From the
Raf-1 is a serine/threonine kinase poised at a key relay
point in mitogenic signal transduction pathways from the cell surface
to the nucleus. Activation of the transforming potential of Raf-1 has
been associated with N-terminal truncation and/or fusion to other
proteins, suggesting that the Raf-1 N-terminal half harbors a negative
regulatory domain. Seven internal deletion mutants that together scan
the entire N-terminal half of human Raf-1 protein were generated to map
functional regions in this regulatory domain. Effects of the deletion
mutations on kinase activity of Raf-1 were evaluated using a
baculovirus/insect cell overexpression system and an in vitro kinase assay with the known physiological substrate of Raf-1,
mitogen-activated protein kinase kinase. Deletion of amino acids
276-323 in the unique sequence between conserved regions 2 and 3
leads to modest elevation of Raf-1 basal kinase activity, whereas
deletion of amino acids 133-180 in conserved region 1 results in
diminished kinase activity. Surprisingly, none of the Raf-1 N-terminal
deletion mutants, including a truncated version that is transforming in
rodent fibroblasts, exhibits greatly increased levels of basal kinase
activity. In addition, while activation of Raf-1 kinase by Ras requires
sequences in conserved region 1, only the C-terminal half containing
the kinase domain of Raf-1 is required for activation by Src. These
findings demonstrate that N-terminal deletions in Raf-1 do not
necessarily result in constitutively elevated basal kinase activity and
that the N-terminal regulatory domain is completely dispensable for
Raf-1 activation by Src.
The 74-kDa cytoplasmic serine/threonine protein kinase, Raf-1,
has a central role in transduction of proliferation and differentiation
signals initiated by diverse extracellular
factors
(1, 2) . Activation of Raf-1, in response to
extracellular stimuli transmitted through membrane tyrosine kinases and
Ras, initiates activation of a kinase cascade involving the
mitogen-activated protein kinases (MAP kinases,
Raf-1 is encoded in the c-raf-1 gene
(13) , which
belongs to a family of protooncogenes that includes two other active
members, A-raf-1
(14) and B-raf(15) .
A-raf-1 and B-raf genes are expressed specifically in
urogenital and brain tissues, respectively, whereas the
c-raf-1 gene is ubiquitously expressed in all tissues
examined
(16) . c-raf-1 was first identified in its
tumorigenic form, v-raf, the oncogene of the acutely
transforming retrovirus, murine sarcoma virus 3611
(17) .
v-raf is expressed as a myristylated Gag-Raf fusion protein in
which the N-terminal half of Raf-1 protein is replaced by Gag
sequences. Activation of the transforming potential of Raf-1 has also
been associated with N-terminal truncation and fusion to other
sequences
(18, 19, 20) . These observations
suggest that the Raf-1 N-terminal sequences contain a negative
regulatory domain, deletion of which results in deregulation of the
C-terminal kinase domain.
Sequence comparisons among members of the
raf family reveal the presence of three blocks of conserved
sequences: conserved regions (CR) 1, 2, and 3
(21) . CR1 contains
a cysteine-rich ``zinc finger'' motif, related to a similar
structure in protein kinase C
(22) , that has been shown to
coordinate 2 mol of zinc
(23) . CR2 consists of a stretch of 20
amino acids rich in serine and threonine residues; these are targets of
phosphorylation by Raf-1 autokinase activity as well as by other
serine/threonine-specific protein kinases, including protein kinase
C
(24, 25) . CR3, which spans the C-terminal half of
Raf-1, comprises the catalytic domain. Interspersed among these
conserved regions are sequences unique to Raf-1. Current models of
Raf-1 regulation postulate that the N-terminal regulatory domain
represses the transforming potential and kinase activity of the
C-terminal catalytic domain
(26) .
