(Received for publication, November 2, 1995; and in revised form, December 27, 1995)
From the
Ras proteins have multiple effectors of distinct structures that
do not share significant structural homology at their Ras interaction
sites. To prove possible differences in their recognition mechanisms of
Ras, we screened 44 human Ha-Ras proteins carrying mutations in the
effector region and its flanking sequences for interaction with human
Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces
cerevisiae adenylyl cyclase. The Ras binding specificities were
largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S,
and A59E, had their affinities for Byr2 selectively reduced. The only
exception was Ras(D38N), which lost the ability to bind Raf-1 while
retaining the activity to bind Byr2 and complement the Byr2 phenotype of S. pombe. On the other hand, adenylyl
cyclase had quite distinct requirements for Ras residues; mutations
P34G and T58A selectively abolished the ability to bind and activate it
without considerably affecting the interaction with Raf-1 and Byr2.
Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2,
could activate adenylyl cyclase efficiently. In addition, V45E mutation
was found to impair the ability of Ras to activate both Raf-1 and
adenylyl cyclase without significantly affecting the binding affinities
for them. These results demonstrate that significant differences exist
in the recognition mechanisms by which the three effector molecules
associate with Ras and suggest that a region of Ras required for
activation of the effectors in general may exist separately from that
for binding the effectors.
The ras genes are widely conserved in a variety of
eukaryotic organisms from yeasts to mammals. Their protein products
belong to a family of small guanine nucleotide-binding proteins and
operate in key processes of intracellular signal transduction systems
that are involved in regulation of cell growth and differentiation. In
higher eukaryotes including Caenorhabditis elegans, Drosophila
melanogaster, and vertebrates, Ras proteins are involved in
intracellular signaling from receptor tyrosine kinases, which results
in activation of a phosphorylation cascade comprising Raf proteins, MAP ()kinase kinases (MEKs), and MAP kinases (ERKs) (for
reviews, see (1) and (2) ). Recent studies
demonstrated direct binding of Raf-1 and its homologue B-Raf to
Ras(3, 4, 5, 6, 7, 8, 9) ,
but the precise mechanism of the Raf activation remains to be
clarified. In addition, other mammalian proteins have been shown to
associate directly with Ras in a GTP-dependent manner. These include
Ral-guanine nucleotide dissociation
stimulator(10, 11, 12) , phosphatidylinositol
3-OH-kinase(13) , the
-isoform of protein kinase
C(14) , and Rin1(15) . However, the significance of
their interaction with Ras is presently unknown. In the fission yeast, Schizosaccharomyces pombe, its single Ras homologue, Ras1, is
involved in signal transduction from mating pheromone receptors, and
its function is mediated by a protein kinase cascade that ultimately
regulates a member of the MAP kinase family (for a review, see (16) ). Protein kinase Byr2 was recently shown to be a direct
downstream target of Ras1(4, 17, 18) . On the
other hand, in the budding yeast Saccharomyces cerevisiae,
adenylyl cyclase is an immediate downstream effector of a pair of Ras
proteins, Ras1 and Ras2, which are structural, biochemical, and
functional homologues of vertebrate Ras (for a review, see (19) ).
Mutational studies of the effector molecules identified discrete Ras-binding regions in them. The Ras-binding site of mammalian Raf-1 was mapped to an 81-amino acid segment in its N-terminal regulatory domain(7, 20, 21, 22) . Recently another Ras-binding site was identified in Raf-1 corresponding to the cysteine-rich region(23, 24) . In yeast adenylyl cyclase, the leucine-rich repeats domain, which is composed of tandemly repeated 23-amino acid leucine-rich motifs(25) , was shown to bind directly to Ras(7, 26) . Byr2 contains a Ras-binding site within its N-terminal 206 amino acid residues(18) , but further mapping has not been attempted. Comparison of the primary structures of these Ras-binding domains revealed virtual absence of homologous sequences among them.
