From the The protein kinase KSR-1 is a recently identified
participant in the Ras signaling pathway. The subcellular localization
of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To
identify the mechanism that mediates KSR-1 translocation, we performed
a yeast two-hybrid screen. Three clones that interacted with KSR-1 were
found to encode the full-length The Ras signaling pathway affects many aspects of cell physiology,
including cell growth, proliferation, movement, and differentiation (1). Recently, KSR-11 was
identified as a component of the Ras signaling cascade by genetic
screens in Drosophila melanogaster and Caenorhabditis elegans (2-4). Inactivating mutations in the
ksr-1 gene blocked the phenotypic effects of activated Ras
in these animals, suggesting that KSR-1 is a positive regulator of
Ras-mediated signaling. Genetic epistasis experiments in
Drosophila demonstrated that KSR-1 acts downstream of Ras
but upstream of or parallel to Raf (4).
Mammalian forms of KSR-1 have been identified on the basis of sequence
homology (4), but the role of mammalian KSR-1 in Ras-mediated signaling
is controversial. Overexpression of KSR-1 in Xenopus oocytes
was found by two groups (including ours) to weakly promote MAP kinase
activation (5-7). In one study (6), overexpression of KSR-1 in
cultured mammalian cells was found to promote MAP kinase activation; it
was shown to inhibit Ras-mediated signaling at the level of MEK
activation in several studies (8-10), and it was found to inhibit
Ras-mediated signaling at the level of transcription factor (Elk-1)
activation (11). In the absence of loss-of-function studies in
mammalian cells, the definitive role of KSR-1 in Ras-mediated signaling
remains unclear.
Both invertebrate and mammalian forms of KSR-1 consist of a putative
amino-terminal regulatory portion and a carboxyl-terminal serine/threonine kinase domain. Five functional domains of KSR-1 have
been identified, including a unique amino-terminal CA1 domain, a
proline-rich CA2 domain, a cysteine-rich zinc finger-like CA3 domain, a
serine/threonine rich CA4 domain, and the amino-terminal protein kinase
CA5 domain (4). KSR-1 is most homologous to Raf-1 kinase, but there is
no evidence that KSR-1 can bind to Ras or phosphorylate MEK. Indeed,
the in vivo substrate(s) of the kinase domain of KSR-1 is
unknown (7, 11).
One model of KSR-1 action purports that it is a molecular scaffold that
behaves like the budding yeast protein ste5, functionally linking the
protein kinases ste11, ste7, and fus3/kss1 (6). Indeed, KSR-1 has been
shown to interact with 14-3-3 protein, Raf-1, MEK, and MAP kinase in
coimmunoprecipitation experiments and yeast two-hybrid assays (5-10).
It is not clear, however, whether KSR-1 links these associated proteins
to promote signal transduction. Mutational analysis of KSR-1 has
revealed that separable domains bind to distinct signaling proteins.
For example, in vitro binding assays have demonstrated that
the CA4 domain of KSR-1 interacts with MAP kinase (12). Furthermore,
yeast two-hybrid assays have shown that the CA5 domain of KSR-1 binds
to MEK (8, 9).
The subcellular localization of KSR-1 is dependent on the activation
state of cells. We previously demonstrated that KSR-1 is a cytoplasmic
protein in serum-starved cells, but that KSR-1 translocates to the
plasma membrane after stimulation with serum (5). The time course of
this translocation is similar to that observed for Raf-1 kinase, which
binds to activated Ras at the plasma membrane. Work by Michaud et
al. (7) has established that the cysteine-rich CA3 domain of KSR-1
is essential for translocation to the plasma membrane. One explanation
for this observation is that KSR-1 accompanies Raf-1 to the plasma
membrane, but inactive Raf-1 does not bind to KSR-1 (5). Another
possibility is that KSR-1 directly binds to a constitutively
membrane-bound target after serum stimulation. To further explore this
possibility, we performed a yeast two-hybrid screen using KSR-1 as bait.
