Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal Region Interferes with Ras-dependent Activation of Raf-1

Abstract

Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functionsin vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR. Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functionsin vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR. Ras belongs to a family of small GTP-binding proteins playing essential roles in cell proliferation and differentiation. Mammalianras genes carrying activating mutations are found in many types of neoplastic tissue and are able to induce morphological transformation in vitro when transfected into fibroblast cell lines. However, the rap1A gene (1Pizon V. Chardin P. Lerosey I. Olofsson B. Tavitian A. Oncogene. 1988; 3: 201-204Google Scholar), encoding a 21-kDa GTP-binding protein with high homology to Ras, has been shown to to induce reversion of the transformed phenotype in Ki-ras-transformed NIH3T3 cells (2Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Google Scholar). In addition to the overall structural homology, Rap1A shares two important structural features with Ras. One is that Rap1A has an identical effector domain (amino acids 32–40) to that of Ras. The effector domain of Ras is essential for the association with and activation of its effectors (3Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Google Scholar). The other is that Rap1A undergoes similar post-translational modification to Ras at its C terminus except that Ras is farnesylated and Rap1A is geranylgeranylated (4Buss J.E. Quilliam L.A. Kato K. Casey P.J. Solski P.A. Wong G. Clark R. McCormick F. Bokoch G.M. Der C.J. Mol. Cell. Biol. 1991; 11: 1523-1530Google Scholar). This modification is essential for the function of Rap1A as observed for Ras (5Kitayama H. Matsuzaki T. Ikawa Y. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4284-4288Google Scholar, 6Sakoda T. Kaibuchi K. Kishi K. Kishida S. Doi K. Hoshino M. Hattori S. Takai Y. Oncogene. 1992; 7: 1705-1711Google Scholar). Raf-1, a serine/threonine kinase regulating the mitogen-activated protein kinase cascade, is a major mammalian Ras effector and is thought to play a key role in Ras-induced cellular transformation (7Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Google Scholar). Although the precise mechanism of Ras-dependent Raf-1 activation remains unclear, it is known that the effector domain of Ras interacts with the N-terminal RBD 1The abbreviations used are: RBD, Ras-binding domain; CRR, cysteine-rich region; MBP, maltose-binding protein; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GST, glutathione S-transferase; MEK, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase kinase; KNERK, a kinase negative mutant of ERK2; PAGE, polyacrylamide gel electrophoresis. (amino acids 51–131) of Raf-1 and that this interaction is essential for physical association between these proteins as well as for the activation of Raf-1 (7Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Google Scholar). Rap1A, too, has been shown to associate with Raf-1 N-terminal fragment in vivo (8Zhang X.-F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Google Scholar), and a recent x-ray diffraction study of the crystal of the complex between Rap1A and Raf-1 RBD has provided evidence for this association at the atomic level (9Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Google Scholar). These studies suggest the possibility that the suppression of Ras function by Rap1A is due to the competitive inhibition of Ras-RBD interaction (10Burgering B.M.T. Boss J.L. Trends Biochem. Sci. 1995; 20: 18-22Google Scholar), although it is unclear why Rap1A cannot activate Raf-1. We have recently identified Raf-1 CRR (amino acids 152–184) as another Ras-binding domain and demonstrated that interaction of Ras with both RBD and CRR is necessary for the activation of Raf-1 (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). Two mutations, N26G and V45E, were found to abolish the interaction of Ha-Ras with CRR and attenuate the activation of Raf-1 by Ha-Ras. The fact that both of these mutations replaced Ha-Ras residues with corresponding Rap1A residues prompted us to examine the possibility that the inability of Rap1A to activate Raf-1 is due to its failure to interact with Raf-1 CRR. Contrary to the expectation, we found that Rap1A exhibited a greatly enhanced ability to bind to CRR. Rap1A cDNA was amplified from a human lung fibroblast cDNA library by polymerase chain reaction (12Saiki R.K. Scharf S. Faloona F. Mullis K.B. Horn G.T. Erlich H.A. Arnheim N. Science. 1985; 230: 1350-1354Google Scholar) using a pair of primers, 5′-CGGGATCCGATATGCGTGAGTACAAGCTAG-3′ and 5′-AACTGCAGCAGCTAGAGCAGCAGACATGATTTC-3′. After cleavage withBamHI and PstI in the primer sequences, it was cloned into matching cleavage sites of the baculovirus transfer vector pBlueBac III (Invitrogen Inc., San Diego, CA). The cDNA for an activated Rap1A, Rap1AV12, was prepared by oligonucleotide-directed mutagenesis (13Kramer W. Fritz H.