To fine map functional
regions in the N-terminal regulatory domain of Raf-1 protein, we
engineered a series of internal deletion mutants that together scan the
entire N-terminal half of human Raf-1. These deletions were designed to
systematically disrupt conserved as well as unique sequences in the
N-terminal half of Raf-1 protein. Wild-type and mutant Raf proteins
were expressed in a baculovirus/Sf9 insect cell system to examine
effects of these mutations on regulation of Raf-1 kinase activity
toward MKK, which is the only known physiological substrate of
Raf-1
(27, 28, 29) . Significantly, results show
that none of the Raf-1 deletion mutants display greatly increased
levels of basal kinase activity. Previous studies demonstrated that
Raf-1 protein expressed in insect cells can be synergistically
activated by Src and Ras proteins expressed from co-infected
recombinant baculoviruses
(30) . Analysis of our deletion mutants
revealed that, while Ras activates Raf-1 kinase through sequences in
CR1 as previously reported
(31, 32) , only the C-terminal
half of Raf-1 containing CR3 is required for activation by Src.
Together, these findings suggest that the function of the Raf-1
N-terminal domain is not merely to repress activity of the C-terminal
kinase domain and that regulation of Raf-1 kinase activity is more
complex than predicted from current models.
As shown in Fig. 6, A and C, Raf-d2,
Raf-d3, and 22W lost the ability to be activated by Ras, whereas the
other deletion mutants and Raf-1 showed 2-4-fold elevated kinase
activities in response to Ras. Surprisingly, the basal kinase activity
of 22W was consistently lower than that of wild-type Raf-1. The common
residues deleted in Raf-d2, Raf-d3, and 22W are amino acids
53-132. Since this region is also required for interaction of
Raf-1 with Ras
(32, 39, 40) , these results are
consistent with the conclusion that the physical interaction of Raf-1
and Ras is essential for Raf-1 activation by Ras.
Genetic analysis of v-Raf and other Raf-1 mutants showed that
deletions in the N-terminal half, in some cases accompanied by fusion
to unrelated sequences, activate the oncogenic potential of
Raf-1
(19, 20) . These findings led to the hypothesis
that the N-terminal half of Raf-1 contains a negative regulatory
domain, which represses the catalytic domain contained in the
C-terminal half of the protein. Direct evidence for specific regions in
the N-terminal domain that demonstrably repress Raf-1 protein kinase
activity, however, is lacking from previous studies. To explore the
mechanism of Raf-1 kinase regulation by the N-terminal domain, we
engineered a panel of internal deletion mutants designed to
systematically dissect potential regulatory elements in the N-terminal
half of Raf-1. Analysis of the Raf-1 deletion mutants revealed that,
contrary to expectations from current models, the N-terminal domain
does not function to simply repress the C-terminal kinase domain.
Significantly, targeted deletion of various conserved and unique
regions in the N-terminal domain of Raf-1 protein did not substantially
affect its basal kinase activity, with the notable exceptions of Raf-d4
and Raf-d7. The Raf-d4 mutant has a deletion that removes the zinc
finger motif in CR1. This cysteine-rich motif in Raf-1 is of interest
because homologous zinc finger structures in protein kinase C have been
shown to be essential for phorbol ester binding and kinase activation
and have been implicated in translocation of the kinase to the membrane
(41, 42). While deletion of the zinc finger in Raf-d4 did result in
partially defective kinase activity, a larger deletion in Raf-d3
encompassing the entire CR1 had little effect on the Raf-1 basal kinase
activity. One possible explanation for this apparent contradiction is
that the zinc finger might have a conformational or functional role
only in the context of the intact CR1.
In the case of Raf-d7,
deletion of amino acids in the unique sequence between CR2 and CR3
resulted in modestly (2-3-fold) elevated kinase activity.
