From the Department of Neurobiology,
Pharmacology and Physiology, ** Committee on Cancer
Biology, and § Ben May Institute for Cancer Research,
University of Chicago, Chicago, Illinois 60637 and
Lilly Research Laboratories, DC1453,
Indianapolis, Indiana 46285
Received for publication, September 30, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Protein kinase C (PKC) regulates
activation of the Raf-1 signaling cascade by growth factors, but the
mechanism by which this occurs has not been elucidated. Here we report
that one mechanism involves dissociation of Raf kinase inhibitory
protein (RKIP) from Raf-1. Classic and atypical but not novel PKC
isoforms phosphorylate RKIP at serine 153 (Ser-153). RKIP
Ser-153 phosphorylation by PKC either in vitro or in
response to
12-O-tetradecanoylphorbol-13-acetate or
epidermal growth factor causes release of RKIP from Raf-1, whereas
mutant RKIP (S153V or S153E) remains bound. Increased expression of PKC
can rescue inhibition of the mitogen-activated protein (MAP) kinase
signaling cascade by wild-type but not mutant S153V RKIP. Taken
together, these results constitute the first model showing how
phosphorylation by PKC relieves a key inhibitor of the Raf/MAP kinase
signaling cascade and may represent a general mechanism for the
regulation of MAP kinase pathways.
The MAP1 kinase cascade,
an evolutionarily conserved signaling module, stimulates numerous
biological processes including growth and differentiation. The known
elements of the pathway include a MAP kinase kinase kinase that
phosphorylates and activates a MAP kinase kinase, which, in turn,
phosphorylates the threonine-X-tyrosine (TXY) activation domain of MAP kinase (reviewed in
Ref. 1). The first characterized subfamily of MAP kinases, termed
extracellular signal-regulated kinases (ERKs), is activated by growth
factors and other stimuli via a cascade involving Ras, Raf-1 kinase,
and MEK/ERK kinase (MEK). Activation of MAP kinase is under
exquisite regulatory control, particularly at the level of Raf-1
activation. The N-terminal regulatory domain of Raf-1 interacts with
Ras leading to dephosphorylation at negative regulatory sites,
conformational changes to expose the kinase domain, and subsequent
phosphorylation at activating sites such as serine 338 (Ser-338) and
tyrosine 341 (Tyr-341) (reviewed in Ref. 2). A variety of studies have shown that protein kinase C (PKC) isozymes are also capable of activating Raf-1 (3-5) and/or the downstream MEK (6), but the
mechanism has not been elucidated.
The PKC family of serine/threonine kinases are key mediators of several
physiological processes including growth, death, differentiation, and
transformation (reviewed in Ref. 7). There are three major classes of
PKCs that are distinguished by their physiological activators. The
classical PKCs ( Multiple hypotheses have been proposed to explain how PKCs activate the
ERK cascade, including direct phosphorylation of either MEK (12) or
Raf-1. Although the phosphorylation of MEK by PKCs is controversial
(6), Raf-1 is phosphorylated by PKCs at multiple sites. For example,
PKC Recent studies from our laboratory suggest that ERK activation
is mediated by specific PKC isoforms in response to different growth
factors (10, 11). In both the conditionally immortalized hippocampal
cell line H19-7 (15) and primary E16 rat hippocampal cells, two
different PKC isoforms, PKC The Raf-1 kinase inhibitor protein (RKIP), was identified recently (17)
by yeast two-hybrid cloning utilizing the kinase domain of Raf-1 as
bait. RKIP was found to be a member of the phosphatidylethanolamine-binding protein (PEBP) family, a ubiquitously expressed protein with homologues in Arabidopsis
thaliana, Saccharomyces cerevisiae,
Caenorhabditis elegans, and Drosophila
melanogaster that display high degrees of interspecies sequence
similarity (18). There are at least three RKIP-like PEBPs in rat, two
in mice, and one in human (19). Also, amino acids 2-12 of PEBP are
identical to the hippocampal cholinergic neurostimulatory peptide
(HCNP) that stimulates acetylcholine synthesis in rat septal nuclei
(20). PEBPs are distinct from other known proteins, and their function
has remained largely enigmatic. In addition to binding phospholipids,
PEBPs bind nucleotides and opioids and were shown recently (21) to
inhibit thrombin. A role for PEBP in signaling was demonstrated when it
was shown that RKIP binds to Raf-1. RKIP itself is neither a kinase nor
a substrate for Raf-1 or MEK (17) but has been reported to be a
specific inhibitor of MEK binding to Raf-1 (22), suppressing both
Raf-1-induced transformation and AP-1-dependent transcription.
In the present study we demonstrate that one mechanism by which PKCs
activate Raf-1 signaling to ERK involves the loss of inhibition by
RKIP. Although both phorbol esters and PKCs have been well established
as activators of MAP kinases, the targets for PKCs in this pathway are
surprisingly unknown. Because phorbol esters are tumor promoters, and
several components of the MAP kinase pathway are mutated in human
cancers, the mechanism of PKC-mediated Raf/MAP kinase activation is of
paramount importance. These results provide an explanation of how PKCs
can physiologically regulate Raf-1 signaling.
Plasmid Construction--
All constructs were made by the
PCR using pCMV-HA-RKIP as template (a gift from K. Yeung, Brown
University). The forward (F) and reverse (R) primers were as follows:
F, 5'-atg gcc gcc gac atc agc cag tgg-3'; R, 5'-ctt ccc agc cag ctg atc
gtg cag-3'. For the pCR-HA-RKIP and FLAG-RKIP constructs the forward
primers were as follows: F-HA, 5'-ggc tcc atc atg tac cca tat gac gtt cca gac tac gct gcc gcc gac atc agc cag tgg-3'; F-FLAG, 5'-ggc tcc atc
atg gac tac aag gac gac gac gac aag gcc gcc gac atc agc cag tgg-3'. The
reverse primer (R-stop) was 5'-cta ctt ccc agc cag ctg atc gtg-3'. The
PCR products were ligated into the pCR3.1 vector (Invitrogen) and
sequenced in both directions to confirm fidelity of the reactions. To
make pGEX-RKIP, the open reading frame of RKIP was excised from
pCR-RKIP by EcoRI digestion and ligated into
EcoRI-digested pGEX-2T (Amersham Biosciences).
Site-directed mutagenesis of Ser-153 to valine was done utilizing
pGEX-RKIP as template and the unique site elimination (U.S.E.)
kit (Amersham Biosciences). The following mutagenic primer was used:
5'-gta ctt ctt tcg aaa gac ctc cac ctt gaa ctt-3'. This primer binds to
the negative strand of pGEX-RKIP and introduces a silent mutation resulting in the creation of a unique BstBI site and
conversion of Ser-153 to valine. The NarI/NheI
selection primer was from Amersham Biosciences. The mutation was
confirmed by sequencing in both directions. The Ser-153 to glutamic
acid (S153E) mutagenesis was done utilizing pGEX-RKIP as template and
the QuickChange kit (Stratagene). The mutagenic primer was as follows:
5'-ggc aag ttc aag gtg gag gag ttt cga aag aag-3'. This primer
introduces a silent mutation, resulting in the creation of a unique
BstBI site and conversion of Ser-153 to glutamic acid. The
mutation was confirmed by sequencing in both directions. The HA- and
FLAG-tagged RKIP(S153V) and RKIP(S153E) plasmids were constructed by
PCR utilizing the appropriate pGEX-RKIP plasmid as template. The F-HA
and F-FLAG forward primers were used, along with the R-stop reverse
primer, and ligated into the pCR3.1 vector to make pCR-HA- and
FLAG-RKIP(S153V) and -RKIP(S153E).