-J. Methods Enzymol. 1987; 154: 350-367Google Scholar) and cloned into pBlueBac III as for the wild-type cDNA. pV-IKS, another baculovirus transfer vector for expressing proteins as GST fusions, was provided by Dr. D. Midra (University of California, San Francisco, CA) through Dr. A. Kikuchi (Hiroshima University, Hiroshima, Japan) (14Kikuchi A. Williams L.T. J. Biol. Chem. 1994; 269: 20054-20059Google Scholar). For expression of Ha-Ras fused to GST, Ha-Ras cDNA was amplified by polymerase chain reaction using a pair of primers, 5′-CGCGTCTAGAATGACGGAATATAAGCTGGTG-3′ and 5′-GCCGGAATTCTCAGGAGAGCACACACTTG-3′. After digestion withXbaI and EcoRI in the primer sequences, it was cloned into matching cleavage sites of pV-IKS. Structures of the constructs were confirmed by DNA sequence analysis. Preparation of recombinant baculoviruses expressing Ha-Ras, Rap1A, and their mutants and the purification of the post-translationally modified proteins from infected Sf9 cells were carried out as described (15Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Google Scholar, 16Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Google Scholar). MBP fusion proteins of Raf-1 N-terminal fragments were expressed in Escherichia coli and immobilized on amylose resin as described (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). Binding reaction was carried out by incubating 20 μl of the resin carrying various amounts of MBP-Raf-1 proteins with various amounts of GTPγS- or GDP-bound Ha-Ras or Rap1A in a total volume of 100 μl of buffer A (20 mm Tris/HCl, pH 7.4, 40 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5 mmMgCl2, and 0.1% Lubrol PX) as described (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). After incubation at 4 °C for 2 h, the resin was washed, and the bound proteins were eluted with buffer A containing 10 mm maltose and subjected to SDS-PAGE followed by Western immunoblot detection with anti-Ras monoclonal antibody Y13-259 (Oncogene Science Inc., Manhasset, NY) or anti-Rap1A polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, California). Both anti-Ha-Ras and anti-Rap1A antibodies exhibited little cross-reactivity to Rap1A and Ha-Ras, respectively (data not shown). The assay for competitive inhibition of Ras binding to the MBP-Raf-1 fusion proteins by Rap1A were carried out by including a fixed amount of Ha-Ras and various amounts of Rap1A in the same binding reaction. For the in vitro co-association of Rap1A with GST-Ha-Ras, GST-Ha-Ras in Sf9 cell lysate was first immobilized on glutathione-Sepharose and then loaded with GTPγS. The resin was then incubated with GTPγS-bound Rap1A in the absence or the presence of purified MBP-Raf-1(51–131) or MBP-Raf-1(48–206). The binding condition was the same as described above except that the bound proteins were eluted with 10 mm glutathione in buffer A. Monolayers of Sf9 cells (2 × 107 cells) were triply infected with the recombinant baculoviruses expressing the full-length Raf-1 and Ha-RasV12, along with that expressing Ha-RasV12(E31K) or Rap1AV12 (1 × 108 plaque-forming units each). After 72 h post-infection, the cells were lysed by sonication in 1 ml of buffer B (20 mm Tris/HCl, pH 7.5, 137 mm NaCl, 1% Nonidet P-40, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 1 mm sodium vanadate) and centrifuged at 13,000 × g for 30 min. Raf-1 was immunoprecipitated from the supernatant (200 μl) with the anti-Raf-1 antibody C12 (2 μl) (Santa Cruz Biotechnology Inc.) and protein A-agarose. The Raf-1 activity was determined by incubating the immunoprecipitates in the presence of GST-MEK (13 μg) and GST-KNERK2 (1 μg) in 30 μl of kinase reaction mixture (20 mmTris/HCl, pH 7.5, 10 mm MnCl2, 10 mm MgCl2, 20 mmβ-glycerophosphate, and 50 μm[γ-32P]ATP (4,000 cpm/pmol)) for 30 min at 25 °C. After the incubation, proteins in the reaction mixture were fractionated by SDS-PAGE and subjected to autoradiography to detect phosphorylation of GST-KNERK2 as described (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar, 14Kikuchi A. Williams L.T. J. Biol. Chem. 1994; 269: 20054-20059Google Scholar). In the previous study, we have shown that Ha-Ras binds to immobilized MBP-Raf-1(132–206), representing CRR, demonstrating that Raf-1 CRR acts as another Ras-binding domain independently of RBD (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). This binding was GTP-independent in contrast to the GTP-dependent binding to MBP-Raf-1(50–131), representing RBD. Ha-Ras mutations N26G and V45E abolished binding to CRR without affecting binding to RBD. The fact that these two mutations replaced Ha-Ras residues with those of Rap1A prompted us to examine if Rap1A lacks the ability to bind to Raf-1 CRR. In the same in vitrobinding assay, Rap1A bound to RBD in a GTP-dependent manner (Fig. 1 A, lanes 1 and2), although the GTP dependence of this binding was less clear compared with that of