Interestingly, the deleted region of Raf-d7 contains a consensus MAP
kinase phosphorylation sequence, Pro-X-Thr*-Pro (amino acids
308-311). MAP kinases have been shown to phosphorylate Raf-1 both
in vitro(43) and in vivo in
insulin-stimulated cells
(44) . Consistent with phosphorylation
events occurring in the region deleted in Raf-d7 is the finding that
this mutant protein, unlike wild-type Raf-1, migrated as a single
species upon electrophoresis through SDS-polyacrylamide gels. It has
been suggested that phosphorylation of Raf-1 by MAP kinases may have an
important physiological role, such as in an inhibitory feedback
loop
(8) . While any regulatory effect that might result from
phosphorylation of this site remains to be demonstrated, our results on
Raf-d7 support the suggestion that a negative regulatory element exists
in this region between CR2 and the catalytic domain. Another deletion
mutant, Raf-d6, which has the entire CR2 deleted, also appeared as a
single species on Western blots. This conserved region is rich in
serine and threonine residues that are potential phosphorylation sites,
including Ser
Our finding
that none of the N-terminal deletion mutants possess greatly elevated
kinase activity is surprising in light of the prediction, from earlier
genetic studies on activation of Raf-1 oncogenic
potential
(19, 20) , that at least some of these mutants
would have acquired deregulated kinase activity. For example, the 22W
Raf-1 mutant, which resembles v-Raf in that it lacks most of the
N-terminal regulatory domain, is oncogenic in rodent
fibroblasts
(19) . Deletion of CR2 also has been associated with
activation of the Raf-1 transforming potential
(18) . In earlier
genetic studies, however, the kinase activities of transforming Raf
mutants could not be measured reliably because an appropriate
physiological substrate had not been identified yet. In the present
study, we find that neither 22W nor Raf-d6, which contains a deletion
of CR2, exhibits elevated kinase activity toward MKK. While the in
vitro kinase assay that we employ here is highly specific and
sensitive to a wide range of Raf-1 kinase activities, we cannot exclude
the possibility that this assay is not an accurate measure of in
vivo kinase activities. For instance, mutant Raf-1 proteins might
be unstable in cell lysates or during incubation in the in vitro kinase assay; however, the fact that all of the deletion mutants
examined here can be substantially activated by Src argues against this
possibility. Alternatively, the kinase activities of Raf proteins
expressed in insect cells might not reflect their activities in
mammalian cells due to crucial differences in cellular regulatory
molecules. Moreover, it remains possible that oncogenic activation of
Raf-1 involves subtle changes in substrate specificity rather than
elevated levels of kinase activity.
Earlier studies established that
Ras binds with high affinity through its effector domain to the Raf-1
N-terminal domain in a GTP-dependent
manner
(39, 45, 46, 47, 48) .
This physical interaction has been shown to be essential for Raf-1
activation by Ras (31, 32). Studies by Williams et al.(30) showed that co-expressing Raf-1 with either Ras or Src in
insect cells led to moderate activation of Raf-1 kinase activity, and
that maximal activation required both Src and Ras together. These
findings raised the possibility of the presence of both Ras-dependent
and Ras-independent mechanisms for Raf activation. Using our panel of
mutants, we dissected the N-terminal half of Raf-1 for regions involved
in responding to Ras and Src in an attempt to gain further insight into
how these activators cooperate. Raf-1 N-terminal deletions that affect
amino acids 53-132, which are required for Raf-1 to bind Ras
in vitro and in
vivo(32, 39, 40) , abolished activation of
Raf-1 kinase by Ras. It is notable that no other region outside of CR1
is essential for Raf-1 activation by Ras. On the other hand, our
finding that all seven deletion mutants and 22W are activable by Src
provides direct evidence that the N-terminal domain is completely
dispensable for activation of Raf-1 kinase by Src. Consistent with
other recent studies
(31, 32) , this finding demonstrates
that Src can activate Raf-1 through a Ras-independent mechanism. The
Src-response element in Raf-1 might involve the previously described
C-terminal tyrosine residues, Tyr
Recent reports have suggested that
Ras functions in the activation of Raf-1 by recruiting it to the plasma
membrane
(49, 50) . In this context, our findings are
consistent with a model in which the N-terminal domain has a role in
mediating interactions with other molecules that regulate the kinase
activity or subcellular localization of Raf-1 rather than simply
repressing its kinase activity. Once localized to the membrane, other
activation events, such as tyrosine phosphorylation of the C-terminal
catalytic domain, may activate Raf-1 kinase activity. In the case of
Raf-1 N-terminal mutants that do not bind Ras, overexpression of Src
kinase together with these mutants in insect cells may overcome the
requirement for Ras. Under normal circumstances in mammalian cells,
however, signal transduction from membrane-associated tyrosine kinases
may still depend on activated Ras to target Raf-1 to the membrane. This
model predicts that Ras and tyrosine kinases normally function in
concert for Raf-1 activation, a prediction that is consistent with
classical experiments in mammalian cells
(51, 52) as
well as the finding that maximal activation of Raf-1 kinase in insect
cells requires co-expression of both Ras and Src
(30) .
