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Biochemistry and Biophysics |
Activation of Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase in Intestinal Epithelial Cells1
Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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receptor 1 regulation of intestinal
epithelial cell proliferation. However, the mechanism of TNF-
mediated activation of extracellular signal-regulated kinase (ERK)/MAP
kinase has not been established clearly. Both TNF- and
cell-permeable ceramide have been reported to increase the kinase
activity of kinase suppressor of Ras (KSR). To determine the role of
KSR in TNF--induced ERK1/ERK2 activation, we studied young adult
mouse colon cells expressing a dominant-negative, kinase-inactive
(ki) KSR. We report that TNF-, a
cell-permeable ceramide, and sphingomyelinase stimulate ERK1/ERK2
activation and increase the phosphoserine content of KSR, which are
inhibited by kiKSR expression in intact cells.
Furthermore, TNF--induced Raf-1 threonine phosphorylation, kinase
activity toward MEK1, and association with KSR are also inhibited by
kiKSR expression. Our data also show by sequential
in vitro kinase assays that TNF- enhances KSR
phosphorylation of Raf-1 on threonine, enhancing Raf-1 kinase activity
toward MAP kinase kinase. We therefore conclude that KSR is an
essential upstream regulator of TNF--stimulated ERK1/ERK2
activation, most likely mediated via direct phosphorylation of Raf-1. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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to inhibit intestinal epithelial cell proliferation requires sustained
ERK1/ERK2 activation (4)
. In fact, by changing sustained
ERK1/ERK2 activation to transient, TNF- is converted from an
antiproliferative to proliferative ligand (4)
. Others have
also reported manipulation of ERK1/ERK2 kinetics to alter intestinal
cell proliferation and differentiation programs (5)
.
Growth factor activation of ERK1/ERK2 is regulated via the Ras
signaling pathway, whereby GTP-Ras binds to Raf-1 at the plasma
membrane, promoting Raf-1 activation. Posttranslational modification of
Raf-1 at the membrane includes phosphorylation on conserved serine,
threonine, and tyrosine sites, a process that regulates its kinase
activity (6, 7, 8, 9, 10)
. In addition, serine phosphorylation on
Raf-1 has been shown to regulate interaction with 14-3-3 proteins and,
consequently, kinase activity (8)
. MEK1, which is
phosphorylated and activated by Raf, directly activates MAP kinase by
dual phosphorylation of its highly conserved threonine and tyrosine
residues (11
, 12)
. Activated MAP kinase then
phosphorylates critical cytoplasmic and nuclear substrates, thereby
regulating cellular responses (13)
. However, the signaling
pathway linking TNF- receptor to MAP kinase activation remains
unclear.
Understanding the mechanisms of TNF- signal transduction in
intestinal epithelial cells is important for both normal development
and the pathogenesis of diseases, such as inflammatory bowel disease.
Anti-TNF- antibodies reverse disease activity in patients with
inflammatory bowel disease (14)
and in animal models of
inflammatory bowel disease (15)
. In fact, TNF-
overexpression is sufficient to induce inflammatory bowel disease in a
mouse model (16)
. A candidate second messenger for TNF-
signal transduction is ceramide. TNF- binding to the TNFR1 in
hematopoietic cells initiates activation of neutral sphingomyelinase,
perhaps through factor associated with neutral
sphingomyelinase, which increases hydrolysis of sphingomyelin to
produce ceramide (17)
. TNF-, sphingomyelinase, and
cell-permeable ceramide have all been shown to stimulate MAP kinase
activation in these cells (18)
. We have also shown that
TNF- activates ERK1/ERK2 through TNFR1, and that cell-permeable
ceramide can mimic this effect in intestinal epithelial cells (4
, 19)
.
One mediator of ceramide action is the ceramide-activated protein
kinase, KSR, a Mr 97,000
proline-directed serine/threonine protein kinase (20
, 21)
.
