(Received for publication, April 21, 1997, and in revised form, June 10, 1997)
From the Department of Pharmacology and the
¶ Department of Biochemistry and Biophysics, Lineberger
Comprehensive Cancer Center, University of North Carolina, Chapel Hill,
North Carolina 27599 and the
Department of Pharmacology, Emory
University School of Medicine, Atlanta, Georgia 30322
Although Raf-1 is a critical effector of Ras
signaling and transformation, the mechanism by which Ras promotes Raf-1
activation is complex and remains poorly understood. We recently
reported that Ras interaction with the Raf-1 cysteine-rich domain
(Raf-CRD, residues 139-184) may be required for Raf-1 activation. The
Raf-CRD is located in the NH2-terminal negative
regulatory domain of Raf-1 and is highly homologous to cysteine-rich
domains found in protein kinase C family members. Recent studies
indicate that the structural integrity of the Raf-CRD is also critical
for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction
may mediate Raf-1 function has not been determined. In the present
study, we demonstrate that 14-3-3 binds directly to the isolated
Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding
of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that
impair 14-3-3 binding resulted in full-length Raf-1 mutants with
enhanced transforming activity. Thus, 14-3-3 interaction with the
Raf-CRD may serve in negative regulation of Raf-1 function by
facilitating dissociation of 14-3-3 from the NH2 terminus
of Raf-1 to promote subsequent events necessary for full activation of
Raf-1.
Substantial genetic, biochemical, and biological evidence supports the critical role of the Raf-1 serine/threonine kinase as a key downstream effector of Ras signaling and transformation (1, 2). Ras interaction with Raf-1 promotes the activation of Raf-1 in vivo, in part by facilitating its translocation from the cytoplasm to the plasma membrane. Activated Raf-1 phosphorylates and activates the mitogen-activated protein kinase kinases (MAPK1 kinases; also referred to as MEKs), which in turn phosphorylate and activate the p42 and p44 MAPKs. Activated MAPKs translocate to the nucleus where they regulate the activity of transcription factors such as Elk-1 (3).
Ras interaction with Raf-1 alone is not sufficient to cause full activation of Raf-1, but rather binding of Ras to Raf-1 initiates other events that lead to full activation. These additional events include tyrosine (4, 5) and serine/threonine (6-9) phosphorylation, phospholipid binding (10, 11), and interactions with other proteins that include members of the 14-3-3 protein family and 14-3-3 associated proteins (12-17). Hence, full kinase activation involves a complex multistep process that remains to be elucidated fully.
An additional complexity of Ras-mediated activation of Raf-1 is that the Ras/Raf-1 interaction is more convoluted than originally believed. We and others have shown recently that Ras interacts with two distinct Ras-binding domains in the NH2-terminal regulatory region of Raf-1 (18, 19). The first Ras-binding domain encompasses Raf-1 residues 55-131 (20, 21) and appears to interact with Ras prior to exposure of the second binding site (19). This second binding region is contained within the Raf-1 cysteine-rich domain (residues 139-184, designated the Raf-CRD; also called Raf-Cys or Raf-C1) that resembles the C1 domains of protein kinase C family members (22). Although the precise role of this second Ras/Raf-1 interaction is unclear, we recently determined that Ras interaction with the Raf-CRD is required for Ras transforming activity (19). We hypothesized that Ras interaction with Raf-1 residues 55-131 promotes Raf-1 association with the plasma membrane while Ras interaction with the Raf-CRD stabilizes this membrane association and/or relieves the regulatory effects of the NH2 terminus. Hence, additional interactions between Ras and Raf-CRD may promote further activation events that could involve interactions with 14-3-3 proteins or phospholipids.
14-3-3 proteins bind directly with Raf-1 in vitro and in vivo (13-17). However, the role of this interaction in Raf-1 function is unclear. Whereas some studies suggest that 14-3-3 may serve a role in activation (14, 16, 17, 23, 24), others support a negative regulatory role (25), and yet another indicates that 14-3-3 proteins are not essential for Raf-1 function (26). Moreover, there are conflicting reports regarding the relationship between the activated state of Raf-1 and its ability to bind 14-3-3 proteins (14, 15, 17).
