(Received for publication, September 20, 1995; and in revised form, October 25, 1995)
From the
The interaction between ``switch I/effector domain'' of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the ``activator domain'' (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the ``cysteine-rich region'' (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.
Ras belongs to a family of small GTP-binding proteins and plays
essential roles in the regulation of cell proliferation and
differentiation (for a review, see (1) ). Like other
GTP-binding proteins, the GTP-bound form of Ras is active and able to
interact with its effectors whereas the GDP-bound form is not. X-ray
crystallographic studies showed that, on the protein surface of
mammalian Ha-Ras, the conformation of two regions named ``switch
I'' (Asp-Asp
) and ``switch
II'' (Gly
-Glu
) (Fig. 1A) changes significantly depending on the nature
of bound guanine nucleotide(2, 3) . The switch I
overlaps with the ``effector domain''
(Tyr
-Tyr
) identified by mutational
studies as a region essential for effector activation but not for
GTPase activity and membrane localization, and is thought to mediate
GTP-dependent interaction with effectors(4, 5) .
However, residues (Asn
, Tyr
,
Val
, and Gly
) flanking the switch I have also
been shown to be critical for effector
activation(6, 7, 8, 9) . These
residues have been proposed to constitute the ``activator
domain'' (Fig. 1A) (for a review, see (10) ). Additional residues (Lys
,
Ile
, and Glu
) belonging to this domain have
been identified recently by Fujita-Yoshigaki et
al.(11) . The activator domain is also exposed on the
surface of Ha-Ras but its conformation is not much altered by GDP/GTP
exchange(10, 11) . In addition, post-translational
modifications of Ha-Ras have also been shown to be crucial for its
biological function (for a review, see (12) ). However, it is
presently unclear how these individual structural features are involved
in effector activation.
Figure 1: Structures of the wild-type Ha-Ras, Raf-1, and its mutant proteins. A, various structural and functional domains of Ha-Ras and its post-translationally modified C-terminal structure are schematically shown. See the text for definition of these domains. The effector domain almost overlaps with the switch I. The activator domain is split into two subregions surrounding the effector domain. B, shown are the structure of the full-length Raf-1 protein (a) and an enlargement of its N-terminal region (b), on which three conserved regions (CR1, CR2, and CR3) and the two subregions (RBD and CRR) in CR1 are indicated. See the text for the definition of these regions and subregions. The structures of various Raf-1 mutants used in this study are also depicted (c-i). Numbers on the bars represent amino acid positions in both A and B.
Raf-1, a 74-kDa cytoplasmic serine/threonine
protein kinase regulating the mitogen-activated protein kinase cascade,
is one of the major effectors of Ha-Ras (for a review, see (13) ). Raf-1 shares three regions of conservation, termed CR1, ()CR2, and CR3, with other Raf isoforms and homologs (Fig. 1B)(13) . CR1 and CR2 are located in the
N-terminal half of Raf-1, and CR3 corresponds to the C-terminal kinase
domain. Activation of Raf-1 by N-terminal truncations indicates that
the N-terminal portion plays an important regulatory role(14) .
The minimal region of Raf-1 responsible for the interaction with Ha-Ras
has been mapped into 81 amino acids in CR1, RBD (amino acids
51-131)(15) , and mutational analyses have suggested that
RBD interacts directly with the switch I of
Ha-Ras(16, 17, 18, 19) . However, a
mutation (C168S) located in another region of CR1, CRR (amino acids
152-184), has been shown to impair physical interaction of Raf-1
with Ha-Ras and to render Raf-1 unresponsive to
Ha-Ras(20, 21) . These observations indicate that not
only RBD but also CRR is critical for Ras-dependent activation of
Raf-1. However, the exact mechanism of involvement of Raf-1 CRR in
Ras-dependent activation remains to be determined. Here we report that
CRR interacts with Ha-Ras independently of RBD and that the activator
domain of Ha-Ras is critical not only for this interaction but also for
the Ha-Ras-dependent activation of Raf-1. These results suggest that
CRR participates in Raf-1 activation through interaction with the
activator domain of Ha-Ras. Evidence is also presented that
post-translational modifications of Ha-Ras C terminus are necessary for
this interaction.
Figure 2:
Direct association of deletion mutants of
Raf-1 CR1 with wild-type and mutant Ha-Ras. The amounts of Ha-Ras
proteins bound to various MBP-Raf-1 fusion proteins immobilized on
amylose resin in vitro were measured by Western immunoblotting
with the anti-Ras antibody Y13-259. A, 10 pmol of
GTPS-bound Ha-Ras (T) or GDP-bound Ha-Ras (D)
were incubated with 25 pmol of MBP-Raf-1 fusion proteins or MBP alone.
The numbers on the top indicate the range of Raf-1,
in amino acid position, expressed as fusions with MBP. B,
various amounts of GTP
S-bound Ha-Ras (T) or GDP-bound
Ha-Ras (D) were incubated with either 100 pmol of
MBP-Raf-1(132-206) (CRR), 50 pmol of
MBP-Raf-1(50-131) (RBD), or 100 pmol of MBP alone (MBP). The numbers indicate the amounts of Ras proteins used
for the assays in pmol. C, 10 pmol of GTP
S-bound Ha-Ras
were incubated with 50 pmol of MBP-Raf-1(50-131) (RBD)
or its mutant MBP-Raf-1(50-131, R89L) (R89L), and with
100 pmol of MBP-Raf-1(132-206) (CRR) or its mutant
MBP-Raf-1(132-206, C168S) (C168S). D, 10 pmol
each of GTP
S-bound Ha-Ras
(WT) or its
mutants (N26G, V45E, A59E, and D38N) were incubated with 25 pmol of MBP-Raf-1(50-131) (RBD), 100 pmol of MBP-Raf-1(132-206) (CRR) or
100 pmol of MBP alone (MBP). The mobilities of the Ha-Ras
mutants differ one another as reported previously(4) . E, 20 pmol of GTP
S-bound post-translationally unmodified
Ha-Ras were incubated with 25 pmol of MBP-Raf-1(50-131) (RBD) or 100 pmol of MBP-Raf-1(132-206) (CRR).