KSR has been shown to phosphorylate and activate Raf-1 in
vitro and in intact myelomonocytic HL-60 cells in response to
TNF- (10)
. Serum stimulates KSR to translocate from
cytoplasmic to plasma membrane fractions in mouse fibroblasts, where it
is associated with Raf-1 (22)
. Autophosphorylation of KSR
and the phosphorylation of Raf-1 are both stimulated by TNF- and
cell-permeable ceramide (23)
. Human, murine,
Drosophila melanogaster, and Caenorhabditis
elegans KSR contain four highly conserved domains, CA1–CA4, on
the NH2 terminus, whereas the COOH-terminal
region of KSR contains the putative kinase domain
(24, 25, 26)
. Importantly, KSR has been suggested to function
upstream of, or in parallel with, Raf (27)
.
In the present study, we have focused on the role of KSR in the
regulation of TNF- induced ERK1/ERK2 activation by expressing either
wt or a dominant-negative kiKSR in YAMC cells.
Our findings demonstrate a requirement for KSR kinase activity in
TNF--stimulated ERK1/ERK2 activation. Moreover, expression of
dominant-negative KSR blocked TNF--induced Raf-1 threonine
phosphorylation, kinase activity, and association with KSR, implicating
KSR as a regulatory kinase between TNFR1 and ERK1/ERK2 MAP kinase.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inducible
promoter, were maintained at 33°C under permissive conditions in RPMI
1640 (pH 7.4) with 5% FBS and 5 units/ml of murine IFN- and
supplemented as described previously (28)
. Confluent
monolayers were serum-deprived (0.5% FBS) without IFN- for 24 h under nonpermissive conditions (37°C) prior to all experiments.
Cellular Transfections.
pFlag-cDNA3-wtKSR, pFlag-cDNA3-kiKSR, and pcDNA3
vector were provided by Richard Kolesnick (Memorial Sloan-Kettering
Cancer Center, New York, NY), and pcDNA3-CrmA was provided by Vishva
Dixit (Genentech, South San Francisco, CA). The kiKSR
plasmid was generated by substitution of alanine residues for two
aspartates (D683 and D700) in the conserved kinase domain
(29)
of mouse KSR with Flag sequence fused to the
NH2 terminus as described (23)
. YAMC
monolayers (90% confluent) were incubated with 25 µl of Cellfectin
(Life Technologies, Inc., Grand Island, NY) and 10 µg of appropriate
plasmid DNA in 4 ml of DMEM at 33°C for 24 h and then incubated
with RPMI 1640 with 5% FBS and IFN- overnight. Transfected cells
were selected by incubating cells in RPMI 1640 containing G418 (500
µg/ml) for 
3 days. Nontransfected YAMC cells incubated with G418
died in <3 days. Single cells resistant to antibiotic were collected
using Cloning Cylinders (Bellco Glass, Inc., Vineland, NJ) for
establishing stably expressing clonal cell lines. Transfected cells
were cultured in the presence of G418 until 24 h prior to
experiments. Flag-wtKSR, Flag-kiKSR, or CrmA
expression were verified by Western blot analysis with anti-Flag M2
(Sigma Chemical Co., St. Louis, MO) or anti-CrmA (PharMingen, San
Diego, CA) antibodies, respectively.
Preparation of Cellular Lysates.
Cellular lysates were prepared from cells treated with murine TNF-
(Pepro Tech, Inc., Rocky Hill, NJ), cell-permeable
C8-ceramide (Biomol, Plymouth Meeting, PA),
sphingomyelinase (Sigma), or murine EGF (gift from Stanley Cohen,
Vanderbilt University, Nashville, TN). Cell monolayers were
rinsed twice on ice with ice-cold PBS and then scraped into cell lysis
buffer {20 mM HEPES (pH 7.5), with phosphatase inhibitors
(1 mM orthovanadate, 50 mM
ß-glycerolphosphate, and 10 mM sodium
PPi), and protease inhibitors [leupeptin (10
µg/ml), aprotinin (10 µg/ml), phenylmethylsulfonyl fluoride (18
µg/ml)] and 1% Triton X-100}. The scraped suspensions were
centrifuged (16,000 x g for 10 min) at
4°C, and the protein content was analyzed using DC protein assay
(Bio-Rad Laboratories, Hercules, CA). Equal amounts of cellular lysate
protein were mixed with Laemmli sample buffer (30)
,
separated by SDS-PAGE for Western blot analysis with
anti-phospho-ERK1/ERK2 (Promega Corp., Madison, WI), anti-ERK1/ERK2
(Transduction Laboratory, San Diego, CA), anti-phospho-SAPK/JNK (New
England BioLab, Inc., Beverly, MA), or anti-SAPK/JNK (New England
BioLab). Western blot recycling kit (Chemicon International, Inc.,
Temecula, CA) was used in some Western blot analysis. The relative
density of detected proteins was determined by densitometric analysis
using Gel-Pro Analyzer software for Macintosh (Media Cybernetics,
Silver Springs, MD). Fold stimulation controlled for protein loading
was determined, where indicated.