Yeast Two-hybrid Screen--
A cDNA encoding the CA2 through
CA5 domains (amino acids 195 to 873) of murine KSR-1 (mKSR-1) was
inserted into the vector pAS1-CYH (gift of Stephen Elledge, Baylor
University, Houston, Texas) as an in-frame fusion with the
transactivation domain of GAL4 (pAS1/CA2-5) as described previously
(12). A human HeLa cell cDNA two-hybrid library (gift of David
Beach, Cold Spring Harbor Laboratory, New York) was screened and
pAS1/CA2-5 was used as the bait. A yeast strain (Y190) was
cotransfected with pAS1/CA2-5 and the HeLa cell library and yeast were
plated onto medium lacking histidine, tryptophan, and leucine (13).
Colonies that grew in the absence of histidine were assayed for
mKSR-1 Deletion Analysis--
The CA2 domain of mKSR1 (residues
234-300), and the CA3 domain (residues 303-397) were inserted into
pAS1-CYH as in-frame fusions with the DNA binding domain of GAL4
(residues 1-147) (13). The two-hybrid construct containing the CA5
domain (residues 541-873) of mKSR1 was a gift of Wendy Fantl (Chiron
Corporation, Emeryville, CA). Human Mammmalian Expression Constructs--
The full-length
mKSR-1 cDNA (gift of Marc Therrien and Gerald Rubin, University of
California, Berkeley) was subcloned into the pTarget (Promega)
mammalian expression vector (4). The human wild-type Antibodies--
The rabbit polyclonal anti- In Vitro Association Experiments--
cDNAs encoding the CA1
and CA2 (amino acids 1-311), CA3 (amino acids 312-392), CA4 (amino
acids 384-519), or CA5 (amino acids 520-873) domains of mKSR-1 were
inserted in-frame into pGEX-4T (Amersham Pharmacia Biotech) that
contained the coding sequence for glutathione S-transferase
(GST). GST fusion proteins were produced in Escherichia coli
and were immobilized on glutathione beads (Sigma). NIH/3T3 fibroblasts
were lysed in Nonidet P-40 lysis buffer (0.5% Nonidet P-40, 137 mM NaCl, 50 mM NaF, 10 mM Tris, pH
7.5, 3 mM dithiothreitol, 6 mM
MgCl2, 2 mM phenylmethylsulfonyl fluoride, 0.2 units/ml aprotinin, 25 µM leupeptin). Lysates were cleared by low speed centrifugation (12,000 × g for 5 min) and then added to immobilized GST fusion protein. Protein lysate
derived from approximately 106 cells was added to 0.5 µg
of immobilized GST fusion protein. Beads were washed three times
with lysis buffer, and adherent proteins were boiled in gel sample
buffer. Proteins were separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) and electrophoretically transferred to
nitrocellulose filters. Filters were blocked in 3% nonfat dry milk and
3% bovine serum albumin in TBS/T (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20). After incubation in primary
antibody, bound antibody was visualized with
alkaline-phosphatase-conjugated secondary antibody and color-developing
agents (Promega).
Cell Culture and Transfections--
COS7 cells were
maintained in Dulbecco's modified Eagle's medium with 10% fetal calf
serum. Cells were split 24 h before transfection with the
indicated cDNAs. All transfections were carried out with the use of
LipofectAMINE Plus reagent (Life Technologies, Inc.) in serum-free
medium (OptiMEM, Life Technologies, Inc.).
Coimmunoprecipitation Assays--
For triple transfection
experiments, 1-3 × 106 COS7 cells in 100-mm culture
plates were transfected with the cDNAs encoding Subcellular Fractionation--
Two days before fractionation,
COS7 cells in 100-mm culture plates were transfected with the cDNAs
encoding mKSR-1 (2.5 µg) or mKSR-1 (2.5 µg) and N17 Ha-Ras (2.5 µg) (as described above). Transfected cells were allowed to grow for
24 h and were then serum starved (Dulbecco's modified Eagle's
medium with 1% bovine serum albumin) overnight with or without added
pertussis toxin (PTX) (Life Technologies, Inc., 200 ng/ml). After serum
starvation, some COS7 cells were stimulated with 10 µM
lysophosphatidic acid (LPA) or 10% FCS for 10 min at 37 °C. Cells
were washed two times with phosphate-buffered saline, scraped into
detergent-free lysis buffer (137 mM NaCl, 50 mM
NaF, 6 mM MgCl2, 10 mM Tris, pH
7.5, 2 mM phenylmethylsulfonyl fluoride, 2 µg/ml
leupeptin, 4 µg/ml aprotinin, and 1 mM
Na3VO4), and sonicated to disrupt cell
membranes. Lysates were cleared by low speed centrifugation
(12,000 × g for 5 min), cleared lysates were separated
by high speed centrifugation (100,000 × g for 1 h) (16, 17). Supernatants were reserved, and pellets were washed twice
in detergent-free lysis buffer and resuspended in an equal volume of
lysis buffer with 1% Triton X-100. Fractions were resolved by use of
SDS-PAGE and immunoblotting as above.
