From the Diabetes and Metabolism Research Unit,
Endocrinology Section, Evans Department of Medicine and
Department of Biochemistry, Boston University School of
Medicine, Boston, Massachusetts 02118, the § Diabetes Unit
and Medical Services and the Department of Molecular Biology,
Massachusetts General Hospital, Harvard Medical School, Boston,
Massachusetts 02114, and the ¶ Department of Ophthalmology,
University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
Received for publication, January 8, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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Growth factors activate Raf-1 by
engaging a complex program, which requires Ras binding, membrane
recruitment, and phosphorylation of Raf-1. The present study employs
the microtubule-depolymerizing drug nocodazole as an alternative
approach to explore the mechanisms of Raf activation. Incubation of
cells with nocodazole leads to activation of Pak1/2, kinases downstream
of small GTPases Rac/Cdc42, which have been previously indicated to
phosphorylate Raf-1 Ser338. Nocodazole-induced
stimulation of Raf-1 is augmented by co-expression of small GTPases
Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block
activation of Raf-1 by nocodazole, but not by epidermal growth factor
(EGF). Thus, our studies define Rac/Cdc42/Pak as a module upstream of
Raf-1 during its activation by microtubule disruption. Although it is
Ras-independent, nocodazole-induced activation of Raf-1 appears to
involve the amino-terminal regulatory region in which the integrity of
the Ras binding domain is required. Surprisingly, the Raf zinc finger
mutation (C165S/C168S) causes a robust activation of Raf-1 by
nocodazole, whereas it diminishes Ras-dependent activation
of Raf-1. We also show that mutation of residues Ser338 to
Ala or Tyr340-Tyr341 to Phe-Phe immediately
amino-terminal to the catalytic domain abrogates activation of both the
wild type and zinc finger mutant Raf by both
EGF/4 The proto-oncogene raf-1, first identified as a
cellular counterpart of the oncogene v-raf, encodes a
serine/threonine protein kinase. Raf-1 is ubiquitously expressed and
plays an important role in cell proliferation and differentiation
(1). The mechanism by which Raf-1 is activated by growth factors is
still incompletely understood, although it is known to be preassembled
as a complex with 14-3-3 and heat shock proteins 90/50 (2-6). It
involves multiple steps including Ras-GTP binding, membrane
recruitment, and phosphorylation.
Raf-1 consists of an amino-terminal regulatory domain and a
carboxyl-terminal kinase domain. The amino-terminal moiety of Raf-1
exerts an inhibitory effect on the catalytic activity, since amino-terminal truncations lead to progressive increases in its transforming ability (7, 8). The amino-terminal regulatory region of
Raf-1 contains a Ras binding domain
(RBD)1 and a cysteine-rich
zinc finger domain (CRD), both of which participate in binding to Ras.
The first interaction engages Raf RBD ranging from aa 50 to 150 and the
effector loop of Ras-GTP, which is essential for activation of Raf-1
(9-11). Raf CRD, located between aa 139 and 184, binds to an epitope
involving Ras residues Asn26 and Val45 outside
the effector loop in prenylated Ras in a GTP-independent manner
(12-15). The strength of this second interaction is much lower than
the first one (12, 13). Nevertheless, it is crucial for the formation
of a productive complex, as the mutation of residues necessary for this
interaction on either proteins abolishes their functions, such as the
ability to transform cells (14) and to activate MAPK kinase (15). Thus,
these studies suggest that growth factor-stimulated GTP charging of Ras
initiates the association of the Raf RBD with the effector loop of Ras,
which ensures the second productive interaction between the CRD and Ras.
Considerable evidence indicates that phosphorylation plays an important
role in Raf activation. Incubation of Raf-1 activated in
vivo with serine/threonine or tyrosine protein phosphatases leads
to inactivation of Raf-1 (16-19). Indeed, a number of
serine/threonine and tyrosine protein kinases have been implicated in
the activation of Raf-1 (3). Raf-1 Ser259 and
Ser621 are the major phosphorylation sites which are
critical determinants for binding to 14-3-3 (20). It appears that
binding of 14-3-3 to the amino-terminal site (Ser(P)259)
plays an inhibitory role, whereas the binding to the carboxyl-terminal site (Ser(P)621) is indispensable for the Raf kinase
activity (21-23). Other crucial residues include
338SSYY341, whose mutation to alanine or
phenylalanine severely inhibits Raf-1 activation and to aspartic acid
or glutamic acid results in an increase in the basal Raf-1 activity
(24-27). In B-Raf, the two residues corresponding to Raf-1
Tyr340-Tyr341 are replaced by aspartic acids,
which may account for the increased kinase activity (27).
p21-activated kinases (Paks), mammalian homologs of Ste20-like Ser/Thr
protein kinases, are activated by signals that increase the level of
GTP-bound form of Rac and Cdc42 GTPases, although the
GTPase-independent pathway also exists (28, 29). The Pak family,
consisting of Pak1 (Pak The requirement of multiple factors for receptor tyrosine
kinase-stimulated activation of Raf-1 has greatly challenged us in
precise elucidation of its regulation. Recent studies on Raf-1 activation by disrupting microtubule integrity that can elude the Ras
binding and membrane recruitment may shed a light on uncovering this
mystery (36, 37). We have shown that nocodazole, a
microtubule-depolymerizing drug, activates Raf-1 and increases its
binding to 14-3-3, while inducing mitosis and hyperphosphorylation of
Raf-1 (38). Furthermore, the activation of Raf-1 is necessary for entry
of the cell cycle into mitosis (38). In the present study, we have
characterized this Ras-independent, nocodazole-induced activation of
Raf-1. Here we first show that nocodazole utilizes Rac/Cdc42/Pak to
activate Raf-1, in which a crucial step is the phosphorylation of
Ser338 by Pak, while EGF activates Raf-1 by a different
Ser338 kinase. We also find that, although Raf-1 activation
by nocodazole is Ras-independent, the integrity of the Raf RBD, but not
the CRD, is still required. Moreover, mutation of
Cys165-Cys168 to Ser-Ser within the CRD causes
a robust activation of Raf-1 by nocodazole and an increased ability of
Raf to be phosphorylated in vitro by Pak, suggesting that
the zinc finger structure plays an inhibitory role in Raf activation.