We thank C.-L. Yu for constructing the baculovirus
transfer vector containing wild-type c-raf-1 cDNA, R. Nairn
for suggesting the use of human IgG as carrier for peptide antigen, D.
Retallack for assistance in preparing the anti-Raf antibody, N.
Williams and T. Roberts for v-Ras baculovirus, J. Fabian and D.
Morrison for the ATPM-Raf and Raf340D baculoviruses, V. Stanton and G.
Cooper for the 22W plasmid DNA, and members of the laboratory for
stimulating discussion.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
also known as extracellular signal-regulated kinases or
ERKs)
(3, 4) . The intermediaries between Raf-1 and MAP
kinases are the dual specificity tyrosine/serine/threonine kinases, MAP
kinase kinases (MKKs or MEKs) (5, 6). Activated MAP kinases are
responsible for regulating the functions of a variety of substrates
that ultimately control gene expression and DNA
synthesis
(7, 8) . The findings that multiple isoforms of
MKKs and MAP kinases exist suggest complex signaling networks that
respond to diverse signals in different cell types (9-12).
Mutagenesis of c-raf-1 Gene
Plasmid p627
containing human c-raf-1 cDNA
(13) was obtained from
the American Type Culture Collection. Dodecameric XhoI linker
(sequence CTCGAGCTCGAG) was ligated into restriction enzyme cleavage
sites of c-raf-1 according to the linker insertion mutagenesis
strategy described previously
(33) . Locations of the unique
XhoI insertion sites, which are entirely predictable, were
identified by detailed restriction mapping. Using selected pairs of
XhoI linker insertion mutants, a second generation of mutants
was constructed by deleting intervening sequence between two insertion
sites.
Recombinant Baculoviruses
A 2.3-kilobase
Bsu36I/ApaI fragment of full-length c-raf-1
cDNA from p627 was subcloned into the baculovirus transfer vector,
pBlueBac (Invitrogen). BamHI/NsiI fragments of the
seven deletion mutants and a 2-kilobase EcoRI/EcoRI
fragment encoding the truncation mutant 22W that lacks the N-terminal
305 amino acids
(19) were subcloned into a similar baculovirus
transfer vector, pBlueBacIII (Invitrogen). The recombinant transfer
vectors were then cotransfected with linear wild-type Autographa
californica nuclear polyhedrosis virus DNA into Sf9 insect cells.
Recombinant baculoviruses containing Raf-coding sequences were screened
for Raf protein expression by immunoblot analysis, and positive clones
were plaque-purified using standard procedures
(34) .
Antibodies
Rabbit polyclonal anti-Raf antibodies
were prepared against a peptide corresponding to the extreme C-terminal
12 amino acids (sequence CTLTTSPRLPVF), which was coupled to purified
human IgG with
m-maleimidobenzoyl-N-hydroxysuccinimide ester (35).
Monoclonal anti-Raf antibody was obtained from Transduction
Laboratories. Monoclonal anti-Ras antibody, LA-069, was obtained from
the NCI Repository (Quality Biotech). The murine hybridoma cell line
producing monoclonal anti-Src antibody (monoclonal antibody 327) was
kindly provided by J. S. Brugge (ARIAD Pharmaceuticals).