In Vitro PKC Kinase Assays--
1 µg of GST-RKIP or
GST-RKIP(S153V) was combined with 200 ng of baculovirus-derived PKC
(Panvera) in 50 µl of kinase buffer. For PKCs Cell Culture--
The immortalized H19-7 cells were generated
from embryonic rat hippocampal cells and grown as described previously
(15). H19-7 cells were maintained in 10% fetal bovine serum, 50 units/ml penicillin/50 µg/ml streptomycin, and 200 µg/ml G418 at
33 °C. Cells were serum-starved at 39 °C in N2 medium or DMEM
overnight prior to treatment. COS-7 cells were grown in DMEM with 10%
fetal bovine serum and 50 units/ml penicillin/50 µg/ml streptomycin.
In Vitro HA-ERK2 Activation Assays--
H19-7 cells were
transfected with 10 µg of DNA per 10-cm plate using TransIT-LTI
(Panvera). DNA in all transfections was added to equal amounts by
addition of empty vector where necessary. 24 h following
transfection, cells were starved overnight followed by treatment with
800 nM
12-O-tetradecanoylphorbol-13-acetate (TPA) or 100 ng/ml EGF. HA-ERK2 kinase assays were performed in vitro as
described previously (11) with either GST-Elk-1 or myelin basic protein
(MBP) as substrates. The amount of HA-ERK2 in each sample was
determined by immunoblot analysis with an anti-HA monoclonal antibody
(3F10; Roche Molecular Biochemicals). Relative kinase activity
was measured with a phosphorimager and normalized to the amount of
HA-ERK2 in each sample. For down-regulation of endogenous RKIP, H19-7
cells were transfected with empty vector, HA-ERK2, or HA-ERK2 plus the
RKIP antisense construct AS-C143 and pHACT (17). 24 h
later, cells were starved overnight, and activation of HA-ERK2 was
assayed as above. In some experiments, ERK activation was determined by
immunoblotting with anti-phospho-MAP kinase antibodies. Samples were
quantified by digital analysis (Alpha Innotech).
In Vitro GST Binding Assays--
In vitro binding
assays were carried out as described (23) with modifications.
Glutathione-Sepharose 4B was blocked with 10% normal goat serum
followed by incubation with 2 µg of GST or GST-RKIP. Coupled GST
fusion proteins were then incubated with 100 ng of PKC Raf and RKIP Co-immunoprecipitations--
Co-immunoprecipitation
of Raf and RKIP were carried out in a modification of a procedure
described previously (17). To observe association, immunoprecipitations
were done with similarly mild conditions of sonication in
phosphate-buffered saline without detergent. 7 µg of Myc-Raf-1
and 3 µg of HA-RKIP or HA-RKIP(S153V) were transfected into COS-7
cells. 24 h later, cells were starved overnight and subsequently
treated as indicated in the figures. Cells were lysed by sonication in
cold PBS plus protease inhibitors and cleared by centrifugation. To
immunoprecipitate myc-Raf-1, 1 µg of anti-Myc monoclonal
antibody (9E10; Upstate Biotechnology) was added to 250 µg of cell
lysate proteins and rotated at 4 °C followed by addition of 25 µl
of protein G-Sepharose (1:1 slurry). The complex was washed three times
with cold PBS and boiled in 3× PAGE sample buffer. The proteins were
separated in 12.5% polyacrylamide gels, transferred to nitrocellulose,
and immunoblotted with anti-HA antibodies (12CA5-HRP; Roche Molecular
Biochemicals) to detect HA-RKIP. The blot was then stripped and
re-probed with anti-Raf-1 antibodies (Santa Cruz Biotechnology, Inc.)
to document amounts of Myc-Raf-1 in all samples.
Antibody Production--
Anti-RKIP antisera were made by
immunization of rabbits with purified GST-RKIP ( Endogenous RKIP Is a Physiological Inhibitor of EGF- and
TPA-induced ERK Activation--
To determine whether RKIP is able to
inhibit ERK activation by EGF or TPA in H19-7 cells, exogenous
RKIP was introduced into cells. Plasmids expressing HA-ERK2 and
FLAG-RKIP were co-transfected into H19-7 cells, and activation of
HA-ERK upon cell stimulation was measured by an in vitro
kinase assay using Elk-1 as a substrate. As shown in Fig.
1A, overexpression of RKIP is
able to block HA-ERK2 activation by EGF and TPA. These results indicate
that RKIP is capable of inhibiting the activation of ERK by these
stimuli. To determine whether endogenous RKIP normally regulates ERK
activity, an antisense RKIP construct, pAS-C143, that was shown
previously (17) to suppress endogenous RKIP, was co-transfected, along with HA-ERK2, into H19-7 cells, and HA-ERK2 activation was assayed before and after EGF or TPA stimulation (Fig. 1B). The
ability of the antisense RKIP to selectively suppress RKIP expression in H19-7 cells was confirmed by co-transfection of RKIP and antisense RKIP (data not shown). Expression of the antisense RKIP construct significantly increased both EGF- and TPA-stimulated HA-ERK2 activity, suggesting that RKIP physiologically regulates signaling to MAP kinase
triggered by EGF, as well as other activators of protein kinase C, in
H19-7 cells.
PKCs Are RKIP Kinases--
Having established that RKIP regulates
the EGF and TPA stimulation of ERK (see Fig. 1A) and
previously demonstrated a requirement for PKC
Because PKC Phosphorylation of RKIP by PKC Causes Release of RKIP from Raf-1
and Increased ERK Activation--
Because PKC
To test the effect of PKC on the interaction of RKIP with full-length
Raf-1 in cells, Myc-Raf-1 was co-transfected into COS-7 cells with
HA-RKIP or HA-RKIP(S153V), and the association of the two molecules was
determined before and after TPA stimulation. In agreement with results
published previously (22), we observed that transfected Myc-Raf-1 and
HA-RKIP could be co-immunoprecipitated from starved COS-7 cells and
that activation of PKC by TPA alone induced the release of HA-RKIP from
Myc-Raf-1 (Fig. 2B). Analysis of the cell lysates by
immunoblotting indicated that both TPA-treated and untreated cells
contain comparable levels of HA-RKIP (data not shown). Treatment of
H19-7 cells with physiological PKC activators such as EGF also induced
dissociation of endogenous RKIP from endogenous c-Raf-1 by 59%, as
shown using an horseradish peroxidase-tagged anti-RKIP antibody for
immunoblotting to eliminate background from light chain antibody (Fig.
