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Molecular and Cellular Biology, September 1999, p. 6057-6064, Vol. 19, No. 9
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Strength of Interaction at the Raf Cysteine-Rich Domain Is a Critical Determinant of Response of Raf to Ras Family Small GTPases

Tomoyo Okada, Chang-Deng Hu, Tai-Guang Jin, Ken-ichi Kariya, Yuriko Yamawaki-Kataoka, and Tohru Kataoka*

Department of Physiology II, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan

Received 30 December 1998/Returned for modification 17 February 1999/Accepted 3 June 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Raf-1 is a serine/threonine kinase that plays a pivotal role in conveying a signal from receptor tyrosine kinases and Ras to the mitogen-activated protein kinase (MAPK) cascade. Although it is well established that Raf-1 interacts directly with the GTP-bound active form of Ras, the precise mechanism by which Raf-1 is activated by interaction with Ras is not known (for reviews, see references 1, 4, and 27). In addition to Raf-1, which is found in a variety of mammalian tissues, two close homologues, B-Raf and A-Raf, have been identified. These exhibit more limited tissue distribution: B-Raf expression is confined to the brain and testis, and A-Raf is expressed most abundantly in the ovary and epididymis (42). All Raf kinases possess three distinct regions designated conserved region 1 (CR1), CR2, and CR3 (for a review, see reference 10). The N-terminal regulatory domain contains CR1 and CR2, while the C-terminal CR3 corresponds to the protein kinase catalytic domain. CR1 consists of two distinct structural modules called the Ras-binding domain (RBD; residues 51 to 131) and the cysteine-rich domain (CRD; residues 139 to 184). RBD has a ubiquitin superfold (30) and constitutes a principal interface for GTP-dependent interaction with Ras and Rap1A (5, 46, 49). CRD consists of two zinc finger motifs resembling the C1 domain of protein kinase C, a phorbol ester-binding site (28). Constitutive activation of Raf-1 by N-terminal truncations indicated that the N-terminal regulatory domain functions to inhibit and regulate the activity of the C-terminal catalytic domain (1, 4, 27).

Numerous studies have indicated that to be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras. One is a high-affinity interaction between its RBD and the effector region (residues 32 to 40 of mammalian Ras) of Ras, which is dependent on the GTP-bound configuration of Ras (5, 46). Recent X-ray crystallographic study of the three-dimensional structure of Raf-1 RBD complexed with the Ras homologue Rap1A solved the molecular basis of this interaction (29, 30). This interaction, when made with the posttranslationally modified Ras, induces translocation of Raf-1 from the cytoplasm to the plasma membrane (20, 22, 40). The other is a low-affinity interaction through CRD, whose function and structural basis are less well characterized; conflicting reports have appeared on the structural requirements for this interaction (3, 9, 12, 13, 15, 17, 21). Although membrane recruitment by the RBD-Ras interaction, which can be experimentally reproduced by attachment of a membrane localization signal to Raf-1, causes a moderate activation of Raf-1 (20, 22, 40), the CRD-Ras interaction is required for full activation of the Raf-1 kinase activity (17, 21, 25, 36, 43). This conclusion is based on the findings that substitutions of certain residues of Raf-1 CRD, including Cys-165 and Cys-168 within the zinc finger, which impaired its ability to associate with Ras, caused substantial reduction in activation of Raf-1 by Ras (12, 21) and that a Ras mutant carrying an N26G or V45E mutation in its activator region (approximately corresponding to residues 26 to 31 and 41 to 53 of mammalian Ras flanking the effector region), which lost the ability to interact with CRD, exhibited an impaired ability to activate Raf-1 while retaining the ability to induce membrane translocation of Ras (17, 43). Moreover, Raf-1 CAAX, which had been activated by recruitment to the plasma membrane by attachment of a CAAX motif to its C terminus, was found to be activated further by Ras when S257L or Y340D/Y341D mutations were introduced (25, 36).

