 |
INTRODUCTION |
Activation of the extracellular signal-regulated kinases
(ERK)1 1 and 2 is required
for cellular proliferation in many cell types and is also a requisite
event in neoplastic transformation (1-4). The activities of ERKs 1 and
2 are regulated through the activation of a sequentially acting protein
kinase cascade, known generically as a mitogen-activated
protein kinase module. At the top of this cascade is the
serine/threonine protein kinase Raf-1. Once activated, Raf-1
phosphorylates and activates the mitogen and extracellular signal-regulated kinase kinases 1 and 2 (MEKs 1 and 2), which subsequently phosphorylate and activate ERKs 1 and 2. ERKs then phosphorylate a large number of nuclear and cytoplasmic substrates that
ultimately regulate the ability of a cell to grow and divide (2).
The Raf-1 activation mechanism is a complex process that involves
binding to the small GTP binding protein Ras, phosphorylation on
multiple residues, altered protein-protein interactions, and perhaps
direct interaction with lipids (5-7). Binding to active Ras
relocalizes Raf-1 from the cytosol to the plasma membrane. This
interaction is initially mediated by a conserved domain in the Raf-1
amino terminus known as the Ras binding domain (RBD). Binding of Ras to
the RBD then promotes contact with an adjacent domain known as the
cysteine-rich domain (CRD), and binding of Ras to both of these domains
is required for full activation of Raf-1 (5). Interaction between Ras
and Raf-1 also stimulates the release of 14-3-3 from its
amino-terminal binding site, which is centered on serine 259, and the
subsequent dephosphorylation of this site (8-10). This is thought to
contribute to the separation of the catalytic domain from the
amino-terminal regulatory domain.
Activation of Raf-1 by Ras is also accompanied by phosphorylation on
multiple residues, including serines 338 and 339 and tyrosines 340 and
341. Phosphorylation of these sites is essential for Raf-1 activation
by extracellular ligands such as epidermal growth factor, phorbol
esters, and integrin binding (11-13). Kinases that may catalyze
the phosphorylation of these sites in the cell are the p21-activated
kinases (PAKs) 1-3, which phosphorylate serines 338 and 339 (13, 14),
and the Src family of tyrosine kinases, which phosphorylate tyrosines
340 and 341 (15, 16). In addition, Raf-1 activation may require
phosphorylation on two conserved sites within the activation loop of
its kinase domain (threonine 491 and serine 494) (17). Kinases that
phosphorylate these residues have not been identified. Despite the
clear importance of phosphorylation of Raf-1 on each of these sites, it
is not yet understood how these events contribute to Raf-1 activation.
Previously it was shown (18) that the amino terminus of Raf-1 contains
an autoinhibitory domain that can block the function of the Raf-1
catalytic domain in Xenopus oocytes. This supports a model
in which the catalytic activity of inactive Raf-1 is inhibited by
interaction with the autoinhibitory domain. The mechanism whereby the
catalytic domain is released from the autoinhibitory domain has not
been determined. In this report we show that the autoinhibitory domain
of Raf-1 can block the ability of a separately expressed Raf-1
catalytic domain to stimulate ERK2 activity in mammalian cells. We also
show that this domain minimally consists of the first 147 amino acids
of Raf-1 and encompasses portions of both the RBD and the CRD. We also
demonstrate that phosphorylation of Raf-1 on serine 338 and tyrosines
340 and 341 relieves this autoinhibitory effect and that this occurs
through a reduction in the affinity of the amino terminus for the
phosphorylated catalytic domain. Furthermore, we demonstrate that
putative phosphorylation sites within the kinase loop (threonine 491 and serine 494) are not likely to regulate autoinhibition. These data
demonstrate that phosphorylation of Raf-1 on serines 338 and 339 and
tyrosines 340 and 341 contributes to Raf-1 activation by blocking the
ability of the autoinhibitory domain to regulate Raf-1 catalytic activity.