Immune Complex Kinase Assays
Sf9 cells were
infected with wild-type baculovirus (A. californica nuclear
polyhedrosis virus) or the indicated Raf recombinant viruses at a
multiplicity of infection of 5, alone or in combination with c-Src or
v-Ras recombinant viruses at multiplicities of infection of 15.
Infected cells were harvested at 48-54 h postinfection and frozen
at -80 °C until ready to be lysed. Cell pellets were lysed in
modified insect lysis buffer (MILY buffer; 20 mM Tris-HCl, pH
7.4, 100 mM KCl, 1 mM MgCl, 10% glycerol,
1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 1
µM leupeptin, 1 µM antipain, 0.1
µM aprotinin, and 5 mM sodium
orthovanadate)
(25, 32) for 15 min on ice. Clarified
lysates (equivalent to 1
10
cells) were incubated
with polyclonal anti-Raf antiserum for 1 h on ice, followed by
collection of the immunoprecipitates with protein A-Sepharose
(Pharmacia Biotech Inc.). Immune complexes were washed twice with MILY
buffer and one time with kinase reaction buffer (25 mM HEPES,
pH 7.4, 10 mM MgCl
, 5 mM
MnCl
), and then incubated for 20 min at 25 °C with 200
ng of purified, polyhistidine-tagged MKK1 or MKK2 proteins (chemically
inactivated by FSBA treatment)
(36) in 30 µl of kinase
reaction buffer supplemented with 1 mM dithiothreitol, 15
µM ATP, and 10 µCi [
-
P]ATP
(Amersham Corp.). Reactions were terminated by addition of 4
SDS sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 50%
glycerol, 0.002% bromphenol blue) and boiling for 5 min. Reaction
products were then electrophoresed through 12% SDS-polyacrylamide gels,
transferred electrophoretically onto nitrocellulose membranes, and
autoradiographed. These nitrocellulose membranes were analyzed in a
Molecular Dynamics PhosphorImager or an AMBIS 4000 radioisotope
detector to directly quantify the phosphorylation levels. To confirm
that equivalent amounts of Raf proteins were precipitated in the kinase
reactions, these same membranes were then probed with anti-Raf
antibodies, which were detected using anti-rabbit immunoglobulin
followed by enhanced chemiluminescence (ECL; Amersham Corp.).
Generation and Expression of Raf-1 Mutants
To
assess the contributions of Raf-1 N-terminal conserved and unique
sequences (Fig. 1A) to regulation of its kinase
activity, 24 XhoI linker insertion mutations were introduced
at predicted enzyme cleavage sites in human c-raf-1 cDNA
(Fig. 1B). From these linker insertion mutants, seven
internal deletion mutants were derived in which segments between
combinations of two linker insertion sites were removed. These in-frame
deletions were designed to target blocks of conserved or unique
sequences in the N-terminal half of Raf-1, as diagrammed in
Fig. 1C. To analyze the kinase activities of these
mutants, a baculovirus/insect cell system was used that allows
expression of high levels of kinase-active Raf-1
(30) .
Full-length Raf-1 and deletion mutants of the expected sizes were
expressed at comparable levels from recombinant baculoviruses in
infected Sf9 cells (Fig. 2). Raf-1 and deletion mutants d1
through d5 appear as multiple bands on immunoblots, whereas d6 and d7
appear as well defined single bands. Multiple species of Raf-1 have
also been detected in mammalian cells and probably result from
phosphorylation
(21) . For analysis of kinase activity, an in
vitro kinase assay was developed using two isoforms of MAP kinase
kinase, MKK1 and MKK2, which are physiologically relevant substrates of
Raf-1
(27, 28, 29) .
