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INTRODUCTION |
Tumor necrosis factor-
(TNF),1 a pleiotropic
cytokine produced mainly by macrophages, regulates many aspects of the
inflammatory response and host defense mechanisms against pathogenic
microorganisms (1, 2). TNF interacts with and induces
oligomerization of two cell surface receptors, CD120a (p55) and CD120b
(p75), that are collectively responsible for signaling the many
distinct cellular responses that are induced by this cytokine (3, 4). Following ligand binding, the adapter molecule TRADD binds through its
death domain to the death domain of CD120a (p55) (5) thereby forming a
platform for the assembly of other signaling molecules including TRAF2
and RIP (6, 7). These interactions set in motion the activation of
downstream signaling cascades, including the activation of members of
the mitogen-activated protein kinase (MAPK) (8) and I-
B kinase
families (9-11), culminating in the trans-activation of
transcription factors, especially AP-1 and NF-B, that regulate the
expression of genes involved in the inflammatory response.
Although RIP has been implicated in the initiation of NF-B
activation, these findings do not, as yet, invoke other
receptor-associated protein Ser/Thr or Tyr kinases in the functions of
CD120a (p55). However, several reports have shown protein Ser/Thr
kinase activities to be bound by sequences located within the
intracellular domain of both TNF receptors (12, 13), as well as by the
related lymphotoxin-
receptor (14). In the case of CD120b (p75), the receptor-associated kinase has been identified as casein kinase I, and
its activity has been shown to prevent apoptotic cell death (15). A
serine-kinase activity that co-immunoprecipitates with CD120a (p55) in
human U937 cells has also been shown to phosphorylate receptor-associated substrates of molecular masses 125, 97, 85, and 60 kDa in a ligand-dependent fashion (16). Darnay and
colleagues (12) have employed fusion proteins between glutathione
S-transferase (GST) and various truncation and deletion
mutants of the cytoplasmic domain of CD120a (p55) to investigate a
Ser/Thr kinase activity designated p60 TRAK and have shown that p60
TRAK is capable of phosphorylating the cytoplasmic domain of CD120a
(p55) but not that of CD120b (p75). The only CD120a (p55)-associated
Ser/Thr-kinase to be cloned is RIP, a death domain-containing
receptor-interacting protein (6, 17). However, although its kinase
activity has been implicated in downstream signaling leading to the
activation of I-B kinases (18), it has not been shown to stimulate
phosphorylation of CD120a (p55) itself. Although these reports have
provided intriguing and important insights into CD120a (p55)-associated
kinases and their mode of interaction with the cytoplasmic domain of
CD120a (p55), the identity and function(s) of CD120a (p55)-Ser/Thr
kinase activities remain largely unknown.
The MAPKs currently comprise three major subfamilies in mammalian cells
as follows: 1) the p38mapk subfamily (19, 20); 2) the
extracellular signal-regulated kinases (ERK) p44mapk/erk1 and
p42mapk/erk2 (21); and 3) the c-Jun NH2-terminal
kinases (JNK) which consist of multiple isoforms of both
p46jnk1 and p54jnk2 (22, 23). All family members are
proline-directed Ser/Thr kinases, and while p38mapk and JNK
sub-family members appear to phosphorylate the basic kinase consensus
motif, (S/T)P (24), ERK-dependent phosphorylation events
are directed toward the consensus sequence
PX1,2(S/T)P, where X represents any
non-acidic residue (25, 26). In work previously reported from this
laboratory, we have shown that specific members of each MAPK subfamily
are rapidly and transiently activated in response to stimulation of
mouse macrophages with TNF (27-29). However, although the targets
of these kinases include transcription factors involved in the
inflammatory response, we have questioned if they also include CD120a
(p55) itself. The 218 amino acids of the cytoplasmic domain of mouse
CD120a (p55) are organized into the death domain (residues 326-411)
(30) and a Ser-, Thr-, and Pro-rich membrane proximal region broadly
located between residues 230 and 280. Within this region are seven
basic Pro-directed kinase motifs and four sequences bearing the
consensus ERK phosphorylation motif. Based on these collective
observations we have investigated (i) if MAPKs interact with and
phosphorylate the cytoplasmic domain of CD120a (p55) and (ii) the
consequence of this event on CD120a (p55) subcellular localization.
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EXPERIMENTAL PROCEDURES |
Materials--
C3H/HeJ mice were used throughout the study and
were bred in the Biological Resource Center at National Jewish Center.
Phospho-specific anti-p42mapk/erk2 antibody (Ab V6671) was
purchased from Promega (Madison, WI). Rabbit polyclonal anti-
p42mapk/erk2 (Ab 93 and 154), anti-p46jnk1 (Ab 154),
anti-p38mapk (Ab 535), phosphospecific anti-p46jnk1 (Ab
6254), and goat polyclonal anti-CD120a (p55) (Ab 1069) antibodies, and
activating transcription factor-2 (ATF-2) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). The anti-p38mapk antibody
used for immunoprecipitation was a kind gift from Dr Gary L. Johnson
(National Jewish Medical and Research Center, Denver, CO). The hamster
monoclonal anti-CD120a (p55) antagonist antibody (Ab 80-4005-01) used
for immunoprecipitation and confocal microscopy was purchased from
Genzyme (Cambridge, MA). The phosphospecific anti-p38mapk
antibody and the mitogen-activated protein kinase kinase (MEK) inhibitor (PD098059) were purchased from New England Biolabs (Beverly, MA). The anti-MEK-1 antibody was from Transduction Laboratories (Lexington, KY). The p38mapk inhibitor (SB203580) and the
recombinant-activated p42mapk/erk2 (Ab 454855) were purchased
from Calbiochem. Rabbit polyclonal anti-p42mapk/erk2 antibodies
(Ab 06-182) were purchased from Upstate Biotechnology, Inc. (Lake
Placid, NY). Anti-vesicular stomatitis virus G-protein (VSV-G) and
anti-adapter protein-1 (AP-1) antibodies were from Sigma. Fluorescent
secondary antibodies were purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA).
Macrophage Isolation, Culture, and Labeling--
Monolayers of
mouse bone marrow-derived macrophages were prepared from femoral and
tibial bone marrow as described previously (27). To label the
endogenous CD120a (p55), bone marrow-derived macrophages (~2 × 106 cells) were washed with phosphate-free medium and
incubated with 1 mCi of [32P]orthophosphate in
phosphate-free Eagle's minimal essential medium for 6 h. Cells
were stimulated with 1 µM okadaic acid during the last
3 h of the incubation period, and lysed in 1 ml of Nonidet P-40
buffer. Lysates were treated with 5 units of DNase for 15 min at
4 °C, before being pre-cleared with 15 µl of protein A/G-Plus Sepharose beads. CD120a (p55) was then immunoprecipitated overnight at
4 °C with 2.5 µg of anti-CD120a (p55) antagonist hamster
monoclonal antibody or 2.5 µg of H57 anti-T-cell receptor hamster
monoclonal antibody as a control, and 25 µl of protein
A/G-Plus-Sepharose beads. The beads were washed 12 times with Nonidet
P-40 buffer. Duplicates of each condition were pooled and resolved by
SDS-PAGE, transferred onto nitrocellulose, and subjected to autoradiography.