 |
MATERIALS AND METHODS |
Plasmids and Recombinant Proteins--
Hemagglutinin
epitope-tagged ERK2, Raf BXB (amino acids 1-25 and 304-648), and
constitutively active PAK1 (PAK1 L107F) were as previously described
(19, 20). Constitutively active chicken Src (21) was subcloned into
pCMV5. Point mutations in pCMV5/Raf BXB plasmids were produced by PCR
and sequenced to confirm correct amplification. Raf-1 1-330, 1-330
C165/168S, 1-256, 1-186, 1-147, 51-131, and 139-186 were produced
by PCR and cloned into pCMVFlag6B (Sigma). Recombinant baculovirus
expressing hexahistidine-tagged RafCAT (amino acids 304-648)
was produced using the plasmid pFastBacHTb, essentially as described by
the manufacturer (Invitrogen). GST·Raf-1 1-330 protein was produced
in BL21DE3 Escherichia coli. Specifically, cells were grown
to A600 = 0.8 at 37 °C and induced
with 400 µM isopropyl-1-thio-
-D-galactopyranoside for 4 h at
37 °C. Cells were frozen and subsequently lysed by sonication in 10 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride). Insoluble protein
was pelleted by centrifugation at 35,000 × g for 30 min at 4 °C. GST·Raf-1 1-330 was purified from the soluble lysate
by affinity chromatography using glutathione-agarose. Specifically, 2 ml of a 50% slurry of pre-swelled glutathione-agarose was added to the
soluble lysate and incubated at 4 °C for 1 h. Bound GST·Raf-1
1-330 protein was precipitated by centrifugation, and the slurry was
washed three times with lysis buffer. The beads were then resuspended
in an equal volume of phosphate-buffered saline and stored at 4 °C. Hexahistidine-tagged RafCAT protein was produced in Sf9 cells grown in Sf-900 II SFM (Invitrogen) by infection with a recombinant baculovirus for 48 h. Cells were pelleted at 500 × g and stored at
80 °C. Cell pellets were lysed by
homogenization in hypotonic lysis buffer (20 mM Tris, pH
8.0, 1 mM EDTA, 80 mM
-glycerophosphate, 1 mM sodium orthovanadate, 10 µg/ml pepstatin, 10 µg/ml
leupeptin, 2 µg/ml aprotinin, and 1 mM
phenylmethylsulfonyl fluoride). Unbroken cells and nuclei were pelleted
by centrifugation. Triton X-100 was added to the lysate to a final
concentration of 0.5%, and insoluble material was pelleted by
centrifugation at 35,000 × g for 30 min at 4 °C.
RafCAT protein was purified by Ni2+-NTA affinity
chromatography using a 10-250 mM imidazole
gradient, essentially as described by the manufacturer (Qiagen).
Transfection, Immunoprecipitation, and in Vitro Kinase
Assays--
HEK 293 cells were maintained in Dulbecco's modified
Eagle's medium plus 10% fetal bovine serum and 10 µg/ml
penicillin/streptomycin (all cell culture reagents from Invitrogen).
Cells in 60-mm dishes were transfected by calcium phosphate
coprecipitation (19). For all assays when shown, 2 µg of HA-tagged
ERK2 and 20 ng of Raf BXB plasmids were transfected. The amounts
transfected for other constructs were as noted in the corresponding
figure legends. Twenty hours after transfection, the culture
medium was replaced with Dulbecco's modified Eagle's medium
without serum, and the cells were incubated for a further 24 h.