MAP Kinase Assays--
Two days before their use, COS7 cells in
6-well dishes (Falcon) were transfected with cDNAs encoding
We performed a yeast two-hybrid screen using a cDNA that
encoded the CA2 through CA5 domains of mKSR-1 (CA2-5) as bait with a
HeLa cell two-hybrid library. Three positive clones were found to
encode the In living cells, Center for Cardiovascular Research,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES
10 subunit of heterotrimeric G-proteins. KSR-1 also interacted with
2
and
3 in a two-hybrid assay. Deletion analysis
demonstrated that the isolated CA3 domain of KSR-1, which contains a
cysteine-rich zinc finger-like domain, interacted with
subunits.
Coimmunoprecipitation experiments demonstrated that KSR-1 bound to
1
3 subunits when all three were
transfected into cultured cells. Lysophosphatidic acid treatment of
cells induced KSR-1 translocation to the plasma membrane from the
cytoplasm that was blocked by administration of pertussis toxin but not
by dominant-negative Ras. Finally, transfection of wild-type KSR-1
inhibited
1
3-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least
in part, by an interaction with
and that this interaction may
modulate mitogen-activated protein kinase signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
-galactosidase activity by use of X-gal (5-bromo-4-chloro-3-indolyl
-D-galactopyranoside) as a substrate. Yeast that
contained only the cDNA library plasmid were mated with yeast
strain Y189 containing either pAS1/CA2-5 or pAS1/lamin, grown on
selective medium, and re-assayed for
-galactosidase activity. Clones
that specifically interacted with CA2-5 were sequenced and BLAST
searches were performed (National Center for Biotechnology Information).
10 was used as an
in-frame fusion with the transactivation domain of GAL4 (residues
768-881) in the vector pGAD GH (13). Human
2 and
3 were used as in-frame fusions with the transactivation domains of GAL4 in the vector pACTII (14). Yeast strain Y190 was
cotransformed with
subunits and the mKSR-1 mutants or lamin. Colonies were assessed for
-galactosidase activity by use of X-gal
as a substrate (13).
1
and
3 cDNAs were subcloned into the mammalian expression vector pCB6+ (14). A 5'-FLAG-tagged version of mKSR-1 was
obtained by use of polymerase chain reaction and was subcloned directly
into pTarget (Promega) and sequenced. The N17 Ras cDNA was a gift
from Dwight Towler (Washington University, St. Louis, MO).
1
subunit antibody (BN-1) has been previously described (15). The rabbit
polyclonal anti-pan-
subunit, the goat polyclonal anti-KSR-1, and
the rabbit polyclonal anti-ERK1 antibodies were obtained from Santa
Cruz Biotechnology. The alkaline phosphatase-conjugated rabbit
anti-goat IgG secondary antibody was obtained from Zymed
Laboratories Inc. The alkaline phosphatase-conjugated goat
anti-rabbit IgG secondary antibody was obtained from Santa Cruz Biotechnology.
1
(2.5 µg),
3 (2.5 µg), and mKSR-1 (2.5 µg). Two
days later, transfected COS7 cells were lysed in Nonidet P-40 lysis
buffer and cleared by low speed centrifugation (5). Goat anti-KSR-1 antibody (Santa Cruz Biotechnology, 1:100 dilution) or goat anti-rabbit IgG (Santa Cruz Biotechnology) was added to lysates and incubated for
90 min at 4 °C. Protein A/G Agarose (Santa Cruz Biotechnology) was
used to immobilize antibody-bound proteins. Immunoprecipitates were
washed three times with Nonidet P-40 lysis buffer and analyzed by
SDS-PAGE and immunoblotting as above.
1 and
3 subunits (0.5 µg each) with or
without the cDNA encoding mKSR-1 (0.5 µg) as described above.