2C). If the mechanism for RKIP dissociation from Raf-1
involves phosphorylation of RKIP by PKC, then the mutant RKIP(S153V)
should remain bound to Raf-1 independent of PKC activation. Consistent
with this prediction, HA-RKIP(S153V) co-immunoprecipitated with
myc-Raf-1 in both resting and TPA-treated COS-7 cells (Fig.
2B). Similar results were obtained when GST-Raf rather than
myc-Raf was co-expressed with RKIP, and the complexes were isolated
using glutathione beads (data not shown). These results indicate that
PKC can regulate RKIP binding to Raf-1 kinase in vivo by
phosphorylating RKIP at residue Ser-153.
Endogenous RKIP Is Phosphorylated at Ser-153 in Vivo--
Because
the above phosphorylation studies used exogenous RKIP, we determined
whether endogenous cellular RKIP is phosphorylated similarly at Ser-153
in response to PKC activation. Therefore, we generated antibodies
against phosphorylated Ser-153 RKIP (
Because phorbol esters are the most robust and specific activators of
PKCs, we initially examined the state of RKIP Ser-153 phosphorylation
in cells following TPA stimulation utilizing the
To determine whether the physiological activator EGF can induce
phosphorylation of RKIP by PKC, two approaches were utilized. In one
set of experiments, H19-7 cells were transfected with FLAG-tagged RKIP
or RKIP(S153V). Following treatment of cells with 100 ng/ml nM EGF for 2-15 min, FLAG-RKIP was isolated from
cell lysates using an anti-FLAG affinity column and eluted with FLAG
peptide. After separation by SDS-PAGE, the induction of phosphorylated RKIP was determined by immunoblotting with the Glutamate Does Not Act as a Phosphomimetic When Substituted at
Residue Ser-153--
Because the crystal structure of bovine PEBP
(RKIP) has been solved to a resolution of 1.84 Å (26), we used
molecular modeling to explore the relationship of Ser-153 to the
functional domains of RKIP. Purified RKIP crystallizes as a dimer (26),
and the monomer is shown in Fig.
4A, with the evolutionarily
conserved phosphatidylethanolamine binding (PEB) domain (residues
64-86) colored in green, and the PKC phosphorylation site
(Ser-153) colored in blue. The PEB domain consists of
hydrophobic residues that form a
Because glutamic acid residues can mimic phosphoserine residues under
some circumstances, we determined whether a mutant RKIP with glutamic
acid substituted at residue 153 (S153E) would still bind to Raf-1. In
fact, GST-RKIP(S153E) bound to the Raf-1 kinase domain (CBP-CR3) at
least as well as GST-RKIP or GST-RKIP(S153V) in vitro (Fig.
4B), suggesting that steric hindrance by the phosphate group
rather than the negative charge is responsible for the inability of
phospho-RKIP to bind. To determine whether an RKIP mutant with glutamic
acid replacing Ser-153 might also promote binding to full-length Raf-1
in vivo, we transfected COS-7 cells with a vector expressing HA-RKIP, HA-RKIP(S153E), or HA-RKIP(S153V). The use of
highly transfectable COS-7 cells enabled us to titrate the amount of
transfected RKIP cDNA to maximize inhibition. Consistent with the
in vitro binding results, HA-RKIP(S153E) was as effective in
blocking EGF-induced ERK activation as HA-RKIP or HA-RKIP(S153V) (Fig.
4, C and D). Similar results were observed with
H19-7 cells (data not shown). Taken together, these data demonstrate
that Ser-153 is situated on the surface of RKIP and modulates RKIP binding to Raf-1.
PKC Phosphorylation Rescues Inhibition of ERK by RKIP but Not by
the S153V RKIP Mutant--
If PKC phosphorylation of Ser-153 causes
the physical release of RKIP from Raf-1, then enhanced expression and
activation of PKC should be sufficient to overcome the RKIP-mediated
inhibition of ERK in cells. However, if the site of PKC phosphorylation
is removed, then no significant rescue by PKC should be observed. To
test this hypothesis, FLAG-RKIP or FLAG-RKIP(S153V) were co-expressed with HA-ERK2 in H19-7 cells, and activation of HA-ERK2 following EGF
stimulation was assayed by in vitro kinase assays.
Expression of the mutant FLAG-RKIP, as well as the wild-type FLAG-RKIP,
blocks EGF-induced HA-ERK2 activation. Co-transfection of FLAG-PKC Model for Regulation of RKIP by PKC--
The results described
here can be summarized in a relatively simple model that explains how
PKC Protein kinase C is a key activator of the Raf/MAP kinase cascade,
but the mechanisms by which it promotes Raf-1 signaling either directly
or in response to growth factor stimulation have not been clear. Here
we demonstrate that RKIP inhibits MAP kinase activation in response to
growth factors or PKC activators in neuronal cells and that PKC can
regulate Raf-1 signaling through phosphorylation of RKIP. Classical and
atypical PKCs phosphorylate RKIP on a serine residue, Ser-153, which
results in the displacement of RKIP from Raf-1. This phosphorylation of
RKIP at Ser-153 has been observed in vivo in response to
both TPA and EGF. A mutant RKIP that has Ser-153 mutated to valine
continues to associate with Raf-1 following PKC stimulation. Taken
together, these results indicate that PKC can regulate MAP kinase
activation under physiological conditions by phosphorylating RKIP and
provide a more general model for the regulation of MAP kinase cascades
by inhibitory factors.
The fact that RKIP reportedly associates not only with Raf-1 but also
weakly with MEK and possibly ERK (17) raises the possibility that RKIP
may play a role as a scaffolding protein. However, it should be pointed
out that the extremely mild methods for cell disruption required to
isolate complexes of RKIP and Raf-1 is not direct evidence for a
scaffold function, because the interactions in vivo,
particularly with MEK or ERK, could be indirect. Other scaffold
proteins for the MAP kinases such as kinase suppressor of Ras (KSR) and
JNK-interacting protein (JIP) have been reported to be
inhibitors or potentiators dependent upon whether they act to sequester
or bring together different components of the MAP kinase cascade
(28-31). To date, RKIP has been characterized as a competitive
inhibitor of the substrate MEK. Our results showing that
phosphorylation of RKIP by the activator PKC causes release from Raf-1
and up-regulation of ERK are also therefore consistent with a role for
RKIP as an inhibitor of the Raf-1 signaling cascade. However, we cannot
rule out the possibility that RKIP will also be found to function as an
activator under other circumstances.
Although our results implicate RKIP as a key regulator of ERK
activation by PKCs, removal of RKIP is not sufficient for ERK activation, indicating that other events must also occur. Activation of
Raf-1 by growth factors requires interaction with Ras (reviewed in Ref.
2). In several cell types, expression of a dominant negative
N17Ras does not block PKC activation of ERK. However, Marshall
and co-workers (32) demonstrated that PKC activation of Raf-1 is
Ras-dependent. They further showed that a mutant of Raf-1
that cannot associate with Ras (R89L) can be activated by TPA if it is
membrane-bound via a CAAX box, indicating that the primary
role of Ras is to translocate Raf-1 to the membrane. Similarly, targeting the R89LRaf-1 to the membrane overcomes the inability of tyrosine kinases to activate Raf (33, 34). The association of Raf-1
and Ras in response to TPA requires GTP-activated Ras, but the
mechanism by which PKC activates Ras is not clear. Taken together with
the results presented here, these data suggest that PKC activates Raf-1
signaling by at least two discrete events, one involving transport to
the membrane via Ras, and the second involving the dissociation of
RKIP.