Materials--
Nocodazole and
4 DNA Construction--
cDNAs encoding Raf-1 variants, wild
type, Ras binding site mutant (84-87AAAA), zinc finger mutant
(165S/168S) and kinase-dead mutant (K375M) were constructed into
pMT2Myc as described previously (15). cDNAs for wild type Raf,
amino-terminally truncated kinase domain (BXBRaf, aa 1-25/303-648)
(39), amino-terminal regulatory region (C4, aa 1-259) (40), and Pak1
variants were inserted into pEBG (40). cDNA for Raf-1
84-87AAAA was also inserted into pBJMFPK3E in which Src myristoylation
sequence was tagged to the amino terminus of three copies of FKBP
followed by the cDNA encoding a hemagglutinin epitope and the Raf
mutant (41). Double site mutants, 165S/168S-380A/339A and
165S/168S-340F/341F, were made by replacing the wild type
SalI fragment in pMTMyc-Raf 165S/168S with the one
containing the mutation S338A/S339A or Y340F/Y341F. N17HaRas, V12Rac
and V12Cdc42 were constructed in pCMV5flag plasmid (15). N17Rac and
N17Cdc42 in pRK5Myc were obtained from W. Xiong, and Pak2 constructs
(in pRK5Myc) were from G. M. Bokoch. Raf 338A/339A and 340F/341F
constructs were from M. S. Marshall.
Transfections, Immunoprecipitation, and Western Blot--
Human
embryonic kidney 293 cells (HEK293T) and COS7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Transfection of plasmid DNA into HEK293T cells was
carried out using the calcium phosphate precipitation method.
LipofectAMINE reagents (Life Technologies, Inc.) were used for
transfection of plasmids into COS7 cells according to manufacturer's
protocol. Forty-eight hours after transfection, cells were
serum-starved in Dulbecco's modified Eagle's medium containing 0.1%
fetal bovine serum for 16-20 h and were treated with nocodazole, EGF,
or TPA as indicated in figure legends. Cells were then lysed in a lysis
buffer (20 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4,
25 mM
For immunoprecipitation, cell lysates were incubated with specific
antibodies and protein A/G-agarose at 4 °C overnight. The precipitates were washed once with the lysis buffer, twice with the
buffer containing 0.5 M NaCl, and twice with a kinase
buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol). For
purification of recombinant GST-Raf, the lysates were incubated with
GSH beads and washed as immunoprecipitation.
For Western blotting, samples were separated by 8% SDS-PAGE and
electrophoretically transferred to polyvinylidene difluoride membranes
(Millipore) in a transfer buffer consisting of 154 mM glycine, 20 mM Tris, and 20% methanol. The membranes were
blocked and incubated with specific antibodies and then with a
horseradish peroxidase-conjugated second antibody. Immunoreactive bands
were visualized by enhanced chemiluminescence (ECL) detection system.
Raf-1 Kinase Assay--
Raf-1 kinase activity was measured by a
coupled enzyme assay in which bacterially expressed recombinant
GST-MEK1 and kinase-dead mutant of ERK2 were sequentially added to the
Raf preparation in the presence of [ Pak Kinase Assay--
Recombinant GST-Pak1 and Myc-tagged Pak2
were transiently expressed in HEK293T cells. After stimulation with 300 nM nocodazole for 1 h, cell lysates were prepared as
described above. GST-Pak1 was purified by GSH beads and Myc-Pak2
immunoprecipitated with anti-Myc antibody (9E10). Pak activity was
analyzed at 30 °C for 30 min using 1 µg of myelin basic protein
(MBP) as a direct substrate. For in vitro phosphorylation of
Raf-1, recombinant GST-Pak1 was purified by GSH beads and eluted with 5 mM GSH. The immunoprecipitated Myc-Raf-1 was incubated with
Pak1 and ATP. The phosphorylation was visualized by
anti-Ser(P)338 blotting (27).
Nocodazole Activates Raf-1 through Ras-independent
Mechanism--
Recently, we have shown that nocodazole activates Raf-1
in a time-dependent manner and such activation is necessary
for transition of the cell cycle from G2 to M phase (38).
To further study the mechanism of nocodazole-induced Raf activation, we
first determined the dose effect of nocodazole on activation of Raf-1.
In doing so, cDNAs encoding the wild type and the kinase-defective
mutant (K375M) of Raf-1 were transiently expressed in HEK293T cells, and Raf-1 kinase activity was assayed after cells were treated with
nocodazole in different doses as indicated in Fig.
