©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Constitutive Effector Region on the C-terminal Side of Switch I of the Ras Protein (*)

(Received for publication, March 28, 1994; and in revised form, November 16, 1994)

Junko Fujita-Yoshigaki (1) (2) Mikako Shirouzu (1) (3) Yutaka Ito (1) (4) Seisuke Hattori (5) Shunsuke Furuyama (2) Susumu Nishimura (6) Shigeyuki Yokoyama (1) (3)(§)

From the  (1)Department of Biophysics and Biochemistry, School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, the (2)Department of Physiology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo-shi, Chiba 271, the (3)Cellular Signaling Laboratory and the (4)Biodesign Research Group, the Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01, the (5)Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, and the (6)Banyu Tsukuba Research Institute in Collaboration with Merck Sharp and Dohme Research Laboratories, Banyu Pharmaceutical Co., Ltd., 3 Ohkuba, Tsukuba-shi, Ibaraki 300-33, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The ``switch I'' region (Asp-Asp) of the Ras protein takes remarkably different conformations between the GDP- and GTP-bound forms and coincides with the so-called ``effector region.'' As for a region on the C-terminal side of switch I, the V45E and G48C mutants of Ras failed to promote neurite outgrowth of PC12 cells (Fujita-Yoshigaki, J., Shirouzu, M., Koide, H., Nishimura, S., and Yokoyama, S.(1991) FEBS Lett. 294, 187-190). In the present study, we performed alanine-scanning mutagenesis within the region Lys-Ile of Ras and found that the K42A, I46A, G48A, E49A, and L53A mutations significantly reduced the neurite-inducing activity. This is an effector region by definition, but its conformation is known to be unaffected by GDP GTP exchange. So, this region is referred to as a ``constitutive'' effector (E(c)) region, distinguished from switch I, a ``switch'' effector (E(s)) region. The E(c) region mutants exhibiting no neurite-inducing activity were found to be correlatably unable to activate mitogen-activated protein (MAP) kinase in PC12 cells. Therefore, the E(c) region is essential for the MAP kinase activation in PC12 cells, whereas mutations in this region only negligibly affect the binding of Ras to Raf-1 (Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, H., Toyama, Y., Yamasaki, K., Fuhrman, S. A., Villafranca, E., Kaziro, Y., and Yokoyama, S.(1994) Oncogene 9, 2153-2157).


INTRODUCTION

Activated Ras proteins have the signal-transducing activity to cause transformation of NIH 3T3 cells, differentiation of PC12 cells, and maturation of Xenopus oocytes ((2, 3, 4, 5) : for review, see (1) ). Ras is also known to induce activation of c-Raf-1 and mitogen-activated protein kinase (MAP kinase) (^1)or extracellular signal-regulated kinase (ERK)(6, 7, 8) . Such signal transducing activities are abolished by mutations in the ``effector region'' (Tyr-Tyr)(9, 10, 11, 12) . Mutations in the effector region affect neither guanine-nucleotide binding nor GTPase activity, so the effector region is considered to be the region that interacts with the target effectors of the Ras protein.

From x-ray crystallographic and nuclear magnetic resonance (NMR) analyses, the three-dimensional structure of the Ras protein has been shown to change upon GDP GTP exchange(13, 14, 15, 16) . In particular, the conformations of the Asp-Asp and Gly-Glu regions change significantly, and these regions are called ``switch I'' and ``switch II,'' respectively(14) . The switch I region essentially overlaps with the effector region. In the switch I region, therefore, there are ``effector residues'' that are involved in the target interaction. GTPase activating proteins (p120-GAP, p100-GAP, and neurofibromin; (17, 18, 19, 20) ), c-Raf-1(21, 22, 23, 24, 25) , and phosphatidylinositol-3-OH kinase (PI-3 kinase; (26) ), which are candidates for target effectors, directly and selectively bind to the GTP-bound form of Ras. Some mutations in the switch I region of Ras have been reported to diminish the interaction with these proteins(19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31) , indicating that the switch I region is a binding site for GAPs, Raf-1, and PI-3 kinase.