On the other hand, extensive mutational studies on Ras identified amino acid residues whose substitution abolished the ability of Ras to transform NIH3T3 cells, to induce neurite outgrowth in PC12 cells, to induce germinal vesicle breakdown in Xenopus laevis oocytes, or to associate with Raf-1, without affecting the guanine nucleotide binding properties(20, 27, 28, 29, 30, 31, 32, 33, 34, 35) . First, many of these residues were concentrated in a region corresponding to amino acid residues 32-40 of mammalian Ras, so that this region was designated the ``effector region.'' Actually, determination of the tertiary structure of Ras showed that this region almost matches to ``switch I,'' one of the two regions that take significantly different conformations between the GTP- and GDP-bound forms of Ras(36, 37, 38) . In contrast, the residues 26, 31, 42, 45, 46, 48, 49, and 53, flanking the effector region, were recently found to be critical for the Ras signaling even though they do not change their conformations upon ligand exchange from GDP to GTP (29, 30, 31, 34, 35) . Therefore, these residues, which are likely to be involved in activation of the Ras effectors, were designated the ``activator region'' (34) or ``constitutive effector region'' (35) .
Presently, there exists an assumption based on studies using a very limited number of the effector region mutants of Ras that all three distinct effectors recognize a similar structure located in the effector region and possibly in its immediately flanking regions for their interaction with Ras. However, the observed absence of structural conservation among the effectors suggests that they may interact with Ras in a somewhat different mode. This prompted us to systematically survey possible differences in the recognition mechanisms of Ras by the three effector molecules. We use a series of human Ha-Ras mutants bearing amino acid substitutions in the effector region and its flanking region as molecular probes for dissecting differential requirements of particular Ras residues. In addition to semi-quantitative estimations by a yeast two-hybrid assay and by in vitro binding measurements, we employ a method based on inhibition of the Ras-dependent activity of yeast adenylyl cyclase for quantitative analysis of the binding reactions between the Ras mutants and the effectors. Results of these binding studies are compared with those of measuring the biochemical and biological activities of the effector proteins.
Figure 1:
In vitro binding of Ha-Ras
mutants to Raf-1 and Byr2. The Ha-Ras mutant proteins, which exhibited
differential interaction with Raf-1 and Byr2 in the two-hybrid assay,
were loaded with GTPS (T) or GDP (D) and
examined for their abilities to bind directly to purified
MBP-Raf-1-(1-257) and GST-Byr2-(1-287) polypeptides as
described under ``Experimental Procedures.'' One pmol each of
Ha-Ras
proteins carrying the indicated mutations or no
mutation (WT) was used for the assay. The bound Ha-Ras and
one-tenth of the amount of the input of Ha-Ras used for each binding
reaction were separated by SDS-PAGE (12% gel) and subjected to Western
immunoblotting with the anti-Ha-Ras antibody F235. The mobilities of
the Ha-Ras mutants differ from one another as reported
previously(27, 33) .
Figure 2:
Measurements of interaction of various
Ha-Ras mutants with yeast adenylyl cyclase. A, adenylyl
cyclase activities were measured in the presence of increasing
concentrations of the GTPS-bound forms of various Ha-Ras
proteins. Panel a, wild type (
), I21A (
),
L23F (
), I24V (
), N26G (
), F28A (
), and V29A
(
); panel b, wild type (
), D30E (
), E31K
(
), Y32F (
), P34G (
), T35S (
), and D38N
(
); panel c, wild type (
), Y40W (
), R41A
(
), Q43A (
), Q43H (
), V44A (
), and V45E
(
); panel d, wild type (
), I46A (
), D47A
(
), L52M/D54E (
), T58A (
), A59E (
), Y64F
(
), and S65T (
). One unit of activity is defined as 1 pmol
of cAMP formed in 1 min of incubation with 1 mg of membrane protein at
32 °C under standard assay conditions. B, 10 pmol each of
Ha-Ras
protein carrying the indicated mutations or no
mutation (WT) was loaded with GTP
S (T) or GDP (D) and examined for its ability to bind directly to purified
GST-CYR1-(606-2026) polypeptide as described under
``Experimental Procedures.'' The bound Ha-Ras and one-tenth
of the amount of the input of Ha-Ras used for each binding reaction
were separated by SDS-PAGE (12% gel) and subjected to Western
immunoblotting with the anti-Ha-Ras antibody
F235.
