivation of a reporter gene, it is possible that there are differences in transactivation efficacy which are intrinsic properties of the two-hybrid constructs and are independent of the Ras-Raf interaction. To compare the relative abilities of an N-terminal fragment and full-length Raf-1 to bind GTP-Ras, we constructed a recombinant baculovirus (NTE) that encodes amino acids 1 to 290 of Raf-1 protein fused to the extreme C terminus of Raf-1 (residues 633 to 648), which is the epitope region for our anti-Raf antibodies (Fig. 1). Full-length Raf-1 and NTE were expressed in Sf9 cells and were tested for the ability to bind GTP-loaded GST-Ras. As shown in Fig. 3, incubation of both lysates resulted in a dose-dependent binding of Raf proteins to GST-Ras. In contrast to the results found in the two-hybrid system, however, densitometric quantification of the Western blots (after normalization for differences in the Raf protein levels in the lysates) revealed little difference in the ability of the two proteins to bind GST-Ras. From these experiments, it seems likely that the Ras-binding domain within Raf-1 is not sequestered under ‘‘basal’’ conditions but, rather, exists in a conformation capable of binding with high affinity to Ras. This suggests that primary regulation
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FIG. 4. Amino acids 53 to 132 are required for binding of Raf-1 to Ras in vitro. (A) Sf9 cells infected with recombinant baculoviruses encoding Raf protein were lysed in MILY buffer and clarified. Aliquots of soluble lysates were analyzed by immunoblotting for expression of Raf proteins. On the basis of quantification of immunoblots, lysates were diluted with MILY buffer to achieve approximate normalization of Raf protein levels. This immunoblot, which was performed with lysates prepared in parallel with those used in panel B, shows the typical levels of Raf proteins detected in the soluble lysates. (B) Aliquots of Sf9 cell lysates expressing Raf proteins were incubated with GTP-loaded GST-Ras (lanes 2 to 9) or GST alone (lane 1). After being washed, bound proteins were detected by immunoblotting with anti-Raf antibodies.
of the Raf-Ras interaction probably takes place at the level of GDP-GTP exchange on Ras. Nevertheless, we cannot exclude the possibility that Raf-1 expressed in Sf9 cells undergoes a constitutive ‘‘activation’’ with respect to Ras binding and that in other systems the affinity of the full-length protein is reduced. The N-terminal region of CR1 is essential for binding of Raf to GST-Ras. Previous studies with bacterially expressed fragments of Raf-1 have implicated both the N-terminal portion of CR1 and the conserved zinc finger motif in the C-terminal portion of CR1 in stable binding to Ras (14, 41, 42, 49). We sought to distinguish between contributions by these two regions of CR1 to Ras binding by using a full-length, kinaseactive version of Raf-1 and testing the ability of mutations in each region to disrupt binding. In addition, it was important to determine if any other regions of Raf-1 might significantly contribute to the binding of Ras. For this purpose, a panel of unique Raf-1 mutants with deletions spanning the N terminus (Fig. 1) were tested for the ability to bind GST-Ras in an in vitro Ras-binding assay. Infected Sf9 cell lysates expressing the various deletion mutants were corrected for differences in soluble Raf protein and incubated with GST-Ras. Bound Raf proteins were then analyzed by Western blotting with anti-Raf antibodies (Fig. 4). As previously reported for other systems, we found binding only to Raf proteins containing the N-terminal portion of the protein (40, 41). Analysis of the N-terminal deletion constructs revealed only two constructs, Raf-d2 and Raf-d3, which were not able to bind Ras. Raf-d2 codes for a protein lacking amino acids 53 to 132; Raf-d3 removes amino acids 53 to 205. Thus, these results demonstrate that the sequence between residues 53 and 132 is strictly required for Ras-Raf interaction. Interestingly, Raf-d1 reproducibly showed enhanced binding; perhaps as a result of perturbation around serine 43, which has been implicated in regulating Ras-Raf interactions (47). An-