Other residues outside the switch I region may also be required for the interaction with these proteins. Krev-1 (Rap1, smg p21), which is the product of a suppressor gene for transformation induced by an activated K-ras gene(32, 33, 34) , shares a strong similarity with the Ras protein. In particular, the amino acid sequence of Tyr-Val in the Krev-1 protein is identical to the corresponding sequence in the Ras protein(32, 33, 34) . Therefore, the transforming activity requires other residue(s). A chimeric Ras protein, consisting of residues 41-60 of Krev-1, was devoid of transforming activity(35) , and the putative effector residues are expected to exist in this region.

To determine the effector residues essential for signal transduction, and to identify the binding sites for the putative target effectors, such as Raf-1 and GAP, we have performed mutational analyses of the Ras protein. We have previously prepared mutant Ras proteins with an amino acid residue replaced by the corresponding residue of the Krev-1 protein (Krev-1-type mutants; (36) and (37) ). The Glu Lys (E31K), V45E, and G48C mutants had no activity, and the N26G mutant was partially defective in a PC12 differentiation assay(36, 37) . The E31K, V45E, and N26G mutations also abolished the transforming activity in NIH 3T3 cells(38, 39) . Thus, the region Asn-Gly was reported to be an extended effector region(40) .

In this study, we prepared mutants in residues 42-55 and examined the effects of the mutations on the differentiation of PC12 cells. We also examined the affinity of GAP for these mutant proteins, and the activation of MAP kinase in PC12 cells. In addition, we compared these properties of the mutants with their abilities to bind the Raf-1 protein.


MATERIALS AND METHODS

Mutagenesis of the c-Ha-ras Gene

Each mutation was introduced into synthetic human c-Ha-ras genes (41) with and without the oncogenic Gly Val (G12V) mutation, by oligonucleotide-directed mutagenesis using the Muta-Gene(TM) kit (Bio-Rad).

PC12 Cells Transfected with a Mutant ras Gene or the v-raf Gene

The stable transfectant clones of PC12 cells harboring the ras gene with a Krev-1-type mutation on the oncogenic G12V background, or the v-raf gene, in the mammalian expression vector pMAM-neo (Clontech) were reported previously(36, 42) . Each of the newly prepared ras genes with a second mutation introduced on the G12V background (``V12 type'') was also subcloned into pMAM-neo. PC12 cells were transfected with DNA (5 µg) by the calcium phosphate precipitation method, and G418-resistant clones were selected. For each gene, three or more independent clones were obtained and used for the following experiments. Expression of the ras or v-raf gene in the PC12 cells was induced by addition of dexamethasone at the final concentration of 1 µM in the culture medium. After 24 h, the number of cells that have extended neurites was counted. MAP kinase activation was examined as described below, for the cells 8 h (unless otherwise noted) after the induction.

Preparation of the Mutant Ras Proteins

The mutant Ras proteins were expressed in Escherichia coli and purified as described, with the following modification(41) : 50 mM Tris-HCl buffer (pH 7.5) containing 5 mM MgCl(2), 5 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF) was used. The purified proteins were bound with GDP. To exchange the bound nucleotide from GDP to guanylyl-(beta,-imido)diphosphonate (GMPPNP), the Ras protein (1 mg) was incubated with GMPPNP (0.2 µmol) and 1 mM EDTA for 5 min at 37 °C. The free nucleotides were removed by ultrafiltration using centricon-10 units (Amicon). This cycle was repeated three times. By high-performance liquid chromatographic analysis, it was confirmed that 95% of the binding nucleotides were converted to GMPPNP.

Microinjection of the Ras Proteins in the GMPPNP-bound Form

PC12 cells were replated 24 h before microinjection at a density of 1 times 10^5 cells/dish. We injected Ras mutants (10 mg/ml) in the GMPPNP-bound form into 100-200 cells using an injectoscope, model IMF-2 (Olympus).

GTPase Activities in the Absence and Presence of GAP

The Ras protein (0.3 nmol) was incubated with 1 mM EDTA and [8,5`-^3H]GTP (0.28 nmol, 43.6 Ci/mmol) for 5 min at 37 °C. Free nucleotides were removed by ultrafiltration, and the final concentration of Ras was adjusted to 3 µM. This solution of [8,5`-^3H]GTP-bound Ras protein (25 µl) was mixed with purified recombinant rat GAP (0.01-2 µM) and incubated at 10 °C. At different times, 5-µl aliquots were removed and boiled with 0.25% SDS and 5 mM EDTA. These mixtures were chromatographed on polyethyleneimine-cellulose TLC plates (Merck) in 0.5 M LiCl and 1 M formic acid. The radioactivities in the GTP and GDP spots were counted. From a semilogarithmic plot of the amounts of bound GTP versus time, the rates of GTP hydrolysis were obtained.