1A. Raf-1 was progressively
activated by increasing doses of nocodazole with a maximal effect at
300 nM under the condition of equal expression of the
polypeptides. The inability of the kinase-defective mutant Raf to be
activated indicated that the nocodazole-increased kinase activity was
not due to kinases copurifying with Raf-1. The
dose-dependent activation of Raf-1 is consistent with the
ability of nocodazole to disrupt the microtubule structure (data not
shown). To differentiate nocodazole-induced activation of Raf-1 from
the Ras-dependent one by EGF, Myc-tagged Raf-1 was
co-expressed with RafC4 (aa 1-259), the amino-terminal regulatory
region containing RBD and CRD, or N17HaRas, a dominant negative mutant
of Ras. This experiment was done in COS7 cells since this cell line
exhibits better response to EGF than HEK293T cells. As shown in Fig.
1B, co-expression of C4 or N17Ras reproducibly inhibited EGF
activation of Raf-1 by about 40%, which is similar to our previous
findings (41). In contrast, these mutants did not have significant
effect on Raf-1 activation by nocodazole, despite the fact that the
immunoblot showed equal expression of C4 or N17Ras under these two
treatments (data not shown). Interestingly, we observed a 40% increase
in Raf activation by nocodazole in the presence of N17 Ras, as compared with that in its absence (Fig. 1B, lanes 1 and 3). Overall, our results indicate that nocodazole
up-regulates Raf kinase via a Ras-independent mechanism, which is
consistent with previous publications (36, 37).
Rac/Cdc42 GTPases and Pak Participate in Raf-1 Activation by
Nocodazole--
Since Ser338 has been previously shown to
be phosphorylated by Pak (33-35), we attempted to evaluate the role of
Rac/Cdc42 and Pak in nocodazole-induced Raf-1 activation. In the first
assay, we introduced GST-Pak1 and Myc-Pak2 into HEK293T cells and
examined activation of these kinases by nocodazole (Fig.
2A). The recombinant Pak1 was
activated by 3-fold, whereas Pak2 activity was increased by about 50%.
The lesser activation of Pak2 might be due to its high basal activity
or partial activation caused by the basic substrate MBP (49).
In the next experiment, a constitutively active mutant of Rac, V12Rac
was co-expressed with the wild type Raf and its activation was
examined after treatment of cells with nocodazole. Coexpression of
V12Rac led to about 2.5-fold increase in basal kinase activity of Raf
(Fig. 2B, lanes 1 and 4) and more
potent activation by nocodazole so that the Raf activity was increased
by an additional 7-fold (Fig. 2B, lanes 1, 4, and
5). Similar activation profiles were obtained by
expression of a constitutively active mutant of Cdc42, Pak1, and Pak2
(Fig. 2, C and D).
To further establish the role of Rac/Cdc42/Pak pathway in activation of
Raf-1 by disrupting the microtubule integrity, we next co-transfected
Raf with a dominant negative mutant of Rac, N17Rac, or a
kinase-defective mutant of Pak2. Fig. 3
shows that, whereas the mutants had no effect on EGF-induced activation
of Raf-1, they strongly inhibited Raf-1 activation by nocodazole. The
same inhibitory effect was also achieved by co-expression of dominant
negative mutants of Cdc42 and Pak1 with Raf-1 (data not shown). These
results clearly place the Rac/Cdc42/Pak lineage as an upstream module
in the activation of Raf-1 by microtubule depolymerization and suggest
that growth factor-dependent activation of Raf-1 occurs
through different regulators.
To evaluate the role of Ser338-Ser339 and
Tyr340-Tyr341 in nocodazole-induced activation
of Raf-1, we transfected Raf mutants 338A/339A and 340F/341F into COS7
cells and examined their kinase activities, as compared with the wild
type Raf-1 (Fig. 4A). Both
mutations greatly inhibited the activation of Raf-1 by EGF and
nocodazole. Additionally, co-expression of Pak was without effect on
Raf-1 activity if Ser338 was mutated to Ala (Fig.
4B). Thus, these results suggest that phosphorylation of
this region is critical to both Ras-dependent and
independent activation of Raf-1.
To verify whether Raf residue Ser338 is phosphorylated
during Raf activation, endogenous Raf-1 was immunoprecipitated and
blotted with phospho-Ser338 antibody. Fig.
5A shows that the
phosphorylation of Ser338 was enhanced by both TPA and
nocodazole. When Raf-1 was co-expressed with the active mutant of Pak2,
Raf Ser338 was highly phosphorylated (Fig. 5B),
which held with its stimulatory role in Raf-1 activation by nocodazole
(Figs. 2 and 3). The phosphorylation of Raf-1 by Pak seemed to be
site-specific, as the phosphorylation of the 14-3-3 binding sites was
not altered under the same conditions (Fig. 5B). To
ascertain whether the phosphorylation and activation of Raf-1 by
nocodazole is a specific cellular event, we examined the
phosphorylation of Akt Ser473, an indicator for its
activation. Fig. 5C shows that phosphorylation of Akt
Ser473 was not changed in HEK 293T cells treated with
nocodazole and TPA, while MAPK/Erk was significantly activated. Another
piece of experimental evidence was the failure of nocodazole to
activate cAMP-dependent protein kinase (data not
shown).
Distinct Requirement of RBD and CRD for Raf Activation by Both
Ras-dependent and -independent Mechanisms--
To assess
whether the amino-terminal moiety is necessary for nocodazole-induced
Raf-1 activation, we compared the activation of full-length Raf-1 and
BXB-Raf-1 containing the entire carboxyl-terminal kinase domain. Fig.
6A shows that the full-length
Raf-1was activated well by both EGF and nocodazole, whereas the
activity of BXB-Raf-1 was barely affected by these agents. The same
results were obtained by using both HEK293T and COS7 cells. Thus, our
findings demonstrate that the amino-terminal regulatory domain
encompassing the Ras binding site is also necessary for Ras-independent
activation of Raf-1.