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other construct, Raf-d4, lacking the entire cysteine-rich motif, reproducibly bound to GTP-loaded GST-Ras, implying that the zinc finger is not necessary for Raf-Ras interactions. Furthermore, because the cysteine-rich motif was present in the Raf-d2 mutant that failed to bind, it can be stated that the zinc finger motif is neither necessary nor sufficient to mediate the binding of Raf to Ras in vitro. Raf-d4 did show partially reduced in vitro binding, however, suggesting that there may be sequences in the C-terminal portion of CR1 that contribute to the binding of Raf to Ras. Alternatively, the deletion in Raf-d4 may in some way adversely affect the local protein conformation such that the adjacent region necessary for Ras binding, i.e., residues 53 to 132, is disrupted. Coprecipitation of Raf-1 with Ras requires amino acids 53 to 132 but not the cysteine-rich region. We wished to extend our in vitro studies of the requirements for Raf-1 interaction with Ras to an in vivo assay. Previous experiments have shown that it is possible to coprecipitate Raf-1 molecules with antiRas antibodies from cells containing activated Ras (13, 16). Therefore, coinfection and coprecipitation experiments were done to determine if the domains found to be necessary in the in vitro binding assay were necessary in vivo. These experiments were also a prerequisite to the assessment of functional Raf-1 activation in later experiments (see below). To assay in vivo binding, Sf9 cells were infected with a virus encoding v-Ras in combination with Raf viruses coding for Raf-1, Rafd2, Raf-d4, or CRM-Raf. The latter construct contains a double point mutation at critical cysteine residues (analogous to those made previously in protein kinase C to disrupt phorbol ester binding [30]) to more directly target the zinc finger and to reduce the risk (30) of disrupting other potentially important elements in the CR1 region. Lysates from infected cells were immunoprecipitated with anti-Ras (LA-069) antibodies, which react with an epitope outside the Ras effector domain, and then probed for the presence of coprecipitating Raf proteins (Fig. 5). Infection with viruses encoding either v-Ras alone or Raf-1 alone resulted in no detectable coprecipitation of Raf-1, demonstrating the specificity of the assay and the absence of endogenous Raf and Ras proteins. When cells were coinfected with Raf-1 and v-Ras viruses, however, Raf-1 protein was coprecipitated with Ras. In agreement with our findings in the in vitro binding assay, deletion of the region encoding amino acids 53 to 132 (Raf-d2) abolished coprecipitation of Raf with Ras. Conversely, neither deletion of the entire C-terminal portion of CR1 (Raf-d4) nor targeted disruption of the zinc finger (CRMRaf) abolished the ability to coprecipitate Raf with anti-Ras antibodies. As observed in the in vitro studies described above, coprecipitation of the Raf-d4 mutant was partially reduced. Significantly, the CRM-Raf mutant bound equally well compared with wild-type Raf-1, suggesting that the zinc finger motif is not essential for high-affinity in vivo interactions between Raf-1 and activated Ras. Physical interaction of Raf and Ras is necessary for activation of Raf-1 by v-Ras. Although it is well established that Ras and Raf-1 can physically interact, the evidence that this interaction is important for the activation of Raf-1 is indirect. It is possible that Raf-1 activation proceeds through a mechanism quite distinct from physical interaction with Ras. For example, it could be argued that binding of Raf-1 to Ras is important in the negative-feedback regulation of Ras, because Raf-1 has been shown to have Ras GTPase-stimulating activity in addition to the ability to compete with GTPase-activating protein for interactions with Ras (42, 49). Raf-1 can be activated in Sf9 cells by coexpressing either Raf and Ras or Raf and Src (10, 25, 45). We therefore took advantage of this system, in combina-
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FIG. 5. The N-terminal region of CRI, but not the zinc finger, is essential for coprecipitation of Raf with Ras. Sf9 cells were infected with baculoviruses encoding v-Ras and Raf-1 or the indicated Raf-1 mutants, either alone (lanes 1 and 2) or in combination (lanes 3 to 6). Ras protein was precipitated by incubating clarified cell lysates from 1.25 3 105 cells with anti-Ras antibody (LA-069). (A) Aliquots were analyzed by immunoblotting with anti-Raf antibodies for the presence of coprecipitating Raf proteins (top panel) or with anti-Ras antibodies (middle panel) to ensure equivalent precipitation of Ras protein. Whole-cell (WC) lysates (2.5 3 104 cells per lane) from Sf9 cells expressing both Ras and Raf proteins (coinfections only) were probed with anti-Raf antibodies to ensure equivalent expression of Raf-1 and Raf mutants (bottom panel, lanes 3 to 6). IP, immunoprecipitate; IgG, immunoglobulin heavy chain. (B) Densitometric quantification of the relative amounts of Raf proteins coprecipitating with Ras.