To examine whether the loss of
activation was ascribable to failure in binding adenylyl cyclase, we
analyzed in vitro binding of the Ha-Ras mutant proteins to
purified GST-CYR1-(606-2026) (Fig. 2B). As
expected, the mutants, E31K, Y32F, and T35S, all of which exhibited low K values, bound to GST-CYR1-(606-2026) in a
GTP-dependent manner as efficiently as wild type. In contrast, all of
the activation-negative Ha-Ras mutants, P34G, D38N, T58A, and A59E,
exhibited no detectable binding, indicating that the loss of adenylyl
cyclase activation reflects the absence of the binding activity. F28A
exhibited very low binding as predicted from its high K
value. The binding activities of the Ha-Ras mutants were
generally in parallel with the K
values of the
cyclase activation. Thus, the Ha-Ras mutants, P34G, T58A, and to a
lesser extent F28A, selectively lost the ability to bind adenylyl
cyclase while retaining the ability to bind Raf-1 and Byr2. These data
indicate that yeast adenylyl cyclase possesses a considerable
difference in the binding requirement for Ras from that of Raf-1 or
Byr2.
Figure 3:
Determination of the Kvalues of Raf-1 and Byr2 for various
Ha-Ras mutants by the adenylyl cyclase inhibition assay. Adenylyl
cyclase activities dependent on various concentrations of the
GTP
S-bound forms of Ha-Ras
proteins were measured
with the addition of varying amounts of MBP-Raf-1-(1-257) (5 pmol
(
), 10 pmol (
), and 20 pmol (
) shown in panels
A, C, and E, respectively) or of
GST-Byr2-(1-287) (0.5 pmol (
), 1 pmol (
), 2 pmol
(
), and 4 pmol (
) shown in panels B, D,
and F, respectively). The Ha-Ras
proteins
carried no mutation (WT) (panels A and B),
Y32F mutation (panels C and D), or T35S mutation (panels E and F). The amount of free Ha-Ras and that
bound to MBP-Raf-1-(1-257) or GST-Byr2-(1-287) were
calculated as described in the text, and a reciprocal of the
concentration of the free Ha-Ras was plotted against a reciprocal of
the concentration of the bound Ha-Ras. The K
values for Ha-Ras were calculated from the points of
intersection with the horizontal axis as described before (26) .
Figure 4:
Measurements of the activities of Ha-Ras
mutants to activate Raf-1. Sf9 cells were infected with the recombinant
baculovirus expressing the full-length Raf-1, alone or in combination
with that expressing either Ha-Ras (WT) or one
of its mutants (A59E, Y32F, D38N, and T35S). The
amounts of Raf-1 and Ha-Ras proteins present in Nonidet P-40 extracts
of the infected Sf9 cells were determined by Western immunoblotting
with the corresponding antibodies. Raf-1 was immunoprecipitated from
the extracts by the anti-Raf-1 antibody and examined for its activity
to induce phosphorylation of GST-KNERK in the presence of GST-MEK as
described under ``Experimental Procedures.'' Shown are an
autoradiogram of an SDS-PAGE gel separating the phosphorylated proteins (upper panel) and the immunoblots of the Sf9 extracts
subjected to detection of Raf-1 (middle panel) or Ha-Ras (lower panel). The Ha-Ras bands formed doublets; the upper
represents the post-translationally unmodified form and the lower
represents the modified form.