Cells were lysed in 0.5 ml of Triton lysis buffer (20 mM
Tris-HCl, pH 8.0, 100 mM NaCl, 80 mM
-glycerophosphate, 1 mM sodium orthovanadate, 1 mM EDTA, 0.5% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, 1 mM
phenylmethylsulfonyl fluoride). Insoluble proteins were pelleted by
centrifugation at 16,000 × g for 10 min at 4 °C,
and the soluble supernatant was stored at
80 °C. Immunoprecipitation-kinase assays measuring hemagglutinin
epitope-tagged ERK2 activity were performed as previously described
(19). Briefly, HA-ERK2 was immunoprecipitated from soluble cell lysates
using 2 µg of mouse anti-HA antibody (Santa Cruz Biotechnology) and 40 µl of a 50% slurry of protein A-Sepharose (Amersham Biosciences). Immunoprecipitates were washed three times with 20 mM
Tris-HCl, pH 8.0, 500 mM NaCl, and once with 20 mM Tris-HCl, pH 8.0. The kinase activity of
immunoprecipitated HA-ERK2 was assayed using 10 µg of myelin basic
protein (MBP) (Sigma) as a substrate. Incubations were for 30 min at
30 °C in kinase buffer (20 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM dithiothreitol, 100 µM ATP, 2 µCi of [
32P]ATP (ICN
Biomedicals)). Phosphorylated MBP was resolved by 15% SDS-PAGE.
After drying, the gel was exposed to autoradiography film, and the
phosphorylation of MBP was quantitated by scintillation counting of the
excised MBP.
RafCAT Binding Assays--
RafCAT protein (1 µg) was
phosphorylated or not by incubation in kinase buffer (20 mM
Tris-HCl, pH 8.0, 10 mM MgCl2, 100 µM ATP) for 30 min at 30 °C with or without
recombinant GST·PAK1 (22) or recombinant Src (Upstate Biotechnology
Inc.). It was previously determined that after 30 min the
phosphorylation of RafCAT by either kinase had reached a maximum. After
phosphorylation, one-tenth of the phosphorylated RafCAT (100 ng) was
mixed with 10 µg of GST·Raf-1 1-330 bound to glutathione-agarose,
and phosphate-buffered saline was added to a final volume of 300 µl.
Proteins were mixed for 1 h at 4 °C.
Glutathione-agarose-protein complexes were then pelleted by
centrifugation, and the supernatant was saved to test for the presence
of unbound RafCAT protein. The pellets were washed three times with 1 ml of ice-cold phosphate-buffered saline. Pelleted complexes were
solubilized with an equal volume of 2× Laemmli sample buffer. Bound
and unbound proteins were loaded on 12% SDS-PAGE and transferred to
nitrocellulose. Western blots were then performed for RafCAT (rabbit
anti-Raf-1, C-12, Santa Cruz Biotechnology), phospho-serine 338 (rat
anti-Raf-1 phospho-serine 338, Upstate Biotechnology Inc.), and
phospho-tyrosine (mouse anti-phospho-tyrosine 4G10, Upstate
Biotechnology Inc.). Western blots were quantitated by densitometry.
 |
RESULTS |
Characterization of the Raf-1 Autoinhibitory Domain in Mammalian
Cells--
Cutler et al. (18) reported that expression of
the Raf-1 amino terminus (amino acids 1-330) in Xenopus
oocytes blocked both germinal vesicle breakdown and mitogen-activated
protein kinase activation stimulated by coexpression of the Raf-1
catalytic domain. To test whether the Raf-1 amino terminus could
function as an autoinhibitory domain in mammalian cells, we tested its
ability to block ERK2 activation stimulated by expression of the Raf-1 catalytic domain. HEK 293 cells were cotransfected with hemagglutinin epitope-tagged ERK2 (HA-ERK2) and a constitutively active Raf-1 catalytic domain construct (Raf BXB, amino acids 1-25 and 304-648) (19), either with or without increasing amounts of the Raf-1 amino
terminus (1-330). After starving the cells for 24 h, the cells
were lysed, and the kinase activity of immunoprecipitated HA-ERK2 was
assayed using MBP as a substrate (19). As shown in Fig.