We next tested whether RBD is required for the action of nocodazole. To
this regard, Raf-1 with the 84KLAK87 to AAAA
mutation in the RBD was assayed for its kinase activity. Although the
wild type Raf-1 was activated normally by both TPA and nocodazole, the
mutation blunted its activation (Fig. 6B), even in the
presence of co-expressed Pak1 (Fig. 6C). To ascertain whether the inability of this mutant to respond to nocodazole was due
to the propagated misfolding of the catalytic domain engendered by the
mutation, we engineered a cDNA expressing a fusion protein (see
"Experimental Procedures") containing Raf 84-87AAAA tagged by the
Src myristoylation sequence, which enables Raf to be constitutively targeted to the plasma membrane. When this mutant was expressed in
cells (Fig. 6D), its activity was dramatically stimulated by nocodazole, indicating that mutation has not altered the conformation of the catalytic domain. Therefore, we conclude that RBD is required for both Ras-dependent and independent activation of Raf.
In addition to Ras binding, the inhibitory role of the zinc finger in
Raf activation was uncovered by the ability of the zinc finger mutant
Raf to be potently activated by nocodazole. When C165S/C168S mutant was
expressed in HEK 293T cells, the response of the mutant to TPA and
nocodazole was different. Whereas the activation of the mutant by TPA
was decreased (Fig. 7A,
columns 2 and 5; p < 0.01), nocodazole caused a marked increase in the activity of the
mutant Raf which was 74% greater than the wild type Raf (Fig.
7A, columns 3 and 6;
p < 0.01). Similar results were obtained in COS7 cells
by comparing nocodazole with EGF and TPA. The results again demonstrate
that nocodazole and EGF/TPA activate Raf-1 through different
mechanisms, and the integrity of CRD is necessary for
Ras-dependent, but not for Ras-independent activation of
Raf-1. They also suggest that the zinc finger plays an inhibitory role
in Raf activation.
According to the observation that mutation of the CRD resulted in an
increased response of Raf to nocodazole and while the S338A/339A or
Y340F/Y341F mutation severely impaired Raf activation, we attempted to
explore the interrelationship between these two sites by considering
two possibilities. First, the CRD exerts its inhibitory effect on the
catalytic domain and such inhibition could be counteracted by
phosphorylation of Ser338/Ser339 or
Tyr340/Tyr341. If so, phosphorylation of this
region would not be necessary for the activation of the CRD mutant. To
test this, we made double site mutations, 165S/168S and 338A, or
165S/168S and 340F/341F. The result in Fig. 7B revealed that
the double site mutants were not activated at all, in contrast to the
control, Raf-1 165S/168S, which was greatly stimulated by nocodazole.
This suggests that the outcome of phosphorylating these residues is not
primarily to relieve the inhibition of catalytic function of Raf-1
imposed by CRD and instead is required for the secondary step for Raf activation. Second, the zinc finger might inhibit phosphorylation of
338SSYY341, which is necessary for the next or
final step for Raf activation. To test this hypothesis, in
vitro phosphorylation of Raf-1 variants, wild type, 165S/168S, and
165S/168S/338A was performed by incubation with recombinant Pak1. As
shown in Fig. 8, the wild type Raf was moderately phosphorylated by Pak1, while phosphorylation of the zinc
finger mutant was 40% higher than that of the wild type Raf (phospho-signal/Raf-1 polypeptide determined by scan densitometry, p < 0.05). However, the Raf kinase activity was not
correspondingly altered after phosphorylation in vitro (data
not shown), suggesting that phosphorylation of Ser338 is
not the final step, albeit necessary, for Raf activation.
Recent studies have demonstrated that such small GTPases as Ki-Ras
and Rac associate with microtubules (43, 44), and a novel guanine
nucleotide exchange factor specific for Rac and RhoA has been isolated
and documented to bind to microtubules (45). However, the biological
significance of these associations has yet to be determined. We are the
first to show here that disrupting microtubules activates Pak and hence
leads to an activation of Raf-1. Thus, our studies provide new evidence
for the link between the functionally undefined associations of
microtubules with the small GTPases and Raf/MEK/MAPK pathway as well as
microtubule dynamics. An important and challenging task in this line of
research will be to elucidate the mechanism by which Raf-1 is regulated by microtubule integrity.
The complexity of the interrelationship between microtubules and
intracellular signaling events has been indicated recently (46).
Microtubules can act as a scaffold for relay of signals from cell
surface to nucleus or to other intracellular compartments or
sequestering the bound signaling modules to repress their functions. Conversely, intracellular signaling events also regulate the dynamic balance between polymerization and depolymerization of microtubules. The application of the microtubule-interfering drugs has advanced our
understanding of the role of microtubules in intracellular signaling
events. However, it also raises the concern about the specificity of
these drugs and correlation of their effects with the microtubule
integrity, since they influence so many signaling molecules and some of
these events are redundant. This question will be fully understood only
when the mechanism by which the individual signaling pathway is
regulated by different drugs is elucidated. Nonetheless, existing
evidence indicates that different microtubule-interfering drugs exert
their effects specifically and differently. 1) Paclitaxel, a
microtubule-polymerizing drug, activates JNK/SAPK only in cells when
its binding site in microtubules is intact (47). 2) Vincristine,
another microtubule-depolymerizing drug, and paclitaxel promote
apoptosis of tumor cells by inducing hyperphosphorylation of the
antiapoptotic protein Bcl2, an event mediated by
cAMP-dependent protein kinase (48). In contrast, nocodazole
is not proapoptotic and fails to activate cAMP-dependent protein kinase (Ref. 48; data not shown). 3) Vincristine and paclitaxel
have been shown to stimulate Ras binding to GTP, one of the avenues
leading to activation of JNK/SAPK pathway (47), whereas our current
data indicate that nocodazole activates Raf-1 through a Ras-independent
mechanism. 4) The present results indicate that the phosphorylation of
Akt is not stimulated by nocodazole treatment.