Contradictory reports regarding the role of 14-3-3 proteins in Raf-1
activation may be due, in part, to the existence of multiple 14-3-3 binding sites in distinct NH2- and COOH-terminal sequences of Raf-1 (13-15). A 14-3-3 recognition consensus motif has been identified (27), and Raf-1 contains two phosphorylation sites that fit
the consensus sequence: serine 259 in the NH2-terminal regulatory sequence downstream of the Raf-CRD and serine 621 in the
COOH-terminal kinase domain. However, disruption of the structural integrity of the Raf-CRD can abolish 14-3-3 interactions (26), indicating that 14-3-3 binding sites distinct from this consensus motif
may be present in the NH2 terminus of Raf-1. In fact,
NH2-terminal fragments of Raf-1 that contain the Raf-CRD
showed binding to 14-3-3 by yeast two-hybrid studies (25). However,
whether 14-3-3 proteins can interact directly with sequences in the
Raf-CRD, and what role, if any, such an interaction may play in the
regulation of Raf-1 function has not been established.
In this report, we demonstrate that the isolated Raf-CRD binds directly
to the isoform of 14-3-3 and that mutations of residues 143-145
cause a loss of 14-3-3, but not Ras, binding in vitro. We
also show that mutations that impair the 14-3-3/Raf-CRD association result in activation of Raf-1 transforming potential. Thus, 14-3-3 interaction with Raf-CRD may serve as a negative regulator of Raf-1
function. When taken together with our observation that Ras interaction
with the Raf-CRD is essential for Ras transforming activity, we propose
that Ras interaction with the Raf-CRD may disrupt the 14-3-3/Raf-CRD
interaction to promote subsequent events that lead to full activation
of Raf-1.
Oligonucleotide-directed mutagenesis of a cDNA
sequence encoding Raf-1 residues 136-187 (encompassing the Raf-CRD)
was utilized to generate sequences encoding specific mutants of
Raf-CRD. Briefly, oligonucleotide primers containing the appropriate
mutations were elongated by polymerase chain reaction, and the
resulting products were ligated into the pCRTMII vector
(Invitrogen) using 5 BamHI or HindIII and 3
EcoRI restriction sites. Dideoxy sequence analysis was done
to confirm the introduction of the correct mutations. The mutated
cDNA sequences encoding Raf-1 residues 136-187 were isolated by
digesting with HindIII/EcoRI or
BamHI/EcoRI and ligated in-frame into the pGEX2T vector for expression of recombinant glutathione
S-transferase (GST) Raf-CRD fusion proteins in the
Escherichia coli strain BL21 (10).
To transfer the mutations into the full-length protein, a cassette was
generated containing the 5 half of the raf-1 cDNA in
which the sequence encoding the Raf-CRD is encompassed by unique BclI and SalI sites. The raf-CRD
variant sequences were amplified to carry the BclI and
SalI sites at their termini, sequenced to confirm their
fidelity, and cloned into the raf-1 cassette. The 5
fragments of raf-1 containing the desired mutations were
spliced to the 3
half of the cDNA using the unique
BstXI site in the raf-1 cDNA sequence.
Sequences encoding mutant Raf-CRDs were employed to generate sequences
in wild type Raf-1 (28) and were introduced into the pCGN-hyg or
pZip-NEO SV(X)1 (29) mammalian expression vector.
An ELISA protocol
was employed to monitor Raf-CRD interactions with 14-3-3 and Ras.
All assays were performed at least twice in triplicate.
Recombinant 14-3-3 was expressed as an NH2-terminal
polyhistidine-tagged fusion protein in E. coli as described
previously (30). Following immobilization of 14-3-3
to a
medium-binding polystyrene plate (Costar), a 1:1500 dilution of an
anti-GST monoclonal antibody (Santa Cruz Biotechnology, Inc.) was
employed to assess binding of each GST-Raf-CRD variant. Corresponding
amounts of GST were used to control for background interactions.
Recombinant Ha-Ras was expressed and purified as described previously (31). Formation of the Ras and GMP-PCP (a nonhydrolyzable GTP analog, Boehringer Mannheim) complex for use in the ELISA experiments is described elsewhere (32). Ras/Raf-CRD interactions were assessed as described previously (18).
14-3-3 Binding AssayRecombinant GST-Raf-CRD proteins were
immobilized to glutathione-coated agarose beads and incubated with
equimolar amounts of 14-3-3 dimer. Raf-CRD proteins were separated
from bound 14-3-3 proteins by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis after washing with 10 mM
Na2HPO4, 100 mM NaCl, 0.05% Tween
20, and 20 mM zinc. To identify 14-3-3
, proteins were
transferred to a polyvinylidene difluoride membrane, probed with a
1:2500 dilution of polyclonal 14-3-3
antibody (Santa Cruz
Biotechnology), and detected by an antibody bound to alkaline
phosphatase. The amount of 14-3-3
captured was assessed by scanning
an equivalent gel stained by Coomassie Brilliant Blue and comparing the
intensity of each band to the intensities of known protein
concentrations. Nonspecific interactions between 14-3-3 proteins and
GST were determined by the amount of captured 14-3-3
with
equivalent amounts of GST protein. All variants were analyzed in
duplicate in at least two independent assays.