tion with selected Raf-1 mutants, to directly test whether the activation by Src or Ras required the direct interaction of Ras and Raf-1. Anti-Raf immunoprecipitates were tested for the ability to phosphorylate purified recombinant MKK substrate (7) in immune-complex kinase reactions. In these experiments (Fig. 6), activation of Raf kinase (three- to fivefold) was seen when wild-type Raf-1 virus was coinfected with either v-Ras or c-Src virus. Coinfection with virus encoding the Raf-d2 mutant, which fails to bind Ras both in vitro and in vivo (see above), resulted in a complete abrogation of the activation by Ras. In striking contrast, Raf-d2 exhibited no significant loss of activation by Src in comparison with wild-type Raf-1. Because the zinc finger has been implicated as a critical region for Raf-1 function, we also tested the ability of the mutant with the zinc finger point mutation, CRM-Raf, to be activated. Surprisingly, this mutant showed normal activation in response to both Src and Ras. Together, these results demonstrate that (i) physical interaction between Raf-1 and Ras is necessary for functional activation of the Raf-1 kinase activity, (ii) Ras and Src can activate Raf-1 by distinct mechanisms, and (iii) the zinc finger
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FIG. 7. Effect of Raf mutations on dominant negative properties of kinasedefective Raf in Xenopus oocytes. Oocytes were given preinjections of 30 ng of capped transcripts encoding WT-Raf, KD/Raf-1, or mutant KD/Raf proteins. After 8 h, oocytes were given injections of 15 ng of capped transcript coding for c-H-RasV12. At 18 to 24 h postinjection, oocytes were scored for germinal vesicle breakdown (GVBD). The data represent the percentage of the c-H-RasV12induced GVBD, as determined from three independent experiments. For controls, the overall percentage of oocytes undergoing GVBD in response to c-HRasV12 alone was 91%; microinjection of a transcript encoding wild-type Raf-1 produced no GVBD.
FIG. 6. Activation by Src and Ras of in vitro kinase activity of Raf-1 and mutant Raf proteins. (A) Sf9 cells were infected with recombinant baculoviruses encoding Raf-1 or mutant Raf proteins, either alone or in combination with viruses coding for c-Src or v-Ras, as indicated. Expressed proteins in lysates from 1.25 3 105 cells were precipitated with anti-Raf antibodies, and an in vitro immune-complex kinase reaction was performed, with recombinant, purified MKK-1 (chemically inactivated by FSBA treatment) as a substrate. Reactions were terminated, and proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. Nitrocellulose filters were routinely probed with anti-Raf antibodies to ensure equivalent precipitation of Raf proteins (results not shown). (B) In vitro kinase assays were quantified by direct counting of 32P incorporated into MKK-1 in an AMBIS 4000 radioisotope imager. Values are net counts adjusted for nonspecific MKK-1 phosphorylation as determined by phosphorylation in the presence of the NTE protein (,25% of Raf-1 basal activity), which lacks a catalytic domain.