Construction of Expression Vectors--
The
GST-CD120a-(207-425) fusion protein was created by fusing the COOH
terminus of glutathione S-transferase (GST) to the full-length intracellular domain (amino acids 207-425) of mouse CD120a
(p55). A 654-base pair EcoRI/SalI cDNA
fragment encoding these residues was amplified by polymerase chain
reaction (PCR), digested with EcoRI and SalI, and
ligated into EcoRI/SalI-digested pGEX-5X-1. The
cDNA encoding amino acids 1-425 (i.e. the entire receptor except for the signal peptide) was amplified by PCR, digested
with EcoRI, and subcloned into EcoRI-digested
pFLAG-CMV-1 downstream of the signal sequence and the sequence encoding
the FLAG epitope (GYKDDDDK). CD120a (p55) T236A, S240A, S244A, S270A and CD120a (p55) T236D, S240E, S244E, S270E mutants were constructed using overlapping PCR and also ligated into pFLAG-CMV-1. The fidelity of all the constructs was verified by restriction enzyme analysis and
nucleotide sequencing. The expression vectors for HA-tagged Xenopus p42mapk/erk2 (HA.ERK2.WT) and a
constitutively active mutated form of Xenopus MEK-1
(MEK1.CA) in the pcDNA3 vector (Invitrogen, Carlsbad, CA) were a
generous gift from Dr. Lynn Heasley, Department of Renal Medicine,
University of Colorado Health Sciences Center, Denver, CO. The vector
expressing VSV-G bearing a COOH-terminal KKTN motif was kindly provided
by Dr. Alexander van der Bliek, Los Angeles, CA.
Expression and Purification of GST Fusion Proteins--
The
expression and purification of GST and the GST-CD120a-(207-425) fusion
protein were carried out as described (31). The amount of fusion
protein was estimated by SDS-PAGE under reducing conditions using
varying amounts of bovine serum albumin as a standard, followed by
staining with Coomassie Blue.
Separation of Kinase Activities by Fast Protein Liquid
Chromatography--
Unstimulated or TNF-stimulated macrophage
monolayers (~5 × 107 cells) were scraped into 2 ml
of ice-cold lysis buffer (50 mM Tris buffer, pH 8.0, containing 1% Nonidet P-40, 50 mM NaCl, 1 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 5 µg/ml aprotinin, and 1 mM
Na3VO4). Nuclear material was pelleted by
centrifugation at 14,000 × g for 10 min at 4 °C.
The supernatant was loaded onto an anion-exchange column (Mono-Q,
BioLogic Systems, Inc.) equilibrated in 50 mM
-glycerophosphate, pH 7.2, containing 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM
Na3VO4, and 0.05 M NaCl. The
elution profile consisted of a flow rate of 1 ml/min for 40 ml with a
linear gradient of 0.05 to 0.2 M NaCl in equilibration buffer, followed by a flow rate of 1 ml/min for 4 ml with a linear gradient of 0.2 to 1 M NaCl, then by an isocratic flow of 1 M NaCl at a rate of 1 ml/min for 20 ml. Two ml fractions
were collected, and 1-ml aliquots were tested for in vitro
kinase activity using GST-CD120a-(207-425) beads as substrate as
described below.
In Vitro Binding of Macrophage Lysates to
GST-CD120a-(207-425)--
Macrophage monolayers (~2 × 106 cells) were rinsed with ice-cold 20 mM
Hepes-buffered saline, pH 7.4, and lysed on ice in 500 µl of Nonidet
P-40 lysis buffer (50 mM Tris buffer, pH 8.0, containing 1% Nonidet P-40, 137 mM NaCl, and protease inhibitors as
described above). Nuclear material was pelleted by centrifugation at
14,000 × g for 10 min at 4 °C. Supernatants were
transferred to a new tube and precleared with 20 µl of washed
GST-coated beads for 20 min at 4 °C on a rotator. The beads were
spun down at 3000 rpm for 3 min, the supernatants were transferred to
new tubes, and incubated with 20 µl of washed
GST-CD120a-(207-425)-coated beads for 150 min at 4 °C on a rotator.
After incubation, the beads were spun down at 3000 rpm for 3 min,
washed twice with Nonidet P-40 lysis buffer, and then twice with PAN
buffer (10 mM Pipes buffer, pH 7.0, containing 100 mM NaCl and 10 µg/ml aprotinin). After the last wash, the
beads were resuspended in 40 µl of PAN buffer to bring the mixture up
to 60 µl and then used in the in vitro kinase assay as
described below. In experiments in which the binding of cytosolic
proteins to GST-CD120-(207-425) was determined by Western blotting,
~5 × 107 cells were lysed and prepared as described
above before being directly subjected to SDS-PAGE and transferred to
nitrocellulose membranes.
In Vitro Kinase Assays--
Kinase assays were performed at
30 °C for 30 min in kinase buffer (10 mM Pipes buffer,
pH 7.2, containing 10 mM MnCl2 and 20 µg/ml aprotinin) together with 10 µCi of
[
-32P]ATP. The kinase reactions were terminated by the
addition of 20 µl of 5× Laemmli sample buffer containing 100 mM dithiothreitol. The mixtures were boiled for 5 min,
separated by SDS-PAGE, and transferred to nitrocellulose for
autoradiography and Western analysis. In in vitro kinase
assays with recombinant p42mapk/erk2, 50 ng of recombinant
MEK-1-activated p42mapk/erk2 (i.e. constitutively
active) were incubated for 150 min at 4 °C with 20 µl of washed
GST-CD120a-(207-425) beads resuspended in 40 µl of PAN buffer. The
kinase reactions were conducted directly or after the beads were washed
4 times with Nonidet P-40 lysis buffer and 4 times with PAN buffer as
described above.
Immune Complex Kinase Assays and Immunodepletion--
Mouse
macrophages were lysed as described above, and post-nuclear
supernatants were precleared with 15 µl of protein A-Sepharose beads.
Individual MAPKs were immunoprecipitated by incubation at 4 °C for
2 h with 5 µg of p38mapk, p42mapk/erk2, and
p46jnk1 antibodies and 25 µl of protein A-Sepharose beads.
The beads were then washed twice with Nonidet P-40 lysis buffer and
twice with PAN buffer. Kinase activity was assayed as described above with either the GST-CD120a-(207-425) fusion protein or recombinant ATF-2 as substrate.
For immunodepletion experiments, the cells were lysed as described
above. The supernatants were precleared with 15 µl of protein A-Sepharose beads, and the MAPKs were immunoprecipitated by incubation at 4 °C for 2 h with 5 µg of p38mapk,
p42mapk/erk2, and p46jnk1 antibodies or non-immune
rabbit IgG, and 25 µl of protein A-Sepharose beads. The beads were
then spun down at 3000 rpm for 3 min, and the supernatants were
transferred to a new tube, precleared with 20 µl of washed GST beads
for 20 min at 4 °C, and incubated with 20 µl of washed
GST-CD120a-(207-425) beads for 150 min at 4 °C on a rotator. After
incubation, an in vitro kinase assay was conducted as
described above.
Transfections and in Vivo Labeling with
[32P]Orthophosphate--
COS-7 cells were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine. ~2 × 105 COS-7 cells/well
were seeded in 6-well (35 mm) dishes and grown in 7.5%
CO2. Cells were transfected the following day using the LipofectAMINETM reagent (Life Technologies, Inc.) according
to the recommendations of the manufacturer. 1.5 µg of DNA were
transfected with 10 µl of LipofectAMINETM reagent in a
total volume of 1 ml of Opti-MEM® I reduced serum medium (Life
Technologies, Inc.). The amount of transfected DNA was kept constant
with pcDNA3 empty vector. Eighteen h after transfection, the cells
were washed with phosphate-free medium and metabolically labeled by
incubation in phosphate-free Eagle's minimal essential medium
containing 1 mCi of [32P]orthophosphate for 6 h. At
the end of the incubation period, the cells were lysed in 250 µl of
Nonidet P-40 buffer, and post-nuclear supernatants were precleared with
25 µl of protein A-Sepharose beads. CD120a (p55) was
immunoprecipitated overnight at 4 °C with 10 µg of anti-CD120a
(p55) antagonist monoclonal antibody and 50 µl of protein A-Sepharose
beads. The beads were washed 12 times with Nonidet P-40 lysis buffer
and resolved by SDS-PAGE.
