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* Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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heavy chain locus
and to induce synthesis of IgE (14, 15).
Structurally, CD40 and mIg are very different receptors that associate
with distinct signal transduction molecules in the plasma membrane. The
BCR associates with Ig
and Igß chains that contain tyrosine
activation motifs (TAMs) that are phosphorylated upon mIg cross-linking
(16, 17, 18, 19). Src family tyrosine kinases and Syk associate with the mIg
receptor complex, are activated upon mIg cross-linking, and mediate the
association and activation of other effector molecules in the mIg
receptor complex (16, 20, 21, 22, 23, 24, 25, 26). The role of tyrosine kinases in CD40
signal transduction is controversial. Marked increases in tyrosine
phosphorylation of cellular substrates following CD40 ligation has been
reported in some systems, but not in others (27, 28, 29, 30, 31). CD40 is a member
of the TNF receptor family and associates with novel signal
transduction molecules termed TNF receptor-associated factors (TRAFs)
(32, 33, 34). TRAF molecules contain zinc ring and zinc finger-like domains
and in some cases an additional isoleucine zipper domain. It is
important to note that TRAFs do not contain TAMs that would enable
effector molecules containing SH2 domains to associate with the CD40
receptor complex. Truncated forms of TRAF molecules lacking the
ring/finger/zipper domains inhibit CD40 signaling when overexpressed in
cells, indicating that the zinc-containing structures and the
isoleucine zipper are important for transduction of CD40 signals.
TRAF-2, -3, -5, and -6 have been implicated in coupling of CD40 to
NF-
B (35, 36), and TRAF2 may also couple CD40 to the Jun kinase
(JNK) pathway (37).
Despite structural differences between mIg- and CD40-associated
molecules, these receptors signal through some of the same signaling
intermediates. Membrane Ig and CD40 activate a similar array of
trans-acting factors, including NF-B, NF-AT, and
activating protein-1 (38, 39, 40). Early reports suggested that mIg and
CD40 were specifically coupled to the ERK and JNK (MAPK) pathways,
respectively (41, 42, 43). However, subsequent studies revealed that
signals through either CD40 or mIg can activate both ERK and JNK in
murine B cells, suggesting that differential coupling to these pathways
is quantitative rather than qualitative (31, 44). Although both CD40
and mIg activate NF-B and MAP kinases, selective inhibition of mIg
responses by depletion of protein kinase C suggests that these
receptors use distinct mechanisms for coupling to the same signal
transduction pathway (31, 38, 45). Selective inhibition of mIg-induced
DNA synthesis by elevation of cAMP also suggested utilization of
distinct signaling pathways by CD40 and mIg in the regulation of B cell
function (8, 46). In other receptor systems, elevation of cAMP inhibits
receptor-mediated activation of the ERK pathway (47, 48, 49, 50, 51, 52). In this study
we examined coupling of CD40 to the ERK pathway and found that in
contrast to mIg, activation of ERK by CD40 involves a novel signaling
pathway that is insensitive to PK-A.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Affinity-purified Abs were obtained from the following sources:
anti-IgM plus IgD heavy and light chain (Fab')2,
anti-IgM, Fc fragment, Jackson ImmunoResearch Laboratories (West
Grove, PA); anti-murine CD40 (hamster IgM), PharMingen (San Diego,
CA); polyclonal rabbit anti-Raf-1 (C-12; sc-133), MEK-1 (C-18;
sc-219), JNK1 (sc-571), and polyclonal rabbit or goat ERK (C-14;
sc-154-G), Santa Cruz Biotech (Santa Cruz, CA); anti-human SHC and
anti-phosphotyrosine (4G10), Upstate Biotechnology, Inc. (Lake
Placid, NY). Murine CD40L was derived from culture supernatants of
J558L cells transfected with a plasmid encoding an mCD40L-CD8 fusion
protein, provided by Dr. Peter Lane (Basel, Switzerland) (53). J558L
transfectants were cultured at 8 x 105/ml in
Iscove’s modified Dulbecco’s medium, 1% FCS, 2 mg/ml G418 (Life
Technologies, Gaithersburg, MD), and antibiotics. Supernatants were
harvested after 24 h and extensively dialyzed against PBS followed
by RPMI 1640 (Sigma Chemical Co., St. Louis, MO). Control supernatants
were derived from untransfected J558L cells cultured under similar
conditions in the absence of G418. PMSF was obtained from Sigma
Chemical Co. (St. Louis, MO). Pefabloc SC
(4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride), aprotinin,
leupeptin, pepstatin, and bestatin were purchased from Boehringer
Mannheim (Indianapolis, IN). PMA and myelin basic protein (MBP) for
in-gel kinase assays were purchased from Sigma Chemical Co. Ultrapure
MBP for immune complex kinase assays was purchased from Upstate
Biotechnology. Activators of adenlylate cyclase, including cholera
toxin, forskolin (6-O-[3'-piperidino)propionyl]
hydrochloride), the inactive analogue, forskolin (1.9-dideoxy-),
dibutyryl cAMP, and the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine were purchased from Calbiochem (La
Jolla, CA).