Cells were serum starved overnight (Dulbecco's modified Eagle's
medium with 1% bovine serum albumin) before they were lysed in Nonidet
P-40 lysis buffer. Lysates were cleared by low speed centrifugation and
then incubated at 4 °C for 90 min with murine monoclonal anti-ERK
antibody (Santa Cruz Biotechnology, 1:100) in 500-µl reactions
containing 100 µg of total protein. After incubation with anti-ERK
antibody, 20 µl of protein A/G-agarose (Santa Cruz Biotechnology) was
added to each reaction to immobilize antibody-bound proteins.
Immunoprecipitates were washed twice in Nonidet P-40 lysis buffer to
which NaCl had been added (1 M final concentration) and
once in 25 mM HEPES (pH 7.45) plus 2 mM
phenylmethylsulfonyl fluoride, and 1 mM
Na3VO4. Immunoprecipitates were then
resuspended in kinase buffer (25 mM HEPES (pH 7.45), 10 mM MgCl2, 1 mM dithiothreitol, and
50 µM ATP) and incubated with 2 µg of myelin basic
protein (Sigma) and 20 µCi [
-32P]ATP for 20 min at
room temperature. Samples were resolved by SDS-PAGE and
visualized by autoradiography.
RESULTS
10 subunit of heterotrimeric G-proteins (18); the subunit did not interact with the protein kinase Mos or with nuclear lamin on two-hybrid assay. In subsequent experiments with the
two-hybrid assay to determine whether the interaction between CA2-5
and
subunits was specific for
10, we found that
2 and
3 also bound to CA2-5 (Fig.
1). Several additional deletion mutant forms of mKSR-1 were then generated to identify the portion of mKSR-1
that was interacting with these subunits (Fig. 1A). The cysteine-rich zinc finger-like CA3 domain of mKSR-1 interacted with
2,
3, and
10 in the
two-hybrid assay (Fig. 1B), whereas neither the proline-rich
CA2 domain nor the protein kinase CA5 domain of mKSR-1 interacted with
any of the subunits. A construct that contained the CA4 domain was
found to be transcriptionally active on its own in yeast.
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Fig. 1.
The CA3 domain of mKSR-1 interacts with
G-protein subunits by two-hybrid assay.
A, mKSR-1 constructs used for two-hybrid analysis. The
full-length mKSR-1 cDNA was used as a template to generate several
deletion constructs for two-hybrid analysis that were subcloned as
in-frame fusion proteins with the transactivation domain of the GAL4
transcription factor in the vector pAS1. B, detection of
interactions between mKSR-1 and
subunits. The cDNAs encoding
human
subunits were subcloned as in-frame fusion proteins with the
DNA-binding domain of the GAL4 transcription factor in the vector pGAD.
The DNA-binding domain and transactivation domain fusion constructs
were cotransfected into a yeast strain (Y190) and grown on medium
lacking leucine and tryptophan. Colony lifts were analyzed for
-galactosidase activity by use of X-gal, and colonies that exhibited
detectable activity were scored positive (+).
and
subunits are obligatorily bound to each
other (19). We therefore investigated the ability of the isolated CA3
domain of mKSR-1 to bind to
subunits derived from cultured cell
protein lysates. Recombinant GST fusion proteins that contained the CA1
and CA2 domains, the CA3 domain, the CA4 domain, or the CA5 domain of
mKSR-1 were immobilized on glutathione beads and were incubated with
NIH/3T3 cell protein lysates. Adherent proteins were analyzed by
anti-
subunit immunoblotting, because the larger
subunit is more
readily detectable on immunoblots than the
subunit, and revealed
that
subunits bound to immobilized GST-CA3, but not to
GST-CA1CA2, GST-CA4, or GST-CA5 (Fig.
2A). We have previously
demonstrated that GST-CA4 specifically interacts with MAP kinase (12),
and that GST-CA5 binds to
MEK.2
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Fig. 2.