1, expression of Raf BXB potently
stimulated ERK2 activity in these cells (compare lanes 1 and
2). However, ERK2 activation by Raf BXB was effectively
blocked by coexpression of increasing amounts of Raf-1 1-330
(lanes 3-5). Thus, the amino terminus of Raf-1 can inhibit
the activity of the Raf-1 catalytic domain in mammalian cells.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Coexpression of the Raf-1 regulatory domain
blocks ERK2 activation by the Raf-1 catalytic domain. HEK293 cells
were transiently transfected with HA-tagged ERK2 and Raf BXB, with or
without increasing amounts of Raf-1 1-330 (1, 3, or 10 µg). Two days
later the HA-ERK2 was immunoprecipitated and assayed for kinase
activity (IP kinase assay) using myelin basic protein (MBP)
as a substrate. The panel shown is an autoradiograph of the
phosphorylated MBP. The data shown are representative of four
independent experiments.
|
|
Because the first 330 amino acids of Raf-1 contain several conserved
domains, we next examined which regions of the Raf-1 amino terminus
were required for autoinhibition. Conserved sequences present in the
amino terminus include the CR1 domain, which contains the RBD, (amino
acids 51-131), the CRD, (amino acids 139-186), and the CR2 domain
(amino acids 255-268) (Fig.
2A) (5). Thus, we tested
different amino-terminal constructs for their ability to inhibit
HA-ERK2 activation stimulated by coexpression of Raf BXB. In these
experiments, similar amounts of each amino-terminal protein were
expressed, as judged by Western blot using an antibody that recognizes
an N-terminal FLAG epitope present in each protein (data not shown). As
shown in Fig. 2B, coexpression of Raf-1 1-330 blocked ERK2
activation by Raf BXB in a dose-dependent manner. Deletions
within the carboxyl-terminal end of the regulatory domain did not
affect autoinhibition, because Raf-1 1-256, 1-186, and 1-147 were as
effective as 1-330 at inhibiting ERK2 activation. However, expression
of the isolated RBD of Raf-1 (55-131) did not block ERK2 activation,
demonstrating that the RBD alone cannot mediate autoinhibition.
Similarly, the isolated CRD (amino acids 139-186) was also relatively
inefficient at blocking ERK2 activation by Raf BXB. Interestingly,
mutation of cysteines 165 and 168 to serine, which disrupts the zinc
finger and blocks the function of the autoinhibitory domain in
Xenopus oocytes (18), only slightly affected the inhibition
of ERK2 activation by the Raf-1 autoinhibitory domain in these cells
(Fig. 2B, Raf-1 1-330 CC/SS). We interpret these
data to indicate that the autoinhibitory domain is minimally encompassed by residues 1-147 but also includes sequences within the
CRD amino-terminal to residue 147. Furthermore, an intact zinc finger
appears not to be required for autoinhibition, because expression of
the CC/SS mutant still blocked ERK2 activation. Because mutation of
these cysteines also blocks the ability of the CRD to bind Ras, 14-3-3, and phosphatidylserine (5, 6, 23, 24), it appears that interaction with
these molecules is not required for autoinhibition in mammalian
cells.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Characterization of the Raf-1 autoinhibitory
domain. A, schematic description of Raf-1 domains and
phosphorylation sites. Numbers refer to the amino acid
residues. The three regions conserved among Raf proteins
(CR1, CR2, and CR3) are shown.
RBD, the Ras binding domain. CRD, the
cysteine-rich domain. Arrows denote the activating
phosphorylation sites in the CR3 (catalytic) domain. B,
deletion analysis of the Raf-1 autoinhibitory domain. HEK 293 cells
were transiently transfected with HA-ERK2, Raf BXB, and the Raf-1
amino-terminal constructs shown. HA-ERK2 activity was monitored by IP
kinase assay using MBP as a substrate. MBP phosphorylation was
quantified by scintillation counting. Values are listed as the percent
of activation achieved by expression of Raf BXB alone. The Raf-1
amino-terminal constructs expressed are shown on the bottom of the
graph. 1-330 CC/SS, Raf-1 1-330 containing the double
C165/168S substitution, which inactivates the zinc finger. For each
Raf-1 amino-terminal construct transfected, dark bars refer
to 1× plasmid transfected, light bars refer to 3× plasmid
transfected. Results are the average of at least three independent
experiments. Errors are the S. E. of the mean.
|
|
Autoinhibition of Raf-1 Activity Is Blocked by Coexpression of
Constitutively Active PAK1 or Constitutively Active Src--
Raf-1 is
phosphorylated on serines 338 and 339 and tyrosines 340 and 341 in
response to extracellular stimuli (15, 16, 25). Among these residues,
serine 338 and tyrosine 341 appear to be the major phospho-acceptor
sites because their phosphorylation is critical for Raf-1 activation
(12, 25). However, the role that phosphorylation of these sites plays
in the Raf-1 activation mechanism is unknown.