Figure 1:
Structures of Raf proteins. Shown in
panelA are the linear structures of cellular Raf-1
protein and the Raf sequences retained in v-Raf and the N-terminal
truncation mutant, 22W. CRI, CRII, and
CRIII, conserved regions 1, 2, and 3, respectively. The
cross-hatched box in the v-Raf structure depicts the fused Gag
sequence. The cysteine-rich, zinc finger motif is shown as a darkbar in CRI. The 12 amino acids at the extreme C terminus,
corresponding to the antigen against which the anti-Raf antiserum was
raised, are indicated by wave pattern-filled boxes. PanelB summarizes the positions of the 24 linker insertion
mutations, and panelC diagrams the primary
structures of the deletion mutants in which the deleted regions are
indicated as solidblackboxes. Amino acid
positions deleted for each mutant are as follows: Raf-d1 (18-52),
Raf-d2 (53-132), Raf-d3 (53-205), Raf-d4 (133-180),
Raf-d5 (207-228), Raf-d6 (250-275), and Raf-d7
(276-323).
Figure 2:
Expression of Raf proteins in
baculovirus-infected insect cells. Sf9 cells were infected with
indicated recombinant baculoviruses, and proteins in whole-cell lysates
were separated through a 7.5% SDS-polyacrylamide gel and blotted
electrophoretically onto nitrocellulose membrane. The membrane was
probed with anti-Raf serum and then with anti-rabbit horseradish
peroxidase-conjugated secondary antibody, followed by detection with
enhanced chemiluminescence (ECL) reagents. Mobilities of all the Raf
proteins are consistent with the predicted molecular
masses.
Characterization of in Vitro Kinase Activity and
Substrate Specificity of Raf-1 Protein
Fig. 3A shows our standard in vitro kinase assay using purified
MKK1 protein, irreversibly inactivated with FSBA, which possesses no
kinase activity of its own (lane4). Significant
levels of phosphorylation on MKK1 were seen in reactions containing
anti-Raf immunoprecipitates prepared from lysates of Raf-1
virus-infected Sf9 cells (lane6), and this kinase
activity could be competed out when the anti-Raf serum was preincubated
with the corresponding synthetic peptide antigen (lane5). These results demonstrate that MKK1 is specifically
phosphorylated by Raf-1 in this reaction. A kinetic analysis of Raf-d7
kinase activity on MKK1 shows that the in vitro reaction rate
of Raf-d7, which has an elevated basal kinase activity (see below),
remains linear for at least 45 min under our standard reaction
conditions (Fig. 3B). For all the experiments described
below, we used conditions similar to this assay and terminated the
reactions at 20 min to ensure that the kinase activities of Raf
proteins were analyzed in the linear range. Experiments were also
performed using insect cell-expressed Raf340D, a Raf mutant with highly
activated kinase
(37) , showing that at least 30-fold elevation
of the basal kinase activity of Raf-1 can be detected under our in
vitro kinase reaction conditions (data not shown).
Figure 3:
Characterization of Raf in vitro kinase activity. PanelA, anti-Raf
immunoprecipitates (Raf IP) from lysates of Sf9 cells infected with
wild-type (A. californica nuclear polyhedrosis virus)
baculovirus (lanes 1-3) or with recombinant virus
encoding Raf-1 (lanes 5-7) were subjected to an in
vitro kinase reaction using purified, chemically inactivated MKK1
as substrate. A kinase reaction containing only MKK1 (lane4) confirms that the substrate lacks intrinsic kinase
activity. Phosphorylated MKK1 is indicated on the autoradiograph.
Controls for background activities include competition with the
synthetic peptide antigen and reactions without added MKK. PanelB, kinetics of Raf-d7 in vitro kinase activity
was analyzed. Aliquots of an in vitro kinase reaction mixture
containing Raf-d7 immunoprecipitates were removed and the reactions
were stopped at the indicated times. Control reactions with MKK1 or
Raf-d7 alone were incubated for 45 min. The graph depicts
quantification of radioactivity incorporated into MKK1 as detected by a
Molecular Dynamics PhosphorImager.