Immunoprecipitation.
For immunoprecipitation of KSR, YAMC cells were rinsed twice on ice
with ice-cold PBS and scraped into membrane isolation buffer [25
mM HEPES (pH 7.4), 5 mM EGTA, and 50
mM NaF plus protease inhibitors]. The cells were lysed by
Dounce homogenization, and then membrane fractions were isolated by
differential centrifugation (1,000 x g for
10 min; 14,000 x g for 7 min; and
25,000 x g for 30 min) and solubilized in 25
µl of ice-cold cell lysis buffer. Equal amounts of cellular membrane
protein were precleared with protein A-Sepharose 4B suspension (Sigma)
for 1 h at 4°C. The supernatant was incubated with rabbit
polyclonal anti-KSR antibody (a gift from Deborah Morrison, National
Cancer Institute, Bethesda, MD) for 2 h at 4°C, followed by
incubation with protein A-Sepharose 4B suspension overnight at 4°C.
Immunoprecipitates were recovered by centrifugation (14,000 x g for 1 min), washed with ice-cold cell lysis buffer
containing 1 M NaCl, and solubilized in Laemmli
sample buffer (30)
for SDS-PAGE and Western blot analysis
with anti-phosphoserine (Zymed Laboratories, Inc., San Francisco, CA)
or anti-phosphothreonine (Zymed Laboratories), anti-phosphotyrosine
(Transduction Laboratory), or anti-KSR antibodies. To test the
anti-phosphoserine specificity, phosphoserine, phosphothreonine, or
phosphotyrosine peptides (Zymed Laboratories) were incubated with the
antibody for 1 h prior to performing Western blot analysis.
For ERK1/ERK2 immunoprecipitation, cells were rinsed on ice with ice-cold PBS and then scraped into ice-cold 50 mM Tris (pH 7.5), 10 mM EDTA, 2 mM EGTA, phosphatase inhibitors, and protease inhibitors with 1% Triton X-100. The lysate was centrifuged (14,000 x g for 5 min) at 4°C, and an equal amount of supernatant was precleared by incubating with 10% (vol/vol) Staphylococcus aureus cell suspension (Sigma) for 1 h at 4°C and then centrifuged (16,000 x g for 1 min) prior to immunoprecipitation with monoclonal p44/42 MAP kinase antibody (New England BioLab) and incubated at 4°C overnight. The antibody/lysate mixture was incubated with 10% (vol/vol) S. aureus cell suspension for 3 h at 4°C. Immunoprecipitates were recovered by centrifugation and washed with the same solubilization buffer containing 0.5 mM NaCl and used for in vitro kinase assay with Elk-1.
For Raf-1 immunoprecipitation, anti-c-Raf (Raf-1) antibody (Upstate Biotechnology) was incubated with protein G-Sepharose 4B (Zymed Laboratories) in PBS at 4°C for 2 h. The protein G-Sepharose 4B was pelleted and washed with PBS by centrifugation and then incubated with equal amounts of cellular lysate protein solubilized in 50 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, phosphatase inhibitors, protease inhibitors, and 1% Triton X-100 for 2 h at 4°C. Immunoprecipitates were recovered by centrifugation and washed with the same solubilization buffer containing 1 M NaCl. Immunoprecipitates were used for in vitro kinase assays or prepared for Western blot analysis with anti-phosphothreonine, anti-c-Raf p-Tyr340/341 (Biosource International, Camarillo, CA), or anti-Raf-1 (Santa Cruz Biotechnology) antibodies.