It has previously been shown that mutation of tyrosine 340 to aspartic
acid, which is thought to mimic the phosphorylation of this site,
attenuates the autoinhibitory activity of the Raf-1 amino terminus in
Xenopus oocytes (18). Thus, we examined whether phosphorylation of serine 338 and tyrosine 341 blocks autoinhibition by
the Raf-1 amino terminus in mammalian cells. HEK 293 cells were
transfected with Raf BXB, with or without Raf-1 1-330, and the
activation of epitope-tagged ERK2 was measured. To stimulate the
phosphorylation of serine 338 and tyrosine 341, the cells were also
transfected with increasing amounts of constitutively active PAK1 or
constitutively active Src, respectively. As shown in Fig.
3A, expression of
constitutively active forms of either PAK1 (PAK1*,
lanes 5-7) or Src (Src*, lanes 8-10)
blocked the autoinhibitory effect of Raf-1 1-330 on ERK2 activation in
a dose-dependent manner. This was accompanied by
phosphorylation of Raf-1 on serine 338 (PAK1 transfectants) or
tyrosines 340 and 341 (Src transfectants), as determined by Western
blotting with antibodies specific for phospho-serine 338 and
phospho-tyrosine (data not shown). The results of three independent
experiments are quantified in Fig. 3B. Thus, these data
indicate that phosphorylation of serine 338 by PAK1, and tyrosines 340 and 341 by Src, block the ability of the autoinhibitory domain to
function.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of constitutively active PAK1 or
constitutively active Src blocks autoinhibition by the Raf-1 amino
terminus. A, HEK 293 cells were transiently transfected
with HA-ERK2 and Raf BXB, with or without Raf-1 1-330. Where indicated
the cells were also transfected with increasing amounts of
constitutively active PAK1 (PAK1*) or constitutively active
Src (Src*). HA-ERK2 activity was measured by IP kinase
assay. The panel is an autoradiograph of phosphorylated MBP. Fold
refers to the fold increase in ERK2 kinase activity relative to cells
expressing ERK2 alone. B, quantification of the relief of
autoinhibition by constitutively active PAK1 or Src. Results represent
the mean of three independent experiments. Errors are S. E. of the
mean.
|
|
To determine whether expression of active PAK1 or active Src relieved
autoinhibition through a mechanism other than phosphorylation of Raf
BXB, we examined whether substitution of the PAK or Src phosphorylation
sites with non-phosphorylatable residues (S338A or Y340/341F,
respectively) affected the ability of active PAK1 or Src to block
autoinhibition. As shown in Fig.
4A, expression of active PAK1
or active Src effectively relieved autoinhibition of wild type Raf BXB
by the Raf-1 amino terminus (lanes 3-6). However,
expression of active PAK1 only marginally rescued ERK2 activation in
cells expressing Raf BXB S338A (S/A), whereas coexpression of active Src still blocked autoinhibition (lanes 7-10).
The modest rescue of autoinhibition by active PAK1 in cells expressing
Raf BXB S338A may be due to a low level of phosphorylation of serine 339 or an increased efficiency of coupling between Raf and MEK1 caused
by the phosphorylation of MEK1 by PAK1 (22, 26, 27). On the other hand,
expression of active Src did not relieve autoinhibition in cells
expressing Raf BXB Y340/341F, whereas active PAK1 was fully able to
block autoinhibition (lanes 11-14). Fig. 4B
represents the mean of four independent experiments. Thus, these
results indicate that expression of active PAK1 or active Src blocks
Raf-1 autoinhibition through the direct phosphorylation of serine 338 and tyrosines 340/341, respectively.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 4.