Cells
The murine B lymphoma line, WEHI-231, was maintained in RPMI 1640 and 5% FCS/5% CS, supplemented with 50 µM 2-ME, 2 mM glutamine, and antibiotics. The WEHI-231 cell line used in these studies exhibited anti-Ig-induced growth arrest followed by apoptosis and rescue from apoptosis by concomitant signaling through CD40 (54). Normal B cells were isolated from spleens obtained from C57BL/6, C57BL/10, or BALB/c mice (8–20 wk of age) and depleted of T cells by incubation at 4°C with a mixture of anti-T cell Abs followed by complement-mediated lysis at 37°C (55). Small, resting B cells were obtained from T-depleted spleen cells by either centrifugal elutriation or centrifugation through discontinuous Percoll gradients as described previously (56, 57). Cells were washed three times with serum-free medium (RPMI 1640 supplemented with 1% BSA (fraction V), 50 µM 2-ME, 2 mM glutamine, and antibiotics) to remove serum-containing protease activity. After washing, cells were resuspended in serum-free medium and equilibrated to 37°C for 15 to 30 min before stimulation.
Cell lysates
For immune precipitation of SHC, cells were lysed by the addition of 4x modified RIPA buffer (final concentration of 1x RIPA buffer: 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 1 mM sodium vanadate, 1 mM PMSF or Peflabloc SC, and 1 µg/ml of aprotinin, leupeptin, and pepstatin). For immune precipitation of ERK, MEK-1, and Raf-1, cells were lysed by addition of 1% Triton X-100, 150 mM NaCl, 10 mM Tris (pH 7.4), 400 µM EDTA, 1 mM PMSF, or Peflabloc SC, 2 µg/ml of aprotinin, leupeptin, and pepstatin, 1 mM benzamidine, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 400 µM sodium vanadate, and 20 mM ß-glycerophosphate. For in-gel kinase assays, cells were lysed in whole cell extract buffer (WCE buffer: 25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 20 mM ß-glycerophosphate, 0.1 mM sodium vanadate, 1 mM PMSF, and 2 µg/ml of aprotinin, leupeptin, and pepstatin). Cell suspensions were incubated on ice for 30 min and then clarified by centrifugation at 15,000 x g for 10 min.
In-gel kinase assay
Activity of MAP kinases in SDS-PAGE gels was determined as
described by Kameshita and Fujisawa with minor modifications (58).
Polyacrylamide gels were cast with 0.5 mg/ml MBP (Sigma) added before
gel polymerization. Following electrophoresis of cell lysates (10–50
µg total protein), SDS was removed by rocking the gel in 20%
isopropanol and 50 mM Tris-HCl, pH 8.0 (three times, 100 ml each time,
1-h total wash time), followed by 50 mM Tris-HCl, pH 8.0, and 1 mM DTT.
Proteins in the gel were denatured by incubation in 100 ml of 6 M
guanidine-HCl (Ultrapure, Life Technologies), 20 mM DTT, 2 mM EDTA, and
50 mM Tris-HCl, pH 8.0, for 1 h. Proteins were renatured by
incubation at 4°C, without agitation, in 250 ml of 50 mM Tris-HCl, 1
mM DTT, 2 mM EDTA, and 0.04% Tween 20 for 12 to 18 h. Kinase
assays were performed by equilibrating the gel in 100 ml of kinase
buffer (1 mM DTT, 0.1 mM EGTA, 20 mM MgCl2, 40 mM
HEPES (pH 8.0), and 100 µM sodium vanadate) for 30 min, followed by
incubation at room temperature in 10 to 25 ml kinase buffer
supplemented with 50 µM ATP and [
-32P]ATP (5–10
µCi/ml, 3000 Ci/mmol; DuPont-New England Nuclear, Boston, MA) for an
additional 30 min. The gels were washed extensively in 5% (w/v) TCA
plus 1% sodium pyrophosphate (Sigma). Autoradiography of dried gels
was performed using Kodak X-OMAT film (Eastman Kodak, Rochester,
NY).