The mechanism by which the zinc finger structure regulates Raf-1
activation is still poorly defined. In addition to binding to processed
Ras (15), the CRD interacts with 14-3-3 (49) and phosphatidylserine
(50). Several recent studies suggest that it imposes an inhibition on
the catalytic domain (49, 51, 52), probably through a intramolecular
interaction (50). Thus, it is rational to propose that Ras-GTP plays
dual role in Raf activation, on the one hand, by recruiting Raf to the
plasma membrane to allow additional modifications, and on the other
hand, by interacting with the Raf RBD and CRD such that the tight
structure of Raf is held open and Raf then becomes accessible to other
regulators such as kinases. It is difficult to dissect these processes
by using mammalian cells, however, mainly for two reasons. First, the
loss-of-function mutation of CRD sequesters Raf in the cytosol by
disrupting its interaction with prenylated Ras, separating it from a
growth factor-regulated kinase in the plasma membrane. As a result, the
activation of Raf is inhibited, even though the mutant Raf-1 might
serve as a better substrate for the kinase. Second, it is almost
impossible to achieve the trans-inhibition of Raf catalytic
domain by the amino-terminal regulatory region when they are both
co-expressed in mammalian cells, as the expression level can not be
manipulated to be as great as that in Xenopus oocytes by
microinjection (51). Our current study employs the approach to disrupt
the microtubule integrity to bypass the requirement of Ras and the
membrane recruitment for Raf activation and demonstrates that the
C165S/C168S mutation does not cause a significant change in the basal
kinase activity, but yields a more robust activation of Raf-1 by
nocodazole (more than 74% increase above the wild type level on
average) (Fig. 7A), suggesting that the CRD inhibits Raf
activation. This argument is strengthened by the finding that the
ability of the Raf to be phosphorylated by Pak was increased by the
zinc finger mutation (Fig. 8).
In the present study, we find that mutation of
84KALK87 to AAAA in RBD inhibits Raf activation
by nocodazole (Fig. 6), similar to its response to EGF (15). Our
results also show that, in contrast to the full-length Raf-1, the
catalytic kinase domain is not activated by nocodazole, suggesting that
the amino-terminal regulatory region is required for Raf activation by
nocodazole. However, overexpression of the amino-terminal regulatory
region does not affect nocodazole activation of Raf-1 as it does the EGF-induced activation, implying a weak interaction between the amino-terminal regulatory region with the nocodazole-induced activator such that the inhibition is effective only when it is overwhelmingly expressed.
We have noticed that our results differ somewhat from previous
publications (35, 36). The authors showed that another Ras binding site
mutant, Raf R89L was activated by fourteen-hour nocodazole treatment
(35), whereas activation of Raf 165S/168S was inhibited (36). Two
factors may account for the discrepancy. First, the difference may be
attributed to nocodazole incubation time. We have observed that Raf-1
activity reaches maximum after brief (1-3 h) exposure to nocodazole
and decreased with extended incubation, followed by
hyperphosphorylation and increased ability to bind to 14-3-3 (38).
Thus, Raf may be regulated differently after prolonged incubation with
nocodazole. Second, it is possible that mutations of Raf
84KALK87 to AAAA and Arg89 to Leu
might have different effects, which can be distinguished by nocodazole,
even though both the mutations impede Ras-dependent activation of Raf-1.
Our study establishes Rac/Cdc42/Pak as an upstream module for Raf-1
activation in association with the integrity of microtubules, but not
for EGF activation of Raf. A study by Frost et al. (32) has
demonstrated that Pak1 can phosphorylate MEK1, thereby enhancing MEK
binding to Raf-1 and consequently its ability to be phosphorylated by
Raf-1. However, dominant negative mutants of Pak and Rac has no effect
on EGF activation of MAPK in their studies, suggesting that Rac and
Pak1 do not operate upstream of MAPK in the EGF pathway. Investigations
by other laboratories suggest that Rac/Cdc42/Pak could serve as an
upstream regulator for Raf under certain circumstances (30, 31,
33-35). The inability of Pak to mediate EGF in activation of Raf
implies that EGF recruits a different kinase to phosphorylate Ser338 of Raf-1 during its activation. In keeping with
this, Mason et al. (27) have shown that mutation of Raf
Tyr341 to Ala does not affect phosphorylation of
Ser338 in response to EGF and TPA, but it abolished the
Ser338 phosphorylation induced by expression of
constitutively active mutants of Ras and Src. This argues that the Raf
Ser338 kinase in response to EGF and TPA is different from
that regulated by constitutively expressed V12Ras and v-Src. The latter
may be Paks. Although many studies including ours indicate that Pak
participates in Raf activation by phosphorylating Ser338,
our results demonstrate that in vitro phosphorylation of Raf Ser338 by Pak1 is not apparently as impressive as the
in vivo study (Figs. 5B and 8; in vivo
data for Pak1 not shown). This difference could be attributed to two
reasons; one is that efficient phosphorylation by Pak may depend on a
scaffold protein, and another one is that the amino-terminal portion of
Raf-1 inhibits its phosphorylation (e.g. by the zinc finger domain).