Another line of evidence for the importance of CRD in Raf-1 regulation came from studies on the interaction of Raf-1 CRD with the antioncogene product Rap1A/Krev-1 (15, 16), which belongs to the Ras family GTPases and has the same effector region as Ras (34). Despite its structural homology with Ras, Rap1A suppresses the Ras-induced malignant transformation of the NIH 3T3 fibroblast cell line (18). Consistent with this suppressive activity toward Ras, Rap1A cannot activate Raf-1 and even antagonizes its Ras-dependent activation (7). We have shown that Rap1A exhibits greatly enhanced interaction with Raf-1 CRD compared to Ras and that this property of Rap1A may account for its loss of the ability to activate Raf-1 as well as for its antagonistic effect on Ras-dependent Raf-1 activation (15). The antagonistic effect of Rap1A is determined by the nature of residue 31, Lys in Rap1A and Glu in Ras, since Ha-Ras(E31K) exhibited an activity resembling Rap1A (15). This observation led to the hypothesis that the strength of interaction at CRD is crucial for determining the mode of regulation of the Raf-1 kinase activity by Ras and Rap1A. The strength of the Ras-CRD interaction must be adequate to cause Raf-1 activation: too strong interaction as observed for Rap1A and Ha-Ras(E31K) or too weak interaction as observed for the activator region mutants and the posttranslationally unmodified form of Ha-Ras as well as for C168S and C165S/C168S mutants of Raf-1 all resulted in loss of Ras-dependent Raf-1 activation (15, 17). This inference is further supported by observation of a good correlation between the strength of interaction with CRD and the ability to activate Raf-1 in a number of mutants of Ras or Rap1A, including Ha-Ras carrying an E31K, E31R, or E31A mutation (38) and Rap1A bearing phosphorylation at Ser-180 or an S180E mutation (16).

Although B-Raf is structurally a close homologue of Raf-1, recent studies have identified a notable difference in their modes of regulation (2, 23, 31, 33, 37, 47, 51). B-Raf activation induced by cyclic AMP and nerve growth factor in PC12 cells was reported to be mediated by Rap1A, not by Ras (47, 51). Rap1B, having a structure almost identical to that of Rap1A, was reported to be capable of activating B-Raf in a cell-free system derived from bovine brain (31). Thus, Rap1A appears to exert a regulatory effect on B-Raf opposite that of Raf-1, although Raf-1 and B-Raf are activated equally by Ras. According to our hypothesis that the strength of interaction at CRD is a crucial determinant for the mode of regulation by Ras and Rap1A, a difference in their CRDs may provide a structural basis for the differential regulation of Raf-1 and B-Raf by Rap1A. To this end, we have compared the interactions of CRDs of Raf-1 and B-Raf with Ha-Ras and Rap1A and carried out a domain-shuffling experiment between Raf-1 and B-Raf to determine what portions of Raf proteins are responsible for the differential regulation by Rap1A. Further, we carried out a screening to isolate a Raf CRD mutant whose interaction with Ras or Rap1A is selectively altered in order to demonstrate the significance of the strength of interaction at CRD in determining the response to Ras and Rap1A.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Plasmid construction and mutagenesis. For exchange of CR1 between Raf-1 and B-Raf, a unique ApaI cleavage site was created by oligonucleotide-directed mutagenesis in the full-length c-raf-1 cDNA at a location corresponding to amino acid residue 235, which is equivalent to an inherent unique ApaI cleavage site at residue 326 in B-raf cDNA. For exchange of CRDs, unique XbaI and KpnI cleavage sites were created by oligonucleotide-directed mutagenesis in the c-raf-1 and B-raf cDNAs at locations corresponding to residues 132 and 206, respectively, of Raf-1 and to residues 227 and 299, respectively, of B-Raf surrounding the respective CRDs without introducing any amino acid change. These cleavage sites were used to create various chimeric constructs between Raf-1 and B-Raf (Fig. 1) and to construct expression vectors for various subfragments of Raf-1 and B-Raf designated Raf-1/B-Raf(x-y), where x-y represents the range of encoded polypeptide in amino acid positions. Full-length Raf-1 and B-Raf and their chimeric constructs were tagged with a FLAG epitope (DYKDDDDK) at their N termini and expressed in COS7 cells by using the simian virus 40-based expression vector pH8 (43). Raf-1(51-131) and Raf-1(132-206), harboring RBD and CRD of Raf-1, respectively, and B-Raf(144-226) and B-Raf(227-299), harboring RBD and CRD of B-Raf, respectively, were expressed as fusions with maltose-binding protein (MBP) in Escherichia coli by using the pMal-c vector (New England Biolabs, Inc.). A C260S/C263S double mutation was introduced into B-Raf CRD by oligonucleotide-directed mutagenesis using a QuickChange site-directed mutagenesis kit (Stratagene). A pool of randomly mutagenized B-Raf(227-299) was created by an error-prone PCR (53) by using 5'-CGGGATCCTCTAGAGAATGTTCCACTTACAACAC-3' (containing BamHI and XbaI cleavage sites) and 5'-CCGCTCGAGCTAGGGTACCGGGTGTGTTCAAAGAACTTG-3' (containing XhoI and KpnI cleavage sites) as sense and antisense primers, respectively. The PCR products were cleaved by BamHI and XhoI in the primer sequences and inserted into pMal-c for expression as MBP fusions in E. coli. B-Raf(227-299) carrying appropriate mutations was excised from the resulting pMal-c plasmids by cleavage with XbaI and KpnI and used to replace the corresponding portion of the full-length B-Raf. pEF-BOS-Ha-RasVal-12 and pSRalpha -Rap1AVal-12 (16) were used to express human Ha-RasVal-12 and Rap1AVal-12, respectively, both of which carry an activating mutation of Gly to Val at amino acid position 12, in COS7 cells. Glutathione S-transferase (GST) fusion proteins of MAPK kinase/ERK kinase (MEK) and a kinase-negative mutant of extracellular signal-regulated kinase 2 (KNERK) were produced in E. coli and purified as described previously (32).


