(Received for publication, September 27, 1995; and in revised form, November 13, 1995)
From the
Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).
Ras proteins are molecular switches controlled by GDP/GTP
cycling (1) . These proteins are transiently activated in
response to ligand-stimulated receptor tyrosine
kinases(2, 3) . Upon activation, Ras complexes with
and promotes activation of the Raf-1 serine/threonine
kinase(4, 5) . Raf-1 then activates mitogen-activated
protein kinase (MAPK) ()kinases (MEKs), which in turn
phosphorylate and activate MAPKs. Activated MAPKs translocate to the
nucleus where they regulate the activities of Elk-1 and other nuclear
transcription factors(6) .
Although substantial evidence supports the importance of Ras-Raf-1 interactions for Ras-mediated signaling and transformation, the precise role of Ras in activating Raf-1 remains unresolved(7) . The potent transforming activity of membrane-targeted forms of Raf-1 suggests that Ras-mediated translocation of Raf-1 to the plasma membrane is an important step in Raf-1 activation(8, 9) . However, it is clear that Ras-Raf-1 interaction alone is not sufficient, and subsequent Ras-independent events are required for full Raf-1 kinase activation. For example, there is evidence that Raf-1 interaction with 14-3-3 proteins(10, 11, 12, 13, 14) , lipids(15) , and protein kinases(16, 17, 18, 19) contributes to full Raf-1 kinase activation. Whether Ras simply promotes Raf-1 membrane association or also modulates the subsequent activation events is presently unclear.
Yeast two-hybrid and in vitro binding studies demonstrated that Raf-1 residues 55-131 are sufficient for stable association with Ras(20, 21, 22) . Additionally, recent structural studies conducted with both Ras and the Ras-related protein, Rap1A, indicate that Raf-1 residues 55-131 interact with residues 33-41 in the Ras effector region(23, 24) . Finally, the critical role for Raf residues 55-131 in Ras-mediated activation of Raf-1 is demonstrated by the ability of a point mutation, Raf (R89L), to disrupt Ras-Raf-1 binding and Raf-1 kinase activation(25) . However, observations that mutations outside the Ras effector domain impair Ras-Raf-1 binding and Ras-mediated cell signaling (26, 27, 28) suggest that other Ras recognition elements may contribute to Raf-1 kinase regulation.
We have recently characterized a second Ras-GTP-binding site, located in the cysteine-rich domain of Raf-1 (residues 139-184), which interacts with Ras both in vitro and in vivo(29) . Additionally, peptides from this region blocked Ras-mediated activation of MAPKs(30) . The importance of a second Ras-binding domain in Raf-1 is supported by a report that mutations in the Raf-1 cysteine-rich region reduced Raf-1-Ras binding by 55% and Raf-1 kinase activity by 60-90%(27) . Moreover, specific mutations in the cysteine-rich or kinase domains of D-Raf reversed the loss of function of a D-Raf variant containing a mutation analogous to c-Raf-1(R89L), leading Perrimon and co-workers (31) to speculate that the cysteine-rich domain formed negative regulatory contacts with the kinase domain, which are relieved upon binding Ras. However, it is presently unclear if the Raf-1 cysteine-rich domain recognizes Ras-GTP binding determinants distinct from those that interact with Raf-1 residues 55-131 and if both Ras-Raf-1 interacting sites are required for Ras signaling and transformation.
Both Ras recognition sites of Raf-1 preferentially bind Ras-GTP. Therefore, mutations that impair Ras transforming activity by causing disruptions in regions of Ras whose conformation differs between Ras-GDP and Ras-GTP (switch I and II) may disrupt interactions with either Ras-binding site of Raf-1. To investigate the significance of the two Ras-binding sites in Raf-1 and to elucidate the regions of activated Ras-GTP important for interactions with the Raf-1 cysteine-rich region, we determined if mutations that abolished Ras transforming activity also perturbed Ras interaction with the two distinct Ras-interacting fragments of Raf-1. Our observations suggest that the Raf-1 cysteine-rich domain contains a cryptic binding site, which recognizes region(s) of Ras different from those that bind Raf-1 residues 55-131, and both of these Ras-interacting sites may be necessary for Ras-mediated transformation.