Association of mKSR-1 with
subunits in vitro
and in vivo. A, in
vitro association of the CA3 domain of mKSR-1 with
subunits. Bacterially expressed GST fusion proteins were immobilized on
glutathione beads, and samples were incubated with NIH/3T3 cell protein
lysates. The beads were washed and adherent proteins were analyzed by
immunoblotting by use of an an anti-pan-
subunit antibody (Santa
Cruz Biotechnology). The fusion proteins contained protein fragments
corresponding to GST alone (GST), the CA1 and CA2 domains
(GST-CA1CA2), the CA3 domain (GST-CA3), the CA4
domain (GST-CA4), and the CA5 domain (GST-CA5) of
mKSR-1. B, in vivo association of mKSR-1with
1
3 subunits in transfected cells. COS7
cells were triple transfected with mammalian expression vectors
encoding wild-type mKSR-1, human
1, and human
3. Anti-KSR or control (rabbit IgG) immunoprecipitates
were analyzed by immunoblotting by use of an anti-
1
subunit antibody (BN-1). Protein lysates derived from 5 × 105 cells were used for each immunoprecipitation. This
immunoblot is representative of the results of three separate
experiments. C, in vivo association of mKSR-1
with
subunits in untransfected cells. Untransfected COS7
cells were cultured in the absence of serum for 24 h and then some
were stimulated with 10% fetal bovine serum for 10 min. Anti-KSR or
control (rabbit IgG) immunoprecipitates were analyzed by immunoblotting
by use of an anti-pan-
subunit antibody. Protein lysates derived
from 107 cells were used for each immunoprecipitation. This
immunoblot is representative of the results of four separate
experiments.
The ability of KSR-1 to interact with subunits in
vivo was examined in coimmunoprecipitation experiments. COS7 cells
were triple transfected with
1,
3, and
mKSR-1. Anti-KSR immunoprecipitates obtained from transfected cell
protein lysates were analyzed by anti-
subunit immunoblotting, and
this showed that
1
3 and KSR-1 form a
complex in vivo (Fig. 2B). The efficiency of this
interaction was determined in three separate experiments by
densitometric analysis of anti-KSR and anti-
subunit immunoblots:
57% ± 15% (S.E.) of
1
3 bound to
KSR-1.
The ability of KSR-1 to interact with subunits in untransfected
cells was also investigated. Untransfected COS7 cells were cultured in the absence of serum for 24 h and then some cells were
stimulated with 10% fetal calf serum for 10 min. Anti-KSR immunoprecipitates obtained from untransfected cell protein lysates were analyzed by anti-pan-
subunit immunoblotting and this showed that
and KSR-1 form a complex in serum-stimulated but not in serum-starved cells (Fig. 2C).
The ability of subunits to form a complex with KSR-1 in
vivo suggested that the liberation of free
subunits on
G-protein activation could cause KSR-1 to translocate to the plasma
membrane. We have previously demonstrated that serum stimulation of
cultured NIH/3T3 cells results in a redistribution of a significant
proportion of KSR-1 from the cytoplasmic fraction of cell lysates to
the plasma membrane fraction (5). Because LPA is an important component of serum that binds to Gi-coupled receptors, we examined
whether LPA could also induce this translocation of KSR-1 (20).
Cultured COS7 cells were treated with LPA, and the subcellular
localization of KSR-1 was examined by differential centrifugation
followed by immunoblotting. LPA treatment of cells resulted in robust
redistribution of KSR-1 to the membrane fraction of cell lysates that
was blocked by pretreatment with PTX, which specifically inhibits
Gi (Fig. 3). Because LPA
stimulates Gi-coupled receptors that can activate Ras (20),
we wished to evaluate whether KSR-1 translocation was dependent on Ras
activation. Transfection of cells with dominant-negative (N17) Ras did
not inhibit LPA-induced redistribution (Fig. 3B) (21).
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To evaluate the biological significance of the interaction
between subunits and KSR-1, we evaluated MAP kinase activity in
transfected cells. Cotransfection of cultured cells with
and
subunits has previously been shown to result in MAP kinase activation
in the absence of serum stimulation (22). It has been demonstrated that
free
subunits interact with and activate phosphatidylinositol
3-kinase
, and this interaction is thought to eventually
lead to activation of Ras and MAP kinase (23). In this study the
additional transfection of cultured cells with full-length mKSR-1
markedly inhibited
1
3-induced MAP kinase activation without affecting
1 subunit protein levels
(Fig. 4).
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DISCUSSION |
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The subcellular localization of many signaling proteins is highly
regulated and is often an important determinant of their activity.