Confocal Immunofluorescence Microscopy--
~105
HeLa cells were seeded in 12-well plates containing 18-mm glass
coverslips and grown in 5% CO2. Cells were transfected with 100 ng of DNA the following day using the
LipofectAMINETM reagent (Life Technologies, Inc.). Fourteen
h after transfection, the cells were washed with PBS, fixed for 15 min
at room temperature in a solution containing 3% (w/v) paraformaldehyde
and 3% (w/v) sucrose in PBS, pH 7.5, washed again, and permeabilized
with 0.2% (v/v) Triton X-100 for 10 min. The cells were then washed,
blocked for 30 min in Hank's balanced solution containing 10% normal
donkey serum, and then incubated with the primary antibodies (1:100) in
blocking solution for 2 h. After washing with PBS, the cells were
incubated with Cy3-conjugated donkey anti-goat or Armenian hamster IgG
and/or fluorescein-conjugated donkey anti-mouse IgG (1:200). The
coverslips were washed with PBS, incubated overnight in PBS
supplemented with 0.02% sodium azide, and mounted in a solution
containing 90% glycerol, 10% Tris-HCl, pH 8.5, and 20 mg/ml
o-phenylenediamine as an anti-fading agent. To stain the endogenous CD120a (p55), HeLa cells were stimulated with 0.1 µM okadaic acid for 150 min, transfected with 100 ng of
DNA for 24 h, or left untreated. Cells were fixed and
permeabilized as above, before being blocked for 60 min in Hank's
balanced solution containing 5% normal donkey serum and 5% normal
goat serum. Cells were then incubated for 2 h with the primary
antibodies in blocking buffer (1:200 of anti-CD120a (p55) hamster
monoclonal antibody or 1:200 of H57 anti-T-cell receptor hamster
monoclonal antibody as a control). Cells were washed with PBS,
incubated for 1 h with Cy3-conjugated goat anti-hamster IgG (1:200
in blocking buffer), washed, and then incubated for 1 h with
Cy3-conjugated donkey anti-goat IgG (1:200 in blocking buffer). The
coverslips were washed, incubated overnight in PBS, and mounted. To
visualize the endoplasmic reticulum, cells were incubated with 5 µg/ml Alexa 488-conjugated concanavalin A (Molecular Probes, Eugene,
OR) together with the secondary antibodies. The specificity of the
secondary antibodies was checked to make sure they recognized only the
appropriate primary antibody. DNA was stained with the Hoechst dye
33342 at 10 µg/ml. Cells were observed with a Leica DMR/XA confocal
immunofluorescence microscope using a 100 × Plan objective.
Digital images were captured using a SensiCam camera, deconvoluted
using the software "Slidebook" (Intelligent Imaging Innovations,
Inc., Denver, CO) to remove out of focus fluorescence, and processed
using Adobe Photoshop 4.0 (Adobe Systems, Inc.).
Electron Microscopy--
HeLa cell monolayers grown on Thermanox
coverslips were fixed in 4% (w/v) paraformaldehyde and 0.05% (v/v)
glutaraldehyde, permeabilized in 0.2% (v/v) Triton X-100, and blocked
in 10% normal rabbit serum for 10 min at room temperature. The
coverslips were incubated with goat anti-CD120a (p55) (1 µg/ml in
blocking buffer) for 2 h at room temperature. After rinsing, bound
antibody was detected using the Vector ABC Elite kit following the
manufacturer's instructions. The coverslips were then post-stained
with osmium tetroxide and uranyl acetate, dehydrated with graded
solutions of ethanol, and embedded in plastic. Ultrathin sections were
cut on a Reichert ultratome and examined in a Philips CM10 electron microscope at 80 kV.
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RESULTS |
A Kinase Activity That Binds to and Phosphorylates
CD120a-(207-425) Co-elutes with p42mapk/erk2--
The TNF
receptor CD120a (p55) is expressed in low abundance on all cell types
studied, and macrophages are no exception (32). Therefore, to study the
phosphorylation of CD120a (p55) we fused residues 207-425 which encode
the entire intracellular domain of mouse CD120a (p55) to GST and
expressed the GST-CD120-(207-425) fusion protein in Escherichia
coli. The fusion protein was then bound to glutathione-Sepharose
beads and was used to affinity purify receptor-interacting proteins
from lysates of unstimulated and TNF-stimulated mouse macrophages.
Kinase(s) capable of phosphorylating GST-CD120a-(207-425) were then
detected in an in vitro kinase assay. As can be seen in
Figs. 2 and 3, incubation of mouse macrophages with TNF (10 ng/ml
for 10 min) led to the activation of a kinase activity that bound to
and trans-phosphorylated the GST-CD120a-(207-425) fusion
protein but not GST alone. The major phosphoprotein of ~51 kDa
co-localized with the GST-CD120a-(207-425) fusion protein. An
additional phosphoprotein of retarded mobility, believed to be a
multiply phosphorylated species of GST-CD120a-(207-425), was also detected.
To begin to explore the possibility that p38mapk,
p42mapk/erk2, or p46jnk1 contributed to the
phosphorylation of GST-CD120a-(207-425), we separated these MAPKs from
lysates of TNF-stimulated (10 ng/ml for 10 min) and unstimulated
macrophages by fast protein liquid chromatography over a Mono-Q
anion-exchange column and determined if CD120a-(207-425) kinase
activity co-eluted with either p38mapk, p42mapk/erk2,
or p46jnk1. As can be seen in Fig.
1B, three peaks of kinase
activity were detected in response to TNF stimulation, and only two
peaks were detected in lysates from unstimulated cells. The
TNF-inducible GST-CD120a-(207-425) kinase activity (peak 2)
co-eluted with phosphorylated p42mapk/erk2 as shown by
immunoblotting with an antibody that recognized both phosphorylated
p42mapk/erk2 and p44mapk/erk1 (Fig. 1D). It
can also be seen that p42mapk/erk2 eluted at approximately 0.17 M NaCl and was separated from p44mapk/erk1. Peak 1 kinase activity eluted at the beginning of the linear NaCl gradient and
did not co-localize with any of the MAPKs, whereas peak 3 kinase
activity eluted at the beginning of the isocratic flow of 1 M NaCl and co-eluted with p38mapk (and probably
other kinases) during this high salt wash.
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Fig. 1.
The
TNF-inducible kinase activity associated with
the GST-CD120a-(207-425) fusion protein co-elutes with
p42mapk/erk2. Proteins of lysates from unstimulated
cells (open circles) or cells stimulated with TNF (10 ng/ml for 10 min) (filled squares) were separated through a
Mono-Q column (A), and the fractions were assayed for kinase
activity using GST-CD120a-(207-425) beads as a substrate
(B), Western blotting with anti-p38mapk
(C), phosphospecific anti- p42mapk/erk2
(D), and anti-p46jnk1 antibodies (E).
FPLC, fast protein liquid chromatography.
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p42mapk/erk2 Phosphorylates GST-CD120a-(207-425) in
Vitro--
Based on the co-elution of CD120a-(207-425) kinase
activity and p42mapk/erk2, we next questioned if the
TNF-induced phosphorylation of GST-CD120a-(207-425) was dependent
upon p42mapk/erk2 by blocking its activation with a
pharmacologic antagonist of MEK activation, PD098059 (33). Macrophage
monolayers were pretreated with PD098059 (30 µM) for
1 h and then incubated with TNF (10 ng/ml) for 10 min in the
continued presence of the antagonist before being lysed and subjected
to an in vitro kinase assay using GST-CD120a-(207-425)-coated Sepharose beads as substrate. As can be
seen in Fig. 2A, pretreatment
with PD098059 completely inhibited the induction of
GST-CD120a-(207-425) kinase activity in response to TNF. In
contrast, pretreatment of macrophage monolayers with SB203580, a
competitive inhibitor of p38mapk, had no effect on the
phosphorylation of the GST-CD120a-(207-425) fusion protein. To verify
that the inhibition of GST-CD120a-(207-425) kinase activity by
PD098059 was not due to inhibition of other protein kinases that may
act upstream or downstream of p42mapk/erk2,
including phosphatidylinositol-3'-OH kinase or p70 S6 kinase, we
pretreated macrophages with wortmannin, an inhibitor of
phosphatidylinositol-3'-OH kinase, (34) and rapamycin, a specific
inhibitor of the p70 S6 kinase (35) prior to incubation with TNF.