The biochemical and biological consequences of each mutation to Ras are summarized in Table 1. Like wild type Ras, two different transforming mutants of Ras (G12V and Q61L) retained high affinity binding to both Raf-N and Raf-Cys (Fig. 1A). Given the GTP dependence of Ras interactions with the two Ras-Raf-1 interacting domains of Raf-1, taken together with recent data showing that Raf-1 residues 55-131 bind directly to the switch I domain of Ras (23, 24) , we postulated that Raf-Cys requires the switch II domain for its interaction with Ras. Hence, to better characterize the regions of Ras required for Raf-Cys binding, we determined whether a mutation that disrupted switch II, but not switch I, altered Ras binding to Raf-N or Raf-Cys.
Figure 1: Both Ras-binding domains of Raf-1 appear necessary for Ras-mediated transformation. Two distinct Raf-1 fragments were tested for their ability to bind various mutants of Ras as described under ``Experimental Procedures.'' A, transforming Ras mutants bound both Raf-N and Raf-Cys. B, Ras variants with switch I or II defects lacked the ability to interact with at least one Ras recognition fragment.
For
these studies, we used Ras G60A and Y64W switch II mutants that abolish
oncogenic Ras transforming activity(26, 43) . Ras
residue Gly-60 interacts with the -phosphate of GTP, and this
interaction is believed to be crucial for propagating conformational
changes within the switch II domain of Ras. Trypsin cleavage profiles
and fluorescence analysis of mutations analogous to the Ras G60A
substitution in other GTP-binding proteins (G
and
EF-Tu) indicate that this mutation disrupts switch II but not switch
I(44, 45, 46) . Moreover, fluorescence
analysis using 8-anilino-1-naphthalenesulfonic acid dye complexed with
wild type and G60A Ras-GTP provides additional evidence that
substitution of alanine for glycine at position 60 alters the active
conformation of Ha-Ras(26) .
Although we detected an association between Raf-N and both the Ras(G60A) and Ras(Y64W) variants, we did not observe complex formation between these Ras mutants and Raf-Cys (Fig. 1B). Thus, these results show that whereas Raf-N interacts with switch I(23, 24) , Raf-Cys requires an intact switch II region for binding to Ras. This is consistent with a previous observation that mutations in the switch II region of Ras (including residue 64) perturb interactions between Ras and various putative effector proteins(47) . Furthermore, the observation that the G60A and Y64W mutations abolish Ras transforming activity without impairment of Raf-N binding suggests that Ras interaction with Raf-N alone is not sufficient for Ras biological activity.
Figure 2:
Ras(G12V, E37G) abolishes Ras interaction
with Raf-N but not the isolated Raf-Cys domain. Yeast two-hybrid
analysis was done to determine the ability of Ras(G12V, E37G) to
interact with different fragments of Raf-1 by procedures described
previously(32) . The indicated Raf-1 residues were fused to
transcriptional activating domains in the yeast reporter strain L40 as
described below. A positive interaction (+) was determined by
growth on medium lacking histidine and by a positive indication of
-galactosidase activity using filter assays;(-) indicates no
interaction. At least four independent yeast colonies expressing the
indicated pairs were tested. Raf-1 residues 51-131 were fused to
the VP-16-activating domain (a gift from A. Vojtek and J. Cooper) (20) while the remaining Raf-1 sequences were fused to the
GAD-activating domain. The Raf-1 CR2 mutants (R256G, S257L, S257P) were
isolated and characterized previously(32) . WT, wild
type.
The inability of Raf-N to complex with the biologically inactive T35A mutant may result from diminished interactions between position 35, magnesium, GTP, and possibly the Asp-38 residue(48, 49) . It is also not surprising that the E37G mutation disrupted interactions with Raf-N, as the x-ray structure of the binding interface between Raf-1 residues 55-131 and Rap1A showed that the Glu-37 residue of Rap1A was in close contact with this Raf-1 sequence and was involved in water-mediated protein-protein interactions. Rap1A shares complete identity with Ras residues 32-40 and can associate with most Ras effectors, including Raf-1, RalGDS, and Ras GTPase-activating proteins (50, 51, 52) .