Although all reports agree that phosphorylation of Ser338
or Tyr341 is a critical step for Raf activation, it is not
the final step, since (1) conversion of Ser338 or
Tyr340-Tyr341 to acidic residues does not
result in full activation of Raf-1, despite an increase in the basal
activity and these mutants can be further activated by V12Ras and v-Src
(24-27), (2) in vitro phosphorylation of Ser338
by Pak does not change Raf kinase activity (data not shown) and (3)
co-expression of active mutant of Pak only leads to about 2-fold
increase of Raf activity although phosphorylation of Ser338
is markedly stimulated (Fig. 5), while Raf activity is elevated by
co-expression of v-Src by more than 20-fold, a level that can only be
achieved by both active mutant of Pak2 and nocodazole treatment (data
not shown).
Regarding the impact of phosphorylating
338SSYY341 on the Raf catalytic function,
Cutler et al. (51) have shown that mutation of Raf
Tyr340 to aspartic acid in the catalytic domain offsets the
trans-inhibitory effect of CRD on its stimulation of
germinal vesicle breakdown when the amino-terminal regulatory domain
and catalytic domain are co-microinjected into Xenopus
oocytes. Thus, it is possible that phosphorylation of
338SSYY341 is to de-repress the catalytic
domain imposed by the CRD. The present results reveal that Raf
165S/168S becomes unresponsive to nocodazole when Ser338 is
mutated to Ala or Tyr340-Tyr341 to Phe-Phe,
implying that phosphorylation of this region is not solely to override
the inhibition of the catalytic domain by CRD, but is rather required
for secondary steps in Raf activation. Therefore, based on our data and
others, we postulate as depicted in Fig.
9 that Raf is activated through the
following sequence: (a) binding of a modulator
(e.g. Ras-GTP at the plasma membrane or X factor in the
cytoplasm in response to microtubule depolymerization) to the
amino-terminal regulatory region changes Raf conformation so to
overcome the inhibition by CRD, which sets the next step in motion;
(b) phosphorylation of the
338SSYY341 region by kinases such as Pak or Src
family provides a prerequisite for additional modifications, that is;
(c) phosphorylation of other sites locks Raf in an active
state.
-12-O-tetradecanoylphorbol-13-acetate and
nocodazole. Finally, an in vitro kinase assay demonstrates
that the zinc finger mutant serves as a better substrate of Pak1 than
the wild type Raf-1. Collectively, our results indicate that 1) the
zinc finger exerts an inhibitory effect on Raf-1 activation, probably
by preventing phosphorylation of 338SSYY341; 2)
such inhibition is first overcome by an unknown factor binding in place
of Ras-GTP to the amino-terminal regulatory region in response to
nocodazole; and 3) EGF and nocodazole utilize different kinases to
phosphorylate Ser338, an event crucial for Raf activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), Pak2 (Pak
), Pak3 (Pak
), and Pak4,
has been implicated in a variety of cellular functions, including
regulation of cell proliferation, apoptosis, the cell cycle, stress
response, oxidant generation, cell adhesion and motility, and
cytoskeletal dynamics. Pak1 participates in V12Ras-induced transformation (30, 31) and cooperates with Raf-1, leading to a maximal
activation of MEK1 by phosphorylating the latter (32). Recent data
reveal that Pak2 can phosphorylate Ser338 (33) and
contribute to Raf-1 activation by V12Ras or a constitutively active
mutant of phosphatidylinositol 3-kinase (34). In addition, integrin-induced activation of Raf-1 has been shown to be mediated by
Pak1 (35).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-12-O-tetradecanoylphorbol-13-acetate (TPA) were
purchased from Sigma. Human recombinant epidermal growth factor
(EGF) was from Calbiochem (San Diego, CA). Glutathione (GSH)-Sepharose
4B was purchased from Amersham Pharmacia Biotech. Monoclonal antibody
against Raf-1 (E10), monoclonal antibody against GST (B14), and
horseradish peroxidase-conjugated second antibodies and protein
A/G-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA).
Monoclonal antibody against Raf-1 phospho-Ser338 were from
Upstate Biotechnology (Lake Placid, NY). Antibodies against
phospho-14-3-3 pan binding sites, phospho-MAPK (Erk1/2) and MAPK, and
Akt phospho-Thr473 and Akt were from New England Biolabs
(Beverly, MA).
-glycerol phosphate, 1 mM dithiothreitol, 1% Nonidet P-40, and protease inhibitors) (40). Cell
debris were removed by centrifugation at 14,000 × g
for 15 min at 4 °C, and protein concentrations in cell lysates were
measured using a Bio-Rad protein assay kit.