Raf BXB requires intact PAK and Src
phosphorylation sites for relief of autoinhibition by active PAK1 or
active Src. A, HEK 293 cells were transiently
transfected with HA-ERK2 and either Raf BXB, Raf BXB S338A
(S/A), or Raf BXB Y340/341F
(YY/FF). Where indicated, the cells were also
transfected with Raf-1 1-330, constitutively active PAK1, or
constitutively active Src. HA-ERK2 kinase activity was measured by IP
kinase assay. The panel is an autoradiograph of phosphorylated MBP.
Fold refers to the fold increase in ERK2 activity relative to cells
expressing ERK2 alone. B, quantification of the effects of
mutating PAK1 or Src phosphorylation sites on rescue from
autoinhibition. Results are the average of four independent
experiments. Errors are S.E. of the mean.
|
|
Phosphorylation of Residues within the Raf-1 Kinase Activation Loop
Is Not Required to Block Autoinhibition--
Based on homology between
B-Raf and Raf-1, Chong et al. (17) identified threonine 491 and serine 494 as potential phosphorylation sites that are required for
Raf-1 activation. These sites are contained within the activation loop
of the Raf-1 kinase domain, and phosphorylation of the corresponding
residues in B-Raf is required for its activation (28). We therefore
examined whether these sites are involved in the Raf-1 autoinhibition mechanism.
We first substituted these sites with alanine (TS/AA), which precludes
their phosphorylation in the cell. As shown in Fig. 5A, this largely blocked the
ability of Raf BXB to stimulate ERK2 activity (lane 7). This
was most likely due to a reduction in Raf BXB kinase activity, because
these sites were shown to be required for the kinase activity of
full-length Raf-1 (17). However, some ERK2 activation was still
apparent, and this was effectively blocked by coexpression of Raf-1
1-330 (lane 8). In addition, autoinhibition of this Raf BXB
mutant was counteracted by coexpression of active PAK1 or active Src
(lanes 9 and 10). Thus, mutation of threonine 491 and serine 494 to nonphosphorylatable residues does not affect the
autoinhibition mechanism.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
Threonine 491 and serine 494 are not required
for autoinhibition by the Raf-1 amino terminus. A, HEK
293 cells were transiently transfected with HA-ERK2 and either Raf BXB,
Raf BXB T491A/S494A (TS/AA), or Raf BXB
T491E/S494D (TS/ED). Where indicated, the cells
were also transfected with Raf-1 1-330, constitutively active PAK1, or
constitutively active Src. HA-ERK2 activity was measured by IP kinase
assay. The panel is an autoradiograph of phosphorylated MBP. Fold
refers to the fold increase in ERK2 activity relative to cells
transfected with ERK2 alone. B, quantification of the
effects of mutating kinase loop phosphorylation sites on Raf-1
autoinhibition. Results are the mean of seven independent experiments.
Errors are S. E. of the mean.
|
|
To mimic the phosphorylation of these sites, we replaced them with
acidic residues (TS/ED) (17). Expression of this Raf BXB mutant
stimulated ERK2 activity to a greater degree than the TS/AA mutant
(compare lanes 7 and 11). However, this Raf BXB
mutant was still subject to autoinhibition by the Raf-1 amino terminus (lane 12). This suggests that phosphorylation of these sites
is not required for relief of autoinhibition. Furthermore,
autoinhibition of Raf BXB TS/ED was effectively blocked by coexpression
of active PAK1 or active Src (lanes 13 and 14).
Fig. 5B represents the mean of seven independent
experiments. Thus, these results demonstrate that phosphorylation of
threonine 491 and serine 494 is not required to block autoinhibition
and indicate that phosphorylation of these sites is required for other
steps within the Raf-1 activation mechanism.