Neither inhibitor had any effect on the phosphorylation of the
GST-CD120a-(207-425) fusion protein (Fig. 2A).
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Fig. 2.
A, effect of pharmacological antagonists
on the phosphorylation of the GST-CD120a-(207-425) fusion protein by
macrophage lysates. Macrophage monolayers were pretreated with the
MEK-1 inhibitor PD098059 (30 µM in dimethyl sulfoxide
(DMSO)), the phosphatidylinositol-3'-OH kinase
(PI3'K) inhibitor wortmannin (10 µM in
dimethyl sulfoxide), the p70 S6 kinase pathway inhibitor rapamycin (10 µg/ml in ethanol), the p38mapk competitive inhibitor SB203580
(1 µM in dimethyl sulfoxide), dimethyl sulfoxide, or
ethanol, and treated with TNF (10 ng/ml for 10 min). The cells were
lysed as described and lysates were subjected to an in vitro
kinase assay using GST-CD120a-(207-425) beads or control GST beads as
substrate. B, immunodepletion of p42mapk/erk2
suppresses the TNF-induced kinase activity associated with the
GST-CD120a-(207-425) fusion protein. The three MAPKs were
immunoprecipitated from detergent lysates of unstimulated or
TNF-stimulated (10 ng/ml, 10 min) macrophages using specific
antibodies. The residual kinase activity was measured in the
post-immunoprecipitation (Post-IP) lysates in an
in vitro kinase assay as described above. Pre-
(Pre-IP) and post-immunoprecipitation lysates
were subjected to Western blotting with specific anti-MAPK antibodies
as indicated.
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To establish directly a role for p42mapk/erk2 in the
phosphorylation of GST-CD120a-(207-425), p38mapk,
p42mapk/erk2, and p46jnk1 were immunoprecipitated from
detergent lysates of TNF-stimulated macrophages (10 ng/ml for 10 min), and residual GST-CD120-(207-425) kinase activity was quantified
in the post-immunoprecipitation supernatants. Immunodepletion with
anti-p42mapk/erk2 antibodies markedly, although incompletely,
removed p42mapk/erk2 even when combinations of polyclonal
antibodies were used (Fig. 2B, lower panel). Nevertheless,
the majority of the TNF-inducible kinase activity was depleted under
the conditions used (Fig. 2B, upper panel). Quantitative
immunodepletion was achieved with anti-p38mapk and
anti-p46jnk1 antibodies (Fig. 2B, lower panel) but
had no effect on the TNF-induced phosphorylation of the
GST-CD120a-(207-425) fusion protein (Fig. 2B, upper panel).
Conversely, and as shown in Fig.
3A, immunoprecipitated p42mapk/erk2, but not p38mapk or p46jnk1, was
found to efficiently phosphorylate the GST-CD120a-(207-425) fusion
protein in vitro, even though all three immunoprecipitated MAPKs were catalytically active, as illustrated by their ability to
phosphorylate ATF-2 (Fig. 3A). The ability of
p42mapk/erk2 to phosphorylate the GST-CD120a-(207-425) was
also confirmed using purified constitutively active recombinant
p42mapk/erk2 (Fig. 3B). We also conducted
experiments in which the constitutively active recombinant
p42mapk/erk2 was allowed to bind to the
GST-CD120a-(207-425)-coated beads. Unbound kinase was then removed by
extensively washing the beads before conducting the in vitro
kinase reaction. As can be seen in Fig. 3B, phosphorylation
of the GST-CD120a-(207-425) fusion protein was detected under these
conditions, albeit with somewhat reduced efficiency (~50%),
indicating that p42mapk/erk2 was bound to the
GST-CD120a-(207-425) fusion protein in the absence of adapter or other
signaling molecules.
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Fig. 3.
p42mapk/erk2 phosphorylates the
GST-CD120a-(207-425) fusion protein in vitro.
A, the three MAPKs were immunoprecipitated from detergent
lysates of TNF-stimulated macrophages using specific antibodies or
non-immune rabbit IgG as a control. An immune complex kinase assay was
performed, using either GST-CD120a-(207-425) beads or recombinant
ATF-2 as a substrate. B, the active form of the recombinant
p42mapk/erk2 was incubated with GST or GST-CD120a-(207-425)
beads; an in vitro kinase assay was performed directly or
after the beads were washed extensively (last lane,
*).
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Binding of p42mapk/erk2 to the GST-CD120a-(207-425) Fusion
Protein--
The purification of the receptor-associated kinase
activity initially involved affinity adsorption of receptor-interacting proteins to the GST-CD120a-(207-425) fusion protein, thus establishing that p42mapk/erk2 must bind to the fusion protein to be
detected. Given the findings described above, we next investigated the
ligand dependence and specificity of this interaction in lysates from
unstimulated and TNF-stimulated macrophages. Macrophage lysates were
incubated with GST-CD120a-(207-425) fusion protein-coated beads. The
beads were then washed, and the bound proteins were separated by
SDS-PAGE. Western blotting showed that both p42mapk/erk2 and
p44mapk/erk1, but not p38mapk or p46jnk1, were
bound to the GST-CD120a-(207-425) fusion protein (Fig. 4). Moreover, binding of
p42mapk/erk2 and p44mapk/erk1 was detected in lysates
from both unstimulated and TNF-stimulated macrophages suggesting
that both inactive and catalytically active members of the ERK family
of MAPKs were capable of interacting with the fusion protein. None of
the MAPKs were found to bind to GST-coated Sepharose beads.
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Fig. 4.
Binding of p42mapk/erk2 to the
GST-CD120a-(207-425) fusion protein. The soluble fraction of
detergent lysates from unstimulated or TNF-stimulated (10 ng/ml, 10 min) macrophages was incubated with GST or GST-CD120a-(207-425) beads.
After extensive washing of the beads, the bound proteins were
dissociated by boiling, separated by SDS-PAGE, transferred on
nitrocellulose, and detected by Western blotting using specific
anti-MAPK antibodies as indicated.
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Phosphorylation of GST-CD120a-(207-425) by Alternative
p42mapk/erk2 Activators--
Phosphorylation and activation of
p42mapk/erk2 have been described in response to a broad range
of stimuli including cytokines, growth factors, and, in the case of
macrophages, certain phagocytic stimuli (36). p38mapk and
p46jnk1 are activated under stress conditions and in response
to a variety of extracellular stimuli including bacterial
lipopolysaccharide, hyperosmolarity, as well as pro-inflammatory
cytokines (19, 37). We suspected that since p42mapk/erk2 is
activated by a variety of other cell surface receptors and activated
oncogenes, phosphorylation of CD120a (p55) would be expected to be
induced in a broader sense by other activators of p42mapk/erk2.
Such a finding would have important implications for the
cross-regulation of CD120a (p55) by other receptors and signal
transduction cascades. As can be seen in Fig.
5, exposure of mouse macrophages to the phagocytic stimulus, zymosan particles, stimulated the phosphorylation of the GST-CD120a-(207-425) fusion protein (Fig. 5A) and,
as expected, activated p42mapk/erk2, p38mapk, and
p46jnk1 (Fig. 5B). Furthermore, incubation of
macrophage monolayers with granulocyte macrophage-CSF and CSF-1 induced
the phosphorylation of the GST-CD120a-(207-425) fusion protein.