toxicity to microinjected Raf proteins, because microinjection of wild-type Raf-1 has little or no effect. The dominant negative properties of KD-Raf are abolished, however, when constructs missing the Ras-binding domain, i.e., Raf-d2 and Rafd3, are used in the context of the defective kinase domain. These results indicate that Ras-Raf interaction is a dominant and essential signaling process in oocyte maturation induced by Ras. Importantly, disruption of the zinc finger motif by point mutations of cysteines 165 and 168 to serine had no effect on the dominant negative behavior of the kinase-defective Raf-1 construct. This result is consistent with our finding that the double point mutation does not disrupt the physical interaction of Ras and Raf-1. DISCUSSION
motif of Raf-1 is not essential for stimulation of the Raf-1 kinase activity in response to either Src or Ras. Dominant negative Raf requires a Ras-binding domain but not a zinc finger. Insect cells serve as an ideal system to activate Raf-1 in a controlled fashion, because the desired signaling molecules are overexpressed and host cell protein synthesis is suppressed in the infection. Nevertheless, because the insect cell system might not accurately reflect physiological Ras-Raf interactions, we sought to further characterize the functional significance of Ras-Raf physical interaction in a well-defined physiological system. Xenopus oocytes can be induced to undergo meiotic maturation, as measured by germinal vesicle breakdown, by the microinjection of RNA encoding activated Ras protein (c-H-RasV12) (10). This process can be specifically blocked by dominant negative Raf-1 constructs, demonstrating a dependence of this process on signaling through the Raf-1 kinase (11, 26). To test the necessity of Raf-Ras interaction, as well as the zinc finger motif, in a complex physiological response induced by activated Ras, we generated kinase-defective versions of full-length Raf-1, Raf-d2, Raf-d3, and CRM-Raf. The constructs were then tested for their ability to act as dominant negative inhibitors of Xenopus oocyte maturation, induced by the microinjection of c-H-RasV12 (Fig. 7). As previously reported (11), KD-Raf acts as a dominant negative inhibitor, nearly abolishing the germinal vesicle breakdown induced by v-Ras. This block represents a specific effect and not a uniform
Previous studies have shown that GTP-activated c-H-Ras and the Raf-1 protein kinase can interact directly (27, 40–42, 49). Evidence for this was derived largely from the use of the yeast two-hybrid system and N-terminal fragments expressed in bacteria as fusion proteins. Although these systems have provided valuable insights, their utility for characterization of Ras-Raf interaction is limited by properties inherent in these systems. The yeast two-hybrid system is indirect because it relies on bringing together the fused transcription components; thus, changes in protein composition may affect not only the direct protein-protein interaction but also secondary interactions and the transactivation efficiency. In addition, a prokaryotic expression system may lack the cellular machinery, such as appropriate chaperones (36, 43), to fold properly and stabilize the native Raf-1 conformation. On the other hand, binding of Ras to full-length native Raf-1 has been demonstrated to occur in mammalian cell lysates (13, 16, 27). Analysis of Raf-1 mutants in mammalian cells, however, may be complicated by the presence of endogenous Raf and Ras. To avoid some of the potential problems posed by these other expression systems, we chose to use the baculovirus-Sf9 insect cell system. This system allows the expression of full-length Raf-1 kinase capable of phosphorylating its physiological substrate, MKK, both in vitro and in vivo (10, 25). Additionally, infected Sf9 cells have no endogenous mammalian Raf-1 or Ras proteins. Lastly, it has been shown that Raf-1 can be activated by Ras and Src and that this activation is capable of activating the mitogen-
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activated protein kinase cascade in multiply-infected insect cells (25, 44, 45). Thus, this baculovirus system makes possible the analysis of Ras-Raf physical interaction and Raf functional activation within the same experimental paradigm. Our results demonstrate that Sf9 cell-expressed Raf-1 binds to c-H-Ras with characteristics similar to those seen with other assays; i.e., binding of Ras and Raf is dependent on the GTPbound state of Ras and takes place through a domain in the N-terminal portion of the protein. Additionally, we have shown that binding of the full-length Raf-1 protein is quite specific for Ras, because a Ras family member with sequence homology in the effector domain, Rac-1, failed to bind Raf-1. Previous experiments have suggested that there may be a large difference in the ability of full-length Raf-1 and an N-terminal fragment of Raf to bind Ras (41). These findings raised the possibility that the C terminus interacts with, and thereby is regulated by, the N terminus in the full-length molecule. This intramolecular interaction would then compete with the Ras intermolecular binding. Such a model would have an as yet unidentified upstream component ‘‘activating’’ Raf, causing