Measurement of Dissociation Rates of Guanine Nucleotides from Mutant Ras Proteins

After incubation of the Ras protein (2 µM) with [8,5`-^3H]GDP or [8,5`-^3H]GTP (2 Ci/mmol, 10 µM) in 450 µl of 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 1 mM DTT, and 1 mM PMSF for 10 min at 37 °C, 2.5 µl of 1 M MgCl(2) and 10 µl of 100 mM nonlabeled GTP were added to the solution. At different times, 100-µl aliquots were applied to nitrocellulose filters and washed five times with 2 ml of a buffer containing 20 mM Tris-HCl (pH 7.5) and 1 mM MgCl(2). The radioactivities remaining on the filters were counted.

In-gel Kinase Assay

PC12 cells (2 times 10^5 cells/60-mm dish) were washed with ice-cold phosphate-buffered saline and scraped with a scraper in 0.1 ml of an extraction buffer consisting of 20 mM Tris-HCl (pH 7.5), 5 mM EGTA, 0.5% Triton X-100, 12 mM beta-glycerophosphate, 1 mM PMSF, 1 mg/ml leupeptin, 6 mM DTT, and 1 mM sodium orthovanadate at 0 °C. Homogenization and centrifugations were performed as described previously(43) . The supernatant was used as the cell extract. We used 10 µg of each extract per assay.

The in-gel kinase assay was performed according to the method described previously(43) . Briefly, after electrophoresis in an SDS-polyacrylamide gel containing myelin basic protein (MBP), the SDS was removed by washing the gel with 20% 2-propanol. After denaturation with 6 M guanidine HCl and renaturation in a 0.04% Tween 40-containing buffer, the gel was incubated at 25 °C for 1 h with 10 ml of 40 mM Hepes-NaOH buffer (pH 8.0) containing 2 mM DTT, 0.1 mM EGTA, 5 mM MgCl(2), and 25 µM [-P]ATP (2.5 µCi/ml) for kinase reactions. The substrate protein for kinases, MBP (0.5 mg/ml), was added to the separating gel prior to the acrylamide polymerization. Experiments were performed at least three times with each transfectant.

Western Blotting of MAP Kinases

Cell extracts (25 µg/assay) were subjected to electrophoresis on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose sheets. Western blots were probed with anti-ERK1 antibody 956/837 and visualized by using the ECL immunodetection system (Amersham Corp.).


RESULTS

To identify the determinant residues responsible for the signal-transducing activity of Ras, we made a series of mutations in the amino acid residues on the C-terminal side (residues 42-55) of the switch I region of the human c-Ha-Ras protein. These residues are included in the antiparallel beta-sheet that consists of the beta2 and beta3 strands of the Ras protein (Fig. 1). We have already prepared ``Krev-1-type'' mutants, with an amino acid residue replaced with the corresponding one of the Krev-1 protein, and have examined their neurite-inducing activity(36) . In this study, we substituted an alanine for each amino acid residue in this region (``alanine-scanning mutagenesis'').


Figure 1: A schematic presentation of the antiparallel beta-sheet structure consisting of residues 38-57 of human c-Ha-Ras. The mutations that were found to abolish the neurite-inducing activity of Ras are indicated.



Neurite-inducing Activities of Mutant ras Genes

When the ras gene with an oncogenic mutation, Gly Val (G12V), was expressed in PC12 cells, about 80% of the cells extended neurites (Fig. 2). We tested here the Ala mutations for their effects on the neurite induction by the G12V ras gene. Among the 15 mutations that we introduced on the G12V background in this study, we found that two mutations (K42A and L53A) completely diminished the neurite-inducing activity in PC12 cells; essentially none of the cells transfected with these mutant genes (V12 type) extended neurites ( Fig. 2and 3a). In addition, the I46A, G48A, and E49A mutations reduced the efficiency of neurite outgrowth down to 32, 45, and 17%, respectively, of the transfected cells (Fig. 3a). In contrast, other mutant ras genes induced neurite outgrowth for 80-95% of the PC12 cells. By Western blotting, it was confirmed that the neurite induction-deficient mutant proteins were expressed in PC12 cells to the same extent as the other mutant proteins (data not shown).