In the same
reaction conditions, Raf-1 kinase activities toward purified,
FSBA-inactivated preparations of MKK1 and MKK2 were compared
(Fig. 4). Raf-1, either at its basal level or activated by
co-expressed c-Src or v-Ras proteins, consistently shows a substrate
preference toward MKK1 over MKK2. MKK1 was therefore used as exogenous
substrate in all of the subsequent in vitro kinase assays.
Figure 4:
Substrate specificity of Raf kinases.
Lysates of 3 10
Sf9 cells infected with recombinant
Raf-1 baculovirus, alone or together with either c-Src or v-Ras
baculovirus, were incubated with anti-Raf antiserum followed by
collection of the immune complexes with protein A-Sepharose. Beads were
washed, resuspended in kinase reaction buffer, and split into
triplicates before being subjected to in vitro kinase assays
using either 0 or 200 ng of MKK1 or MKK2 as substrate. Following
separation by SDS-polyacrylamide gel electrophoresis, the reaction
products were transferred to nitrocellulose and then visualized by
autoradiography. The nitrocellulose filter was later probed with
anti-Raf antibodies to ensure the presence of equal amounts of Raf
proteins in the triplicates.
Basal Kinase Activities of Raf Deletion
Mutants
Kinase activities of the various Raf mutants were
analyzed to evaluate effects of the deletions on Raf-1 basal kinase
activity (Fig. 5). In order to normalize the levels of Raf-1
proteins in the in vitro kinase assays, small amounts of
immune serum were used so that the anti-Raf antibody would be saturated
and immunoprecipitate similar amounts of Raf proteins, as confirmed by
Western blotting analysis (Fig. 5B). In several
independent experiments, Raf-d7 consistently showed a 2-3-fold
higher kinase activity over wild-type Raf-1 (Fig. 5A).
In contrast, Raf-d4, lacking the cysteine-rich zinc finger motif,
showed only 10-40% the activity of Raf-1. The other deletion
mutants revealed no consistently significant differences in kinase
activity compared with Raf-1 (Fig. 5B). A
kinase-inactive mutant (ATPM) of Raf-1 (25) was used as a negative
control to estimate background activity in the in vitro kinase
assays. Basal kinase activities of the Raf mutants were relatively
unaffected by the presence or absence of 0.5 M NaCl, 0.1% SDS,
or 5 mM sodium orthovanadate in the cell lysis buffer, and
similar results were obtained using a monoclonal anti-Raf antibody for
immunoprecipitation (data not shown). These results demonstrate that
deletions that together span the length of the N-terminal regulatory
domain do not lead to striking activation of Raf-1 kinase activity in
this assay system.
Figure 5:
Basal kinase activities of Raf deletion
mutants. PanelA shows an autoradiograph of in
vitro kinase reaction products from anti-Raf immunoprecipitates
using lysates of Sf9 cells infected with viruses encoding wild-type
Raf-1, various Raf deletion mutants, or a kinase-negative Raf mutant
(ATPM). MKK1 was used as substrate. PanelB, anti-Raf
immunoblot using the same nitrocellulose membrane from panelA was performed to confirm equivalent precipitation of
Raf proteins in the kinase assays (Raf-d3 migrates close to the
immunoglobulin heavy chain band, labeled Ig). PanelC, phosphorylation of MKK1 substrate was quantified by
direct counting in an AMBIS 4000 radioisotope detector. The counts
representing wild-type Raf-1 kinase activity are arbitrarily defined as
1, and the activities of the mutant Raf proteins are expressed as -fold
activity in comparison with Raf-1. Statistically consistent data were
obtained from multiple independent experiments, some of which are shown
in Figs. 6C and 7C.
Activation of Mutant Raf Proteins by Src or
Ras
Earlier studies showed that Raf-1 protein can be activated
by co-expressed Src or Ras proteins in the baculovirus/insect cell
system
(30, 37, 38) . Utilizing a similar system
and our panel of Raf-1 mutants, we assessed the regions in the
N-terminal half of Raf-1 protein involved in regulation of Raf-1 by
these upstream activators. We used constitutively active v-Ras
(30) and chicken c-Src
(34) , which is active because of
lack of the negative-regulating Tyr kinase in insect
cells, as the activators. Ras or Src protein was overexpressed with Raf
proteins in co-infected Sf9 cells (Figs. 6B and 7B)
using multiplicities of infection that ensure favorable activator:Raf
ratios.