Flag-tagged wtKSR and kiKSR proteins were immunoprecipitated by incubating 4.4 µg anti-Flag antibody and 600 µg of cellular lysate solubilized in 25 mM Tris-HCl (pH 7.4), 1% Triton X-100, 25 mM CaCl2, 300 mM NaCl, phosphatase inhibitors, and protease inhibitors for 4 h at 4°C, followed by incubation with protein G-Sepharose 4B suspension for 2 h at 4°C. Where indicated, Flag peptide (Sigma) was added to the cellular lysate during anti-Flag immunoprecipitation, or mouse IgG was used for immunoprecipitation. The immunoprecipitates were washed by centrifugation with the solubilization buffer containing 0.5 M NaCl for detection of Raf-1 coprecipitation by Western blot analysis with anti-Raf-1, anti-KSR, or anti-phosphoserine antibodies. Immunoprecipitates of KSR used for in vitro kinase assays were washed with 1 M NaCl to remove coprecipitating kinases (31) .
In Vitro Kinase Assays.
In vitro GST-Elk-1 phosphorylation assay was performed by
incubating immunoprecipitated ERK1/ERK2 in 30 µl of kinase buffer
[25 mM Tris (pH 7.5), 1% NP40, 5
mM ß-glycerolphosphate, 2
mM DTT, 0.1 mM
Na3VO4, 10
mM MgCl2, and 50
µM ATP and protease inhibitors] and 1 µg of
GST-Elk-1 fusion protein (New England BioLab) with 10 µCi of
[-32P]ATP at 30°C for 30 min, as described
previously (4)
. GST-Elk-1 was recovered by centrifugation
and separated by SDS-PAGE for detection of phosphorylation by
autoradiography. The membrane was blotted with anti-Elk antibody (New
England BioLab) to verify equal protein loading.
In vitro Raf-1 phosphorylation was performed by incubating immunoprecipitated Flag-KSR and 2 units of recombinant Raf-1 (Upstate Biotechnology) in 30 µl of KSR kinase buffer [20 mM MOPS (pH 7.2), 25 mM ß-glycerolphosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM DTT, and 25 mM MgCl2], with 200 µM ATP at 30°C for 30 min (31) . Raf-1 was recovered by centrifugation and prepared for Western blot analysis with anti-phosphothreonine or anti-Raf-1 antibodies.
In vitro MEK1 phosphorylation was performed by incubating 0.5 µg of ki MEK1 (provided by Natalie Ahn, Howard Hughes Medical Institute, University of Colorado, Boulder, CO) and immunoprecipitated Raf-1 or the recombinant Raf-1 recovered from Raf-1 phosphorylation assays as described above, in 30 µl of KSR kinase buffer with 200 µM ATP (31) . MEK1 was separated by SDS-PAGE for Western blot analysis with anti-phospho-MEK1/2 or anti-MEK1/2 (New England BioLab) antibodies. The phospho-MEK1/2 antibody detects phosphorylation of the activating serine 217/221 sites on MEK (32) .
All experiments were performed on at least three separate occasions. Representative Western blots or autoradiograms are shown from each experiment.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Cell-permeable Ceramide, and Sphingomyelinase Stimulate
ERK1/ERK2 Activation in YAMC Cells.,
C8-ceramide, or sphingomyelinase. Total cellular
lysates were separated by SDS-PAGE for Western blot analysis with
anti-phospho-ERK1/ERK2. TNF-, C8-ceramide, and
sphingomyelinase all stimulate ERK1/ERK2 activation in YAMC cells (Fig. 1)
. These findings indicate that either cell-permeable ceramide or the
generation of endogenous ceramide is sufficient to induce ERK1/ERK2
activation.