Figure 5:
Examination of the abilities of Ha-Ras
mutants to complement the Ras1 phenotype of S.
pombe diploid cells. The S. pombe JZ840 cells harboring
pREP3 plasmid (panel A) or that carrying either wild-type
Ha-Ras
(panel B), Ha-Ras
(Y32F) (panel C), Ha-Ras
(T35S) (panel D),
Ha-Ras
(D38N) (panel E), or
Ha-Ras
(A59E) (panel F) were examined for their
ability to undergo sporulation as described under ``Experimental
Procedures.'' Shown are photographs of representative
fields.
Mutational studies of Ras by a number of groups showed that
the effector region (Tyr-Tyr
) is
critical for its biological activity as well as for binding to its
effector, Raf-1 (20, 27, 28, 29, 30, 31, 32, 33, 34, 35) .
This region roughly corresponds to the switch I
(Tyr
-Asp
), whose structure is altered
significantly between the GTP- and GDP-bound
forms(36, 37, 38) . Recent determination of
the crystal structure of a complex between the Ras-binding domain
(residues 51-131) of Raf-1 and the Ras-related protein
Rap1A/Krev-1 indicated that the residues in the effector domain
constitute a principal binding interface(56) . In contrast, for
the abilities to interact with other Ras effectors including Byr2 and S. cerevisiae adenylyl cyclase, only a limited set of Ras
mutants has been studied(4, 18, 27) . The
present study is the first to carry out a systematic survey of a
possible difference in the recognition mechanisms of Ras by its
distinct effectors. The results are summarized in Table 2only
for the Ha-Ras mutants that exhibited differential interactions with
the effectors. Also, available data on the activities of these mutants
to transform NIH3T3 or rat2 cells and to induce neurite outgrowth in
PC12 cells are presented in the same table.
It is shown clearly that
the D38N mutation abolishes the ability of Ha-Ras to associate with
both Raf-1 and adenylyl cyclase without significantly affecting the
ability to bind and activate Byr2. The inability of the D38N mutant to
interact with Raf-1 was reported before (5, 33) .
Mutations at position 38 were shown to abolish the biological activity
without significantly altering the overall conformation of Ras,
suggesting a direct implication of this residue in the effector
activation(57) . A nonconservative substitution at this
position, D38A, was known to abolish interaction with all of the three
effectors(5, 27) . Here, use of the conservative
substitution, D38N, has helped in identifying a subtle difference in
the recognition mechanisms of Ras by the effectors. In addition, our
screening has yielded three mutants, Y32F, T35S, and A59E, which, to a
lesser degree, can bind discriminatively to Raf-1 and Byr2.
Determination of the K values indicated that the
affinity of the Y32F mutant for Byr2 was about 18-fold weaker than that
of wild type, whereas its affinity for Raf-1 was unaffected. Similarly,
the T35S mutation selectively reduced the affinity for Byr2 by about
5-fold. However, when the activities of these mutants to stimulate the
effectors were examined, they did not discriminate between Raf-1 and
Byr2; for both of them, Y32F was inactive, while T35S was active. Y32F
had also been reported to lack the activity to induce neurite outgrowth
in PC12 cells and to transform NIH3T3 cells(58, 59) .
The A59E mutant lost the ability to bind Byr2, but its ability to bind
Raf-1 was also significantly attenuated. This mutant again exhibited
severely attenuated activities to stimulate both Raf-1 and Byr2. Thus,
we could not obtain any mutant that selectively lost the ability to
interact with and activate Byr2. Presently, we do not know the reason
why the Y32F and A59E mutations cause the loss of activation of both
Byr2 and Raf-1.