Figure 2: Morphological change of PC12 cells induced by mutant ras genes (V12 type). The photographs were taken 24 h after the addition of dexamethasone. a, G12V; b, G12V/K42A; c, G12V/I46A; d, G12V/G48A; e, G12V/E49A; f, G12V/L53A.




Figure 3: The neurite-inducing activities of mutant Ras proteins. The numbers of PC12 cells that extended neurites were counted and are shown as percentages of the total number of cells. For each mutant, the value shown is the mean ± S.E. for at least three independent experiments with different transfectant clones. a, transfection with mutant ras genes (V12 type). b, microinjection with GMPPNP -bound mutant Ras proteins (G12 type).



We have already reported that the V45E and G48C mutations abolished the neurite-inducing activity(36) . Thus, the region bearing residues Lys, Val, Ile, Gly, Glu, and Leu is now concluded to be essential for the neurite-inducing activity of Ras.

Neurite-inducing Activities of Ras Mutants in the GMPPNP-bound Form

The Ras protein is thought to transduce signals in the active GTP-bound form(44) . There was a possibility that these inactive mutants could not interact properly with the guanine-nucleotide exchange factor (45) and therefore failed to be converted to the GTP-bound form in the transfected cells. To test this possibility, we exchanged the bound nucleotide of the wild-type and K42A and L53A mutant proteins from GDP to an unhydrolyzable GTP analog, GMPPNP, and microinjected these proteins into PC12 cells (we used here the Ras proteins that have glycine at position 12 (G12 type)). As a result, 67% of the cells that were injected with the GMPPNP-bound wild-type Ras extended neurites. Microinjection of the GMPPNP-bound L53A mutant protein partially promoted neurite outgrowth, but the K42A mutant protein did not induce neurite outgrowth (Fig. 3b). We have already shown that the V45E and G48C mutants bound with GMPPNP had no neurite-inducing activity(36) .

Dissociation Rates of GDP or GTP from Ras Proteins

We investigated the dissociation rates of guanine nucleotides from the mutants lacking the neurite-inducing activity. Ras proteins of the G12 type were used in this experiment. We measured the amounts of [^3H]GTP or [^3H]GDP bound to the Ras proteins after an incubation in the presence of Mg. As for the wild type, the dissociation rate was slightly higher for GDP than for GTP (Table 1). The nucleotide dissociation rates for the mutant proteins were very similar to those of the wild type, except for the L53A mutant. The GDP and GTP dissociation rates of the L53A mutant were 2.8- and 5.0-fold higher than those of the wild type, respectively. The effects of other mutations on the dissociation rates of guanine nucleotide were negligible.



GTPase Activities of Mutant Ras Proteins and Affinities between Ras Mutants and GAP

To investigate the effects of mutations on GTPase activity, the G12-type Ras proteins were used. The intrinsic GTPase activities of the mutant proteins were similar to that of the wild-type Ras, except for the L52M/D54E mutant Ras, which had a 2-fold increased GTPase activity (Fig. 4a).


Figure 4: Intrinsic GTPase activities and GAP affinities of the mutant Ras proteins (G12 type). a, GTP hydrolysis rates of the mutant Ras proteins at 37 °C in the absence of GAP. b, concentrations of GAP that half-maximally increased the GTPase activities of the mutant Ras proteins at 10 °C.



Next, we investigated the GTPase activities in the presence of GAP. At 37 °C, the GTPase activities of all the mutants in the presence of GAP were very similar to that of the wild type (data not shown). We then measured the GTP hydrolysis rates of mutant Ras proteins at 10 °C in the presence of various concentrations of GAP. A plot of the results shows that the GTPase activity of 3 µM of the wild type is half-maximally increased by 0.20 µM GAP (Fig. 4b). The V45E mutant Ras protein had a 1.7-fold higher affinity with GAP, and the affinities of the I46A, G48A, and G48C mutant Ras proteins were reduced to half of that of the wild type (Fig. 4b). The GAP affinities of the other mutants were almost the same as that of the wild type. The fully GAP-enhanced GTP hydrolysis rates of all the mutants were similar to that of the wild type (0.74 min).