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Stimulates KSR Phosphoserine and Phosphothreonine Content. and
C8-ceramide increase KSR phosphoserine and
phosphothreonine content. YAMC cells were treated with TNF-,
C8-ceramide, or EGF, and cellular membranes were
isolated by detergent solubility and differential centrifugation for
KSR immunoprecipitation. TNF- and C8-ceramide
stimulate serine and threonine phosphorylation of KSR (Fig. 2A)
. However, EGF treatment does not alter KSR phosphoserine
and phosphothreonine contents. No increase in KSR tyrosine
phosphorylation is detected by either TNF- or EGF treatment of
cells, although both treatments cause increased tyrosine
phosphorylation of several other cytoplasmic and membrane proteins
(data not shown) on the same Western blot analysis. To test the
anti-phosphoserine specificity, phosphoserine, phosphothreonine, or
phosphotyrosine peptides were incubated with anti-phosphoserine prior
to performing Western blot analysis. Phosphoserine but not
phosphothreonine or phosphotyrosine peptides inhibit detection of
serine phosphorylation on KSR (Fig. 2B)
. These data indicate
that both TNF- and C8-ceramide stimulate
increased KSR serine and threonine phosphorylation.
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Activation of ERK1/ERK2. stimulates phosphorylation of KSR, which has been shown to
activate MAP kinase kinase kinase (Raf-1) in the Raf/MEK/ERK pathway
(23)
. Therefore, we studied the role of KSR in
TNF--regulated ERK1/ERK2 activation. We established stable clonal
YAMC cell lines expressing varying amounts of Flag-tagged,
dominant-negative kiKSR (23)
. Flag-tagged
wtKSR or Flag-tagged kiKSR was immunoprecipitated
from transfected cells with anti-Flag antibody for Western blot
analysis with anti-phosphoserine or anti-KSR. Compared with
wtKSR isolated from TNF--stimulated cells,
kiKSR phosphoserine content is significantly reduced (Fig. 3A)
. To determine the effect of kiKSR on TNF-
activation of ERK1/ERK2, we selected YAMC cell lines expressing various
levels of kiKSR, as determined by anti-Flag immunoblotting.
Nontransfected, kiKSR clonal cell lines, wtKSR,
and vector-only transfected cells were treated with TNF- or EGF, and
cellular lysates were studied by Western blot analysis for dual
phosphorylation on ERK1/ERK2. Dominant-negative kiKSR
expression inhibits TNF--stimulated ERK1/ERK2 phosphorylation in a
manner directly proportional to the level of kiKSR
expression (Fig. 3B)
. However, kiKSR expression
does not alter EGF activation of ERK1/ERK2 (Fig. 3C)
.
Interestingly, TNF- activation of ERK1/ERK2 is unaffected in cells
expressing an equivalent amount of wtKSR (Fig. 3C)
. The wtKSR clone 4 and kiKSR clone
5 were selected for use in Fig. 3, A
and C, and
subsequent experiments comparing the effects of kiKSR and
wtKSR expression because, as shown by anti-Flag staining,
these clonal cell lines express equivalent amounts of the respective
form of KSR. We determined the kinase activity of ERK1/ERK2 in
vitro using immunoprecipitated ERK1/ERK2 and GST-Elk-1 fusion
protein (4)
. Consistent with the phosphorylation data,
kiKSR expression inhibits TNF- increased ERK1/ERK2 kinase
activity toward GST-Elk-1 (Fig. 3D)
. These observations
clearly indicate KSR kinase-dependent regulation of ERK1/ERK2
activation pathways by TNF- but not EGF in intestinal epithelial
cells.
|
. Importantly, expression of
wtKSR has no effect on ERK activation by either ceramide or
sphingomyelinase (Fig. 4C)
. Thus, TNF-, or putative
signaling intermediates, require an intact KSR kinase domain for
ERK1/ERK2 activation.
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-stimulated Raf-1 Kinase Activity toward MEK1 Requires
Intact KSR Kinase. on KSR association
with Raf-1. Cells stably expressing either wtKSR or
kiKSR were incubated with TNF- or EGF. Ectopically
expressed KSR was immunoprecipitated with anti-Flag for Western blot
analysis with anti-Raf-1 or anti-KSR. TNF- enhances Raf-1
coprecipitation with KSR in cells expressing wtKSR but not
kiKSR (Fig. 5A)
. Specificity of Raf-1 association with KSR is shown by
decreased coprecipitation in the presence of Flag peptide and no
precipitation with mouse IgG (Fig. 5B)
.