Immune precipitation and Western blotting
Cell lysates (1 ml) were precleared by rotation with either
protein A- or protein G-Sepharose (50 µl of 50% slurry) for 
2 h
at 4°C. In some experiments lysates were precleared with the
combination of 1 µg/ml nonspecific rabbit IgG and protein
A-Sepharose. Polyclonal rabbit Abs specific for SHC (5 µg/ml), Raf-1
(1 µg/ml), and MEK-1 (1 µg/ml) or a polyclonal goat Ab specific for
ERK (2 µg/ml) were added to cell lysates along with protein
A-Sepharose (50 µl of 50%) for immune precipitations using rabbit
Abs or protein G-Sepharose for immune precipitations with goat Abs and
rotated for 4 to 16 h at 4°C. Beads were washed three times with
lysis buffer followed by either a wash with kinase buffer (see below)
in preparation for immune complex kinase assays or boiling following
the addition of an equal volume of 2x sample buffer (final
concentrations: 50 mM Tris (pH 6.8), 100 mM DTT, 2% SDS, 0.1%
bromophenol blue, and 10% glycerol) in preparation for Western
blotting. Boiled samples were resolved by SDS-PAGE (8–10%
polyacrylamide gels). Gels were equilibrated in transfer buffer (10 mM
CAPS (pH 11.0) and 10% methanol) and transferred (400 mA, for 2
h) to Immobilon-P (polyvinylidene difluoride) membranes (Millipore,
Bedford, MA) using the Panther (HEP-1) semidry electroblotter (Owl
Scientific, Cambridge, MA). Membranes were blocked by incubation
without agitation in 1% BSA/PBS/0.05% sodium azide overnight at
4°C. Membranes were incubated with (0.5–1 µg/ml) biotin-conjugated
anti-phosphotyrosine (4G10), anti-SHC (0.5 µg/ml),
anti-Raf-1 (0.25–0.5 µg/ml), or anti-ERK (1 µg/ml) in
PBS/0.05% Tween 20 for 2 to 4 h at room temperature. Polyclonal
rabbit primary Abs were detected by 2- to 4-h incubation at room
temperature with a 1/10,000 dilution of biotin-conjugated donkey
anti-rabbit secondary Ab (Jackson ImmunoResearch, West Grove, PA).
Incubation with biotin-conjugated Ab was followed by 0.5 to 1 h at
room temperature incubation with (1/1000) streptavidin-horseradish
peroxidase conjugate (Amersham, Arlington Heights, IL). All incubations
were followed by five 100-ml washes with PBS/0.05% Tween 20 over a
period of 30 min. Western blots were visualized by incubation with ECL
(Amersham) or Renaissance chemiluminescence reagents (DuPont-New
England Nuclear) according to the protocols recommended by the
manufacturer followed by exposure (30–120 s) to Reflection film
(DuPont-New England Nuclear).
Immune complex kinase assays
Enzyme-Ab complexes were washed once with 1 ml of kinase buffer
(Raf-1 buffer: 10 mM PIPES (pH 7.0), 100 mM NaCl, 5 mM
MnCl2, and 2 µg/ml aprotonin; ERK/MEK-1 buffer: 25
mM HEPES (pH 7.4), 1 mM DTT, and 10 mM MgCl2). Kinase
reactions were performed by addition of 30 µl of kinase buffer
supplemented with substrate (ERK substrate: 5–10 µg MBP (Upstate
Biotechnology); Raf-1 substrate: 100–200 ng/ml GST-MEK-1 K97R (gift
from K.-L. Guan, University of Michigan); MEK-1 substrate: 100–200 ng
His-ERK (p42; sc-4024; Santa Cruz Biotechnology) plus 5–10 µg MBP
(Upstate Biotechnology), 25–50 µM ATP, and 5 µCi
[-32P]ATP (3000 Ci/mmol)), and the reaction mixtures
were incubated at 30°C for 30 min. Kinase reactions were terminated
by the addition of 2x sample buffer and incubation at 100°C for 5
min. Substrates were resolved in SDS-PAGE gels (10% MBP and 8%
GST-MEK-1), and incorporation of 32P into substrates was
determined by autoradiography of dried gels using Reflection
(DuPont-New England Nuclear) or Kodak X-OMAT film. Exposure time ranged
from 0.5 to 4 h for ERK and MEK-1 assays and from 24 to 48 h
with two intensifying screens for Raf-1 assays. Densitometric analysis
of files generated by scanning autoradiographs (HP Scanjet II,
Hewlett-Packard, Palo Alto, CA) was performed using National Institutes
of Health Image 1.54 software (Bethesda, MD).