Phosphorylation of Raf-1 by PAK1 or Src Decreases the Affinity of
the Autoinhibitory Domain for the Catalytic Domain--
Using purified
proteins, we next examined whether phosphorylation of the Raf-1
catalytic domain by PAK1 or Src affected the ability of the
autoinhibitory domain to bind to the catalytic domain. For these
experiments, Raf-1 1-330 was expressed in bacteria as a GST fusion
protein, and the Raf-1 catalytic domain (RafCAT, residues
304-648) was expressed as an hexahistidine-tagged fusion protein in
insect cells (it is insoluble in bacteria). To measure the binding of
these proteins, an excess of GST·Raf-1 1-330 was incubated
with RafCAT, and the binding was allowed to proceed to equilibrium.
GST·Raf-1 1-330 was then precipitated using glutathione-agarose, and
the amount of RafCAT bound was examined by Western blotting using an
antibody specific for the RafCAT protein. The amount of RafCAT protein
still present in the supernatant was also measured. Under these
conditions, ~10% of the RafCAT protein did not bind to Raf-1 1-330
(Fig. 6). Furthermore, coprecipitation of
RafCAT with the Raf-1 amino terminus required the presence of Raf-1
1-330 because RafCAT did not coprecipitate with glutathione beads
bound to glutathione S-transferase alone (data not shown).
However, when RafCAT was phosphorylated with Src prior to incubation
with Raf-1 1-330, the amount of free RafCAT increased to ~30%. The reduction in binding was even more apparent if one measured only the
tyrosine-phosphorylated RafCAT population (40% unbound). Similar results were observed when RafCAT was phosphorylated by recombinant PAK1 prior to incubation with the Raf-1 amino terminus. Thus, these
data indicate that phosphorylation of Raf-1 on serine 338 or tyrosines
340/341 reduces the affinity of the autoinhibitory domain for the
catalytic domain.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Phosphorylation of the Raf-1 catalytic domain
on serine 338 or tyrosine 341 reduces the binding of the Raf-1
autoinhibitory domain to the catalytic domain.
Hexahistidine-tagged Raf-1 catalytic domain (RafCAT) was
incubated at 4 °C with GST·Raf-1 1-330 prebound to
glutathione-agarose beads. Prior to incubation with the Raf-1 amino
terminus, RafCAT was phosphorylated or not by incubation in kinase
buffer with or without recombinant PAK1 or recombinant Src. After
binding had reached equilibrium, GST·Raf-1 1-330 was precipitated by
centrifugation. RafCAT associated with the GST·Raf-1 1-330, as well
as that still in solution (unbound), was measured by Western blot with
an antibody specific for the Raf-1 catalytic domain. Western blots were
quantified by densitometry. Shown is the average of three independent
experiments. Errors are the S.E. of the mean.
|
|
 |
DISCUSSION |
Raf-1 activation by growth factors is a complex process that
entails recruitment to the plasma membrane by active Ras and the
subsequent phosphorylation of Raf-1 on multiple sites. The role of
phosphorylation within this mechanism has remained unclear. Previous
work has shown that Raf-1 contains an autoinhibitory domain within the
first 330 amino acids of its amino terminus (18). This was demonstrated
by measuring the ability of the Raf-1 amino terminus to inhibit meiotic
maturation in Xenopus oocytes stimulated by expression of
the Raf-1 catalytic domain. It was also shown that regulation of
autoinhibition did not correlate with changes in 14-3-3 binding to the
Raf-1 amino terminus and that it depended on the integrity of the
CRD. In the present study we have shown that the Raf-1
autoinhibitory domain can block the ability of the Raf-1 catalytic
domain to stimulate ERK2 activity in mammalian cells. Furthermore, this
domain minimally consists of the first 147 amino acids of Raf-1,
although sequences within the CRD carboxyl-terminal to residue 147 may
contribute to autoregulation. We also show that phosphorylation of
Raf-1 on serine 338 and tyrosines 340 and 341 blocks autoinhibition and
that this is due to a reduction in the affinity of the autoinhibitory
domain for the phosphorylated, catalytic domain. Thus, these results
indicate that phosphorylation of Raf-1 on these sites is required
for activation because this modification blocks autoinhibition.