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. These findings show TNF--stimulated Raf-1 kinase activity toward
MEK-1 requires intact KSR kinase activity. Consistent with our findings
for ERK1/ERK2 activation, EGF-enhanced Raf-1 kinase activity toward
MEK-1 is not inhibited by kiKSR expression (Fig. 6
,
upper panel, Lane 7).
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Stimulates KSR Kinase Activity toward Raf-1, Enhancing
Raf-1 Kinase Activity toward MEK1. has been shown to increase threonine 268/269 phosphorylation on
Raf-1 (10
, 23)
. Therefore, we asked whether TNF-
enhances threonine phosphorylation of Raf-1 in intestinal cells stably
expressing either wtKSR or kiKSR. We performed an
in vitro kinase assay with immunoprecipitated KSR isolated
from TNF- and EGF-treated cells and recombinant Raf-1 (Fig. 7A)
. Only Raf-1 incubated with wtKSR isolated from
TNF--treated cells shows increased threonine phosphorylation. The
lack of Raf-1 autophosphorylation on threonine is consistent with
Cleghon and Morrison (33)
observation that Raf-1
auto-kinase activity required coexpression with Fyn/Src. The
recombinant Raf-1 was recovered from the above kinase assays for
incubation with kiMEK1 in an in vitro kinase
assay. MEK1 activation was then determined by Western blot analysis
with anti-phospho-MEK1/2 (Fig. 7B)
. These data show that
TNF- stimulates KSR kinase activity toward Raf-1, increasing
threonine phosphorylation and enhancing Raf-1 kinase activity toward
MEK1. No direct kinase activity toward MEK1 is shown by
wtKSR from TNF--treated cells (Fig. 7
B, Lane
8). Furthermore, Raf-1 immunoprecipitation shows that TNF-,
C8-ceramide, and sphingomyelinase all
increase threonine phosphorylation of Raf-1 in wtKSR- but
not kiKSR-expressing cells (Fig. 7C)
. However,
EGF-stimulated Raf-1 phosphorylation of the regulatory tyrosines
340/341 in YAMC and wtKSR-expressing cells is not affected
by kiKSR expression (Fig. 7D)
. These data
indicate that KSR regulates TNF--enhanced ERK1/ERK2 activation at
the level of Raf-1 threonine phosphorylation.
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in YAMC cells
(19)
. The expression of kiKSR has no effect on
SAPK/JNK activation as shown (Fig. 8A)
. Ceramide production by neutral sphingomyelinase, but not
acid sphingomyelinase, has been shown to regulate KSR serine/threonine
kinase activity (34)
. Because cowpox virus CrmA prevents
activation of acid sphingomyelinase in mammalian cells
(35)
, we established stable YAMC clonal lines expressing
CrmA (Fig. 8B)
. Whereas expression of CrmA has no effect on
TNF--induced ERK1/ERK2 phosphorylation (data not shown), SAPK/JNK
MAP kinase activation is inhibited 3-fold (Fig. 8B)
. These
findings demonstrate that TNF- activation of ERK1/ERK2 is dependent
upon KSR kinase activity, but SAPK/JNK activation is not.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activation of ERK1/ERK2 in intestinal epithelial cells. Stable
expression of a dominant-negative kiKSR inhibits ERK1/ERK2
activation induced by TNF-, C8-ceramide, or
sphingomyelinase in a concentration-dependent manner.
TNF--stimulated Raf-1 kinase activity, threonine phosphorylation,
and association with KSR are all inhibited by kiKSR
expression. These data indicate that KSR is a key regulatory molecule
between TNFR binding and enhanced Raf-1 kinase activity mediating
TNF--induced ERK1/ERK2 activation in intestinal epithelial cells.
The results of these experiments strengthen previous observations
in vitro and in mammalian cells demonstrating that TNF-
or ceramide induce MAP kinase via KSR phosphorylation of Raf-1
(10
, 23
, 36) . Only wtKSR immunoprecipitated
from TNF--treated cells stimulates Raf-1 threonine phosphorylation.