Solid phase Jun kinase assays
Activity of JNK in cell lysates was determined as described by
Hibi et al. (59). Briefly, cell lysates prepared using WCE buffer were
adjusted to a binding buffer composition of 20 mM HEPES (pH 7.7), 75 mM
NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100,
0.5 mM DTT, 20 mM ß-glycerophosphate, 0.1 mM sodium vanadate, 1 mM
PMSF, and 2 µg/ml of aprotinin, leupeptin, and pepstatin.
GST-Jun1–223 bound to glutathione-agarose beads (Sigma)
was incubated with cell lysates for 3 to 16 h. Beads were washed
three times with binding buffer (20 mM HEPES (pH 7.7), 75 mM NaCl, 2.5
mM MgCl2, 0.1 mM EDTA, and 0.05% Triton X-100). The
relative amount of JNK enzyme bound to GST-Jun agarose beads for each
sample was determined by Western blotting using polyclonal
anti-JNK1 (Santa Cruz Biotechnology). Jun kinase activity was
determined by incubating beads in 30 µl of kinase buffer (20 mM HEPES
(pH 7.6), 20 mM MgCl2, 2 mM DTT, 20 mM
ß-glycerophosphate, and 0.1 mM sodium vanadate) supplemented with 25
µM ATP and 5 µCi [-32P]ATP for 30 min at 30°C.
Reactions were terminated by the addition of 2x Laemmli sample buffer
and resolved on 8 to 10% SDS-PAGE gels. Incorporation of
32P-labeled phosphate into GST-Jun was quantitated by
autoradiography using Reflection film (DuPont-New England Nuclear).
Exposure time ranged from 0.5 to 4 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Whether CD40 effectively couples to the ERK pathway in B
lymphocytes remains controversial (31, 41, 42, 43, 44). To determine whether
regulation of the ERK pathway is associated with a mitogenic response
to CD40, the activation of ERK was examined in both the WEHI-231 B
lymphoma and normal mouse B cells. Although signals through CD40 are
mitogenic for normal mouse B cells (53), spontaneous cell cycle
progression in WEHI-231 cells is unaffected by CD40 (54). The activity
of ERK2 was induced up to fivefold in splenic B cells
15 min after treatment with anti-CD40 (Fig. 1
A). Levels of
ERK2 activity stimulated by cross-linking of CD40 were
consistently higher than those induced by 1 to 15 µg/ml of
anti-Ig (Figs. 1 and 2). In WEHI-231 cells, anti-CD40 failed to
stimulate ERK2 activity over the time course examined
(5–60 min), while cross-linking of mIg was an effective stimulus for
ERK2 activation, with maximal ERK2 activity
occurring 15 to 30 min after addition of anti-Ig (Fig. 1B). In an earlier study Kashiwada et al. reported
that CD40 cross-linking induced rapid and transient activation of ERK
(within 1–2 min following addition of anti-CD40) in WEHI-231 cells
(44). An examination of ERK activity at points between 30 s and 5
min after addition of anti-CD40 failed to reveal CD40 coupling to
the ERK pathway in the WEHI-231 cells used in this study (data not
shown). In contrast, activation of ERK2 in WEHI-231 cells
was observed as early as 30 s to 1 min following addition of
anti-Ig (data not shown). These WEHI-231 cells do respond to
signals through CD40, since anti-CD40 treatment induces marked
activation of jun kinases (Fig. 3C) and
prevented mIg-induced growth arrest in these cells (data not shown).
These data suggest that coupling of CD40 to the ERK pathway is
associated with a mitogenic response in B lymphocytes. In addition,
these results indicate that transformed B cell lines are not always
suitable models for studying signal transduction in normal B cells.