Although the activation of ERK by EGF and TPA is comparable, the
phosphorylation of RKIP by TPA is significantly higher and of longer
duration. As shown previously (11), addition of exogenous PKC The fact that substitution of glutamic acid for Ser-153 did not mimic
the phosphorylated residue is not surprising in light of the crystal
structure. These results are analogous to those seen with the Ser-259
negative regulatory site of Raf-1. This residue is phosphorylated by
protein kinase B/Akt (35) and correlates with Raf-1 inactivation.
Mutation of this site to a negatively charged amino acid, however, does
not mimic the effects of phosphorylation. Instead, the physical
presence of the phosphate group is required, suggesting that such sites
are involved sterically in protein-protein interactions (2). The
modulation of Raf-1 binding to RKIP by substitution of phosphoserine or
glutamate for Ser-153 indicates that this region of RKIP influences the
binding interaction, possibly by directly participating as another
docking site within the binding pocket.
RKIP is a member of a larger family of PEBPs. In addition to the
RKIP-like PEBPs, there is also one more distant family with orthologs
in humans and mice (19). Whether all members of the PEBP family act in
a similar fashion to regulate kinases is unclear. Interestingly, the
residue corresponding to RKIP Ser-153 in PEBP2, another isoform of RKIP
(GenBankTM accession number AF226629) is an alanine,
suggesting a different mode of regulation. Although the structure and
key residues of the PEB binding domain of RKIP are conserved from
bacteria to mammals, the loop containing Ser-153 is more variable,
consistent with a role in specificity. The most variable region among
the different PEBPs is the N terminus. Interestingly, residues 2-12 of
RKIP correspond to the HCNP (36). Recent studies have suggested that
HCNP may play important role in the development and/or differentiation of the human hippocampus (37), and some learning and memory deficiencies in the hippocampus have been linked to unusually high
levels of either the HCNP peptide or RKIP mRNA (reviewed in Ref.
38). These and other studies suggest that RKIP is a multifunctional
protein, and it is possible that RKIP in either its unphosphorylated or
phosphorylated state may have other roles within the cell.
The regulation of Raf signaling by RKIP and PKC occurs at a key step in
growth factor signaling cascades. Because amplification of the signal
occurs between Raf-1 and MEK (1), it makes sense that this point in the
cascade would be a key target for regulation. Although two different
stimuli might commonly activate Ras and thus relieve inhibition by the
negative regulatory domain of Raf-1, the actual amplification of the
signal in the case of RKIP-bound Raf-1 could be determined by the
extent of PKC activation. Thus, this mechanism enables selective
modulation of the signal. Evidence for activation of other MAP kinase
cascades by classic or atypical PKCs such as the I
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
) require both Ca2+ and diacylglycerol (DAG) for activation whereas the
novel PKCs (
,
,
, and
) are Ca2+-independent
but still require DAG. Both of these classes of PKCs are activated by
phorbol esters that mimic the DAG stimulus. In contrast, the atypical
PKCs,
and
/
, are Ca2+-, DAG-, and phorbol
ester-independent. Not only are PKCs able to activate Raf-1, but in a
number of cell systems they are required for the activation of ERKs by
growth factors (8-11).
phosphorylates Raf-1 at serine 499 (13), but mutation of this
residue did not impede activation of Raf-1 by the physiological
stimulators Ras and Lck. Similarly, both v-Src and phorbol esters were
able to activate Raf-1 even though the PKC phosphorylation sites at
serine 497 and serine 499 were mutated to alanine (14). Thus, although
some PKC phosphorylation sites on Raf-1 have been identified, these
sites do not appear to be required for activation of Raf-1.
and PKC
, mediate ERK activation by
epidermal growth factor (EGF) and fibroblast growth factor,
respectively (10, 11). Both PKCs are required for activation of ERKs at
a step upstream of MEK and either downstream or at the level of Raf-1.
Although these studies suggested that PKC
activates Raf-1 in
response to EGF, phosphorylation of neither Raf-1 nor MEK appeared to
be responsible (3, 6, 16) (data not shown). Furthermore, phosphatase
inhibitors such as okadaic acid did not alleviate the requirement for
PKC (data not shown). We therefore considered the possibility that
Raf-1 regulatory proteins might be potential targets of PKC.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
I,
II, and
the buffer used was 20 mM HEPES, pH 7.4, 100 µM CaCl2, 10 mM
MgCl2, 100 µg/ml L-
-phosphatidylserine, and 20 µg/ml diacylglycerol. The buffer used for novel PKCs
,
,
and
was 25 mM Tris, pH 7.5, 5 mM
MgCl2, 0.5 mM EGTA, 200 µg/ml
L-
-phosphatidylserine, and 20 µg/ml diacylglycerol.
The buffer for PKC
was 25 mM Tris, pH 7.5, 5 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol, and 100 µg/ml
L-
-phosphatidylserine. Samples were aliquoted from
premixed cocktails to ensure that the amounts of CBP-CR3 or
PKC
were the same in different tubes. The reactions were started by
the addition of 5 µCi of [
-32P]ATP, carried out at
30 °C for 30 min, and then stopped by the addition of 6× sample
buffer and heating at 100 °C for 3 min. The samples were loaded onto
a 10% acrylamide gel, transferred to nitrocellulose, and exposed to film.
with 10%
bovine serum albumin as nonspecific competitor for 1 h at 4 °C
followed by extensive washing in TENNS buffer (2.5 mM Tris,
pH 7.4, 2.5 mM EDTA, 250 mM NaCl, 1% Nonidet
P-40, 2.5% sucrose). Bound proteins were resolved in a 12.5%
acrylamide gel, transferred to nitrocellulose, and immunoblotted with
anti-PKC
antibodies (Santa Cruz Biotechnology, Inc.). For in
vitro CBP-CR3 binding assays, GST fusion proteins were coupled to
glutathione-Sepharose 4B as above. 600 ng of bacterially expressed
Raf-1 kinase domain (CBP-CR3) was added and incubated at 4 °C for
2 h. After extensive washing in TENNS buffer, 100 ng of PKC
was
added, and kinase assays were carried out as above. Bound proteins were
resolved in a 12.5% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with anti-Raf-1 antibodies.
-RKIP).
Anti-phospho-Ser-153-RKIP antisera (
-pSer-153-RKIP) were made by
immunization of rabbits with a peptide (United Biochemicals)
corresponding to residues 146-157 of the rat RKIP sequence conjugated
to ovalbumin, NH-RGKFKVES*FRKK-COOH, where the S* indicates a
phosphoserine residue.