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  		FIG. 1.   Schematic representation of the structures of Raf-1, B-Raf, and their chimeric constructs. The top two horizontal bars represent the structures of Raf-1 and B-Raf protein, on which three conserved regions (CR1, CR2, and CR3) and the two subregions (RBD and CRD) in CR1 are indicated. See the text for the definition of these regions and subregions. The structures of chimeric constructs of Raf-1 and B-Raf used in this study are also shown. Numbers below the bars represent the amino acid positions of restriction cleavage sites which were used for exchanging various domains between Raf-1 and B-Raf.

In vitro binding assays. The posttranslationally fully modified forms of Ha-Ras and Rap1A and the unmodified form of Ha-Ras were purified from Spodoptera frugiperda Sf9 cells infected with baculoviruses overexpressing the respective proteins as described previously (15, 19, 32). The in vitro binding reactions were carried out by incubating 20 to 30 µl of amylose resin carrying various immobilized MBP fusion proteins with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S)- or guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S)-loaded Ha-Ras and 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 mM MgCl2, 0.1% Lubrol PX) as described previously (17). 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 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% gel) followed by Western immunoblot detection with anti-Ha-Ras monoclonal antibody F235 (Oncogene Science Inc., Manhasset, N.Y.) or anti-Rap1A polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.). The ECL (enhanced chemiluminescence) system (Amersham Pharmacia Biotech) was used for signal development. For quantification of the amounts of Ha-Ras and Rap1A bound to Raf, Ha-Ras and Rap1A were loaded with [gamma -35S]GTPgamma S to the specific activity of 10,000 cpm/pmol and subjected to the binding reaction with MBP fusion proteins immobilized on amylose resin as described above except that unlabeled GTPgamma S (0.1 mM) was included in the binding reaction. The eluate from amylose resin was counted for 35S label. The results were calculated by subtraction of a background count obtained from MBP-attached resin. Unless otherwise noted, the posttranslationally fully modified forms of Ha-Ras and Rap1A were used for the binding reactions.

Transfection and assay for Raf kinase activity. COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. Cells in 10-cm-diameter dishes (50% confluency) were cotransfected with a combination of a pH8 vector expressing Raf-1, B-Raf, or one of their chimeric constructs and either pEF-BOS-Ha-RasVal-12, pSRalpha -Rap1AVal-12, or pEF-BOS by using Superfect transfection reagent (Qiagen GmbH, Hilden, Germany). Three hours after transfection, cells were transferred to medium containing 0.1% serum and further cultured for 24 h. Cellular extracts were prepared in two ways. Cells in each dish were lysed by sonication in 0.125 ml of lysis buffer (20 mM Tris-HCl [pH 7.5], 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 20 µg of aprotinin per ml, 10 µg of leupeptin per ml, 20 mM beta -glycerophosphate, 1 mM sodium vanadate) containing 1% Triton X-100. After centrifugation at 100,000 × g for 1 h, the resulting supernatant was used as a total cellular extract. Alternatively, cells in each dish were homogenized by sonication in 500 µl of lysis buffer and separated into cytosol and membrane fractions as described before (43). A membrane extract was prepared by suspending the resulting membrane fraction in 0.125 ml of lysis buffer containing 1% Triton X-100 and subsequent centrifugation at 100,000 × g for 1 h. FLAG epitope-tagged Raf-1, B-Raf, and their chimeric proteins in the extracts were immunoprecipitated with anti-FLAG affinity gel (Eastman Kodak). Raf kinase activity was determined by incubating the immunoprecipitates in the presence of GST-MEK (0.1 µg) and GST-KNERK (1 µg) in 50 µl of kinase reaction mixture (20 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 20 mM beta -glycerophosphate, 50 µM [gamma -32P]ATP [2,000 cpm/pmol]) for 20 min at 30°C as described previously (43). After incubation, proteins in the reaction mixture were fractionated by SDS-PAGE. Phosphorylated proteins were visualized and quantified by using a BAS2000 bioimaging analyzer (Fujix, Tokyo, Japan). Raf-1, B-Raf, and their chimeric proteins in the extracts were fractionated by SDS-PAGE and detected by Western immunoblotting with either anti-Raf-1 polyclonal antibody C12 or anti-B-Raf polyclonal antibody C19 (Santa Cruz Biotechnology).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Role of CRD in activation and membrane translocation of B-Raf by Ha-Ras and Rap1A. Although the crucial role of CRD in regulation of the Raf-1 kinase activity by Ras and Rap1A had been determined, its role in regulation of B-Raf remained to be clarified. To analyze the role of CRD in regulation of the B-Raf kinase activity by Ras and Rap1A, we first examined the effect of a zinc finger mutation in B-Raf CRD. Cysteine residues Cys-260 and Cys-263 of B-Raf, corresponding to Cys-165 and Cys-168 of Raf-1, were converted to serines by oligonucleotide-directed mutagenesis, and the resulting mutant, B-Raf(C260S/C263S) (Fig. 1), was expressed in COS7 cells with an N-terminal FLAG epitope tag and examined for regulation of its kinase activity by coexpression of Ha-RasVal-12 or Rap1AVal-12 (Fig. 2A). To determine kinase activity, we immunopurified FLAG-B-Raf and its mutant from the total cellular extracts and measured the induction of phosphorylation of GST-KNERK in the presence of GST-MEK as described in Materials and Methods. As reported by others (47, 51), we observed stimulation of the kinase activity of wild-type B-Raf by both Rap1AVal-12 and Ha-RasVal-12. The stimulatory action of Rap1A on B-Raf was in sharp contrast to its inhibitory action of Raf-1. On the other hand, B-Raf(C260S/C263S) was not activated by either Ha-Ras or Rap1A, indicating that the intact zinc finger structure is required for B-Raf activation. The basal activity of B-Raf(C260S/C263S) appeared to be higher than that of wild-type B-Raf (Fig. 2A). Next, we examined the effect of C260S/C263S mutation on the efficiency of translocation of B-Raf to the plasma membrane by coexpression of Ha-RasVal-12 or Rap1AVal-12 (Fig. 2B). B-Raf(C260S/C263S) was found to be recruited to the membrane by Ha-Ras and Rap1A as efficiently as wild-type B-Raf.