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Treatment of B lymphocytes with agents that elevate cAMP
selectively inhibits DNA synthesis stimulated by signals through mIg,
but not through CD40 (8, 46), suggesting that PK-A activation
differentially inhibits signal transduction by mIg and CD40. The
effects of agents that elevate cAMP on ERK2 activation
by CD40 was examined to determine whether CD40 regulates
ERK2 through a PK-A-sensitive pathway. As shown in Figure 2
, A, B,
C, and E, treatment of normal B cells with either
cholera toxin or dibutyryl cAMP (0.1–1 mM) inhibited ERK2
activation by two- to threefold in response to stimulation with optimal
concentrations of anti-Ig (5–15 µg/ml). In contrast,
ERK2 activation induced by anti-CD40 was unaffected by
treatment with agents that elevate cAMP in cells, as neither cholera
toxin nor dibutyryl cAMP (Fig. 2, A, B,
D, and F) inhibited the response to
suboptimal concentrations of anti-CD40 (0.1 µg/ml). Similar
results were obtained with forskolin-treated cells (not shown). Thus,
CD40 regulates ERK2 activity through a PK-A-insensitive
pathway.
Elevation of cAMP inhibits Raf-1 and subsequent ERK, but not JNK, activation by mIg
To define the mechanism(s) by which elevation of cAMP modulates
signals through mIg, the effect of cholera toxin on mIg coupling to the
ERK pathway was examined. Pretreatment of WEHI-231 cells with cholera
toxin inhibited activation of Raf-1 and ERK by mIg cross-linking (Fig. 3, A and B,
respectively). Similar results were obtained with forskolin (data not
shown). Consistent with results presented in Figure 1, treatment of B
lymphoma cells with either soluble CD40L (Fig. 3B) or
anti-CD40 (Fig. 3A) failed to induce Raf-1 and
ERK activation in the presence or the absence of cholera toxin. In two
independent experiments, treatment of WEHI-231 cells with either
cholera toxin or forskolin did not affect activation of JNK by either
mIg or CD40 (Fig. 3C), demonstrating that elevation
of cAMP does not affect the MEK kinase(s) that functions in the Jun
kinase pathway. Forskolin had no effect on induction of SHC
phosphorylation by anti-Ig, indicating that the effects of cAMP lie
distal to recruitment and tyrosine phosphorylation of SHC (data not
shown). Together these results suggest that inhibition of mIg-induced
DNA synthesis by agents that elevate cAMP may be explained at least in
part by inhibition of the ERK pathway at the level of Raf-1. To date,
efforts to measure Raf-1 activation in normal B cells have failed to
reveal receptor regulation of this enzyme.
Signals through either mIg or CD40 activate MEK-1
The results presented in Figures 2 and 3 indicate that CD40
regulates ERK activity through a Raf-1-independent pathway. In response
to signals from a variety of receptors, activation of ERK is mediated
by dual phosphorylation on tyrosine and threonine residues by MEK-1
(Fig. 6). To determine whether MEK-1 functions in the pathway regulated
by CD40, the effect of receptor cross-linking on MEK-1 activity was
examined. Treatment of normal B cells with either anti-CD40 or
anti-Ig stimulated MEK-1 activity two- to threefold (Fig. 4). Interestingly, MEK-1 activation by
cross-linking of CD40 was delayed relative to the mIg response. MEK-1
activation was induced 10 min following addition of anti-CD40, and
activity further increased by 15 min. Activation of
ERK2 by signals through CD40 exhibited similar
kinetics (data not shown). In contrast, activation of MEK-1 was maximal
2 min following addition of anti-Ig and declined after 10 min.
Interestingly, ERK2 activity in cells treated with
anti-Ig declined only slightly after 15 min, indicating that
ERK2 activation is maintained for several minutes following
down-regulation of MEK-1 activity. (not shown). These data suggest that
regulation of ERK by signals through either receptor is mediated by
MEK-1, and thus, MEK-1 is the point of convergence between CD40 and mIg
signaling pathways leading to ERK activation.
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Differences in the kinetics of MEK-1 activation suggests that mIg
and CD40 couple to the ERK pathway through distinct mechanisms.
Cross-linking of mIg results in the recruitment of SHC to
phosphorylated TAM sequences within the Ig and Igß chains (60).