Primary focus-formation assays were conducted to quantitate the ability of each Raf-CRD mutant of Raf-1 in cooperation with activated RhoA(Q63L) (33) to transform NIH 3T3 cells using procedures that have been described previously (34, 35). Also, secondary focus-formation assays were performed to quantitate the focus-forming potential of full-length Raf-CRD variants. Briefly, NIH 3T3 cells were transfected with 1 µg of plasmid DNA encoding each Raf-1 mutant protein using the calcium phosphate precipitation method as described (34) and were selected in 500 µg/ml G418 (Geneticin, Life Technologies, Inc.). Following selection, multiple drug-resistant colonies were pooled together (>100 colonies) and replated at 5 × 105 cells per 60-mm dish. The appearance of foci of transformed cells was quantitated after 14 days for both primary and secondary assays. Raf(Y340D) is a weakly transforming mutant of Raf-1 that contains a Y340D mutation (4, 35).
Although the Raf-CRD has been implicated in interactions
with 14-3-3 (25), direct binding has not been reported. To evaluate this possibility, we determined whether a bacterially expressed GST
fusion protein that encompasses Raf-CRD (residues 136-187) could
interact with bacterially expressed 14-3-3 in vitro. By ELISA, we detected 14-3-3
binding to GST-Raf-CRD, but not to GST
alone (Fig. 1A). Thus, in
addition to the two 14-3-3 consensus recognition motifs (27), 14-3-3 can interact with sequences contained within the Raf-CRD. Consequently,
the Raf-CRD defines at least a second distinct 14-3-3 binding site in
the NH2-terminal regulatory domain of Raf-1.
Mutation of Raf-1 Residues 143-145 Impair 14-3-3 Interactions with the Raf-CRD
To identify specific residues within the Raf-CRD that
are involved in interaction with 14-3-3 , we introduced amino acid substitutions of conserved, charged, exterior residues in the Raf-CRD.
This mutagenesis approach was employed because conservation of charged
amino acids at the protein surface within protein families often
indicates functional importance. Also, structural evidence suggests
that charged residues in 14-3-3 proteins may be involved in binding
other proteins (36, 37).
The following mutations were introduced into GST-Raf-CRD and then
analyzed for interaction with 14-3-3 by ELISA: R143E, R143E/K144E,
K144Q, T145E, and D153N/K179Q. Although the D153N/K179Q double mutation
did not affect binding, the substitution of R143E/K144E or T145E
severely impaired Raf-CRD/14-3-3 interactions (Fig. 1A and
Table I). The impaired binding of the
R143E/K144E mutant was the combined consequence of each substitution
since the R143E mutation alone showed a partial reduction in
binding.
|
To address the
possibility that the inability of the Raf-CRD mutants to interact with
14-3-3 is due to a collapse of the Raf-CRD structure, we assessed
the structural integrity of the R143E/K144E mutant by nuclear magnetic
resonance (data not shown). These nuclear magnetic resonance
measurements indicated that the structure of the isolated Raf-CRD
variant was unaltered. Moreover, none of the mutations analyzed in this
study impaired expression of these variants as GST-fusion proteins
(data not shown), providing further indication that these mutations do
not diminish the structural integrity of the Raf-CRD. Thus, we conclude
that the failure of the Raf-CRD mutants to bind 14-3-3
is not due
to a collapse of the cysteine-rich domain and instead is mediated by
the isolated mutations.
We showed previously that the Raf-CRD can interact with Ras (18). To address the specificity of these point mutations on 14-3-3/Raf-CRD interactions, we assessed the effects of these substitutions on binding interactions between the Raf-CRD and Ras. ELISA analysis showed that all of the variants retained the ability to bind GTP-complexed, bacterially expressed Ha-Ras protein (Fig. 1B). Thus, mutation of Raf-1 residues 143-145 caused selective impairment of binding to 14-3-3 but not Ras. These results were unexpected since we speculated previously that these residues may represent a consensus Ras-GTP binding sequence shared between Raf-1 and other candidate Ras effectors (38). While these observations do not exclude their role in Ras interaction, it is clear that the integrity of each residue alone is dispensable for Ras binding.