-32P]ATP (100 µM, 2000 cpm/pmol), as described previously (40). The
reaction was stopped by the addition of a SDS-PAGE sample buffer, and
the labeled mixture was resolved by 8% SDS-PAGE, transferred to
polyvinylidene difluoride membranes, and visualized by autoradiography. The radiolabeled Erk2 bands were excised and quantified by liquid scintillation counting.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nocodazole stimulates Raf-1
kinase activity in a Ras-independent mechanism. A,
activation of Raf-1 kinase by nocodazole. Wild type (WT)
Raf-1 and kinase-dead mutant Raf K375M (KD) in pMTMyc and
empty vector were transfected into HEK293T cells. After serum
starvation, cells were treated with or without increasing doses of
nocodazole (NZ) for 1 h as indicated or with EGF (20 ng/ml) for 10 min. Raf-1 was immunoprecipitated with anti-Myc antibody
(9E10), and the kinase activity was assayed as described under
"Experimental Procedures." 32P incorporation into Erk2
was quantified by liquid scintillation counting of the Erk bands after
autoradiography, and immunoblotting was carried out using Raf antibody
(E10). Upper part represents results of three
experiments (means ± S.E.) expressed as -fold of the wild type
basal activity, and lower part shows an example
of Raf-1 blots after immunoprecipitation. B, Ras-independent
activation of Raf-1 by nocodazole. Myc-Raf-1 was co-expressed with a
dominant negative mutant of Ras, N17Ras, or amino-terminal regulatory
domain C4, GST-RafC4 in COS7 cells. Cells were treated with or without
nocodazole (300 nM) for 1 h or EGF (20 ng/ml) for 10 min, and Raf kinase was assayed as in A. The
bottom panel shows Myc-Raf-1 immunoblot after
immunoprecipitation. This figure represents one of three independent
experiments.
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Fig. 2.
Rac/Cdc42/Pak participate in
nocodazole-induced activation of Raf-1. A, Pak kinase
is activated by nocodazole (NZ). HEK 293T cells expressing
GST-Pak1 and Myc-Pak2 were treated without or with nocodazole. Pak
activity was analyzed using MBP after Pak purification, as described
under "Experimental Procedures." The reaction mixture was resolved
on 12% SDS-PAGE, transferred to polyvinylidene difluoride membrane,
and determined by autoradiography. The left and
right panels show Pak1 and Pak2 activities and
their immunoblots from crude extracts, respectively. The activity for
MBP phosphorylation (cpm) by Pak1 is as follows: nocodazole, 4332; + nocodazole, 12,917; and by Pak2:
nocodazole, 60,059; + nocodazole,
90,093. B, effect of co-expression of Rac GTPase on the
nocodazole activation of Raf-1. Plasmid encoding Raf-1 or empty plasmid
were co-transfected into HEK293T cells with a constitutively active
mutant of Rac, V12Rac. Cell treatment (nocodazole, 300 nM,
1 h; TPA, 1 µM, 15 min) and the kinase assay were as
in Fig. 1. WT, wild type. C and D,
Cdc42 GTPase and Pak1/2 augment Raf activation by nocodazole.
Transfection and the kinase assay were carried out as in B
except that plasmids encoding V12Cdc42 and Pak1/2 were used for
co-transfection. This figure represents one of three independent
experiments.
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Fig. 3.
Inhibition of nocodazole activation of Raf-1
by the dominant negative mutants of Rac and Pak2. GST-Raf-1 was
co-expressed in COS7 cells with or without dominant negative mutants of
Rac, N17Rac, and Pak2,KD. Following incubation with or without EGF or
nocodazole (NZ), recombinant GST-Raf-1 was purified by GSH
beads and kinase activity was analyzed as Fig. 1. The specific activity
of GST-Raf-1 was determined by the ratio of cpm to densitometric units
of Raf blot (i.e. Erk2 cpm/Raf quantity) and expressed as
percentage of the activity treated with nocodazole. These data
represent one of three independent experiments. WT, wild
type; KD, kinase-dead.
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Fig. 4.
Ser338-Ser339 and
Tyr340-Tyr341 are critical for both
Ras-dependent and independent activation of Raf-1.
A, mutation of Ser338-Ser339 to
Ala-Ala or Tyr340-Tyr341 to Phe-Phe abolishes
Raf-1 activation by nocodazole (NZ). COS7 cells expressing
Raf-1 wild type (WT), 338A/339A, or 340F/341F were treated
or untreated with EGF or nocodazole. Kinase assay and immunoblotting
were performed as in Fig. 1. B, Pak is not able to stimulate
the activity of Raf S338A. cDNAs encoding Myc-tagged Raf-1, wild
type, and Ala338-Ala339 mutant were
co-transfected with GST-Pak1 in HEK293T cells. Lysates from cells
treated with or without nocodazole were used for purification of
Myc-Raf-1 variants. The Raf-1 activity assay was carried out as Fig. 1.
This figure is a representative of two independent experiments.
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Fig. 5.
Phosphorylation of Ser338 is
specifically stimulated by TPA, nocodazole (NZ), or
Pak2. A, endogenous Raf-1 was immunoprecipitated
(IP) with anti-Raf-1 antibody (E10) from HEK293T cells
treated without or with TPA or nocodazole as Fig. 1 and blotted with
anti-phospho-Ser338
( -pS338) antibody and subsequently
reprobed with E10. B, GST-Raf-1 was co-transfected with the
constitutive active mutant of Myc-Pak2 or an empty vector and purified
with GSH beads. The precipitates were divided into three aliquots for
immunoblotting with anti-phospho-Ser338 antibody,
anti-phospho-14-3-3 binding site antibody, and anti-Raf-1 (E10)
antibody, respectively. C, crude cell extracts (20 µg) of
HEK 293T cells treated with or without nocodazole or TPA were blotted
with antibodies against phospho-Akt Ser473 and Akt, and
phospho-MAPK/Erk and MAPK/Erk, respectively.
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Fig. 6.