Subsequent phosphorylation of SHC on tyrosine residues enables
formation of the SHC-GRB2-SOS complex and activation of Ras through
SOS-mediated guanine nucleotide exchange (61, 62, 63, 64). Whether CD40 uses
SHC as an adapter molecule to couple to the ERK pathway was examined by
monitoring tyrosine phosphorylation of SHC in response to treatment of
cells with anti-CD40. As shown in Figure 5, SHC was phosphorylated on tyrosine
following treatment of splenic B cells with anti-Ig, but not with
anti-CD40. A 130- to 150-kDa phosphoprotein coprecipitated with SHC
from cells treated with anti-Ig, but not with those treated with
anti-CD40. In anti-Ig-treated cells phosphorylation of SHC was
maximal by 5 min, and SHC remained phosphorylated 15 min after addition
of anti-Ig (data not shown). In contrast, SHC was not
phosphorylated on tyrosine 15 min following treatment with
anti-CD40, at which time ERK2 activation by
signals through CD40 were maximal. The results presented in Figure 5
are representative of at least three independent experiments. These
results indicate that CD40 uses adapter proteins other than SHC to
couple to the ERK pathway.
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Discussion |
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B, NF-AT, and
activating protein-1 (39, 40, 45, 65). Receptor-specific regulation of
trans-acting factors has been reported, and as studies of
CD40 and mIg signal transduction continue, more differences are likely
to be revealed (66, 67). Both receptors activate MAP kinases, including
ERK, JNK, and p38 (31, 41, 42, 43, 44). Differential activation of MAP kinases
by signals through mIg and CD40 in B cells appears to be more a
function of the cellular differentiation state than an intrinsic
property of the receptor. Thus, while CD40 is not effectively coupled
to the ERK pathway in transformed B cell lines, CD40 cross-linking
stimulates ERK2 activation in normal, murine B cells (Fig. 1). Although both mIg and CD40 activate ERK, Jun, and p38 kinases,
differences in the kinetics and level of activation suggest distinct
mechanisms of coupling to these pathways.
Characterization of the effects of cAMP elevation on MAP kinase
activation revealed that CD40 and the BCR activate ERK2
through different pathways that converge at MEK-1 (Fig. 6). Both CD40 and mIg induce MEK-1
activation in normal B cells (Fig. 4). A specific inhibitor of MEK-1,
PD98059, blocks ERK2 activation by signals through either
receptor, indicating that MEK-1 activity is required for
ERK2 activation by signals through CD40 or mIg (31).
Cross-linking of mIg induced Raf-1 activity in WEHI-231 cells, and
elevation of cAMP inhibited Raf-1 activation by this receptor (Fig. 3A). The latter result is consistent with reports
demonstrating that phosphorylation of Raf-1 by PK-A inhibits the MEK
kinase activity of Raf-1 (47, 48, 49, 50, 51, 52). Although induction of Raf-1 activity
by cross-linking mIg was not observed in normal B lymphocytes,
elevation of cAMP inhibited ERK2 activation by signals
through mIg (Fig. 2). The inability to measure Raf-1 activity in normal
B cells might be explained by a PK-A-sensitive MEK kinase other than
Raf-1 that functions in these cells; however, since Raf-1 activation
involves dimerization and undefined interactions following
Ras-dependent recruitment to the plasma membrane, immune complex kinase
assays may not detect Raf-1 activity in normal B cells (68, 69, 70, 71). Taken
together, these results indicate that in normal B cells activation of
MEK-1 by mIg is mediated by a PK-A-sensitive MEK kinase that is most
likely Raf-1.
The evidence presented here indicates that CD40 uses a MEK-1 kinase
activity other than Raf-1 in the regulation of ERK (Fig. 6). Elevation
of cAMP did not affect ERK2 activation by signals through
CD40, demonstrating that the CD40 pathway involves a PK-A-insensitive
MEK kinase (Fig. 2). The identity of the enzyme that regulates MEK-1
activity in the CD40 pathway remains to be established. One candidate
enzyme is MEKK-1, a murine homologue of Byr2 and Ste11 kinases in
yeast. Overexpression of MEKK-1 in cells induces activation of both ERK
and JNK; however, at lower levels of expression, MEKK-1 selectively
activates JNK (72, 73). In macrophages, activation of ERK by TNF-
correlates with increases in MEKK-1, but not in Raf-1, activity (74).