Ha-Ras (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). Unexpectedly, Rap1A bound efficiently to CRR (lanes 3 and 4). As observed for Ha-Ras, this binding was GTP-independent (lanes 3 and4) and required an intact zinc finger structure of CRR because Rap1A bound very poorly to MBP-Raf-1(132–206, C168S) (Fig.1 B). Binding of Rap1A to MBP-Raf-1(48–206) containing both RBD and CRR was severalfold stronger and less GTP-dependent than that to RBD (Fig. 1 A, lanes 5 and6). These results suggested that CRR is necessary for efficient association between Rap1A and Raf-1 and that CRR rather than RBD plays a major role in this association. Comparison of the binding properties between Ha-Ras and Rap1A is shown in Fig. 1 C. Ha-Ras bound to CRR yielded roughly 10-fold less signal than that to RBD even though the amounts of CRR in the binding reaction were doubled, indicating that the ability of Ha-Ras to bind to CRR was roughly 20-fold less than that to RBD. This estimation by immunoblot was consistent with our kinetic measurement of the affinity of Ha-Ras for RBD and CRR using a competitive inhibition of Ha-Ras-dependent activation of Saccharomyces cerevisiae adenylyl cyclase (15Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Google Scholar, 16Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Google Scholar). 2C.-D. Hu, K. Kariya, and T. Kataoka, unpublished results. In a striking contrast, Rap1A bound to CRR yielded stronger signal than that to RBD under the same condition, indicating that it has the ability to bind equally to RBD and to CRR (see also Fig.1 A). After taking account of the observation that Rap1A yielded twice as much signal as Ha-Ras on the equimolar basis (Fig.1 D), the ability of Rap1A to bind to CRR was roughly 10-fold greater than that of Ha-Ras. In addition, the ability of Rap1A to bind to RBD was about one-half of that of Ha-Ras after the same normalization. These results indicated that Rap1A has greatly enhanced rather than impaired activity to bind to CRR. In our previous report, we found that the binding of Ha-Ras to CRR required post-translational modification (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). To test if binding of Rap1A to CRR was also dependent on its post-translational modification, we incubated RBD and CRR with a lysate of Sf9 cells expressing Rap1A. As shown in Fig. 1 E, RBD bound both modified and unmodified Rap1A represented by the faster and slower migrating bands, respectively, on the Western immunoblot as described before Proc. Natl. Acad. Sci. U. S. A. 1991; Scholar). In contrast, CRR bound the modified of Rap1A. These results indicated that binding of Rap1A to CRR also its The above are to our previous that binding to CRR is necessary for Raf-1 because Rap1A bind to CRR than Ha-Ras and cannot activate Raf-1. One is that enhanced binding to CRR is to the activation of Raf-1. To test this we first for a Ha-Ras mutant binding to CRR was After than 40 mutants described K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Google Scholar), the mutant was found to roughly 10-fold activity to bind to CRR compared with (Fig. 1 we Raf-1 with Rap1AV12 or Ha-RasV12(E31K) mutant in Sf9 cells. The Raf-1 immunoprecipitates were examined for the activity to induce phosphorylation of in the presence of The results that Rap1AV12 and Ha-RasV12(E31K) not activate Raf-1 (lanes expression of of along with and Raf-1 was found to the activation of Raf-1 by 5 and These suggested that the enhanced binding to CRR is to the activation of Raf-1. also suggested the of this enhanced binding in the of Rap1A in Ras-dependent Raf-1 which is examined in the We that the greatly enhanced ability of Rap1A to bind to CRR found also in competitive inhibition of association by Rap1A. Rap1A association of Ras with a Raf-1 N-terminal region containing both RBD and CRR much efficiently than that with RBD To test this we first incubated with a fixed amount of Ha-Ras and amounts of Rap1A. As the amount of bound Ha-Ras was found to by amounts of Rap1A, that of bound Rap1A was A). We then MBP-Raf-1(48–206) in the same To our the binding of Ha-Ras to MBP-Raf-1(48–206) was found enhanced to even when the amounts of bound Rap1A were (Fig. 3 B). The for these results that Rap1A and Ha-Ras with the same Raf-1 N-terminal through their binding to CRR and RBD, respectively, and that these To test this we immobilized GST-Ha-Ras fusion protein glutathione-Sepharose and incubated it with Rap1A in the absence or the presence of MBP-Raf-1 fusion As shown in Fig. 3 Rap1A was found to with GST-Ha-Ras when were incubated in the absence of MBP-Raf-1 in the presence of not in association of Rap1A with GST-Ha-Ras was with GST-Ha-Ras when incubated in the presence of not MBP-Raf-1(48–206) also Rap1A was found to with GST-Ha-Ras These results indicated that Rap1A and Ha-Ras with Raf-1 N-terminal region when it both RBD and CRR. results if association of GST-Ha-Ras with MBP-Raf-1(48–206) of to binding of Rap1A