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  		FIG. 2.   Ras- and Rap1A-dependent activation and membrane translocation of B-Raf and B-Raf(C260S/C263S). (A) pH8-FLAG-B-Raf (wild type [WT]) or pH8-FLAG-B-Raf(C260S/C263S) (0.5 µg of each) was cotransfected with either pEF-BOS-Ha-RasVal-12, pSRalpha -Rap1AVal-12, or pEF-BOS (3 µg of each) into COS7 cells. FLAG-B-Raf proteins were immunoprecipitated from the total cellular extract and examined for induction of phosphorylation of GST-KNERK in the presence of GST-MEK as described in Materials and Methods. The upper panel shows autoradiograms of phosphorylated GST-KNERK. The intensity of the KNERK bands was quantified with a BAS2000 bioimaging analyzer (lower panel) and expressed as fold increase with respect to the cells cotransfected with pEF-BOS. Immunoblot detection of B-Raf in the extracts is shown in the middle panel. The data shown are the means of three independent experiments. Standard deviations are indicated as error bars. (B) The transfected cells were homogenized and separated into cytosol (C) and membrane (M) fractions. B-Raf proteins present in the two fractions were detected by Western immunoblotting with anti-B-Raf antibody.

The observed effects of C260S/C263S mutation of B-Raf were similar to those observed for the analogous C165S/C168S mutation on Raf-1. The C165S/C168S mutation had been shown to reduce or abolish the Ras-dependent activation of Raf-1 (24, 36). In contrast, the C165S/C168S mutation had no effect on the Ras-dependent membrane translocation of Raf-1, which is mainly brought about by a high-affinity interaction with Ras through RBD (36). These results indicate that CRD plays a crucial role in Ras/Rap1A-dependent activation of B-Raf, which is presumably unrelated to the membrane translocation events. Aside from the difference in regulation of their activity by Rap1A, this function of CRDs appears to be conserved between Raf-1 and B-Raf.

In vitro association of Ha-Ras and Rap1A with B-Raf CRD. Our previous studies had indicated that the strength of interaction at CRD is a crucial factor in determination of the response of Raf-1 to Ras and Rap1A (15, 16, 17). Thus, we examined the strength of interaction of B-Raf CRD with Ha-Ras and Rap1A and compared it with that of Raf-1 CRD, hoping to find a mechanistic explanation for the observed differential action of Rap1A on Raf-1 and B-Raf. As shown in Fig. 3A, MBP-B-Raf(227-299), containing B-Raf CRD, associated in vitro with the posttranslationally fully modified forms of Ha-Ras and Rap1A with comparable strengths. This result is strikingly different from that obtained with MBP-Raf-1(132-206) containing Raf-1 CRD: Rap1A exhibited a much stronger interaction than Ha-Ras (Fig. 3A), as reported before (15). In either case, the associations were independent of the guanine nucleotide configuration of Ha-Ras and Rap1A, as observed before (15, 17). The strength of the interaction was estimated more quantitatively by using Ha-Ras and Rap1A, which were labeled with [gamma -35S]GTPgamma S to the same specific activity (Fig. 3B). The amounts of Ha-Ras and Rap1A bound to B-Raf CRD at the concentration of 200 nM were comparable to that of Ha-Ras bound to Raf-1 CRD but were five- to sixfold less than that of Rap1A bound to Raf-1 CRD. In contrast, MBP-B-Raf(144-226), containing B-Raf RBD, exhibited a strong GTP-dependent association with both Ha-Ras and Rap1A with a strength comparable to that of association of MBP-Raf-1(51-131) containing Raf-1 RBD (Fig. 3A and B). Rap1A bound more weakly to Raf-1 RBD than Ha-Ras, as reported before (14, 15, 38). The binding of B-Raf RBD to Rap1A was also found to be weaker than that to Ha-Ras (Fig. 3B).