dissociation of the internal interactions and a large increase in the affinity for Ras. Our results, however, showed that there was no significant difference in the binding of the N-terminal fragment and fulllength Raf-1. We cannot rule out, however, the possibility that the Sf9 cell-expressed Raf-1 is constitutively activated with respect to Ras binding. All the experimental approaches used to date have identified sequences in the CR1 region as being necessary for Ras-Raf interaction. Nevertheless, there has been some discrepancy in the region within this conserved domain that is required for interaction with Ras. Zhang et al. (49) reported that when the two-hybrid system or a GST-Raf N-terminal (amino acids 1 to 257) construct was used, the zinc finger was essential for Raf-1 binding to Ras. These experiments were based on a single cysteine-to-serine point mutation (at position 168) in the zinc finger motif, which resulted in loss of the ability to bind in both assays. By contrast, Vojtek et al. (41), using the two-hybrid system, isolated six cDNAs encoding Raf sequences in which the overlapping Ras-binding sequence (amino acids 51 to 131) did not include the zinc finger. Warne et al. (42) found that renaturation of GST-Raf (residues 1 to 257) in the absence of zinc decreased the binding to Ras. Interestingly, Ghosh et al. (14) found that constructs with just the cysteine-rich domain (residues 128 to 196) or constructs lacking the zinc finger (amino acids 1 to 130 or 1 to 147) were capable of binding Ras when expressed as GST fusions in Escherichia coli. In contrast to the studies described above, we used deletion mutants of full-length, kinase-active Raf-1 in these experiments. Deletions spanning the region between amino acids 53 and 132 resulted in abrogation of the Ras-Raf interaction as assayed both in vitro and in vivo. This region is nearly overlapping with that identified as the consensus sequence from the cDNAs isolated by Vojtek et al. (41). Notably, no other mutations outside this region of Raf-1 resulted in a loss of binding in our studies. We found, however, that although deletion of the entire cysteine-rich motif did not abolish Ras binding, the deletion mutant lacking residues 133 to 180 (Raf-d4) was partially impaired in its interaction with Ras both in vitro and in vivo. Importantly, this decreased binding was not due to interactions mediated by an intact zinc finger, because CRM-Raf containing the double point mutations (cysteines 165 and 168 to serine) to disrupt the zinc finger bound Ras as well as did wild-type Raf-1. It is unclear why the GST-Raf fusion protein with the single cysteine-to-serine point mutation used by Zhang et al. (49) failed to bind Ras. One possibility is that point mutations in the zinc finger are more disruptive to the
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conformation of surrounding sequences when only the isolated N-terminal portion of the protein is expressed. Similarly, expression in bacteria may result in a protein in which proper folding is more easily disrupted. Finally, we have used a double point mutation in the Raf-1 zinc finger, analogous to those made in the protein kinase C zinc finger that abolished phorbol ester binding (30); it is possible that a single point mutation in this region is much more disruptive to the overall conformation of the protein. Taken collectively, data from our studies and the other experiments mentioned above indicate that the region located between amino acids 53 and 132 in Raf-1 is both necessary and sufficient for Ras-Raf interaction. Although the zinc finger motif does not play an essential role in Ras interaction, sequences N-terminal to the zinc finger, perhaps in the 130 to 147 region, probably contribute to high-affinity stable interactions. This suggestion is based on the findings that (i) a 17amino-acid peptide based on the sequence from 130 to 147 partially blocked the association of a GST-Raf fusion protein with Ras (14) and (ii) Raf-d4 (deletion of residues 133 to 180), although capable of binding Ras, showed reduced affinity, a characteristic not mimicked by targeted disruption of the zinc finger. Similar conclusions were recently reported from an in vitro competition assay that measured the relative affinities of Raf-1 fusion proteins to Ras (4). Although it is widely believed that Ras activates Raf-1 kinase activity directly or indirectly through its physical association, this idea has not yet been critically tested. To examine the functional consequence of disrupting Ras-Raf interactions, we tested whether the kinase activity of selected Raf mutants could be activated by coinfection with either Src or Ras. As previously reported (10, 45), we found that infection with either Src or Ras in combination with Raf-1 resulted in a modest activation (three- to fivefold) of Raf kinase. Activation of Raf kinase activity was abolished in the Raf-d2 mutant, however, which failed to bind Ras both in vitro and in vivo. This mutation did not significantly alter either the Raf-1 basal kinase activity or the activation by Src. These results suggest that the abolition of Raf-1 activation by Ras is the result of a failure to bind Ras rather than an inactivating mutation in Raf-1. Furthermore, activation of