However, although these growth factors induced phosphorylation of
p42mapk/erk2, they failed to stimulate phosphorylation of
p38mapk or p46jnk1. In contrast, incubation of
macrophages with (i) hyperosmolar concentrations of sodium chloride or
sorbitol or (ii) anisomycin induced activation of p38mapk and
p46jnk1 but not p42mapk/erk2 (Fig. 5B).
However, these stress-inducing stimuli failed to induce the
phosphorylation of the GST-CD120a-(207-425) fusion protein (Fig.
5A). These findings thus indicate that the phosphorylation of sequences in the intracellular domain of CD120a (p55) by
p42mapk/erk2 occurs after activation of p42mapk/erk2 by
a variety of cell surface receptors and thus is not simply a response
to TNF.
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Fig. 5.
Factors that activate p42mapk/erk2,
but not factors that activate p38mapk or p46jnk1, can
induce the phosphorylation of the GST-CD120a-(207-425) fusion
protein. Macrophage monolayers were treated with TNF (10 ng/ml,
10 min), sodium chloride (200 mM, 10 min), sorbitol (400 mM, 10 min), anisomycin (10 µg/ml in dimethyl sulfoxide
(DMSO), 1 h), dimethyl sulfoxide (1 h), zymosan A (30 particles/cell, 30 min), or starved for CSF-1 for 18 h
(lanes 8-10) and then stimulated with
granulocyte-macrophage colony-stimulating factor (10 ng/ml, 10 min),
CSF-1 (1000 units/ml), or with a CSF-1-containing medium derived from
the culture of L929 cells (10 min). A, the soluble fraction
of the lysates was subjected to an in vitro kinase assay as
described above. B, the lysates were also separated by
SDS-PAGE, transferred on nitrocellulose, and Western-blotted with
phosphospecific anti-MAPK antibodies as indicated.
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Phosphorylation of CD120a (p55) p42mapk/erk2 in COS-7
Cells--
Collectively, the findings presented support the notion
that (i) p42mapk/erk2 is capable of binding to the
intracellular domain of CD120a (p55) and (ii) following activation,
p42mapk/erk2trans-phosphorylates sites within the
intracellular domain of CD120a (p55). However, since these conclusions
were based on data obtained using a fusion protein encoding the
intracellular domain of CD120a (p55), they raise the question of
whether p42mapk/erk2 is capable of phosphorylating CD120a (p55)
in vivo. To address this question, we employed a
transfection system in which COS-7 cells were co-transfected with
expression vectors encoding CD120a (p55), HA-tagged wild-type
p42mapk/erk2, and a constitutively active mutant of MEK-1 to
drive activation of the transfected p42mapk/erk2. Eighteen h
after transfection, the cells were labeled with
[32P]orthophosphate for 6 h and lysed as described
under "Experimental Procedures." CD120a (p55) was then
immunoprecipitated, separated by SDS-PAGE, electroblotted onto
nitrocellulose membranes, and subjected to autoradiography and
immunoblotting. As can be seen in Fig.
6B, immunoblotting with an
anti-CD120a (p55) antibody showed that CD120a (p55) was expressed at
similar levels following transfection into COS-7 cells. Fig.
6A shows that 32P-labeled CD120a (p55) was
detected in COS-7 cells that were co-transfected with vectors encoding
CD120a (p55), wild-type p42mapk/erk2, and constitutively active
MEK-1. Phosphorylation of CD120a (p55) was not detected in cells that
were co-transfected with CD120a (p55) and the wild-type
p42mapk/erk2 plasmids in the absence of the constitutively
active MEK-1 plasmid (Fig. 6A) nor was it detected in cells
transfected with CD120a (p55) and the constitutively active MEK-1
plasmid in the absence of p42mapk/erk2 (data not shown).
Immunoblotting of whole cell lysates obtained from the same
transfections with an anti-HA antibody showed that the HA-tagged
p42mapk/erk2 was expressed as predicted (Fig. 6C)
and underwent the characteristic mobility shift when COS-7 cells were
also transfected with the constitutively active MEK-1 (Fig.
6C). Interestingly, immunoblotting with anti-CD120a (p55)
antibodies also revealed a mobility shift in the receptor when cells
were co-transfected with expression plasmids encoding CD120a (p55),
p42mapk/erk2, and constitutively active MEK-1. The gel-shifted
CD120a (p55) detected in these immunoblots co-localized with the
32P-labeled receptor shown in Fig. 6A. These
findings demonstrate that activated p42mapk/erk2 is capable of
trans-phosphorylating the cytoplasmic domain of CD120a (p55)
in intact cells.
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Fig. 6.
CD120a (p55) is phosphorylated by
catalytically active p42mapk/erk2 in
vivo. A, COS-7 cells were transfected with
the indicated expression vectors, metabolically labeled with
[32P]orthophosphate and lysed, and CD120a (p55) was
immunoprecipitated. Immunoprecipitates were resolved by SDS-PAGE,
transferred to nitrocellulose, and subjected to autoradiography.
B, the immunoprecipitates were Western-blotted with
anti-CD120a (p55) antibodies. C, the lysates were separated
by SDS-PAGE, transferred onto nitrocellulose, and subjected to Western
blotting with an anti-HA antibody. P-CD120a (p55) and
P-p42mapk/erk2 represent the gel-shifted position of
phosphorylated forms of CD120a (p55) and p42mapk/erk2,
respectively.
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Effect of Phosphorylation by p42mapk/erk2 on the
Subcellular Localization of the CD120a (p55)--
The finding that
p42mapk/erk2 phosphorylates CD120a (p55) both in
vitro and in intact cells raises the question of the functional
consequences of this event. Phosphorylation of receptors modifies their
functions in many ways including altering their subcellular
localization. To address this issue we studied the effect of
phosphorylation of CD120a (p55) on the subcellular localization of the
receptor in HeLa cells transfected with expression vectors encoding
CD120a (p55). Fourteen h after transfection, the cells were fixed,
permeabilized, stained for CD120a (p55), and observed by confocal
fluorescence microscopy. As shown in Fig.
7 (A1-2), transfected cells
consistently showed a punctate and membranous staining of the receptor
as previously reported (58) as well as a localized juxtanuclear
staining. To ensure that the punctate staining of CD120a (p55) was
localized on the plasma membrane, transfected cells were stained for
CD120a (p55) in the absence of detergent permeabilization. CD120a (p55) was still present as a peripheral staining on the plasma membrane (Fig.
7, A3), although tubulin, used as a control to establish the
absence of permeabilization, was not stained (data not shown). We
suspected that the juxtanuclear staining may represent CD120a (p55)
associated with the Golgi apparatus as previously reported (38). When
transfected HeLa cells were co-stained for CD120a (p55) and the Golgi
marker AP-1, the localized juxtanuclear staining of CD120a (p55)
co-localized with AP-1 (Fig. 7, A4-6), confirming that a
significant amount of the receptor is present in the Golgi apparatus.
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Fig. 7.
Phosphorylation of CD120a (p55) by
p42mapk/erk2 abolishes its plasma membrane and Golgi
localization. HeLa cells were transfected as indicated and
observed by confocal fluorescence microscopy. Text in
italics refers to staining, and roman text refers
to transfection. Green represents the staining of CD120a
(p55), red the staining of MEK-1 (A-G3) or AP-1
(A-G5, A-G6), blue the staining of nuclei by
the Hoechst dye 33342. A, localization of CD120a (p55) at
the plasma membrane and in the Golgi apparatus. B and
C, localization of CD120a (p55) in perinuclear and
cytoplasmic curvilinear structures in cells co-transfected with CD120a
(p55), p42mapk/erk2, and constitutively active MEK-1.