In turn, only Raf-1 that is threonine phosphorylated by KSR shows
increased kinase activity toward MEK1 (Fig. 7)
. Although Volle et
al. (37)
reported a faster migrating kinase that
coprecipitates with the NH2 terminus of KSR, no
direct kinase activity toward MEK1 is shown by wtKSR
isolated from TNF--treated cells (Fig. 7
B, Lane 8). Xing
et al. (31)
showed recently that 1
M NaCl washes of immunoprecipitated KSR, which we
used in our kinase assay, are sufficient to remove coprecipitating
kinases. Recovery of Raf-1 kinase activity toward MEK1 from
TNF--treated cells shows inhibition only in
kiKSR-expressing cells. In contrast, Raf-1 from EGF-treated
kiKSR cells shows normal enhanced MEK1 kinase activity (Fig. 6)
and tyrosine 340/341 phosphorylation (Fig. 7D)
. KSR and
Raf-1 have been shown to associate at the plasma membrane and in immune
complex assays (23
, 38)
. Given this background, the
simplest interpretation of our data is that KSR directly phosphorylates
Raf-1 on threonine and increases its kinase activity toward MEK1 in a
TNF--regulated pathway. In our study, we have not defined the
threonine phosphorylation site(s) on Raf-1; however, Zhang et
al. (23)
have shown that Raf-1 threonine 268/269
phosphorylation by KSR is necessary for TNF- activation of Raf-1
kinase activity toward MEK1.
Ceramide generated by neutral sphingomyelinase is a likely second
messenger molecule in this pathway, increasing KSR serine and threonine
phosphorylation, perhaps by initiating KSR via autophosphorylation
(10)
. Although we did not study ceramide in this report,
endogenous ceramide production initiated by the addition of
sphingomyelinase to YAMC cells activated ERK1/ERK2 in a KSR
kinase-dependent manner. Expression of CrmA to prevent acid
sphingomyelinase activation has no effect on TNF--stimulated
ERK1/ERK2 activation; however, TNF--stimulated JNK/SAPK activation
is inhibited. Consistent with these differences, mutations of TNFR1
that impair neutral sphingomyelinase activation have no effect on acid
sphingomyelinase activation (34
, 39)
.
Our findings contrast with those of several groups showing that overexpression of KSR or the isolated kinase domain inhibit this pathway in models of oocyte differentiation, foci formation, and MAP kinase activation (38) . In fact, very high levels of KSR expression block Ras-dependent oocyte maturation and photoreceptor cell differentiation in the Drosophila eye (40) . Ectopic expression of KSR in fibroblasts also inhibits MAP kinase activation by activated Ras or Raf (41) . Interestingly, membrane ruffling initiated by activated Ras was not affected, suggesting a divergence in the requirement for KSR kinase activity in these two Ras-regulated pathways. Because our studies were performed in intestinal cell lines, cell type specificity may provide one explanation for these discrepancies. However, similar results have been shown in epithelial carcinoma cells (31) and fibroblast cell lines (23) .
We report divergence in the regulation of MAP kinase whereby
TNF--initiated but not EGF-initiated ERK1/ERK2 activation requires
an intact KSR kinase domain. In part, this may explain the difference
in Raf-1 phosphorylation seen between TNF- and EGF. Threonine
phosphorylation of Raf-1 is inhibited in kiKSR-expressing
cells treated with TNF-, yet Raf-1 tyrosine 340/341 phosphorylation
stimulated by EGF is not affected. It is unclear why KSR kinase
activity is necessary for MAP kinase activation via one receptor system
and not for another, although both involve Raf-1 kinase. However, we
have reported previously that TNF- and EGF cause differences in both
the duration of activation and the intracellular localization of
ERK1/ERK2 MAP kinase in intestinal cells (4)
.