Mutations That Impair 14-3-3 Binding Cause Activation of Raf-1 Transforming PotentialTo determine the contribution of the 14-3-3/Raf-CRD interaction to Raf-1 function, we assessed the consequences of disrupting Raf-CRD/14-3-3 interactions on Raf-1 transforming potential. For these analyses, we introduced the R143E/K144E, T145E, and D153N/K179Q mutations into wild type, full-length Raf-1 (28).
We determined if the loss of 14-3-3/Raf-CRD interaction may potentiate
the transforming activity of wild type Raf-1. For these analyses we
used the weakly activated Raf(Y340D) mutant as a positive control.
Raf(Y340D) shows strong focus-forming activity when assayed in a
secondary focus-formation assay or when co-transfected with an
expression construct encoding the constitutively activated RhoA(Q63L)
mutant proteins in primary focus-formation assays (33). Raf-1
containing the R143E/K144E and T145E mutations showed a 5-fold enhanced
focus-forming activity when compared with wild type Raf-1 (Fig.
2A). However, this activity
was less than that seen with Raf(Y340D). In contrast, the D153N/K179Q
mutant showed activity comparable with wild type Raf-1. Similarly,
Raf-1 mutants deficient in 14-3-3 binding (R143E/K144E, T145E) showed
3-fold enhanced focus-forming activity when co-expressed with
RhoA(Q63L). The focus-forming activity was comparable with that
observed with Raf(Y340D) (Fig. 2B). Thus, loss of 14-3-3 binding to the Raf-CRD enhanced Raf-1 transforming potential,
suggesting that 14-3-3 acts as a negative regulator of Raf-1 function
via this interaction. These results are consistent with another report
of an activating mutation in the Raf-CRD (26).
Raf-CRD Mutations May Complement Inactivating Mutations of Ras
We showed recently that the transformation-defective
Ras(G12V/G60A) mutant protein retained the ability to bind to Raf-1
residues 55-131 but not to the Raf-CRD (19). Moreover, the inability of v-Ha-Ras(G60A) to stimulate Raf-1 activity (39) suggests that both
Ras binding sites in Raf-1 (Raf-1 residues 55-131 and 139-184) are
required for Raf-1 activation and Ras-mediated transformation. Since we
suspect that Ras interaction with Raf-CRD may be required to overcome
the negative regulatory effects of 14-3-3, the transformation-defective nature of this mutant may be due to its inability to reverse the 14-3-3/Raf-CRD interaction. Therefore, we speculated that co-expression of Ras(G12V/G60A) with Raf-1 mutants impaired in 14-3-3 binding may
bypass the requirement for this Ras mutant to interact with Raf-CRD.
Consistent with this possibility, we observed that co-expression of the
R143E/K144E or T145E mutants of Raf-1, but not wild type Raf-1, with
Ras(G12V/G60A) showed greatly enhanced focus-forming activity (5-20
foci versus 0 foci; Fig. 3).
These results support our hypothesis that Ras interaction with the
Raf-CRD is required, in part, to promote the loss of 14-3-3 interaction
to allow subsequent events that lead to full activation of Raf-1 kinase
function. The observation by Rommel et al. (25) that
activated Ras displaces 14-3-3 from the NH2 terminus of
Raf-1 provides further support for this model.
In summary, we have shown that the isoform of 14-3-3 can interact
directly with the isolated Raf-CRD and that mutations impaired in
Raf-CRD/14-3-3 interactions cause a partial activation of Raf-1
transforming potential. When taken together with our previous
observation that Ras interaction with Raf-CRD is necessary for Ras
transforming activity (19), we propose that Ras interaction with
Raf-CRD is required, in part, to remove the negative regulatory action
of 14-3-3. Our observation that Raf-1 mutant proteins impaired in
Raf-CRD/14-3-3 binding can cooperate with a transformation-defective Ras mutant that has lost the ability to bind to the Raf-CRD is consistent with this model. However, since the loss of 14-3-3 binding
to the Raf-CRD caused only partial activation of Raf-1 transforming
activity, the removal of 14-3-3 interaction with this domain may be a
preactivation step, and additional events are required to promote full
activation. The ability of Ras and 14-3-3 to compete with each other
for binding to the Raf-CRD and the role of other potential
Raf-CRD-interacting components (e.g. phospholipids) in
regulation of Raf-1 function are currently under investigation.
We thank Carol Martin and Sarah Johnson for
technical assistance, Chris Lombardo for valuable discussions, Jennifer
Parrish for preparation of figures, and Lixin Xhang for preparation of 14-3-3 .