Activation of MAP Kinases in PC12 Cells Expressing the Mutant Ras Proteins

As oncogenic ras genes activate MAP kinase in PC12 cells(7, 8) , we analyzed whether the MAP kinase was activated in PC12 cells that were induced to express a mutant Ras protein (V12 type). The in-gel kinase assay tests renatured MAP kinases for their abilities to phosphorylate the embedded MBP (43) as shown in Fig. 5a. It should be noted here that the MAP kinase activation by Ras is much lower than that by nerve growth factor (NGF) as reported(8) . So, by the in-gel kinase assay, it was impossible to analyze the band due to the p42 MAP kinase (ERK2) because of overlapping with an unidentified band. In contrast, the band of the p44 MAP kinase (ERK1) could clearly be distinguished. From the intensities of this band, therefore, we examined the abilities of the mutant Ras proteins to activate p44 MAP kinase in PC12 cells (Fig. 5a) as summarized in Table 2in comparison with their neurite-inducing activities.


Figure 5: The activation of MAP kinases by Ras mutants (a and b) and v-Raf (c) in PC12 cells. a, in-gel kinase assay of the activation of p44 MAP kinase by Ras mutants. Extracts of PC12 cells 8 h after the induction of a mutant Ras protein (V12 type) with dexamethasone were electrophoresed in SDS-polyacrylamide gels containing MBP. After renaturation of the proteins in the gels, the phosphorylation of MBP with [-P]ATP was detected by autoradiography. The autoradiographs shown are representatives of those obtained from at least three independent experiments with different transfectant clones. Only for lane NGF, a very short exposure radioautograph was used. The bands corresponding to the p44 MAP kinase are indicated with arrowheads. b, Western blot analysis of the activation of p42 MAP kinase by Ras mutants. The active and inactive forms of the p42 MAP kinase were detected as two separate bands; the former migrates more slowly than the latter, as indicated. In a and b, control, the normal PC12 cells were treated with dexamethasone; lane NGF, the normal PC12 cells were treated with NGF; others, the PC12 cells transfected with the ras gene having the indicated mutation on the background of G12V (V12 type) were treated with dexamethasone. c, in-gel kinase assay of the activation of p44 MAP kinase at 4, 8, and 16 h after induction of expression of v-Raf with dexamethasone. The results were obtained and presented as described for a.





First, the K42A, V45E, G48C, and L53A mutations completely abolished the activation of p44 MAP kinase by Ras in the PC12 cells. In addition, the p44 MAP kinase activation was significantly diminished by the E49A mutation. Correspondingly, the neurite-inducing activity was completely impaired by the K42A, V45E, G48C, and L53A mutations and drastically reduced by the E49A mutation. In contrast, the other mutations, which did not affect the neurite-inducing activity, exhibited no effect on the p44 MAP kinase activation. Thus, as far as these mutations of Ras are concerned, the activation of p44 MAP kinase correlates well with the neurite-inducing activity.

On the other hand, we could observe the activation of p42 MAP kinase by Western blotting detection of the phosphorylated, active form which migrates more slowly than the inactive form(8) . As shown in Fig. 5b, the activation of p42 MAP kinase was certainly reduced by the K42A and E49A mutations, but not appreciably by the T50A and C51A mutations. Therefore, there is no significant difference, between the p42 and p44 MAP kinase species, in the effects of the Ras mutations on the activation in PC12 cells.

Activation of MAP Kinases by v-Raf Expression

In NIH 3T3 cells and COS cells, v-Raf has been reported to induce the activation of MAP kinase. To determine whether v-Raf activates p44 MAP kinase in PC12 cells, we utilized PC12 cells transfected by the v-raf gene(42) . For this stable transfectant, we have already reported that the induced expression of v-Raf resulted in neurite outgrowth(42) . On the other hand, it was also reported that a v-Raf expression failed to activate MAP kinases in PC12 cells, although it was unclear whether or not the transfectant cells exhibited v-Raf-dependent neurite outgrowth (8) . We show here that the expression of the v-Raf protein as induced by incubation with dexamethasone certainly promoted the activation of p44 MAP kinase (Fig. 5c). The p44 MAP kinase activity could be detected after a 4-h incubation with dexamethasone and were maximal after 8 h. The kinase activity was even weaker than that induced by the oncogenic Ras, G12V.