KSR was originally cloned as a loss-of-function mutation in the Ras
signaling pathway of Drosophila melanogaster and
Caenorhabditis elegans (26)
. Interestingly, it
was identified previously as a Mr
97,000, plasma membrane-localized, serine/threonine protein kinase
activity inducible by either TNF- or ceramide (20)
. Two
models of KSR function have emerged to explain its role as an effector
of Ras activity in the Ras/Raf/MEK/ERK signal transduction pathway. In
the first model, KSR kinase activity is necessary for Ras activation of
Raf (10
, 23)
. In fact, identification of KSR as a
loss-of-function mutation in Drosophila and C.
elegans suggests that its kinase activity plays a role in the
Ras/Raf/MEK/MAPK pathway because several mutations were found in
conserved regions of the kinase domain (24, 25, 26)
. In the
second model, KSR functions as a scaffolding protein, organizing a
higher order molecular complex at the plasma membrane regulating
Ras/Raf/MEK/ERK signal transduction (40
, 42)
. We observed
Raf-1 recovery with KSR enhanced by TNF-, similar to a previous
report (23)
. Activated Ras also increases KSR/Raf-1
activation (38
, 43)
. MEK1, MEK2, and several members of
the heat shock protein family and p42 ERK2/MAP kinase have been
recovered from KSR immunoprecipitates (40
, 42)
. The 14-3-3
protein family members have been shown to bind to phosphorylated
serines 297 and 392 on KSR (40)
.
Morrison and colleagues (40
, 44
, 45)
have suggested KSR
functions as a scaffolding protein comparable with the osmo-regulatory
pathway in Saccharomyces cerevisiae coordinated by Ste5 and
Pbs2p, in which kinase signaling complexes are interdependent.
Alternatively, Davis and colleagues (46
, 47)
have proposed
that selective activation of JNK is orchestrated by the JNK-interacting
protein group of scaffold proteins. However, we are unaware of an
analogous system where activation within a single pathway such as Raf
to ERK1/ERK2 requires a single intermediate molecule to maintain an
intact kinase domain for signaling by one ligand (TNF-) and to
function as a scaffolding protein for signaling by another ligand (EGF
or activated Ras). It is interesting to speculate that modifiers such
as the recently described connector enhancer of KSR may
provide another level of Raf regulation (48)
. Perhaps, the
increased serine phosphorylation on KSR we demonstrate in response to
TNF-, but not EGF, may be important in determining the role of KSR
in ERK1/ERK2 activation.
In summary, expression of a dominant-negative kiKSR in
intestinal epithelial cells inhibits ERK1/ERK2 MAP kinase activation
and serine phosphorylation of KSR by TNF-, cell-permeable ceramide,
and sphingomyelinase. Furthermore, TNF--stimulated Raf-1 association
with KSR and Raf-1 threonine phosphorylation, but not EGF-induced Raf-1
tyrosine 340/341 phosphorylation, is inhibited by kiKSR
expression. This regulatory role for KSR in TNF- activation of the
Raf-1/MEK/ERK pathway is emphasized by reconstitution of this signal
transduction pathway in vitro. We conclude that KSR is an
upstream regulatory kinase for TNF--stimulated ERK1/ERK2 activation,
implicating ceramide as a second messenger in this pathway. In
addition, we conclude there is divergence in the Raf-1/MEK/ERK
signaling cassette in intestinal cells whereby an intact KSR kinase
domain is required for activation by TNF- but not EGF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grants DK02212, T32 DK07673,
and DK56008; a Research Grant from the Crohn’s and Colitis Foundation
of America; and a Turner Scholar Award. 
2 To whom requests for reprints should be
addressed, at Department of Pediatrics, Division of Gastroenterology,
Hepatology and Nutrition, S4322 MCN, 21st and Garland Avenue,
Nashville, TN 37232-2576. Phone: (615) 322-7449; Fax: (615) 343-8915;
E-mail: d-brent.polk{at}mcmail.vanderbilt.edu
3 The abbreviations used are: MAP,
mitogen-activated protein; ERK, extracellular signal-regulated kinase;
SAPK/JNK, stress-activated protein kinase-c-Jun
NH2-terminal kinase; TNF, tumor necrosis factor; MEK1, MAP
kinase kinase; TNFR, TNF- receptor; KSR, kinase suppressor of Ras;
wt, wild type; ki, kinase inactive; YAMC, young adult mouse colon; EGF,
epidermal growth factor.
Received 8/21/00. Accepted 11/20/00.
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