Experiments were repeated three times giving equivalent
results.
Figure 3:
Abilities of the Ha-Ras mutants to
activate Raf-1 kinase activity. 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 (V45E, A59E, and D38N). A, the amount of Ha-Ras protein present in
Nonidet P-40 extract of the infected Sf9 cells was measured by Western
immunoblotting with the anti-Ras antibody Y13-259. The arrow indicates the position of Ha-Ras on the blot. The upper band in each lane represents the unmodified form, while the lower represents the modified form. B, the amount of Raf-1 was
measured in the same extract by Western immunoblotting with an
anti-Raf-1 antibody C12. The arrow indicates the position of
Raf-1 on the blot. C, Raf-1 was immunoprecipitated from the
same extract by the anti-Raf-1 antibody and examined for its activity
to induce autophosphorylation of Raf-1. The arrow indicates
the position of phosphorylated Raf-1 on the autoradiogram. D,
the immunoprecipitated Raf-1 was examined for its activity to induce
phosphorylation of GST-KNERK in the presence of GST-MEK. The arrow indicates the position of phosphorylated GST-KNERK on the
autoradiogram. Shown are representative of three experiments giving
similar results.
In the present study, we have shown that CRR interacts with Ha-Ras independently of RBD. During the preparation of this manuscript, Brtva et al. also reported that CRR (GST-Raf-1(139-186)) alone can interact with Ha-Ras in a GTP-dependent manner using an ELISA-based assay(29) . However, this result is contradictory to our finding that CRR binds to Ha-Ras in a GTP-independent manner. Considering the GTP-dependent binding of all other Raf-1 mutants to Ha-Ras (Fig. 2, A and B), it seems that the condition of our assay is appropriate to detect GTP-dependent binding. Furthermore, the observations that CRR did not interact with Ha-Ras mutants N26G and V45E, and that the C168S mutation introduced into CRR could abolish its interaction with wild-type Ha-Ras, strongly support the specificity of the interaction between Ha-Ras and Raf-1 CRR. Finally, our results agree with the fact that the conformation of the activator domain is not affected by GDP/GTP exchange(10, 11) . Although the reason for the discrepancy regarding GTP dependence is unknown, it must be pointed out that the source of Ha-Ras protein in our assay is different from that in their assay. We used post-translationally modified Ha-Ras protein purified from infected Sf9 cells, while they used bacterially expressed Ha-Ras protein which was not modified. In our hands, unmodified Ha-Ras protein purified from Sf9 cells did not bind CRR at all (Fig. 2E).
Post-translational modifications of
Ha-Ras at its C terminus have been shown to be necessary for its
membrane-anchoring as well as for Raf-1
activation(12, 28) . Modifications of Ha-Ras may have
an indirect role in Raf-1 activation; modified and membrane-anchored
Ha-Ras may simply recruit Raf-1 to the plasma membrane, where it is
subsequently activated by an unknown membrane-bound
factor(s)(30, 31) . However, it is possible that
modifications may have a direct role in this process; they may be
required for establishing a specific mode of interaction essential for
Raf-1 activation. Previous in vivo studies using an Ha-Ras
C-terminal mutant deficient in modifications could not examine the two
possible roles of modifications in Raf-1 activation
separately(28) . Furthermore, in vitro binding studies
using a Raf-1 fragment containing both RBD and CRR did not reveal any
significant defect of unmodified Ha-Ras in interaction with
Raf-1(21) . In the present study, by separately examining RBD
and CRR, we have shown that unmodified Ha-Ras lacks the ability to
interact with CRR. This represents a novel molecular defect of
unmodified Ha-Ras, which is unrelated to the membrane-anchoring defect.
Recently, we have established an in vitro membrane-free system
in which modified Ha-Ras can activate B-Raf but unmodified Ha-Ras
cannot. ()In another membrane-free system, modifications are
also required for yeast Ras2 to activate its effector, yeast adenylyl
cyclase(32) . These observations employing the membrane-free
systems also indicate that the defect of unmodified Ras proteins in
activating the effectors is unrelated to membrane anchoring.
Previous reports examined effects of mutations in RBD or CRR to study roles of these domains in Ras-dependent activation(17, 20, 21) . However, these manipulations may either alter conformation of Raf-1 or modulate its basal activity(17, 20) . Therefore, use of Ha-Ras mutants that possess the ability to interact differentially with the two Ras-binding domains may provide further insights. In the present study, we actually found these mutants and used them to provide evidence that CRR is involved in Ras-dependent activation of Raf-1. The analysis also showed that residues belonging to the activator domain of Ha-Ras are critical for interaction with CRR, providing a molecular basis for the role of this domain in activation of Raf-1. The binding affinity of CRR for the activator domain is lower than that of RBD for the switch I/effector domain and may not be sufficient for stable association of Raf-1 with GDP-bound Ha-Ras in vivo. However, after establishment of the interaction between RBD and switch I/effector domain of GTP-bound Ha-Ras, the interaction between CRR and the activator domain may take place efficiently and induce further conformational change of Raf-1 to be activated. Post-translational modifications of Ha-Ras C terminus are involved in the latter interaction, although the mechanism of their action remains to be clarified. Further understanding of these interactions will require x-ray crystallographic study of the complex between post-translationally modified Ha-Ras and Raf-1 including both RBD and CRR.