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  		FIG. 3.   Association of Ha-Ras and Rap1A with various subfragments of Raf-1, B-Raf, and B-Raf(C260S/C263S). (A) Ha-Ras and Rap1A (10 pmol of each) were loaded with GTPgamma S (T) or GDPbeta S (D) and examined for association with 25 pmol of MBP-Raf-1(51-131) (Raf-1 RBD) or MBP-B-Raf(144-226) (B-Raf RBD) and with 100 pmol of MBP-Raf-1(132-206) (Raf-1 CRD) or MBP-B-Raf(227-299) (B-Raf CRD), which were immobilized on amylose resin, as described in Materials and Methods. The bound Ha-Ras and Rap1A were fractioned by SDS-PAGE (12% gel) and subjected to Western immunoblotting with anti-Ha-Ras and anti-Rap1A antibodies, respectively. One-tenth of the amount of Ha-Ras or Rap1A used for the binding reactions was applied on the same gel (Input). All Western blots were exposed to X-ray film for the same period. The sensitivities of anti-Ha-Ras and anti-Rap1A antibodies were almost equal, as observed before (15, 16). (B) Ha-Ras and Rap1A (20 pmol for CRD binding and 10 pmol for RBD binding) were loaded with [gamma -35S]GTPgamma S and examined for association with 60 pmol of MBP fusion proteins immobilized on amylose resin as described in Materials and Methods. The results shown are the means of three independent experiments performed in duplicate and expressed as fold differences where the amount of Ha-Ras bound to Raf-1 CRD (1.8 pmol) (left) or to Raf-1 RBD (3.8 pmol) (right) is defined as 1. Standard deviations are indicated as error bars. (C) In vitro binding reactions carried out as for panel A by using 100 pmol each of MBP-B-Raf(227-299) (wild type [WT]) and MBP-B-Raf(C260S/C263S). (D) Ten picomoles of posttranslationally unmodified Ha-Ras was loaded with GTPgamma S (T) or GDPbeta S (D) and incubated with 25 pmol of MBP-B-Raf(144-226) (B-Raf RBD) or 100 pmol of MBP-B-Raf(227-299) (B-Raf CRD) as described for panel A.

We next examined the effect of the C260S/C263S double mutation on the binding of B-Raf CRD to Ha-Ras and Rap1A. As shown in Fig. 3C, MBP-B-Raf(227-299) carrying this mutation exhibited a grossly impaired ability to associate with both Ha-Ras and Rap1A. As observed for Raf-1 CRD (17), the association of B-Raf CRD with Ha-Ras was dependent on prenylation of Ha-Ras, since the unmodified form of Ha-Ras exhibited no detectable association with MBP-B-Raf(227-299) while retaining the ability to associate with MBP-B-Raf(144-226) (Fig. 3D). The results presented so far fit very well with our hypothesis that the strength of interaction at CRD with Ras or Rap1A is a crucial determinant for regulation of the Raf kinase activity and that it must be at a level adequate to cause Raf activation (15). The stimulatory effect of Rap1A on B-Raf activity is accounted for by the fact that the strength of interaction of B-Raf CRD with Rap1A was reduced to the level of interaction with Ha-Ras.