-pSer-153-RKIP was affinity-purified by
passing the crude antisera through a column containing the immunogenic
peptide followed by protein A-Sepharose chromatography for concentration.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Endogenous RKIP is a specific inhibitor of
EGF-induced ERK activation and a substrate for PKCs. A,
overexpression of RKIP blocks ERK activation. H19-7 cells were
transfected with 1 µg of HA-ERK2 plus 4 µg of empty vector or
FLAG-RKIP. 24 h later the cells were switched to 39 °C, starved
overnight in N2 medium, and either left untreated (CTRL) or
stimulated with 100 ng/ml EGF or 800 nM TPA for 5 min.
HA-ERK2 was immunoprecipitated and assayed as described under
"Experimental Procedures." The amount of GST-Elk phosphorylation
was determined by phosphorimaging. The amount of HA-ERK2 in each sample
was determined by immunoblot analysis (anti-HA, 3F10) and quantified by
digital analysis. The graph shown is a plot of mean data ± S.D.
from four independent experiments. B, endogenous RKIP is a
specific inhibitor of EGF-induced ERK activation. H19-7 cells were
transfected with 1 µg of HA-ERK2 plus 4 µg of empty vector or 3 µg of pAS-C143 and 1 µg of pHACT (17). 24 h later the cells
were switched to 39 °C and starved overnight in N2 medium followed
by stimulation with 100 ng/ml EGF or 800 nM TPA for 5 min.
HA-ERK2 was immunoprecipitated and assayed as described under
"Experimental Procedures." The amount of GST-Elk phosphorylation
was determined by phosphorimaging. The amount of HA-ERK2 in each sample was determined by immunoblot analysis (anti-HA, 3F10) and
quantified by digital analysis. The graph shown is a plot of mean
data ± S.D. from four independent experiments. C, PKCs
phosphorylate RKIP Ser-153. 1 µg of GST-RKIP or GST-RKIP(S153V) was
incubated with vehicle (none) or the indicated recombinant
PKC, and kinase assays were carried out as described under
"Experimental Procedures." The amount of GST protein in each sample
was determined by stripping the membrane and re-probing with an -GST
antibody. The results shown are representative of five independent
experiments. D, RKIP binds to PKC
in vitro.
GST or GST-RKIP was coupled to glutathione-Sepharose and incubated with
100 ng of recombinant PKC
or recombinant PKC
pre-incubated with 1 µg of competing
-PKC
antibody (Upstate Biotechnology) as
described under "Experimental Procedures." Following extensive
washing, bound proteins were resolved by 12.5% SDS-PAGE, and PKC
was detected by immunoblot analysis. The amount of GST protein in each
sample was determined by probing with an
-GST antibody (Upstate
Biotechnology). The results shown are representative of three
independent experiments.
in EGF-stimulated ERK
activation, we investigated whether RKIP and PKCs could interact
directly. An analysis of the RKIP protein sequence for PKC consensus
phosphorylation sites (24) identified Ser-153 of the rat RKIP sequence
as a potential target. This site was mutated to a non-phosphorylatable
valine residue (S153V), and the mutant RKIP was cloned into a bacterial
expression vector to produce recombinant GST-RKIP(S153V). Purified
GST-RKIP and GST-RKIP(S153V) were then used as substrates for PKC in
in vitro kinase assays. As shown in Fig.
1C, RKIP is a substrate for PKCs
,
I,
II,
, and
, and substitution of Ser-153 prevents most of this
phosphorylation. Overexposure of the blot reveals some additional
phosphorylation by PKCs
and
, but the amount is low compared
with the phosphorylation of Ser-153. These data demonstrate that
Ser-153 is the major site of RKIP phosphorylation by PKC.
Interestingly, the novel PKCs, including, PKCs
,
, and
, are
not RKIP kinases in vitro, suggesting that RKIP
phosphorylation is not a feature of all PKC isozymes.
is able to phosphorylate RKIP, we determined whether
PKC
and RKIP could associate in vitro and in
vivo. We focused on the PKC
isoform based on our previous work
(11) indicating that PKC
mediates Raf-1 activation by EGF in H19-7
cells; however, because classical PKCs can phosphorylate RKIP in
vitro, we expect that they would also associate physically and
mediate the action of TPA, as well as specific growth factors dependent
upon the particular cell type. As shown in Fig. 1D,
recombinant PKC
binds to GST-RKIP but not GST. These results
indicate that RKIP and PKC
are capable of interacting specifically.
However, no stable association was detected in vivo (data
not shown), consistent with an enzyme-substrate interaction.
potentiates Raf
activity (11), we determined whether direct phosphorylation of RKIP by
PKC
could result in the release of RKIP from Raf-1. To test this
possibility in vitro, GST, GST-RKIP, or GST-RKIP(S153V) were
pre-bound to glutathione-Sepharose beads and incubated with a
bacterially expressed Raf-1 kinase domain (CBP-CR3) (25), the region of
Raf that binds to RKIP. The Raf-1 kinase domain bound to both wild-type
RKIP and RKIP(S153V) (Fig.
2A). Addition of purified
PKC
and ATP led to the release of the Raf-1 kinase domain from
GST-RKIP but not from GST-RKIP(S153V), demonstrating that
phosphorylation of Ser-153 on RKIP by PKC
causes dissociation of the
Raf-1 kinase domain and RKIP.
View larger version (35K):
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Fig. 2.
RKIP Ser-153 mediates Raf-1 binding.
A, PKC phosphorylation of RKIP Ser-153 mediates Raf-1
release in vitro. GST, GST-RKIP, or GST-RKIP(S153V) was
coupled to glutathione-Sepharose and incubated with 600 ng of
bacterially expressed Raf-1 kinase domain (CBP-CR3).
Following extensive washing, 100 ng of recombinant PKC
with or
without cold ATP was added, and kinase assays were carried out as
described under "Experimental Procedures." Further washings were
carried out, bound proteins were separated by 12.5% SDS-PAGE, and
Raf-1 was detected by immunoblot analysis. The amount of GST protein in
each sample was determined by stripping the membrane and re-probing
with an
-GST antibody. The results shown are representative of three
independent experiments. B, phosphorylation of RKIP Ser-153
mediates Raf-1 release in vivo. COS-7 cells were transfected
with 10 µg of either empty vector or HA-RKIP, HA-RKIP S153V, or
Myc-Raf, alone or in combination. At 24 h post-transfection the
cells were serum-starved in DMEM for 16 h. Cells were then treated
with 800 nM TPA for 30 min as indicated. Myc-Raf-1 was
immunoprecipitated, and the bound proteins were separated by 12.5%
SDS-PAGE. Myc-Raf-1 and HA-RKIP were detected by immunoblot analysis.
The results shown are representative of at least four independent
experiments. C, EGF causes release of endogenous RKIP from
Raf. H19-7 cells were starved in DMEM for 16 h and then treated
with 100 ng/ml EGF as indicated. Cells were lysed by sonication in PBS.
Raf-1 was immunoprecipitated from 1 mg of cell lysate protein with 1 µg of anti-Raf-1, and immunoprecipitated proteins were separated by
12.5% SDS-PAGE, along with 20 µg cell lysate (WCE) and 1 µg of anti-Raf-1 antibody alone as a control (IgG). RKIP
was detected by Western blotting with an horseradish
peroxidase-conjugated anti-RKIP, and Raf-1 levels were assessed by
immunoblotting for Raf-1.