to the MBP-Raf-1(48–206) However, of MBP-Raf-1(48–206) was not the because incubation of GST-Ha-Ras and Ha-Ras in the presence of MBP-Raf-1(48–206) same as except that Ha-Ras was used of not in association between GST-Ha-Ras and Ha-Ras (data not shown). The of of Rap1A competitive inhibition by Rap1A of the interaction between Ras and Raf-1 RBD (10Burgering B.M.T. Boss J.L. Trends Biochem. Sci. 1995; 20: 18-22Google Scholar). However, we found in this study that Rap1A greatly enhanced ability to bind to CRR, another Ras-binding domain identified by us (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar) and S. G.M. J. Biol. Chem. 1994; 269: Scholar, S. J. Biol. Chem. 1994; 269: Scholar, S. S. Der C.J. J. Biol. Chem. 1995; 270: Scholar). This binding even in the co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through their binding to CRR and RBD, respectively, and these we have shown that binding of Ras to both CRR and RBD is necessary for Raf-1 activation (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar), it is very that this complex in of Raf-1 activation due to the failure of Ha-Ras to bind to CRR. The previous that unmodified Rap1A cannot Ras function (5Kitayama H. Matsuzaki T. Ikawa Y. Noda M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4284-4288Google Scholar, 6Sakoda T. Kaibuchi K. Kishi K. Kishida S. Doi K. Hoshino M. Hattori S. Takai Y. Oncogene. 1992; 7: 1705-1711Google Scholar) is also consistent with because we found that modification of Rap1A is essential for its binding to CRR. A study with indicated that of Ras both of the two residues and the other Rap1A residues although residues is also required for activity to transformation K. Noda M. Science. 1990; Scholar). residues that are that are the Ras and that are not on the protein and have been to the protein is or (3Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Google Scholar, K. Noda M. Science. 1990; Scholar, M.S. Mol. Cell. Biol. 1991; 11: Scholar, J. Shirouzu M. H. S. Yokoyama S. 1991; Scholar, A. G. H. J. Biol. Chem. 1992; Scholar). In of residues 30 or of activated Ras with those of Rap1A in of activity M.S. Mol. Cell. Biol. 1991; 11: Scholar, A. G. H. J. Biol. Chem. 1992; Scholar). However, it unclear which of these residues plays the and have been to a domain on the of Ras protein (3Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Google Scholar, J. Shirouzu M. Y. Hattori S. S. S. Yokoyama S. J. Biol. Chem. 1995; 270: Scholar). The domain, was suggested to play an important role for activation of effectors through physical interaction with In the previous study, we found that Ha-Ras proteins carrying mutations N26G and V45E failed to bind to Raf-1 CRR (11Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Google Scholar). These mutants were also of activating Raf-1 in Sf9 cells. on these we that the domain interacts with Raf-1 CRR and that this interaction is essential for Raf-1 activation. This that the inability of Rap1A to activate Raf-1 from its failure to bind to CRR, because Rap1A residues and which abolished binding to CRR in the of Ha-Ras. Contrary to this expectation, we found that Rap1A exhibited greatly enhanced rather than binding to CRR. One for this is that of the domain residues as and which are between Ras and Rap1A, not interact with CRR. the domain residues as and which are between Ras and Rap1A, in interaction with CRR. with this and mutations have been shown to of Ras (3Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Google J. Shirouzu M. Y. Hattori S. S. S. Yokoyama S. J. Biol. Chem. 1995; 270: Scholar). In the of Ha-Ras, both and necessary for these residues to their which are by the N26G and V45E the other in the of Rap1A, of and by of other residues that are not with Ha-Ras. In this of Rap1A play a role because we found that mutation enhanced CRR binding in the of Ha-Ras. A for the role of the the domain also from a study of Rap1A recently the structure of mutant with RBD by x-ray N. Horn G. Herrmann C. C. R. Wittinghofer A. Biol. 1996; 3: Scholar). Comparison of this structure with that of wild-type Rap1A with RBD indicated that the mutation led to a by than of a containing residues which with the domain. it is that residues 30 and of Ras the of the domain In addition, of was found to interact with of Raf-1 RBD, that of Ras in the same also that and an activity to from a Ras-dependent to a small and that this is for by a in the affinity of Rap1A for RBD due to the However, the is also consistent with our that the of the CRR The observed of the domain of Rap1A have about a in the affinity of the Rap1A domain for CRR to a level for Raf-1 to the of the Although the structure of CRR has been S. S. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar), of the mechanism of CRR binding the structural of the complex between CRR and Ras or Rap1A. We A. Kikuchi at of for and pV-IKS, K. Kishi at of for human lung fibroblast cDNA and for We also A. and A. for in of this