D, localization of CD120a (p55) T236A, S240A, S244A, and
S270A mutant (p55.4A) at the plasma membrane and in the
Golgi apparatus. E, co-transfection of p42mapk/erk2
and constitutively active MEK-1 does not alter the localization of
CD120a (p55) T236A, S240A, S244A, and S270A mutant (p55.4A).
F and G, localization of CD120a (p55) to
curvilinear structures is reproduced by the CD120a (p55) T236D, S240E,
S244E, and S270E mutant (p55.4D/E). The arrow in
G3 indicates a junction between the nuclear membrane and the
curvilinear structures. The results shown are representative of six
different experiments.
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To determine if phosphorylation of CD120a (p55) altered its subcellular
localization, HeLa cells were co-transfected with expression vectors
encoding CD120a (p55), wild-type p42mapk/erk2, and a
constitutively active mutant of MEK-1 and stained for CD120a (p55) and
MEK-1. As can be seen in Fig. 7 (B1-3 and
C1-3), the punctate membranous expression of CD120a (p55)
had disappeared in cells expressing both CD120a (p55) and MEK-1, and
the receptor was consistently present in curvilinear tubular
structures, which predominated in the perinuclear region of these
cells. These structures were not stained in non-permeabilized cells,
indicating their intracellular location (data not shown) and did not
co-localize with the Golgi marker AP-1 (Fig. 7,
B4-6 and C4-6). In addition, the same
pattern of staining was seen when the cells were fixed with methanol
instead of paraformaldehyde excluding the possibility of a fixation artifact.
These data suggest that the phosphorylation of CD120a (p55) by
p42mapk/erk2 alters its subcellular localization from the
plasma membrane and the Golgi apparatus to intracytoplasmic tubular
structures. The cytoplasmic domain of CD120a (p55) contains four amino
acid sequences that conform to the ERK consensus phosphorylation motif PX1,2(S/T)P. To rule out the possibility that
the change in the subcellular localization of CD120a (p55) observed
when co-transfecting the kinases may be due to an indirect effect of
the activation of p42mapk/erk2 and not to the phosphorylation
of the receptor per se, the four potential ERK
phosphorylation sites were mutated to alanine residues (CD120a
(p55)T236A, S240A, S244A, and S270A or "p55 4A"). Transfection of
the 4-alanine mutant receptor into HeLa cells resulted in a pattern of
localization that was similar to the wild-type receptor (Fig. 7,
D1-6; D3 shows cells that were not permeabilized). However, co-transfection of the 4-alanine mutant receptor along with
p42mapk/erk2 and constitutively active MEK-1 dramatically
reduced the expression of the curvilinear tubular structures as
compared with the wild-type receptor (Fig. 7, E1-6). In
contrast, when the four potential ERK phosphorylation sites were
mutated to Asp and Glu residues to mimic the phosphorylated Ser and Thr
residues (CD120a (p55)T236E, S240D, S244D, and S270D or "p55
4D/E"), the mutant receptor showed a similar curvilinear tubular
intracellular localization when transfected in the absence of kinases
(Fig. 7, F1-3 and G1-3) to that seen when the
wild-type receptor was co-transfected with constitutively active MEK
and p42mapk/erk2. In addition, the p55 4D/E mutant receptor was
not expressed at the plasma membrane and did not co-localize with the
Golgi marker AP-1 (Fig. 7, F4-6 and G4-6).
To establish the identity of the organelle(s) containing or associated
with the phosphorylated receptor, HeLa cells transfected with the p55
4D/E mutant were stained for CD120a (p55) itself and with various dyes
and markers specific for the following: (i) the Golgi apparatus (AP-1,
wheat germ agglutinin); (ii) actin filaments (phalloidin); (iii)
mitochondria (Mitotracker orange); (iv) endosomes
(fluorescein-conjugated dextran); (v) lysosomes (antibody for cathepsin
D); (vi) vimentin intermediate filaments (anti-vimentin antibodies);
(vii) microtubules (antibody for -tubulin); (viii) stable
microtubules (antibody for acetyl-tubulin or treatment for 1 h
with nocodazole and staining for -tubulin); (ix) the plasma membrane
(wheat germ agglutinin); (x) DNA (Hoechst dye 33342); and (xi) kinesin
(anti-kinesin antibodies). As shown in Fig.
8, none of these organelles and
structures co-localized with the mutated receptor. However, the
endoplasmic reticulum, stained with Alexa 488-conjugated concanavalin
A, encompassed curvilinear structures that partially co-localized with
the p55 4D/E mutant receptor (Fig. 9,
A-H). To investigate further the possible association of
the phosphorylated receptor with elements of the endoplasmic reticulum,
HeLa cells were co-transfected with expression vectors encoding the p55
4D/E receptor and the vesicular stomatitis virus glycoprotein bearing a
COOH-terminal KKTN endoplasmic reticulum targeting motif (VSV-G). As
shown in Fig. 9 (I-P), the mutated receptor partially
co-localized with the VSV-G protein, demonstrating that the
intracellular curvilinear and perinuclear tubular structures in which
the receptor is present represent elements of the endoplasmic reticulum. As can also be seen in Fig. 9, the elements of the endoplasmic reticulum containing the p55 D/E receptor appeared swollen
compared with areas of the endoplasmic reticulum from which the
receptor was absent. However, we should emphasize that the
phosphorylated receptor was localized in certain areas of the
endoplasmic reticulum rather than being uniformly distributed throughout the entire endoplasmic reticulum. Thus, much of the endoplasmic reticulum does not contain the phosphorylated receptor, but
most of the phosphorylated receptor is associated with the endoplasmic
reticulum. To confirm further the association of the phosphorylated
receptor with the endoplasmic reticulum, we transfected HeLa cells with
the p55 D/E mutant and subjected the fixed and permeabilized cells to
immunochemistry for CD120a (p55) at the electron microscope level. As
can be seen in Fig. 10, abundant peroxidase reaction product was detected in structures that bear morphologic identity to the rough endoplasmic reticulum, thus confirming the presence of the phosphorylated receptor in elements of
the endoplasmic reticulum. Taken together, these data suggest that the
phosphorylation of CD120a (p55) by p42mapk/erk2 abolishes both
its plasma membrane and Golgi localization and targets the receptor to
tubular elements of the endoplasmic reticulum.
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Fig. 8.
Absence of co-localization of CD120a (p55) to
cell organelles. HeLa cells were transfected with the p55 D/E
mutant TNF receptor to mimic the effects of phosphorylation at the ERK
consensus sites. The cells were then stained with dyes and
representative markers for different intracellular compartments. Text
in italics refers to staining. Green represents
the staining of CD120a (p55); blue represents the staining
of nuclei by the Hoechst dye 33342. Red represents the
staining of the plasma membrane and Golgi apparatus with fluorescein
isothiocyanate-conjugated wheat germ agglutinin (A);
mitochondria with Mitotracker orange (B); the Golgi
apparatus with anti-adaptin-1 antibody (C); endosomes with
lysine fixable FITC-dextran 70 kDa (D); lysosomes with
anti-cathepsin D antibody (E); actin fibers with
tetramethylrhodamine isothiocyanate phalloidin (F);
microtubules with anti--tubulin antibody (G); stable
microtubules with antibody for acetyl-tubulin (H); vimentin
intermediate filaments with anti-vimentin antibody (I); and
kinesin with specific antibody (J). None of the above
organelles colocalized with the cytoplasmic tubular structures
associated with CD120a (p55) 4D/E mutant.
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Fig. 9.
The curvilinear structures to which the
phosphorylated CD120a (p55) is redistributed are part of the
endoplasmic reticulum. HeLa cells were transfected with expression
vectors encoding the CD120a (p55) T236D, S240E, S244E, and S270E mutant
(p55.4D/E) alone (A-H) or together with
VSV-G-KKTN (I-P) and observed by confocal fluorescence
microscopy. Text in italics refers to staining.