-pSer-153-RKIP) by immunizing
rabbits with a 12-amino acid peptide containing residues 146-157 of
the rat RKIP sequence with a phosphoserine at position 153 and
purifying the antibody via peptide affinity chromatography. To test the
specificity of the antibody, GST-RKIP and GST-RKIP(S153V) were
initially incubated with recombinant PKC and ATP to phosphorylate RKIP.
The products of this reaction were then resolved by SDS-PAGE, along
with GST, unphosphorylated GST-RKIP, GST-RKIP(S153V), and
GST-RKIP(S153E), a mutant of GST-RKIP with glutamic acid substituted at
residue 153 to mimic phosphorylation at this site. Immunoblot analysis
with the purified
-pSer-153-RKIP antibody strongly detected a single
48-kDa band corresponding to GST-RKIP that was phosphorylated by PKC
(Fig. 3A). In contrast, the
antibody did not recognize unphosphorylated GST-RKIP, GST, GST-RKIP(S153V), or GST-RKIP(S153E). These data demonstrate that the
purified antibody specifically detects RKIP phosphorylated at
Ser-153.
View larger version (22K):
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Fig. 3.
TPA and EGF induce
phosphorylation of RKIP Ser-153 in vivo.
A, the anti-pSer-153-RKIP antibody specifically detects RKIP
phosphorylated by PKC. 1 µg of GST, GST-RKIP, GST-RKIP(S153V), or
GST-RKIP(S153E) was incubated alone or with 100 ng of recombinant
PKC , and kinase assays were carried out with cold ATP as described
under "Experimental Procedures." Proteins were separated by 10%
SDS-PAGE, and pSer-153-RKIP was detected by immunoblot analysis
(
-pSer-153-RKIP). The amount of GST protein in each sample was
determined by stripping the membrane and re-probing with an
-GST
antibody. The results shown are representative of three independent
experiments. B, TPA induces RKIP Ser-153 phosphorylation
in vivo. H19-7 cells were starved overnight in DMEM at
39 °C and then stimulated with 800 nM TPA for the
indicated times. In one sample, the cells were pre-incubated with
bisindolylmaleimide I (BIM; Calbiochem) for 30 min prior to
stimulation. 100 µg of protein lysates were separated by 12.5%
SDS-PAGE and transferred to nitrocellulose, and the membrane was probed
with anti-pSer-153-RKIP. The membrane was then stripped and re-probed
with anti-RKIP. The results shown are representative of three
independent experiments. C, EGF induces RKIP phosphorylation
in vivo. H19-7 cells were transfected with 10 µg of
FLAG-RKIP. After 24 h, cells were serum-starved in DMEM overnight.
Cells were then treated with 100 ng/ml EGF for 2-15 min as indicated.
Cells were lysed, and FLAG-RKIP was immunoprecipitated using anti-FLAG
(M2; Sigma) antibody. Immunoprecipitated proteins were eluted using 100 µg/ml FLAG peptide, resolved by SDS-PAGE, and blotted for
anti-phospho-Ser-153-RKIP and anti-FLAG-RKIP. D, H19-7 cells
starved in DMEM overnight at 39 °C. Triplicate plates were treated
with 100 ng/ml EGF for 2 min or not (CTRL). The cells were
lysed, and 10 or 20 µg of protein from each lysate were loaded on
12.5% polyacrylamide gels, transferred to membrane, and probed for
pRKIP (
-PRKIP Ser-153). The membranes were then stripped and probed
for
-tubulin (Santa Cruz Biotechnology, Inc.) or RKIP (
GST-RKIP)
to normalize for protein loading. Samples were quantified by digital
analysis. These data are representative of five independent
experiments.
-pSer-153-RKIP
antibody. Western blot analysis of H19-7 cell lysates revealed minimal
pSer-153-RKIP in starved cells (Fig. 3B). Stimulation with
TPA resulted in the induction of a 23-kDa band corresponding in size to
RKIP. Pre-incubation of cells with the PKC inhibitor
bisindolylmaleimide I blocked Ser-153 phosphorylation, demonstrating
that PKC is indeed mediating this phosphorylation. Inclusion of the
immunizing peptide in the buffer inhibited antibody recognition,
verifying the specificity of the recognition site (data not shown).
This data demonstrates that RKIP is phosphorylated on Ser-153 in
vivo in a PKC-dependent manner.
-pSer-153-RKIP antibody. As shown in Fig. 3C, EGF stimulated
phosphorylation of RKIP, and the peak of phosphorylation occurred at 2 min. This time course corresponds to those of Raf and ERK activation,
which also peak at 2 min in response to EGF in H19-7 cells (data not shown). In an alternative approach involving endogenous RKIP, H19-7
cells were incubated with 100 ng/ml EGF for 2 min. pSer-153-RKIP was
measured by immunoblotting cell lysates with
-pSer-153-RKIP antibody
and then normalizing the results to tubulin. As shown in Fig.
3D, EGF treatment caused an increase in phospho-RKIP of ~2-fold in H19-7 cells. These results indicate that RKIP is
phosphorylated at Ser-153 in response to cell stimulation by the
physiological activator EGF.
-sheet and mediate the high
affinity binding of membrane phosphatidylethanolamine (26).
Interestingly, Ser-153 lies on the surface of RKIP in a loop domain
that has more variability than other conserved regions of RKIP. To
address the structural consequences of phosphorylation, we modeled the
RKIP structure with a phosphorylated Ser-153 residue (Fig.
4A). The added phosphate group strikingly extends into the
potential Raf-1 binding pocket, supporting our data that
phosphorylation at this site mediates the release of Raf-1 from RKIP.
Substitution of a glutamic residue at this site (S153E) results in a
slightly larger group than the original serine residue, but one that is
not as bulky as the phosphoserine (Fig. 4A).
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Fig. 4.
Substitution of glutamate for serine at
residue Ser-153 does not mimic phosphorylation of Ser-153.
A, the RKIP crystal structure. The PEB domain is
shown in green, and Ser-153 is in blue. Either
glutamic acid (RKIP(S153E)) or a phosphate group
(RKIP-pSer-153) was substituted for Ser-153 by using the
Swiss-PDB viewer in conjunction with POV-RAY software. B,
RKIP(S153E) is not a phosphomimetic. GST, GST-RKIP, GST-RKIP(S153V), or
GST-RKIP(S153E) was coupled to glutathione-Sepharose and incubated with
1 µg of bacterially expressed Raf-1 kinase domain (CBP-CR3).