In contrast to the situation with Raf-1 and Byr2, we observed clearer differences in the mode of interaction of Ras with S. cerevisiae adenylyl cyclase. The P34G and T58A mutations selectively abolished the association with adenylyl cyclase, whereas the association with Raf-1 or Byr2 was not significantly affected. The A59E mutant behaved similarly although its ability to interact with Byr2 or Raf-1 was also attenuated to a lesser degree. The affinity for adenylyl cyclase was greatly reduced by the F28A and I46A mutations, whereas that for Raf-1 or Byr2 was not so much affected. The Y32F mutant, lacking the ability to activate both Raf-1 and Byr2, could effectively bind and activate adenylyl cyclase. These results raise a possibility that adenylyl cyclase may recognize the residues Thr-58 and Ala-59, immediately flanking the switch II region, in addition to the effector region. Thus, we have demonstrated that significant differences exist in the recognition mechanisms of Ras by Raf-1, Byr2, and adenylyl cyclase and successfully obtained the Ha-Ras mutants, which interact with and activate only a specific set of the effectors. Especially, the D38N and Y32F mutants could selectively activate a single effector out of the three effectors tested. These mutants may be useful in differentially analyzing the individual function of the distinct effector molecules in cells as already proposed(60) .
It has been proposed that some residues of Ras surrounding the
switch I region are involved in the effector activation(34) .
Actually, mutations of residues 26, 31, 42, 45, 46, 48, and 53 were
found to deprive Ras of its activities to cause cellular
events(29, 30, 31, 32, 33, 35) ,
whereas most of these mutations did not affect the ability to associate
with Raf-1(20, 33, 35) . In the present
study, it was demonstrated that mutants, N26G, E31K and V45E, retained
not only the ability to associate with Raf-1 but also those to bind
Byr2 and yeast adenylyl cyclase. However, the V values of adenylyl cyclase attained by the E31K, V45E, and, to a
lesser degree, N26G mutants were considerably reduced compared with
that of wild type, whereas their K
values were not
significantly changed. This suggests that the definition of the
``activator region'' of Ras may also be valid for another
effector, yeast adenylyl cyclase. We have recently shown that the
activator region of Ras binds directly to the cysteine-rich region of
Raf-1, which is located at the C-terminal side of the Ras-binding
domain, in a GTP-independent manner and that post-translational
modifications of Ras are required for this interaction(24) .
Because post-translational modifications of Ras were also critical for
activation of adenylyl cyclase(46) , these data may suggest the
presence in adenylyl cyclase of a separate binding site for the
activator region of Ras from that for the effector region.
While this work was in progress, White et al.(60) reported the isolation of Ha-Ras effector region mutants, which interact differentially with Raf-1 and Byr2 by screening of a pool of randomly mutagenized Ha-Ras with the yeast two-hybrid assay. By using the results of this assay as a sole criterion, they identified the T35S mutant as that interacted with Raf-1 but not with Byr2 and the E37G mutant as that interacted with Byr2 but not with Raf-1. Although we confirmed the two-hybrid assay result with the T35S mutant, it was clearly shown here that this mutant retains a considerable ability to associate with Byr2 and activate it in vivo. This represents a possible pitfall in counting on the two-hybrid assay, which, as we observed, tends to yield false negative results caused by minor alterations in the binding parameters.
Previously, it was found that
the T35S mutation impaired the ability of the Ras protein without
post-translational modifications to coprecipitate with
Raf-1(20, 33) . In fact, we have found that
dissociation of the T35S mutant from the Raf-1 Ras-binding domain is
appreciably faster than the unmodified wild-type Ras. ()In
contrast, the present in vitro binding and competition assays,
which were performed with the post-translationally modified Ras
protein, demonstrated the efficient association of the T35S mutant with
the N-terminal segment of Raf-1 encompassing both the Ras-binding
domain and the cysteine-rich region, as in the case of the two-hybrid
assay with Ras possibly modified post-translationally in yeast cells. A
similar result has been obtained for the E31K mutant ( (33) and
this paper). This discrepancy may be accounted for by our recent
finding that post-translational modifications of Ras are required for
the interaction of Ras with the cysteine-rich region of
Raf-1(24) .
In the present study, we have clearly shown that
the D38N mutant interacts properly with Byr2 but not detectably with
Raf-1. Although we do not know how stringently the E37G mutant (60) distinguishes between Raf-1 and Byr2, it is interesting to
point out that the E37G mutation may impair the functional conformation
of the neighboring Asp, which is indispensable for
association with and activation of Raf-1 rather than Byr2.