Green represents the staining of CD120a (p55);
red represents the staining of the endoplasmic reticulum by
concanavalin A (A-H) or VSV-G (I-P);
yellow, the overlay of the stainings for CD120a (p55) and
endoplasmic reticulum; and blue represents the staining of
nuclei by the Hoechst dye 33342. I-P, co-localization of
the p55.4D/E mutant and the endoplasmic reticulum marker concanavalin
A. I-P, co-localization of the p55.4D/E mutant and the
endoplasmic reticulum marker VSV-G-KKTN. The results shown are
representative of three different experiments.
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Fig. 10.
Immunocytochemical localization of the p554
D/E mutant at the electron microscope level. HeLa cells were
transfected with the p554 D/E mutant, fixed and permeabilized, and
stained with goat polyclonal anti-CD120a (p55). Bound antibody was
detected with biotinylated secondary antibody and
streptavidin-horseradish peroxidase. The bar represents 0.4 µm. CD120a (p55) was detected within ribosome-bound tubular
structures.
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Phosphorylation and Subcellular Redistribution of CD120a (p55) in
Intact Cells--
To establish the physiological relevance of these
observations, we investigated if endogenous CD120a (p55) is
phosphorylated in intact macrophages upon ERK activation and the effect
of phosphorylation on the redistribution of the receptor to
intracellular tubular structures. Given the rapid and transient nature
of MAPK activation in response to stimulation with TNF (27-29) and
the low level of endogenous CD120a (p55) expression, we chose to use a
stimulus that resulted in sustained ERK activation, namely okadaic
acid, a previously reported TNF mimic (59). Bone marrow-derived
mouse macrophages were labeled with [32P]orthophosphate
for 6 h, stimulated with 1 µM okadaic acid for the
last 3 h of labeling, and lysed as described under "Experimental Procedures." CD120a (p55) was immunoprecipitated, separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and subjected to autoradiography. As shown in Fig.
11, a low level of basal
phosphorylation of CD120a (p55) was detected in unstimulated cells. In
contrast, treatment of cells with okadaic acid induced a dramatic
increase in the 32P-labeling of CD120a (p55) and a
subsequent gel mobility shift of the phosphorylated receptor. As a
control, cell lysates were separated by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with antibodies specific for the
phosphorylated form of ERKs, confirming the activation of ERKs in cells
treated with okadaic acid. These findings demonstrate that CD120a (p55)
is phosphorylated in intact macrophages following activation of
p42mapk/erk2.
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Fig. 11.
Phosphorylation of the endogenous CD120a
(p55) in intact macrophages. A, bone marrow-derived
mouse macrophages were labeled with [32P]orthophosphate
for 6 h, stimulated with 1 µM okadaic acid for the
last 3 h of labeling (lane 3), and lysed. CD120a (p55)
was immunoprecipitated, separated by SDS-PAGE, transferred onto
nitrocellulose, and subjected to autoradiography (lanes 2 and 3). The H57 anti-T-cell receptor hamster monoclonal
antibody was used as a control for the immunoprecipitation (lane
1). The arrow indicates basal phosphorylation of CD120a
(p55); the arrow indicates the gel mobility shift of
32P-labeled CD120a (p55). As a control, COS cells were
transfected with CD120a (p55), and the lysate was separated by
SDS-PAGE, transferred onto nitrocellulose, and Western-blotted with
antibodies specific for CD120a (p55) (lane 4). B,
the cell lysates were also separated by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with phosphospecific anti-ERK
antibodies.
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To determine if activation of p42mapk/erk2 results in the
subcellular redistribution of endogenous CD120a (p55) in intact cells,
HeLa cells were treated with okadaic acid for 150 min, fixed,
permeabilized, stained for the endogenous CD120a (p55) as described
under "Experimental Procedures," and observed by confocal
immunofluorescence microscopy. As shown in Fig.
12, untreated cells exhibited a
punctate and membranous staining of the receptor similar to that seen
in CD120a (p55) transfected cells, although the level of staining was
less intense. In contrast, cells treated with okadaic acid showed a
redistribution of the receptor to intracellular tubular structures.
These tubules were not stained in unpermeabilized cells (data not
shown), confirming their intracellular localization. In addition,
staining of the receptor in cytoplasmic tubular structures was also
observed when staining the endogenous CD120a (p55) in HeLa cells
transfected with an expression vector encoding a constitutively active
mutant of MEK-1 to drive activation of endogenous p42mapk/erk2
(Fig. 12). Thus, activation of p42mapk/erk2 in intact cells
leads to the redistribution of the endogenous CD120a (p55) from the
plasma membrane to intracellular tubular structures.
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Fig. 12.
Subcellular redistribution of the endogenous
CD120a (p55) to tubular structures in intact cells. HeLa cells
were left untreated (A-D), transfected with an expression
vector encoding a constitutively active mutant of MEK-1 (E
and F), or stimulated with 0.1 µM okadaic acid
for 150 min (G and H) before being fixed,
permeabilized, stained for endogenous CD120a (p55) (C-H) as
described under "Experimental Procedures," and observed by confocal
fluorescence microscopy. The H57 anti-T-cell receptor hamster
monoclonal antibody was used as a control for the staining
(A and B).
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DISCUSSION |
Although the ability of cytosolic Ser/Thr protein kinase
activities to interact with sequences present in the cytoplasmic domain
of CD120a (p55) has been recognized for several years, their identity
until now has remained enigmatic. By using both primary cultures of
mouse macrophages, and a transfection approach in COS-7 and HeLa cells,
the major findings of the work reported herein are that one of these
kinases is p42mapk/erk2 and that the phosphorylation of CD120a
(p55) alters the subcellular distribution of the receptor from the
plasma membrane and Golgi apparatus to curvilinear tubular structures
that by both confocal and electron microscopy appear to be associated
with elements of the endoplasmic reticulum. These findings thus provide
the following: (i) the first demonstration of inducible phosphorylation of CD120a (p55) in an intact cell system, (ii) defines one of the
responsible kinases, and (iii) identifies a novel functional consequence of this event.
Several approaches were used to establish that the inducible
GST-CD120a-(207-425) kinase present in mouse macrophages was p42mapk/erk2. First, inducible kinase activity co-eluted with
p42mapk/erk2, but not with other MAPKs including
p38mapk, p46jnk1, or p44mapk/erk1. Second,
pharmacologic antagonism of p42mapk/erk2 activation with
PD098059 or specific immunodepletion of p42mapk/erk2
substantially inhibited the phosphorylation of GST-CD120a-(207-425) by
lysates from stimulated macrophages, whereas depletion or pharmacologic inhibition of other related kinases was without effect. Third, immunoprecipitated catalytically active or recombinant constitutively active p42mapk/erk2 were able to trans-phosphorylate
the GST-CD120a-(207-425) fusion protein in an in vitro
kinase assay. These findings therefore suggest that
p42mapk/erk2, in the absence of other adapter or signaling
molecules, is able to interact directly with and phosphorylate
sequences present in the cytoplasmic domain of CD120a (p55). Although
the site of phosphorylation between residues 207-425 was not fully
defined in this study, the cytoplasmic domain does contain four amino acid sequences that conform to the ERK consensus phosphorylation motif
PX1,2-(S/T)P. Three sites, Thr-236, Ser-240, and
Ser-244, are clustered in a Pro-rich region between residues 234 and
245, whereas a fourth site is centered at Ser-270. Significantly, only Ser-244 is conserved within the sequence FSP in all species for which
sequence data are available and may therefore represent a candidate
site of phosphorylation by p42mapk/erk2.