Following extensive washing, bound proteins were separated by 12.5%
SDS-PAGE, and Raf-1 was detected by immunoblot analysis. The amount of
GST protein in each sample was determined by stripping the membrane and
re-probing with an anti-GST antibody. The results shown are
represent-ative of three independent experiments. C,
titration of the ability of RKIP to block ERK activation. COS-7 cells were
transfected with 1.5 µg of HA-ERK2 and no HA-RKIP
(Control) or HA-RKIP at the indicated ratios. 24 h
later the cells were starved overnight by washing once in PBS and
switching the medium to DMEM. The cells were treated with 100 ng/ml EGF
for 3 min. HA-ERK2 was immunoprecipitated and assayed as described
under "Experimental Procedures." The amount of HA-ERK2 in each
sample was determined by immunoblot analysis (anti-HA, 3F10 antibody),
and quantified by digital analysis. The results shown are
representative of four independent experiments. D, COS-7
cells were transfected with 1.5 µg of HA-ERK2 and 7.5 µg of empty
vector (Control) or HA-RKIP, HA-RKIP(S153V), or
HA-RKIP(S153E). 24 h later the cells were starved overnight by
washing once in PBS and switching the medium to DMEM. The cells were
treated with 100 ng/ml EGF for 3 min. HA-ERK2 was immunoprecipitated
and assayed as described under "Experimental Procedures." The
amount of HA-ERK2 in each sample was determined by immunoblot analysis
(anti-HA, 3F10 antibody) and quantified by digital analysis. The
results shown are representative of four independent experiments.
is able to overcome this inhibition in cells expressing
FLAG-RKIP but not mutant FLAG-RKIP(S153V), indicating that PKC can
mediate activation of ERK in vivo by phosphorylation of RKIP
(Fig. 5). Similar results were obtained
using COS-7 cells, and similar levels of PKC
were detected in cell
lysates (data not shown). These results demonstrate that Raf-1
signaling to ERK, as well as its physical association with RKIP, is
regulated by phosphorylation of RKIP at Ser-153.
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Fig. 5.
PKC phosphorylation rescues inhibition of ERK
by RKIP but not by the S153V RKIP mutant. H19-7 cells were
transfected with 10 µg of empty vector, 2 µg of HA-ERK2 plus 8 µg
of empty vector, 4 µg of empty vector and 4 µg of FLAG-RKIP or
FLAG-RKIP(S153V), or 4 µg of FLAG-PKC and 4 µg of FLAG-RKIP or
FLAG-RKIP(S153V). 24 h later, the cells were switched to 39 °C,
starved overnight in N2 medium, and either left untreated or stimulated
with 10 ng/ml EGF for 5 min. HA-ERK2 was immunoprecipitated and assayed
as described under "Experimental Procedures." The amount of HA-ERK2
in each sample was determined by immunoblot analysis, as shown in the
lower panel. The results shown are representative of four
independent experiments.
can activate the Raf-1 signaling cascade in response to EGF
(Fig. 6). Dephosphorylation of Raf-1 at
residue Ser-259 upon association with EGF-activated Ras results in
release of 14-3-3 (2). PKC
can be recruited to Raf-1 by binding to
14-3-3 and subsequently released from Raf-1 by phosphorylating 14-3-3 (27). Our results suggest that PKC
can also associate with RKIP and
phosphorylate residue Ser-153 on RKIP, causing RKIP to be released from
Raf-1. However, if a S153V or S153E mutant is expressed, then RKIP
inhibition would not be overridden by growth factor signaling via PKC.
Because RKIP prevents MEK phosphorylation, release of RKIP from Raf-1
also enhances downstream signaling to ERKs.
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Fig. 6.
A scheme depicting regulation of Raf-1
signaling by RKIP and PKC.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
increases ERK activation by EGF suggesting that the EGF
receptor-associated PKC
is rate-limiting. It is likely that the
phosphorylation of RKIP by EGF involves a small subset of the cellular
RKIP by PKC
that is in close proximity to the EGF receptor and
subject to rapid dephosphorylation. ERK activation is limited by the
amount of Raf-1 activated in response to EGF. In contrast, TPA would be
able to phosphorylate significantly more RKIP by activating both
classical and novel PKCs that are widely distributed in the cells;
therefore, it is not surprising that TPA is a more potent inducer of
RKIP phosphorylation.
B kinase
(IKK) cascade (39-41), combined with recent evidence that RKIP
can regulate the IKK cascade (42), suggests that a similar regulatory
mechanism might exist for other MAP kinases either involving RKIP or
other inhibitory proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Anning Lin, Mitchell Villereal, Aaron Fox, and Wei-Jen Tang for helpful discussions, Jane Booker for assistance with the manuscript, Matthew Clark and Suzana Gomes for expert technical assistance, Kam Yeung for pCMV-HA-RKIP and pAS-RKIP constructs, and Xiaojing Yang for assistance with structure modeling.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Pharmacological Sciences Training Grant T32GM07151 (to K. C. C.), Cancer Biology Training Grant 5T32CA09594 (to N. T.), National Institutes of Health Grant NS38846 (to M. R. R.), and a gift from the Cornelius Crane Trust for Eczema Research (to M. R. R.).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.
¶ Both authors contributed equally.
Current address: Stanford University, Stanford, CA 94305.
§§ To whom correspondence should be addressed: Ben May Cancer Inst. for Cancer Research, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 773-702-0380; Fax: 773-702-4634; E-mail: m-rosner@uchicago.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210015200
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ABBREVIATIONS |
---|
The abbreviations used are: MAP, mitogen-activated protein; RKIP, Raf kinase inhibitory protein; PEBP, phosphatidylethanolamine-binding protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; PKC, protein kinase C; DAG, diacylglycerol; EGF, epidermal growth factor; HCNP, hippocampal cholinergic neurostimulatory peptide; HA, hemagglutinin; F, forward; R, reverse; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; TPA, tetradecanoylphorbol-13-acetate; PBS, phosphate-buffered saline; pSer, phosphoserine; PEB, phosphatidylethanolamine binding; CBP, calmodulin binding protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Pearson, G.,
Robinson, F.,
Beers Gibson, T.,
Xu, B.,
Karandikar, M.,
Berman, K.,
and Cobb, M. H.
(2001)
Endocr. Rev.
22,
153-183 |
2. | Kolch, W. (2000) Biochem. J. 351, 289-305[CrossRef][Medline] [Order article via Infotrieve] |
3. | Sözeri, O., Voller, K., Liyanage, M., Frith, D., Kour, G., Mark, G. E., III, and Stabel, S. (1992) Oncogene 7, 2259-2262[Medline] [Order article via Infotrieve] |
4. | Cacace, A. M., Ueffing, M., Philipp, A., Han, E. K., Kolch, W., and Weinstein, I. B. (1996) Oncogene 13, 2517-2526[Medline] [Order article via Infotrieve] |
5. |
Ueda, Y.,
Hirai, S.,
Osada, S.,
Suzuki, A.,
Mizuno, K.,
and Ohno, S.
(1996)
J. Biol. Chem.
271,
23512-23519 |
6. |
Schonwasser, D. C.,
Marais, R. M.,
Marshall, C. J.,
and Parker, P. J.
(1998)
Mol. Cell. Biol.