Figure 6:
Activation of mutant Raf proteins by Ras.
Sf9 cells were singly infected with various recombinant Raf viruses or
doubly infected with the Raf viruses and v-Ras virus as indicated.
Kinase activities on MKK1 were analyzed in immune complex assays, and
the autoradiograph is shown in panelA. Consistent
high levels of Ras protein expressed in the doubly infected cells were
confirmed by anti-Ras Western blot of whole-cell lysates shown in
panelB. Western blots of whole-cell lysates were
also performed to ensure comparable expression levels of the various
Raf proteins in singly and doubly infected cells, and equivalent
amounts of Raf proteins present in each kinase assay were further
confirmed on the nitrocellulose membrane following autoradiography (not
shown). PanelC, quantification of MKK1
phosphorylation data in the kinase assays detected with the AMBIS 4000.
Kinase activities of the mutant Raf proteins and of various Raf
proteins co-expressed with Ras are shown as -fold activity in
comparison with wild-type Raf-1 basal activity. Results shown are the
means with standard errors of assays done in three independent
experiments.
On the other hand,
the MKK1 phosphorylating activities of Raf-1, 22W, as well as all seven
deletion mutants were activated 5-7-fold by co-expressed Src
protein (Fig. 7, A and C). Variable
phosphorylation of Raf proteins and immunoglobulin heavy chain, which
might partly be due to co-precipitated Src kinase, was observed but did
not correspond to the extent of MKK1 phosphorylation. In addition, some
of the Raf phosphorylation could be due to autokinase activity enhanced
by Src, as previously reported
(30) . Raf-1 proteins, but not
co-precipitated Src protein, account for the elevated MKK1 kinase
activity, as evidenced by the low level of phosphorylation on MKK1
despite substantial immunoglobulin heavy chain phosphorylation in the
case of ATPM/Src co-infection. These results demonstrate that the
sequences required for activation by Src do not reside in the
N-terminal 323 amino acids.
Figure 7:
Activation of mutant Raf proteins by Src.
Sf9 cells were singly infected with various recombinant Raf viruses or
doubly infected with the Raf viruses and c-Src virus as indicated.
PanelA, anti-Raf immunoprecipitates were analyzed
for in vitro kinase activity on MKK1. Arrowheads denote phosphorylated Raf proteins, and Ig indicates
phosphorylated immunoglobulin heavy chain. PanelB,
anti-Src immunoblot of whole-cell lysates prepared from doubly infected
cells. Comparable expression levels of the various Raf proteins in
singly and doubly infected cells and equivalent amounts of Raf proteins
present in each kinase assay were also confirmed by Western blotting
(not shown). PanelC shows quantification of MKK1
phosphorylation data in the kinase assays analyzed by the AMBIS 4000.
Kinase activities of the mutant Raf proteins and of various Raf
proteins co-expressed with Src are shown as -fold activity in
comparison with Raf-1 basal activity. Results shown are the means with
standard errors of assays done in three independent experiments, except
for ATPM, Raf-1, and d7, which are from five independent
experiments.
phosphorylated by protein kinase C
(24) and Thr
phosphorylated by Raf-1 autokinase
activity (25). Because Raf-d6 does not show a significantly different
kinase activity compared with wild-type Raf-1, however, no regulatory
function of CR2 is required for Raf-1 kinase activity.
and Tyr
,
which have been identified as the major tyrosine phosphorylation sites
of Raf-1 when co-expressed with activated tyrosine kinases in insect
cells (37). Additional studies will be required to determine whether
phosphorylation on these tyrosine residues of Raf-1 is directly
responsible for its activation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.