Our analyses of mutations that impair Ras
transformation as a consequence of perturbations to either the Ras
switch I or switch II domain suggest that Ras transforming activity
requires interaction with two distinct NH-terminal Raf-1
sequences. Furthermore, Raf-N and Raf-Cys demonstrated opposing binding
profiles with Ras proteins containing mutations that disrupt switch I
(T35A and E37G) versus switch II (G60A and Y64W), indicating
that they recognize distinct Ras GTP-binding elements. These
observations, together with our demonstration that peptides containing
a consensus Ras binding sequence from Raf-Cys can block Ras activation
of MAPKs (30) , provide strong evidence that Ras interaction
with Raf-Cys is a critical step in Ras-mediated activation of Raf-1.
Previous studies determined that Raf-N represents a minimal Ras binding sequence and that a mutation in this Raf-1 domain (R89L) prevented Ras interaction and activation of full-length Raf-1(25) . Therefore, we speculated that the Ras-binding elements in Raf-Cys are cryptic in the intact unstimulated Raf-1 protein(29) . In support of this hypothesis, we have shown in the present study that two Ras effector domain mutations (T35A and E37G), which impair Ras binding to both full-length Raf-1 and Raf-N, did not abolish Ras binding to the isolated Raf-Cys domain.
Recent
observations that addition of the Ras membrane-targeting sequence onto
Raf-1 caused activation of Raf-1 transforming activity suggested that
Ras binding to Raf-N was important for Ras-mediated translocation of
Raf-1 from the cytosol to the plasma membrane(8, 9) .
Once at the membrane, Ras-independent events have been proposed to
trigger Raf-1 kinase activation. However, our observation that the G60A
and Y64W mutations retained interaction with Raf-N yet abolished
oncogenic Ras transforming activity suggests that Ras interaction with
Raf-Cys is also required for Raf-1 activation. Therefore, Ras
interaction with both Raf-N and Raf-Cys may be necessary to promote
Raf-1 association with the plasma membrane. However, several lines of
evidence suggest that the NH-terminal half of Raf-1 serves
to negatively regulate the activity of the COOH-terminal kinase domain
because mutation, insertion, or deletion of these regions results in
oncogenic activation of the Raf-1
kinase(54, 55, 56) . Therefore, Ras binding
to Raf-Cys may relieve the negative regulatory action of the Raf-1
NH
terminus to allow other events to activate Raf-1. This
hypothesis is consistent with our observation that the Ras-binding site
in Raf-Cys is cryptic in full-length Raf-1. Additionally, it provides
an explanation for previous findings that specific mutations in the
cysteine-rich and kinase domains of D-Raf reverse the loss of function
associated with a D-Raf mutation in the Raf-N site that abolishes Ras
binding.
In light of our observations, we propose a model for the
role of Ras-mediated activation of Raf-1 via two distinct Ras binding
sequences in Raf-1 (Fig. 3). Binding of the Ras-GTP switch I
domain to Raf-N promotes both membrane localization of Raf-1 and
exposes residues in Raf-Cys for binding Ras-GTP and possibly other
membrane components. Interactions with Raf-Cys then trigger loss of the
negative regulatory activity of the Raf-1 NH terminus to
allow subsequent Ras-independent events to promote Raf-1 kinase
activation.
Figure 3: Model for the role of the Raf-1 cysteine-rich region in Raf-1 kinase activation. Binding of Ras-GTP to Raf-N promotes membrane localization of Raf-1 and exposes Ras-binding elements in the adjacent Raf-Cys domain for interaction with Ras-GTP and possibly phosphatidylserine (PS). These interactions, in turn, facilitate the removal of any autoinhibitory contacts with the kinase domain. Following phosphorylation of Raf-1 residues, Raf-1 kinase is stabilized in its active configuration and stimulates the MEK/MAPK cascade.
The Raf-Cys zinc finger motif also contains binding
determinants for phosphatidylserine (15) and 14-3-3
proteins(7) . Hence, Ras interaction with Raf-Cys may modulate
their activities to mediate Raf-1 activation. By analogy with the
cysteine-rich domain of protein kinase
C(35, 57, 58, 59) , the Ras-binding
site in Raf-Cys may functionally substitute for diacylglycerol, and
synergistic binding of Ras and phosphatidylserine may be involved in
release of negative regulatory constraints between the NH and COOH termini of Raf-1. It is also possible that exposure of
the Ras-binding elements in Raf-Cys may require release of 14-3-3
proteins(14) . In summary, the Ras-Raf-Cys interaction may
induce the removal of negative regulatory action in the Raf-1 NH
terminus and consequently facilitate Raf-1 activation by
additional events such as phosphorylation of select residues in Raf-1.