18,
790-798 |
7. | Nishizuka, Y. (1992) Science 258, 607-614[Medline] [Order article via Infotrieve] |
8. | Berra, E., Diaz-Meco, M. T., Dominguez, I., Municio, M. M., Sanz, L., Lozano, J., Chapkin, R. S., and Moscat, J. (1993) Cell 74, 555-563[Medline] [Order article via Infotrieve] |
9. | Cai, H., Smola, U., Wixler, V., Eisenmann-Tappe, I., Diaz-Meco, M. T., Moscat, J., Rapp, U., and Cooper, G. M. (1997) Mol. Cell. Biol. 17, 732-741[Abstract] |
10. |
Corbit, K. C.,
Foster, D. A.,
and Rosner, M. R.
(1999)
Mol. Cell. Biol.
19,
4209-4218 |
11. |
Corbit, K. C.,
Soh, J. W.,
Yoshida, K.,
Eves, E. M.,
Weinstein, I. B.,
and Rosner, M. R.
(2000)
Mol. Cell. Biol.
20,
5392-5403 |
12. |
Monick, M. M.,
Carter, A. B.,
Flaherty, D. M.,
Peterson, M. W.,
and Hunninghake, G. W.
(2000)
J. Immunol.
165,
4632-4639 |
13. | Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenceller, G., Marne, D., and Rapp, U. (1993) Nature 364, 249-251[CrossRef][Medline] [Order article via Infotrieve] |
14. | Yip-Schneider, M. T., Miao, W., Lin, A., Barnard, D. S., Tzivion, G., and Marshall, M. S. (2000) Biochem. J. 351, 151-159[CrossRef][Medline] [Order article via Infotrieve] |
15. | Eves, E. M., Tucker, M. S., Roback, J. D., Downen, M., Rosner, M. R., and Wainer, B. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4373-4377[Abstract] |
16. | Kieser, A., Seitz, T., Adler, H. S., Coffer, P., Kremmer, E., Crespo, P., Gutkind, J. S., Henderson, D. W., Mushinski, J. F., Kolch, W., and Mischak, H. (1996) Genes Dev. 10, 1455-1466[Abstract] |
17. | Yeung, K., Seitz, T., Li, S., Janosch, P., McFerran, B., Kaiser, C., Fee, F., Katsanakis, K. D., Rose, D. W., Mischak, H., Sedivy, J. M., and Kolch, W. (1999) Nature 401, 173-177[CrossRef][Medline] [Order article via Infotrieve] |
18. | Serre, L., Pereira de Jesus, K., Zelwer, C., Bureaud, N., Schoentgen, F., and Benedetti, H. (2001) J. Mol. Biol. 310, 617-634[CrossRef][Medline] [Order article via Infotrieve] |
19. | Simister, P. C., Banfield, M. J., and Brady, R. L. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 1077-1080[CrossRef][Medline] [Order article via Infotrieve] |
20. | Ojika, K., Kojima, S., Ueki, Y., Fukushima, N., Hayashi, K., and Yamamoto, M. (1992) Brain Res. 572, 164-171[Medline] [Order article via Infotrieve] |
21. |
Hengst, U.,
Albrecht, H.,
Hess, D.,
and Monard, D.
(2001)
J. Biol. Chem.
276,
535-540 |
22. |
Yeung, K.,
Janosch, P.,
McFerran, B.,
Rose, D. W.,
Mischak, H.,
Sedivy, J. M.,
and Kolch, W.
(2000)
Mol. Cell. Biol.
20,
3079-3085 |
23. | Takayama, S., Sato, T., Krajewski, S., Kochel, K., Irie, S., Millan, J. A., and Reed, J. C. (1995) Cell 80, 279-284[Medline] [Order article via Infotrieve] |
24. |
Nishikawa, K.,
Toker, A.,
Johannes, F. J.,
Songyang, Z.,
and Cantley, L. C.
(1997)
J. Biol. Chem.
272,
952-960 |
25. | King, A. J., Sun, H., Diaz, B., Barnard, D., Miao, W., Bagrodia, S., and Marshall, M. S. (1998) Nature 396, 180-183[CrossRef][Medline] [Order article via Infotrieve] |
26. | Serre, L., Vallee, B., Bureaud, N., Schoentgen, F., and Zelwer, C. (1998) Structure 6, 1255-1265[Medline] [Order article via Infotrieve] |
27. | van Dijk, M. C., Hilkmann, H., and van Blitterswijk, W. J. (1997) Biochem. J. 325, 303-307[Medline] [Order article via Infotrieve] |
28. | Yu, W., Fantl, W. J., Harrowe, G., and Williams, L. T. (1998) Curr. Biol. 8, 56-64[Medline] [Order article via Infotrieve] |
29. |
Morrison, D. K.
(2001)
J. Cell Sci.
114,
1609-1612 |
30. |
Dickens, M.,
Rogers, J. S.,
Cavanagh, J.,
Raitano, A.,
Xia, Z.,
Halpern, J. R.,
Greenberg, M. E.,
Sawyers, C. L.,
and Davis, R. J.
(1997)
Science
277,
693-696 |
31. |
Yasuda, J.,
Whitmarsh, A. J.,
Cavanagh, J.,
Sharma, M.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
7245-7254 |
32. |
Marais, R.,
Light, Y.,
Mason, C.,
Paterson, H.,
Olson, M. F.,
and Marshall, C. J.
(1998)
Science
280,
109-112 |
33. | Marais, R., Light, Y., Paterson, H. F., and Marshall, C. J. (1995) EMBO J. 14, 3136-3145[Abstract] |
34. | Stokoe, D., Macdonald, S. G., Cadwallader, K., Symons, M., and Hancock, J. F. (1994) Science 264, 1463-1467[Medline] [Order article via Infotrieve] |
35. |
Zimmermann, S.,
and Moelling, K.
(1999)
Science
286,
1741-1744 |
36. | Tohdoh, N., Tojo, S., Agui, H., and Ojika, K. (1995) Brain Res. Mol. Brain Res. 30, 381-384[CrossRef][Medline] [Order article via Infotrieve] |
37. | Yuasa, H., Ojika, K., Mitake, S., Katada, E., Matsukawa, N., Otsuka, Y., Fujimori, O., and Hirano, A. (2001) Brain Res. Dev. Brain Res. 127, 1-7[Medline] [Order article via Infotrieve] |
38. | Ojika, K., Mitake, S., Tohdoh, N., Appel, S. H., Otsuka, Y., Katada, E., and Matsukawa, N. (2000) Prog. Neurobiol. 60, 37-83[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Lallena, M. J.,
Diaz-Meco, M. T.,
Bren, G.,
Paya, C. V.,
and Moscat, J.
(1999)
Mol. Cell. Biol.
19,
2180-2188 |
40. | Vertegaal, A. C., Kuiperij, H. B., Yamaoka, S., Courtois, G., van der Eb, A. J., and Zantema, A. (2000) Cell. Signal. 12, 759-768[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Khoshnan, A.,
Bae, D.,
Tindell, C. A.,
and Nel, A. E.
(2000)
J. Immunol.
165,
6933-6940 |
42. |
Yeung, K. C.,
Rose, D. W.,
Dhillon, A. S.,
Yaros, D.,
Gustafsson, M.,
Chatterjee, D.,
McFerran, B.,
Wyche, J.,
Kolch, W.,
and Sedivy, J. M.
(2001)
Mol. Cell. Biol.
21,
7207-7217 |