The nature of CRD determines the mode of regulation of Raf by Rap1A. The experiments described so far indicated that Rap1A has differential regulatory effects on Raf-1 and B-Raf. To locate the region of Raf responsible for these differential responses, we carried out a domain-shuffling experiment. By using unique restriction enzyme cleavage sites created at the boundaries of RBD, CRD, and CR2, we constructed a series of chimeras between Raf-1 and B-Raf (Fig. 1). The chimeric Raf proteins were expressed with an N-terminal FLAG epitope tag in COS7 cells and examined for regulation of their kinase activities by coexpression with Ha-RasVal-12 or Rap1AVal-12 (Fig. 4). FLAG-B-Raf, FLAG-Raf-1, and their chimeras were immunopurified from the total cellular extracts and subjected to measurement of the kinase activity as described in Materials and Methods. B-CR1/Raf-1, a composite of the B-Raf N-terminal region containing CR1 (residues 1 to 326) and the Raf-1 C-terminal region containing CR2 and the catalytic domain (residues 236 to 648), was found to be activated by both Ha-Ras and Rap1A (Fig. 4A), whereas R-CR1/B-Raf, an opposite combination to B-CR1/Raf-1, was activated by Ha-Ras but not by Rap1A (Fig. 4B). Thus, the response of each of these chimeras to Rap1A depended on the nature of the CR1. For a finer mapping, we next constructed additional chimeras with exchanges of CRD between Raf-1 and B-Raf. B-CRD/Raf-1, Raf-1 whose CRD is replaced by B-Raf CRD, became activatable by Rap1A (Fig. 4A), whereas R-CRD/B-Raf, B-Raf whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A (Fig. 4B). Both B-CRD/Raf-1 and R-CRD/B-Raf were activatable by Ha-Ras. Raf-1, B-CR1/Raf-1, and B-CRD/Raf-1 were expressed in the same amounts, as measured by immunoblotting with anti-Raf-1 antibody (Fig. 4A), while B-Raf, R-CR1/B-Raf, and R-CRD/B-Raf were expressed in the same amounts, as measured by immunoblotting with anti-B-Raf antibody (Fig. 4B). These results clearly indicated that the nature of CRD determines the response of Raf to Rap1A.


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  		FIG. 4.   Ras- and Rap1A-dependent kinase activities of Raf-1, B-Raf, and their chimeric constructs. (A) pH8-FLAG-Raf-1, pH8-FLAG-B-CR1/Raf-1, or pH8-FLAG-B-CRD/Raf-1 (3 µg of each) was cotransfected into COS7 cells with either pEF-BOS-Ha-RasVal-12, pSRalpha -Rap1AVal-12, or pEF-BOS (3 µg of each). The kinase activities of Raf immunoprecipitated from the total cellular extracts were measured as described in Materials and Methods. The upper panel shows autoradiograms of the phosphorylated GST-KNERK. The intensity of the KNERK bands was quantified as for Fig. 2A (lower panel). The amounts of FLAG-Raf-1 and its chimeras in the extracts were measured by immunoblotting with anti-Raf-1 antibody (middle panel). (B) pH8-FLAG-B-Raf, pH8-FLAG-R-CR1/B-Raf, or pH8-FLAG-R-CRD/B-Raf (0.5 µg of each) was cotransfected into COS7 cells with either pEF-BOS-Ha-RasVal-12, pSRalpha -Rap1AVal-12, or pEF-BOS (3 µg of each), and their kinase activities were measured as described for panel A. The upper panel shows the autoradiograms of the phosphorylated GST-KNERK. The intensity of the KNERK bands was quantified as for Fig. 2A (lower panel). The amounts of FLAG-B-Raf and its chimeras in the extracts were measured by immunoblotting with anti-B-Raf antibody (middle panel). The data shown are the means of two independent experiments performed in duplicate. Standard deviations are indicated as error bars.

We carried out a series of similar experiments to measure the kinase activity of a population of Raf which was recruited to the plasma membrane by coexpression of Rap1A (Fig. 5). COS7 cells were homogenized and fractionated into cytosol and membrane fractions. FLAG-Raf proteins were immunoprecipitated from Triton X-100 extracts of the membrane fractions and subjected to measurement of kinase activity. The amount of FLAG-B-CRD/Raf-1 recruited to the plasma membrane was similar to that of FLAG-Raf-1. However, the kinase activity of FLAG-B-CRD/Raf-1 was strongly stimulated by coexpression with Rap1A compared to the activity observed with FLAG-Raf-1 (Fig. 5). In contrast, FLAG-R-CRD/B-Raf was not activated by Rap1A even though it was recruited to the plasma membrane as efficiently as FLAG-B-Raf (Fig. 5). These results taken together indicated that the nature of CRD is responsible for determining the mode of regulation of the Raf kinase activity by Rap1A. This regulation does not appear to involve the membrane translocation process of Raf induced by Rap1A.


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  		FIG. 5.   Rap1A-dependent activation of Raf-1, B-Raf, and their chimeras translocated to the membrane. pH8-FLAG-Raf-1, pH8-FLAG-B-CRD/Raf-1, pH8-FLAG-B-Raf, or pH8-FLAG-R-CRD/B-Raf (3 µg of each) was cotransfected into COS7 cells with 3 µg of pSRalpha -Rap1AVal-12. The transfected cells were homogenized and separated into cytosol and membrane fractions. The kinase activities of Raf immunoprecipitated from the membrane extracts were measured as described in Materials and Methods. The upper panel shows the autoradiograms of the phosphorylated GST-KNERK. The amounts of FLAG-Raf-1 or FLAG-B-Raf and their chimeras in the membrane extracts were measured by immunoblotting with anti-Raf-1 antibody or anti-B-Raf antibody (lower panel). Three independent experiments were performed with similar results.

