(Received for publication, March 28, 1994; and in revised form, November 16, 1994)
From the
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
) region,
distinguished from switch I, a ``switch'' effector
(E
) region. The E
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
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).
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) ()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.
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, 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.
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 -sheet that consists of the
2 and
3 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 -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.
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.
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).
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.
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 region (residues
42-49) in the tertiary structure of the Ras protein (G12 type) in
the guanylyl-(
,
-methylene)diphosphate (GMPPCP)-bound form(14) . Red, GMPPCP; yellow, the E
region or switch I (residues
30-40); blue, functionally important residues of the
E
region, Lys
, Val
,
Ile
, Gly
, and Glu
; cyan, other residues of the E
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
-sheet structure displaying several important residues (Fig. 1), as the Gly residue is involved in a
-turn located
in one end of the
-sheet(13, 14, 15, 16) . But,
it is also possible that Gly
is directly recognized
(probably together with the
-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
region). Strictly speaking, in addition to the switch I region
consisting of residues 30-38, Tyr
should be included
in the E
region(13, 14, 46) .
Thus, we call the region consisting of residues 42-49 the
``constitutive effector (E
)'' region.
With
which protein does the E region interact? The E
region overlaps well with the ``activator'' that
potentially activates GAP function(40) . The E
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
region slightly affected the
affinity for GAP, suggesting that the E
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 region of Ras were
shown to completely block the Ras-triggered MAP kinase activation in
PC12 cells. Furthermore, the E
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
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 region of Ras only
slightly reduced the ability to bind to
Raf-1(31, 63) , and other mutants in the E
region binds Raf-1 as efficiently as the wild type (31) . (
)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 region diminished the
binding of Ras to B-Raf without impairing the Raf-1 binding. If this is
the case, the E
region mutants would be useful to
distinguish between two pathways of Raf-1 and B-Raf.
On the other
hand, one of the E 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
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
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 region of Ras is
involved also in the interaction of these target effectors.