DISCUSSION

It has been reported that some residues in the C-terminal side of the Ras switch I region is important for signal-transducing activity. For example, the K42D mutation abolished the transforming activity in NIH 3T3 cells(12) , and the Q43R mutant is a temperature-sensitive mutant for rodent fibroblast transformation(10) . We have already reported that residues Val and Gly are required to promote neurite outgrowth of PC12 cells(36) . Other groups have also reported that the V45E mutation abolished the activity to transform NIH 3T3 cells(38, 39) . In this study, we performed alanine-scanning mutagenesis in this region and found 4 other amino acid residues essential for the induction of differentiation of PC12 cells. Therefore, in this region, there are, in total, 6 residues whose mutations abolish the neurite-inducing activity.

Among these 6 residues, Leu has unique properties. Only the L53A mutant exhibited lower guanine-nucleotide binding activity. Moreover, Leu is buried within the Ras protein, whereas the other residues lie exposed on the surface of the protein (Fig. 6; (14) ). Therefore, it is proposed that Leu does not bind to the target effector(s), but instead plays a role in maintaining the functional three-dimensional structure of the Ras protein. Intriguingly, Leu is spatially close to Lys ( Fig. 1and Fig. 6), and the signal-transducing activities of the L53A mutant are similar to those of the K42A mutant, which will be discussed again below. Therefore, it is possible that the L53A mutation reduced the neurite-inducing activity by affecting the conformation of Lys.


Figure 6: The E(c) region (residues 42-49) in the tertiary structure of the Ras protein (G12 type) in the guanylyl-(beta,-methylene)diphosphate (GMPPCP)-bound form(14) . Red, GMPPCP; yellow, the E(s) region or switch I (residues 30-40); blue, functionally important residues of the E(c) region, Lys, Val, Ile, Gly, and Glu; cyan, other residues of the E(c) region; green, Leu. The molecular graphics image was produced using the MidasPlus software system from the Computer Graphics Laboratory, University of California, San Francisco(71) .



Other neurite induction-defective mutants except L53A have the guanine-nucleotide binding and GTP hydrolysis activities as high as those of the wild type. The effect of the mutation of Gly might be an indirect one through some slight distortion of the beta-sheet structure displaying several important residues (Fig. 1), as the Gly residue is involved in a beta-turn located in one end of the beta-sheet(13, 14, 15, 16) . But, it is also possible that Gly is directly recognized (probably together with the beta-turn structure centering around it) by the target effector(s). Therefore, residues Lys, Val, Ile, Gly, and Glu are probably required for the direct interaction with target effector(s) and constitute a part of the effector region. This region does not change its conformation upon GDP GTP exchange, whereas the conformational change of the switch I region is significant(14, 15) . Therefore, we distinguish this nonswitch effector region from the switch I effector region (E(s) region). Strictly speaking, in addition to the switch I region consisting of residues 30-38, Tyr should be included in the E(s) region(13, 14, 46) . Thus, we call the region consisting of residues 42-49 the ``constitutive effector (E(c))'' region.

With which protein does the E(c) region interact? The E(c) region overlaps well with the ``activator'' that potentially activates GAP function(40) . The E(s) region has been shown to be the interaction site for GAP; residues Asp, Ile, and Asp are essential for enhancement of the GTPase activity by GAP(12, 27, 28, 29, 47) . Some of the mutations in the E(c) region slightly affected the affinity for GAP, suggesting that the E(c) region is also involved in the interaction with GAP. However, the affinity did not correlate with the neurite-inducing activity.

MAP kinase is activated by oncogenic Ras in PC12 cells, although the extent of activation is much less than that by NGF(7, 8) . In this study, several mutations in the E(c) region of Ras were shown to completely block the Ras-triggered MAP kinase activation in PC12 cells. Furthermore, the E(c) region mutations that abolished the MAP kinase activation also impaired the neurite-inducing activity of Ras. So, in this region, the two activities are well correlated with each other. This result is another evidence that the activation of MAP kinase is essential and sufficient for differentiation of PC12 cells (48) . Therefore, the E(c) region of Ras is likely to be involved in interaction with a target effector that causes the MAP kinase activation in PC12 cells.

In general, MAP kinase is activated upon phosphorylation by MAP kinase kinase (MEK; (49, 50, 51) ), and several pathways are proposed for Ras-dependent activation of MEK. First, Raf-1 kinase has been reported to activate MEK in NIH 3T3 cells, COS cells, and in vitro(52, 53, 54) . Raf-1 is essential for the transformation of NIH 3T3 cells (55) and is probably the major kinase to phosphorylate MEK in Ras-mediated signal transduction in NIH 3T3 cells. In PC12 cell, there is another member of the Raf family, B-Raf(56) . It was reported that both Raf-1 and B-Raf were activated(57, 58) , but it was also reported that B-Raf, but not Raf-1, was activated (59) upon NGF stimulation of PC12 cells.