Isolation of a mutant of B-Raf CRD exhibiting enhanced binding to Rap1A. To further demonstrate the relationship between the strength of interaction at CRD and the response to Ras and Rap1A, we undertook a screening for mutants of CRD whose interactions with Ras or Rap1A are selectively altered. To this end, we introduced random mutations into B-Raf(227-299), containing CRD, by an error-prone PCR and expressed them as MBP fusions in E. coli. The resulting mutant MBP-B-Raf(227-299) protein was examined for association with Ha-Ras and Rap1A by the in vitro binding assays using Western immunoblotting (Fig. 6A) or [gamma -35S]GTPgamma S-labeled proteins (Fig. 6B). One mutant which exhibited enhanced binding to Rap1A was found to carry the double mutation K252E/M278T (Fig. 6A and B). Binding of this mutant to Rap1A was 2.5-fold greater than that of wild-type B-Raf CRD, whereas its Ha-Ras-binding activity was almost the same. We next examined the response of B-Raf carrying this mutation to Ha-Ras and Rap1A when expressed in COS7 cells (Fig. 6C). The kinase activity of FLAG-B-Raf(K252E/M278T) was not activated by coexpression of Rap1A but was activated by Ha-Ras as efficiently as wild-type B-Raf. Thus, the response to Rap1A was selectively abrogated by a B-Raf CRD mutation which increased the strength of interaction with Rap1A. This result strongly supports our hypothesis that the strength of interaction at CRD with Ras or Rap1A is a crucial determinant for regulation of the Raf kinase activity and that it must be at a level adequate to cause Raf activation (15, 16).


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  		FIG. 6.   Effects of a K252E/M278T mutation on the interaction of B-Raf-CRD with, and activation of B-Raf by, Ras and Rap1A. (A) MBP-B-Raf(227-299) (wild type [WT]) and MBP-B-Raf (K252E/M278T) were examined for in vitro association with Ha-Ras or Rap1A as described in the legend to Fig. 3. The bound Ha-Ras and Rap1A were fractioned by SDS-PAGE (12% gel) and subjected to Western immunoblotting with anti-Ha-Ras and anti-Rap1A antibodies, respectively. One-tenth of the amount of Ha-Ras or Rap1A used for the binding reactions were applied on the same gel (Input). (B) Ha-Ras and Rap1A (20 pmol of each) were loaded with [gamma -35S]GTPgamma S and examined for association with 60 pmol each of MBP-B-Raf(227-299) (WT) and MBP-B-Raf(K252E/M278T) as described in Materials and Methods. The results shown are the means of three independent experiments performed in duplicate and expressed as fold increases over the binding activities of wild-type B-Raf CRD (1.5 pmol for Ha-Ras and 1.2 pmol for Rap1A). Standard deviations are indicated as error bars. (C) pH8-FLAG-B-Raf or pH8-FLAG-B-Raf(K252E/M278T) (0.5 µg of each) was cotransfected into COS7 cells with either pEF-BOS-Ha-RasVal-12, pSRalpha -Rap1AVal-12, or pEF-BOS (3 µg of each). The kinase activities of B-Raf immunoprecipitated from the total cellular extracts were measured as described in Materials and Methods. The upper panel shows the autoradiograms of the phosphorylated GST-KNERK. The intensity of the KNERK bands was quantified as for Fig. 2A (lower panel). The amounts of FLAG-B-Raf in the extracts were measured by immunoblotting with anti-B-Raf antibody (middle panel). The data shown are the means of three independent experiments. Standard deviations are indicated as error bars.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The mechanism of activation of Raf by association with Ras seems to involve a much more complex process than was originally thought. Although membrane recruitment of Raf-1 was considered to be the only function of Ras in activation of its kinase activity (20, 22, 40), subsequent studies by a number of groups have demonstrated an additional crucial role of Ras in the activation of Raf-1 (25, 36, 43). We and others have demonstrated that this additional role is mediated through interaction of Raf-1 CRD with Ras (12, 15, 17, 21, 25, 36, 43). However, the molecular mechanism of this interaction is unclear; conflicting reports on the structural requirements for this interaction have appeared. We and others consistently observed that the CRD-Ras interaction is independent of the guanine nucleotide configuration of Ras and requires both the C-terminal posttranslational modifications and the intact activator region of Ras (15, 17, 21, 38), a finding confirmed by the present study. This is consistent with the observation that activator region mutants of Ras lack the ability to activate Raf-1 while retaining the ability to associate with RBD (43) as well as with the results of studies using in vitro cell-free systems, which found an absolute dependence of the Raf activation on the C-terminal posttranslational modifications of Ras (32, 41, 50) even though another cellular factor seems to be required for achieving Ras-dependent activation of Raf-1 and B-Raf (11, 26, 41). In contrast, studies by other groups found that the CRD-Ras interaction exhibits a GTP dependence (3, 12), is independent of the posttranslational modifications of Ras (9), and is abolished by mutations in the switch II region of Ras (12). However, none of these studies included a quantitative comparison between the modified and unmodified forms of Ras except for that using a yeast two-hybrid system (9), which obviously tends to be grossly affected by other factors such as the efficiency of nuclear localization of the interacting proteins.