Through extensive deletion analysis we have more precisely defined the
region encompassing the autoinhibitory domain. This domain includes
sequences within the RBD (amino acids 51-131) and the CRD (amino acids
139-186) but does not include the CR2 domain of Raf-1 (amino acids
255-268). A coincidence of autoinhibitory and small G protein binding
domains exists in other kinases. For example, PAK1 contains an
autoinhibitory domain that partially coincides with its Rac/Cdc42
binding domain (20, 29). In addition, the interaction of the PAK1
autoinhibitory domain with the catalytic domain is regulated by
phosphorylation (30, 31).
Interestingly, in contrast to the results of Cutler et al.
(18), we found that an intact zinc finger within the CRD domain was not required for autoinhibition. This is based on our definition of
the minimal autoinhibitory domain (residues 1-147), which does not
include the zinc finger region, and the finding that disruption of the
zinc finger by mutation of two key cysteine residues to serine
(C165/186S) only slightly affected autoregulation by the Raf-1 amino
terminus (Fig. 2B). One possible explanation for these conflicting results is the divergent systems used to assay
autoinhibition (maturation of Xenopus oocytes
versus ERK2 activation in HEK 293 cells). In oocyte
maturation, for example, a requirement for the CRD in autoinhibition
may reflect a role in other aspects of Raf-1 signaling that are not
directly related to mitogen-activated protein kinase activation.
Previous work also suggested that phosphorylation of Raf-1 on tyrosine
340 may block autoregulation. This was proposed because expression of
RafCAT containing a phosphorylation mimic at Y340 (Y340D) precluded
inhibition of meiotic maturation by the Raf-1 regulatory domain (18).
In addition, it was recently published that phosphorylation of residues
between serine 338 and tyrosine 341 was necessary for high affinity
interaction between Raf-1 and MEK1 (32). Our results are consistent
with the idea that phosphorylation of serine 338 and/or tyrosine 340 is
required to block autoinhibition. We cannot, however, preclude a role
for the phosphorylation of these sites in regulating the affinity of
Raf-1 for MEK1. In fact, it is possible that phosphorylation of serine
338 and tyrosine 340 serves a dual role in stimulating the activity of
Raf-1 toward MEK1, namely to block autoinhibition and to increase
interaction between Raf-1 and MEK1.
We have also found that phosphorylation of threonine 491 and serine 494 is not likely to be involved in the regulation of autoinhibition,
because substitution of these sites with acidic residues, which mimics
their phosphorylation, does not affect autoinhibition. These sites are
located within the activation loop of the Raf-1 kinase domain, and in
many kinases phosphorylation of sites within the activation loop
directly affects catalytic activity (33, 34). Thus, given our data, we
would predict that phosphorylation of threonine 491 and serine 494 is
necessary for an increase in Raf-1 catalytic activity rather than a
relief of autoinhibition.
In conclusion, our data detail a role for phosphorylation of Raf-1 on
serine 338 and tyrosines 340 and 341 in the Raf-1 activation mechanism.
Specifically, phosphorylation of these sites prevents interaction
between the autoinhibitory and catalytic domains. Given this data, we
can refine a common model for growth factor-mediated Raf-1 activation
(5, 17). In this model, inactive Raf-1 is recruited to the plasma
membrane by GTP-bound Ras. Ras first binds to the RBD of Raf-1 and then
interacts with the CRD. Binding of Ras to these domains causes 14-3-3
to release from its amino-terminal binding site (serine 259), thereby
allowing Raf-1 to further unfold. Dephosphorylation of serine 259 may
also occur at this time. Raf-1 is then phosphorylated on serine 338 and
tyrosines 340 and 341 by kinases whose activities are also regulated by
Ras. Phosphorylation of these sites locks Raf-1 in an open conformation
by preventing interaction between the catalytic and autoinhibitory
domains. However, full activation of Raf-1 would not occur until it is phosphorylated on threonine 491 and serine 494 by one or more as yet
unidentified kinases. Once Raf-1 is phosphorylated on all four sites it
is fully active and able to phosphorylate its downstream target MEK.
Refinement of this model remains an area for further study.