Raf-1 by Src does not require Ras, even though these two activators can cooperate in activating Raf-1 (10, 44, 45). Consistent with our results with insect cells, other studies with mammalian cells have suggested that Raf-1 kinase can be activated by epidermal growth factor via both Rasdependent and -independent mechanisms (23). Recently, conclusions similar to those described here were reached by authors using a Raf-1 mutant with an arginine-to-leucine substitution at position 89, which abolished Ras binding and activation of Raf-1 kinase by Ras but not by Src in insect cells (12). It is currently unclear exactly how Raf-1 is activated by Ras. Experiments involving Raf-1 targeted to the membrane have suggested that the primary role of the Ras-Raf interaction is to recruit Raf-1 to the membrane (23, 38), where it is activated by an unidentified mechanism. Interestingly, the zinc finger motif has been shown to mediate binding of Raf-1 to phospholipid liposomes in vitro (14), raising the possibility that phospholipid interaction with the zinc finger is an essential step in the activation of Raf-1 at membranes. In earlier studies, however, the functional consequence of zinc finger disruption was addressed in the context of an N-terminal dominant negative fragment (3). Interpretation of these experiments is complicated by the finding that a similar construct loses the ability to bind Ras when tested in vitro (49), making it impossible to distinguish between contributions of Ras binding and independent func-
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tions of the zinc finger. In the present study, we tested a mutant, CRM-Raf, in which the zinc finger was disrupted by point mutations analogous to those that disrupt protein kinase C phospholipid-dependent binding to phorbol esters for the ability to be activated by Ras and Src. Because this Raf mutant shows in vivo Ras binding comparable to that of wild-type Raf-1, these assays are a direct measure of any independent functions of the zinc finger motif. Significantly, both Ras and Src were able to activate CRM-Raf despite the absence of an intact zinc finger. Thus, from these experiments, it seems that the zinc finger is not essential for the activation of Raf-1 by Ras or Src. Nevertheless, we cannot rule out a modulatory role of this region or an essential regulatory mechanism not preserved in the insect cell system. When a kinase-defective CRM-Raf construct was assayed for its ability to act as a dominant negative inhibitor of Rasinduced oocyte maturation, it was nearly equivalent to kinasedefective Raf-1. In contrast, the kinase-defective Raf-d2 construct, which cannot bind Ras in vivo or in vitro, lost the ability to function as a dominant negative inhibitor. Taken together with the other results described above, these experiments suggest that loss of dominant negative function of the N-terminal Raf fragment as a result of a zinc finger mutation reported in previous studies (3) is probably due to a loss in the ability to bind Ras. The present experiments further suggest that kinasedefective Raf-1 is insufficient to block Ras-mediated signaling in the absence of the capacity to bind Ras. This latter finding is particularly important in interpreting experiments with Raf-1 dominant negative constructs, because it implies that they need to function by binding Ras. It is impossible at present to distinguish, by using only experiments with dominant negative constructs alone, whether the observed effects are the result of disruption of signal propogation from Ras to Raf-1 or, alternatively, from Ras to another effector.
ACKNOWLEDGMENTS We thank P. Dent and T. Sturgill for purified MKK1 protein, N. Williams and T. Roberts for v-Ras baculovirus, D. Lowy for a c-H-Ras cDNA; K.-L. Guan and J. Dixon for the pGEX-KG expression vector, and R. Herrera for the GST–Rac-1 expression vector and for comments on the manuscript. K.P. is the recipient of a Parke-Davis/University of Michigan Biotechnology Fellowship. This investigation was funded in part by grant CA55652 from the National Institutes of Health. REFERENCES 1. Blenis, J. 1993. Signal transduction via the MAP kinases: proceed at your own RSK. Proc. Natl. Acad. Sci. USA 90:5889–5892. 2. Bonner, T. I., H. Oppermann, P. Seeburg, S. B. Kerby, M. A. Gunnell, A. C. Young, and U. R. Rapp. 1986. The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene. Nucleic Acids. Res. 14:1009–1015. 3. Bruder, J. T., G. Heidecker, and U. R. Rapp. 1992. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev. 6:545–556. 3a.Chow, Y.-H., K. M. Puniglia, T. Jun, P. Dent, T. Sturgill, and R. Jove. Submitted for publication. 4. Chuang, E., D. Barnard, L. Hettich, X. F. Zhang, J. Avruch, and M. S. Marshall. 1994. Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues. Mol. Cell. Biol. 14:5318–5325. 5. Crews, C. M., and R. L. Erikson. 1993. Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell 74:215–217. 6. Davis, R. J. 1993. The mitogen-activated protein kinase signal transduction pathway. J. Biol. Chem. 268:14553–14556. 7. Dent, P., Y.-H. Chow, J. Wu, D. K. Morrison, R. Jove, and T. W. Sturgill. 1994. Expression, purification, and characterization of recombinant MAP kinase kinases, MKK1 and MKK2. Biochem. J. 303:105–112.