The amino-terminal regulatory region
is required for nocodazole (NZ) activation of
Raf-1. A, nocodazole activates the full-length but not
the carboxyl kinase domain of Raf-1. Full-length and the kinase domain
of Raf-1 were expressed as GST fusion proteins in HEK293T cells and
purified with GSH-beads. Kinase activity was assayed after cells were
incubated with EGF or nocodazole as in Fig. 1. The
bottom panel shows Raf immunoblot with crude
extracts. B, mutation of Ras-binding domain abolishes
nocodazole activation of Raf-1. HEK293T cells expressing Myc-Raf
variants, wild type (WT), and the Ras binding site mutant
84-87AAAA, were stimulated with or without TPA or nocodazole. The Raf
immunoprecipitates were assayed for the kinase activity and
immunoblotted with E10. C, mutation of the Ras binding
domain abrogates the effect of Pak1 on nocodazole activation of Raf-1.
Raf kinase activity was assayed from HEK293T cells co-expressing
Myc-Raf-1 or Myc-Raf 84-87AAAA with wild type GST-Pak1. After
autoradiography, Raf-1 was blotted with E10. D,
myristoylated Raf mutant 84-87AAAA is able to be activated by
nocodazole. Myr-HA-Raf (84-87AAAA) in pBJMFPK3E was transfected into
HEK293T cells. Recombinant Raf was immunoprecipitated with anti-HA
antibody (12CA5) and assayed for the kinase activity after cell
treatment as indicated. Relative levels of Raf-1 in the
immunoprecipitates were assessed by anti-HA blot. Raf activity
(top) was expressed as in Fig. 3. This figure represents one
of three independent experiments.
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Fig. 7.
Role of the zinc finger in Raf-1
activation. A, the zinc finger mutant responds to TPA
and nocodazole differently. Wild type (WT) Raf and the zinc
finger mutant Raf 165S/168S were transiently expressed in HEK 293T
cells. Cell extracts containing equal amount of the Raf polypeptide
were used for immunoprecipitation and kinase assay. Raf specific
activity was expressed as percentage of the wild type stimulated with
nocodazole (NZ). Results represent means ± S.E. of
five independent experiments. B, mutation of
Ser338-Ser339 to Ala-Ala or
Tyr340-Tyr341 to Phe-Phe prevents activation of
Raf 165S/168S by nocodazole. cDNAs encoding Raf mutants, 165S/168S,
165S/168S-338A/339A, and 165S/168S-340F/341F, were transfected into
HEK293T cells. Raf kinase activity was assayed after
immunoprecipitation of recombinant Rafs from cells treated with TPA or
nocodazole or untreated. These data represent one of three independent
experiments.
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Fig. 8.
In vitro phosphorylation of Raf-1
by Pak1. Raf variants, wild type (WT), 165S/168S, and
165S/168S-338A/339A, were expressed in HEK293 cells and
immunoprecipitated with anti-Myc antibody. In vitro
phosphorylation of Raf-1 in the presence or absence of recombinant
GST-Pak1, constitutively active mutant (CA) or kinase-dead
mutant (KD) was conducted as described under "Experimental
Procedures" and visualized by Western blotting with
anti-phospho-Ser338 antibody. The activity was expressed as
percentage of specific phosphorylation of the wild type Raf-1 (ratio of
densitometric units for phospho-signal/Raf-1 polypeptide).
Error bars are mean ± S.E. of a triplicate
experiment. Lower panel shows the purification of
GST-Pak1 from HEK293T cells and Pak activity (autophosphorylation).
Similar results have been reproduced twice.
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EXPERIMENTAL PROCEDURES
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REFERENCES
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Fig. 9.
Model for the mechanism of Raf
activation. The diagram may not necessarily be
topologically accurate. HSP, heat shock proteins.
In summary, a key event in mitogenic signaling is the activation of
Raf-1. This is a complex process that requires participation of
Ras-GTP, 14-3-3, and kinases (such as Pak, Src, protein kinase C, KSR,
and/or yet unidentified kinases) that directly phosphorylate Raf (3).
However, it is still not clear how these factors act in concert to
activate Raf-1. The present study has employed nocodazole and EGF/TPA
to evaluate the importance of the structural elements, the RBD, the
CRD, and 338SSYY341, in
Ras-dependent and independent activation of Raf-1. We find the RBD to be necessary for both Ras-dependent and
independent activation of Raf-1. In contrast, mutation of the CRD
results in nocodazole-stimulated Raf activity that was significantly
higher than that of the wild type Raf-1, but the same mutation
diminishes Ras-dependent activation of Raf-1. The results
indicate that the zinc finger plays an inhibitory role in Raf
activation, at least in part through inhibition of phosphorylation of
Ser338. Our study also defines Rac/Cdc42/Pak as an upstream
module for Raf-1 in response to microtubule depolymerization.
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ACKNOWLEDGEMENTS |
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We thank Drs. W. Xiong, M. S. Marshall, and G. M. Bokoch for plasmids. We also thank Cynthia Hayne for helpful discussions and suggestions during this work.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 57959 (to Z. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Diabetes and Metabolism Research Unit, Endocrinology Section, Evans Dept. of Medicine, Boston University School of Medicine, 650 Albany St., Rm. 820, Boston, MA 02118. Tel.: 617-414-1033; Fax: 617-638-7094; E-mail: zluo@medicine.bu.edu.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M100152200
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ABBREVIATIONS |
---|
The abbreviations used are:
RBD, Ras binding
domain;
CRD, cysteine-rich zinc finger domain;
MAPK, mitogen-activated
protein kinase;
Erk, extracellular regulated kinase;
MEK, mitogen-activated protein kinase and
extracellular regulated kinase kinase;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
EGF, epidermal growth factor;
TPA, 4-12-O-tetradecanoylphorbol-13-acetate;
Pak, p21-activated kinase;
MBP, myelin basic protein;
HA, hemagglutinin;
aa, amino acid(s).
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