Ras directly binds to Raf-1(20, 21, 22, 23, 24, 30) and also to B-Raf(59) . The post-translational modification at the C-terminal region of Ras, which is required for membrane anchoring, was shown to be essential for Raf-1 activation(60) . Furthermore, it was demonstrated that the Raf-1 kinase can be activated upon direct anchoring to the plasma membrane through covalent fusion with only several amino acid residues derived from the C-terminal anchoring site of Ras(61, 62) . Therefore, it was proposed that the most important role of Ras in terms of the Raf-1 activation is to anchor the Raf-1 protein to the plasma membrane using its C-terminal anchoring site(61, 62) . According to this idea, Ras mutants that retain the activity to bind Raf-1 may also retain the Raf-1 activation activity.

However, we have already found that two mutations (V44A and V45E) within the E(c) region of Ras only slightly reduced the ability to bind to Raf-1(31, 63) , and other mutants in the E(c) region binds Raf-1 as efficiently as the wild type (31) . (^2)So, the MAP kinase activation ability of these Ras mutants in PC12 cells does not correlate with their Raf-1 binding activity.

In this context, it has been reported that Raf-1 and B-Raf are activated by NGF to different extents in PC12 cells(59) , indicating that B-Raf is activated by Ras in a different manner from that of Raf-1. One possibility is, therefore, that the neurite-deficient mutations in the E(c) region diminished the binding of Ras to B-Raf without impairing the Raf-1 binding. If this is the case, the E(c) region mutants would be useful to distinguish between two pathways of Raf-1 and B-Raf.

On the other hand, one of the E(c) region mutants (V45E), whose Raf-1 binding ability is not much different from that of the wild type(31, 63) , lacks the transforming activity in NIH 3T3 cells(38, 39) . Note that Raf-1 is mainly responsible for the Ras-mediated signal transduction in NIH 3T3 cells(55) . In addition, v-Raf, which lacks the Ras-binding region and is therefore unable to be anchored through Ras to the membrane, can activate MAP kinase in PC12 cells (Fig. 5b) and in NIH 3T3 cells(52) . Is the membrane anchoring really essential and sufficient for the Raf-1 kinase activation? So, we are now examining the effects of other E(c) region mutations on the transforming activity of Ras in NIH 3T3 cells. The activation of Raf-1 by Ras is supposed to require the 14-3-3 protein(64, 65, 66) . There is a possibility that the E(c) region of Ras is essential for the putative activation process involving the Raf-1, Ras, and 14-3-3 proteins on the plasma membrane.

In addition to the Raf family, MEK kinase, the mammalian homolog of the yeast protein kinase Byr2 (Saccharomyces cerevisiae) and STE11 (Schizosaccharomyces pombe), activates MEK independently of Raf-1(67) . The activation of MEK kinase by growth factors requires the Ras protein(57) . REKS, which is a kinase that phosphorylates MEK in a GTP-bound Ras-dependent fashion, was also reported(68) . Recently, PI-3 kinase was shown to be involved in the growth factor-dependent signal transduction(69) , and to directly bind to the GTP-bound form of Ras(26) , indicating the divergency of Ras-mediated signal transduction. In PC12 cells, Wortmannin, specific PI-3 kinase inhibitor inhibited the neurite outgrowth(70) . Accordingly, it is important to examine whether the E(c) region of Ras is involved also in the interaction of these target effectors.


FOOTNOTES

*
This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a by grant for the Biodesign Research Program from RIKEN (to S. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-3812-1805; Fax: 81-3-5689-5609.

(^1)
The abbreviations used are: MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; GAP, GTPase-activating protein; PI-3 kinase, phosphatidylinositol-3-OH kinase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; GMPPNP, guanylyl-(beta,-imido)diphosphate; MBP, myelin basic protein; NGF, nerve growth factor; MEK, MAP kinase kinase/ERK kinase; GMPPCP, guanylyl-(beta,-methylene)diphosphate.

(^2)
M. Shirouzu, A. Kamiyar, J. Fujita-Yoshigaki, and S. Yokoyama, unpublished result.


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