Localization is thought to influence activity by increasing the
proximity of an enzyme to activating molecules or to substrates. For
example, the protein kinase Raf-1 must translocate to the plasma
membrane to be fully activated (17, 18, 24). The protein kinase KSR-1
also translocates from the cytosol to the plasma membrane, but the
importance of this event in regulating the activity of KSR-1 has not
been determined. In the experiments described here, we evaluated the
mechanism of the translocation of KSR-1 from the cytosol to the plasma
membrane. By use of the yeast two-hybrid assay we confirmed that KSR-1
can interact with the 2,
3, and
10 subunits of heterotrimeric G-proteins. These G-protein subunits are lipid modified and have been shown to be constitutively plasma membrane-bound (19). We also demonstrated that
KSR-1 binds to
subunits in cultured mammalian cells, and that a
ligand that liberates
subunits, LPA, can stimulate the translocation of KSR-1 to the plasma membrane. These findings confirm
that
subunits can mediate the translocation of KSR-1 to the
plasma membrane.
One interesting aspect of the interaction between subunits and
KSR-1 is that
effectors usually bind directly to the larger
subunit (25-27). The surface of the
dimer that interacts with effectors has been examined by x-ray crystallography, demonstrating that there are several distinct areas that interact with effectors (28-30), particularly the amino-terminal coiled-coil domain of
subunit, which is immediately adjacent to the amino-terminal domain of
the
subunit that also forms a coiled-coil (31-33). The amino
termini of both subunits form a continuous surface that could
theoretically interact with effectors although it remains to be
determined whether this is the site of interaction with KSR-1.
We demonstrated by two-hybrid assay that the CA3 domain of KSR-1 can
bind to 2,
3, and
10. This
domain is highly homologous to the cysteine-rich domains of Raf-1,
A-Raf, protein kinase Cµ, and citron kinase, and is less homologous
to the cysteine-rich domains of diacylglycerol kinase, the racGAP
N-chimaerin, and the PTPL1-associated rhoGAP (BLAST search, National
Center for Biotechnology Information). Previous investigations have
demonstrated that Raf-1 can interact with
subunits in
vitro and in vivo (34). The budding yeast scaffolding
protein ste5 interacts with
subunits via its cysteine-rich
ring-H2 domain (35, 36). It will be important to determine whether
other proteins that contain cysteine-rich domains are able to interact
with
subunits because of the possibility that cysteine-rich
domains, as a class, are
effectors.
The ability of subunits to bind specifically to the CA3 domain,
MEK to bind to CA5 (8, 9), and MAP kinase to bind to CA4 (12) supports
the hypothesis that mKSR-1 is a scaffolding protein. In budding yeast,
ste5 links
subunits (ste4, ste18) to the MAP kinase cascade
proteins ste11, ste7, and fus3/kss1 and promotes their activation (35,
36). In marked contrast to findings with ste5, we found that
overexpression of mKSR-1 inhibits
-induced MAP kinase activation.
This discrepancy suggests that mKSR-1 has a unique physiologic role in
the regulation of MAP kinase signaling. Our findings complement recent
work by other investigators demonstrating that overexpression of mKSR-1
in cultured mammalian cells inhibits serum- and ligand-induced MAP
kinase activation (8-10).
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ACKNOWLEDGEMENTS |
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We thank Maurine Linder and Ken Blumer for technical advice and helpful comments. We thank David Beach, Steve Elledge, Wendy Fantl, Gerald Rubin, Marc Therrien, and Dwight Towler for reagents.
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FOOTNOTES |
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* 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.
Supported by grants from the Barnes-Jewish Hospital Foundation,
the National Institutes of Health, and the American Heart Association.
To whom correspondence should be addressed: Center for Cardiovascular
Research, Box 8086, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-3525; Fax:
314-362-0186; E-mail: amuslin{at}imgate.wustl.edu.
2 Heming Xing and Anthony J. Muslin, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
KSR-1, kinase
suppressor of Ras;
mKSR-1, murine KSR-1;
FCS, fetal calf serum;
LPA, lysophosphatidic acid;
PTX, pertussis toxin;
PAGE, polyacrylamide gel
electrophoresis;
GST, glutathione S-transferase;
X-gal, 5-bromo-4-chloro-3-indolyl -D-galactopyranoside;
ERK, extracellular signal-regulated kinase;
MAP, mitogen-activated protein;
MEK, MAP kinase or extracellular signal-regulated kinase kinase.
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