Binding assays using GST-CD120a-(207-425) as an affinity matrix
established that p42mapk/erk2 and p44mapk/erk1 were
capable of physically interacting with the cytoplasmic domain of CD120a
(p55). In addition, we have also detected an interaction between
p42mapk/erk2 and the cytoplasmic domain of CD120a (p55) in
yeast two-hybrid studies.2
These findings are also consistent with previously reported studies in
other receptor systems. For example, Avery and Dupont (39) have
reported that p42mapk/erk2 co-precipitates with the T-cell
receptor complex; and David and colleagues (40) have shown
p42mapk/erk2 to be associated with the interferon
/-receptor and to participate in IFN/-dependent
signal transduction.
Of the previously described kinase activities capable of
phosphorylating residues in the cytoplasmic domain of CD120a (p55), p60
TRAK has been the most thoroughly characterized. p60 TRAK, however,
appears distinct from p42mapk/erk2 based on several criteria.
First, substrate specificity experiments using affinity purified p60
TRAK have shown a preference for histone H1 and casein but not for the
ERK substrate, myelin basic protein (12). Second, the estimated
molecular mass of p60 TRAK of 52 kDa (12) is inconsistent with the
molecular mass of p42mapk/erk2. Third, p60 TRAK directly or
indirectly interacts with residues 344-397 of human CD120a (p55), a
region that is not required for the interaction with
p42mapk/erk2.3 Other
kinases capable of phosphorylating sequences within the cytoplasmic
domain of CD120a (p55) include pp60src and possibly other as
yet to be defined tyrosine kinases that phosphorylate Tyr-331 (41).
Phosphorylation events positively and negatively regulate the functions
of many receptors. For example, phosphorylation of Ser and Thr residues
in the cytoplasmic domains of the G-protein-coupled formyl-methionyl-leucyl-phenylalanine and CXCR4 receptors has been
shown to promote receptor internalization (42, 43). In addition,
protein kinase C has been shown to phosphorylate the epidermal growth
factor receptor at Thr-654, thereby inhibiting the ability of the
receptor to stimulate cellular proliferation in the presence of ligand
(44). In contrast, phosphorylation of the estrogen receptor by
p42mapk/erk2 has been shown to be required to
trans-activate fully estrogen-responsive promoters (45), and
the requirement for Tyr phosphorylation of receptors to enable
recruitment of SH2-domain containing signaling molecules is now
accepted dogma (46, 47).
The results of the present study show that phosphorylation of CD120a
(p55) by p42mapk/erk2 induces a striking alteration in the
subcellular localization of the receptor in which expression at the
plasma membrane and Golgi apparatus is lost, whereas expression within
cytoplasmic curvilinear tubular structures is increased. The tubular
structures in which the phosphorylated receptor is present were shown
to represent distinct elements of the endoplasmic reticulum by three separate methods, namely colocalization with (i) concanavalin A, (ii)
VSV-G-KKTN, and (iii) immunolocalization at the electron microscopy
level. These findings appeared to be a direct effect of phosphorylation
of the receptor itself (as opposed to a change in localization induced
by effects of activated p42mapk/erk2 on other substrates) since
when all four ERK consensus sequences in the cytoplasmic domain were
mutated to Ala, the redistribution of the receptor was largely
prevented. Conversely, when the four ERK consensus sequences were
mutated to Asp and Glu to mimic the charge change induced by
phosphorylation of Ser and Thr residues, the receptor localized to the
curvilinear tubular structures in the absence of activated
p42mapk/erk2. We also showed that the endogenous CD120a (p55)
in mouse macrophages is phosphorylated upon sustained activation of
p42mapk/erk2 and that the endogenous receptor is translocated
from the plasma membrane to the curvilinear tubular structures under
the same conditions of stimulation in HeLa cells. These findings thus
indicate that neither the punctate distribution of the receptor nor the formation of tubular structures are artifacts introduced as a consequence of transient overexpression of CD120a (p55).
Although the spectrum of events affected by the phosphorylation of
CD120a (p55) by p42mapk/erk2 remain to be fully defined,
phosphorylation may serve a role in down-regulating receptor function
by promoting its translocation from the plasma membrane and Golgi
apparatus to the tubular structures. Events analogous to this have been
reported for the autocrine motility factor receptor which traffics
between plasma membrane caveoli and the endoplasmic reticulum in
NIH-3T3 cells (48). Alternatively (or in addition), receptor
phosphorylation may prevent newly synthesized CD120a (p55) from
trafficking from the endoplasmic reticulum to the Golgi apparatus and
subsequently to the plasma membrane. In either situation
(i.e. retention in elements of the endoplasmic reticulum or
trafficking from the plasma membrane to the endoplasmic reticulum),
phosphorylation of the receptor may represent a mechanism to prevent
cell surface expression and subsequent signaling functions. This
conclusion is intriguing given our current finding that activation of
p42mapk/erk2 by a variety of receptor-ligand interactions
results in phosphorylation of GST-CD120a-(207-425). Many receptors
undergo homologous and/or heterologous desensitization upon ligand
binding or following activation of a panoply of kinases and other
signaling molecules (49). In the case of G-protein-coupled receptors
including those for - and -adrenergic stimuli, glucagon-like
peptide-1, and the rhodopsin photoreceptor, desensitization has been
shown to be associated with phosphorylation of Ser- and Ser/Thr-rich
sequences thereby enabling the binding of -arrestin which
subsequently interferes with the ability of the receptor to activate
the appropriate heterotrimeric G-protein (50, 51). Thus, it is
conceivable that activation of p42mapk/erk2 may promote
heterologous desensitization of CD120a (p55) through retrograde
translocation of the receptor to cytoplasmic curvilinear tubular structures.
An additional and possibly related function of phosphorylation of the
cytoplasmic domain of CD120a (p55) by p42mapk/erk2 may be to
recruit additional proteins that promote the accumulation of the
receptor within the curvilinear tubular structures. There are few
examples of signaling proteins that are capable of interacting with
phosphoserine or phosphothreonine although 14-3-3 proteins are a clear
example (52). Alternatively, phosphorylation by p42mapk/erk2
may alter the conformation of the cytoplasmic domain of CD120a (p55) to
reveal novel protein interaction motifs. It is also possible that the
receptor-associated p42mapk/erk2 may serve to phosphorylate
other proteins that directly or indirectly become recruited to CD120a
(p55) and regulate the trafficking of the receptor. It is noteworthy
that whereas the death domain region of the receptor has been shown to
be required for many downstream functions initiated by ligation of the
TNF receptor, the membrane proximal region, which contains the site of
phosphorylation by p42mapk/erk2, is also required for the
expression of nitric oxide synthase (30) although the mechanism of
involvement of this region remains unknown.
What happens to the receptor after its accumulation within the
curvilinear structures remains unknown. However, the endoplasmic reticulum is known to participate in the degradation of several receptors including the T-cell receptor (53). Recent evidence suggests
that these and other receptors probably exit the endoplasmic reticulum
through the Sec61c retrograde transporter (54) and undergo degradation
in the cytosol in a proteasome-dependent fashion. An
additional functional consequence of the phosphorylation of CD120a
(p55) may therefore be to promote its ubiquitination. Other studies
have also shown that when Bcl-2 is expressed in the endoplasmic reticulum it prevents apoptosis of rat-1/Myc fibroblasts following serum deprivation (55). In addition, expression of Bcl-2 in the
endoplasmic reticulum has been shown to assist in the assembly of a
putative apoptotic signaling complex containing caspase 8 (56).
Intriguingly, filamentous cytoplasmic structures (called death-effector
filaments) are formed by transfected FADD, an adapter molecule that is
part of the apoptosis-signaling complex assembled after engagement of
the Fas receptor, as well as by the death-effector domain containing
prodomain of procaspase-8 (57). These death-effector filaments recruit
procaspase-8, and their formation is associated with induction of
apoptosis in Jurkat cells. Thus, perhaps the formation of tubular
structures by the phosphorylated TNF receptor CD120a (p55) may also
serve a physiologic function distinct from its potential for receptor
degradation, such as recruitment of an apoptotic signaling complex.
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ABSTRACT
INTRODUCTION