Based on a good correlation between the strength of interaction with CRD and the ability to activate Raf-1 in Ras, Rap1A, and their various mutants, we propose that the strength of interaction at CRD plays a crucial role in determination of the mode of regulation of the Raf kinase activity by Ras and Rap1A and must be at a level adequate to cause Raf-1 activation. This hypothesis is consistent with our recent observation that phosphorylation of Ser-180 of Rap1A by protein kinase A caused a loss of its enhanced activity to associate with Raf-1 CRD simultaneously with a loss in its ability to antagonize Ras-dependent activation of Raf-1 (16). Data presented in this paper provide further support for our hypothesis. Differential regulation of Raf-1 and B-Raf by Rap1A can be explained in terms of a difference in the strength of interaction of their CRDs with Rap1A, which has been unambiguously demonstrated by the in vitro binding and the domain-shuffling experiments. Furthermore, the importance of the strength of interaction with CRD has been confirmed by isolation of a mutant of B-Raf CRD, whose interaction with Rap1A is selectively enhanced, by screening a pool of randomly mutagenized clones. B-Raf carrying this K252E/M278T mutation was found to have lost the ability to be activated by Rap1A while retaining the normal response to Ha-Ras. It is interesting that Raf-1 Lys-157 and Met-183, corresponding to B-Raf Lys-252 and Met-278, respectively, were identified by Daub et al. (9) as the residues whose mutations caused an alteration of the Ras-dependent activity of Raf-1.

We do not know the molecular mechanism by which the strength of interaction with Ras and Rap1A determines the response of Raf to these small GTPases. Raf-1 CRD is reported to interact with 14-3-3 (6, 24) and phosphatidylserine (4, 13) in addition to Ras and Rap1A. The significance of these interactions in Ras-dependent activation of Raf-1 remains to be clarified, although interactions with 14-3-3 at two positions, the phosphorylated Ser-259 and Ser-621, were shown to be crucial for Raf-1 activation (44, 45). In addition, Raf-1 CRD was shown to effect an intramolecular autoinhibitory interaction with the kinase domain CR3 (8). Although extensive mutational studies on Raf-1 CRD have identified some of the epitopes involved in association with Ras (9), 14-3-3 (6), phosphatidylserine (4), and CR3 (48), the exact binding sites of these factors have not yet been identified. It is possible that some of these factors share a common binding site with Ras/Rap1A and exhibit a competition for binding with Ras/Rap1A. In fact, certain mutations at residues 143 and 144 of Raf-1 CRD were reported to cause an increased binding to Ras simultaneously with a reduced interaction with CR3 (48). This observation was interpreted to indicate that the CRD-CR3 interaction affects the availability of CRD for interaction with Ras. Studies using an in vitro cell-free system for Ras-dependent Raf activation suggested the presence of an unknown factor essential for the activation (11, 26, 41). This factor might also be a candidate for competition with Ras and/or Rap1A. Such a competition may hypothetically occur either at an initial step of Raf activation, at a later step involved in maintaining the active state of Raf, or in a transitional state between these steps. For example, Ras or Rap1A may associate first with CRD to induce a transiently active conformation of Raf and subsequently detach to allow Raf to interact with another protein and assume a different conformation pertinent to its prolonged activation. The appropriate strength of the CRD interaction might be indispensable for such a sequence of attachment and detachment of Ras or Rap1A. Further analyses will be needed to elucidate the molecular mechanism by which the Ras family GTPases regulate the Raf kinase activity dependent on the strength of its interaction with CRD.


    ACKNOWLEDGMENTS

We thank X.-H. Deng for skillful technical assistance and A. Seki and Y. Kawabe for help in preparation of the manuscript.

This investigation was supported by grants-in-aid for scientific research on priority areas, for scientific research (B), and for encouragement of young scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Yamanouchi Foundation for Research on Metabolic Diseases.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-382-5380. Fax: 81-78-382-5399. E-mail: kataoka{at}kobe-u.ac.jp.


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Abstract
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Materials and Methods
Results
Discussion
References

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