405
8. Dickson, B., F. Sprenger, D. Morrison, and E. Hafen. 1992. Raf functions downstream of Ras1 in the Sevenless signal transduction pathway. Nature (London) 360:600–603. 9. Egan, S. E., and R. A. Weinberg. 1993. The pathway to signal achievement. Nature (London) 365:781–783. 10. Fabian, J. R., I. O. Daar, and D. K. Morrison. 1993. Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase. Mol. Cell. Biol. 13:7170–7179. 11. Fabian, J. R., D. K. Morrison, and I. O. Daar. 1993. Requirement for Raf and MAP kinase function during the meiotic maturation of Xenopus oocytes. J. Cell Biol. 122:645–652. 12. Fabian, J. R., A. B. Vojtek, J. A. Cooper, and D. K. Morrison. 1994. A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function. Proc. Natl. Acad. Sci. USA 91:5982–5986. 13. Finney, R. E., S. M. Robbins, and J. M. Bishop. 1993. Association of pRas and pRaf-1 in a complex correlates with activation of signal transduction pathway. Curr. Biol. 3:805–812. 14. Ghosh, S., W. Q. Xie, A. F. Quest, G. M. Mabrouk, J. C. Strum, and R. M. Bell. 1994. The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. J. Biol. Chem. 269:10000–10007. 15. Guan, K. L., and J. E. Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192:262–267. 16. Hallberg, B., S. I. Rayter, and J. Downward. 1994. Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation. J. Biol. Chem. 269:3913–3916. 17. Han, M., A. Golden, Y. Han, and P. W. Sternberg. 1993. C. elegans lin-45 raf gene participates in let-60 ras-stimulated vulval differentiation. Nature (London) 363:133–140. 18. Heidecker, G., M. Huleihel, J. L. Cleveland, W. Kolch, T. W. Beck, P. Lloyd, T. Pawson, and U. R. Rapp. 1990. Mutational activation of c-raf-1 and definition of the minimal transforming sequence. Mol. Cell. Biol. 10:2503– 2512. 19. Heidecker, G., W. Kolch, D. K. Morrison, and U. R. Rapp. 1992. The role of Raf-1 phosphorylation in signal transduction. Adv. Cancer Res. 58:53–73. 20. Ikawa, S., M. Fukui, Y. Ueyama, N. Tamaoki, T. Yamamoto, and K. Toyoshima. 1988. B-raf, a new member of the raf family, is activated by DNA rearrangement. Mol. Cell. Biol. 8:2651–2654. 21. Ishikawa, F., R. Sakai, M. Ochiai, F. Takaku, T. Sugimura, and M. Nagao. 1988. Identification of a transforming activity suppressing sequence in the c-raf oncogene. Oncogene 3:653–658. 22. Kolch, W., G. Heidecker, P. Lloyd, and U. R. Rapp. 1991. Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature (London) 349:426–428. 23. Leevers, S. J., H. F. Paterson, and C. J. Marshall. 1994. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature (London) 369:411–414. 24. Le Guellec, R., A. Couturier, K. Le Guellec, J. Paris, N. Le Fur, and M. Philippe. 1991. Xenopus c-raf proto-oncogene: cloning and expression during oogenesis and early development. Biol. Cell. 72:39–45. 25. Macdonald, S. G., C. M. Crews, L. Wu, J. Driller, R. Clark, R. L. Erikson, and F. McCormick. 1993. Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro. Mol. Cell. Biol. 13:6615–6620. 26. MacNicol, A. M., A. J. Muslin, and L. T. Williams. 1993. Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF. Cell 73:571–583. 27. Moodie, S. A., B. M. Willumsen, M. J. Weber, and A. Wolfman. 1993. Complexes of Ras. GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260:1658–1661. 28. Morrison, D. K., G. Heidecker, U. R. Rapp, and T. D. Copeland. 1993. Identification of the major phosphorylation sites of the Raf-1 kinase. J. Biol. Chem. 268:17309–17316. 29. Nishida, Y., M. Hata, T. Ayaki, H. Ryo, M. Yamagata, K. Shimizu, and Y. Nishizuka. 1988. Proliferation of both somatic and germ cells is affected in the Drosophila mutants of raf proto-oncogene. EMBO J. 7:775–781. 30. Ono, Y., T. Fujii, K. Igarashi, T. Kuno, C. Tanaka, U. Kikkawa, and Y. Nishizuka. 1989. Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc. Natl. Acad. Sci. USA 86:4868– 4871. 31. Park, S., M. S. Marshall, J. B. Gibbs, and R. Jove. 1992. Reconstitution of interactions between the Src tyrosine kinases and Ras GTPase-activating protein using a baculovirus expression system. J. Biol. Chem. 267:11612– 11618. 32. Ridley, A. J., and A. Hall. 1992. Distinct patterns of actin organization regulated by the small GTP-binding proteins Rac and Rho. Cold Spring Harbor Symp. Quant. Biol. 57:661–671. 33. Ridley, A. J., H. F. Paterson, C. L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70:401–410. 34. Roberts, T. M. 1992. A signal chain of events. Nature (London) 360:534–535. 35. Smith, M. R., S. J. DeGudicibus, and D. W. Stacey. 1986. Requirement for
406
36.
37. 38. 39. 40. 41. 42.
PUMIGLIA ET AL. c-ras proteins during viral oncogene transformation. Nature (London) 320: 540–543. Stancato, L. F., Y. H. Chow, K. A. Hutchison, G. H. Perdew, R. Jove, and W. B. Pratt. 1993. Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system. J. Biol. Chem. 268:21711– 21716. Stanton, V. P., Jr., D. W. Nichols, A. P. Laudano, and G. M. Cooper. 1989. Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol. Cell. Biol. 9:639–647. Stokoe, D., S. G. Macdonald, K. Cadwallader, M. Symons, and J. F. Hancock. 1994. Activation of Raf as a result of recruitment to the plasma membrane. Science 264:1463–1467. Storm, S. M., J. L. Cleveland, and U. R. Rapp. 1990. Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene 5:345–351. Van Aelst, L., M. Barr, S. Marcus, A. Polverino, and M. Wigler. 1993. Complex formation between RAS and RAF and other protein kinases. Proc. Natl. Acad. Sci. USA 90:6213–6217. Vojtek, A. B., S. M. Hollenberg, and J. A. Cooper. 1993. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell 74:205–214. Warne, P. H., P. R. Viciana, and J. Downward. 1993. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature (London) 364:352–355.
MOL. CELL. BIOL. 43. Wartmann, M., and R. J. Davis. 1994. The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex. J. Biol. Chem. 269:6695–6701. 44. Williams, N. G., H. Paradis, S. Agarwal, D. L. Charest, S. L. Pelech, and T. M. Roberts. 1993. Raf-1 and p21v-ras cooperate in the activation of mitogen-activated protein kinase. Proc. Natl. Acad. Sci. USA 90:5772–5776. 45. Williams, N. G., T. M. Roberts, and P. Li. 1992. Both p21ras and pp60v-src are required, but neither alone is sufficient, to activate the Raf-1 kinase. Proc. Natl. Acad. Sci. USA 89:2922–2926. 46. Wood, K. W., C. Sarnecki, T. M. Roberts, and J. Blenis. 1992. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell 68:1041–1050. 47. Wu, J., P. Dent, T. Jelinek, A. Wolfman, M. J. Weber, and T. W. Sturgill. 1993. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 39,59-monophosphate. Science 262:1065–1069. 48. Zhang, K., J. E. DeClue, W. C. Vass, A. G. Papageorge, F. McCormick, and D. R. Lowy. 1990. Suppression of c-ras transformation by GTPase-activating protein. Nature (London) 346:754–756. 49. Zhang, X. F., J. Settleman, J. M. Kyriakis, E. Takeuchi-Suzuki, S. J. Elledge, M. S. Marshall, J. T. Bruder, U. R. Rapp, and J. Avruch. 1993. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature (London) 364:308–313.