Although cross-linking of CD40 is a very effective stimulus for JNK
activation in B cells, receptor signals only slightly increase MEKK-1
activity in Ramos cells, an EBV-transformed B cell line (41).
Furthermore, cross-linking of CD40 does not affect MEKK-1 activity in
WEHI-231 cells under conditions that activate Jun kinases (J. M.
Purkerson et al., unpublished observations). Although elevation of cAMP
in PC12 cells inhibits MEKK-1 activation by epidermal growth factor,
nerve growth factor, and tetradecanoyl phorbol acetate (75), elevation
of cAMP does not inhibit JNK activation by CD40 (Fig. 3C). Thus, preliminary efforts to correlate MAPK
activation with increases in MEKK-1 activity have failed to reveal a
role for MEKK-1 in ERK or JNK activation by CD40. B-Raf is another
candidate for a MEK kinase that is regulated by CD40. In PC12 cells
B-Raf activity is positively regulated by elevation of cAMP, and B-Raf
mediates activation of the ERK pathway by cAMP agonists (76). However,
elevation of cAMP alone was not sufficient to activate MAP kinase
pathways in mouse B cells (Figs. 2 and 3), suggesting differences
between PC12 cells and B cells with respect to coupling of PK-A to the
ERK pathway. Interestingly, the CD40-associated molecule, TRAF-2, binds
a kinase, termed NIK, that is related to mammalian and yeast MEK
kinases (77). It is intriguing to speculate that CD40 is directly
coupled to NIK through TRAF-2 and that NIK mediates activation of
ERK2 through phosphorylation of MEK-1. The role of NIK and
other MEK kinases in the regulation of the MAP kinase pathway by CD40
is currently under investigation.
The function of guanine nucleotide binding proteins, such as Ras, in MAP kinase pathways is to recruit MEK kinases to the plasma membrane, where the kinase is activated via an undefined mechanism (68, 78). CD40 triggers guanine nucleotide exchange by Ras and Rac1 in an EBV-transformed B cell line, and expression of a dominant inhibitory mutant of Ras blocks MAP kinase activation by signals through CD40 (79). Since Ras mediates activation of MEK kinases other than Raf-1 (75), the results presented in this study are not inconsistent with Ras-dependent coupling of CD40 to a PK-A insensitive MEK kinase. A direct examination of Ras function in the ERK pathway of normal mouse B cells is under way to confirm the results obtained in human B cell lines.
CD40 and mIg use distinct adapter molecules to couple to GTP binding
proteins in the MAP kinase pathway. As depicted in Figure 6, cross-linking of mIg promotes association of SHC with the Ig and
Igß chains and the formation of the SHC-GRB2-SOS complex that
triggers guanine nucleotide exchange by Ras (61, 62). Signals through
CD40 did not induce tyrosine phosphorylation of SHC, suggesting that
CD40 does not use SHC as an adapter molecule to couple to the ERK
pathway (Fig. 5). Although signals through CD40 induce phosphorylation
of SOS, SOS phosphorylation is subsequent to ERK activation and results
in feedback inhibition of the MAP kinase pathway (31, 49). Thus,
whether SOS functions as a guanine nucleotide exchange factor in the
ERK pathway regulated by CD40 remains to be established. Furthermore,
coupling of CD40 to MAP kinase pathways may involve adapter molecules
whose association with the CD40 receptor complex is not regulated by
tyrosine phosphorylation. In contrast to the Ig and Igß chains
that interact with the BCR, TRAF molecules that associate with CD40 do
not contain TAMs. TRAF-2 has been implicated in coupling of TNF
receptor 1 to the JNK pathway (80, 81). Whether TRAF-2 directly or
indirectly mediates association with GRB2/SOS or other adapter/guanine
nucleotide exchange factors, thereby enabling coupling of CD40 to the
ERK pathway, remains to be established.
Perhaps the most significant finding of this study is that CD40
regulates ERK through a PK-A-insensitive pathway. The absence of
PK-A-mediated inhibition of mitogenic signal transduction through CD40
is significant because signals through receptors that couple to
adenylate cyclase frequently enhance B cell responses to CD40 ligation.
Elevation of cAMP synergizes with CD40 for induction of B7-1 and B7-2
expression (82, 83). In addition, agonists for adenylate cyclase
promote Ig isotype production regulated by CD40 in human B lymphocytes.
ß2 adrenergic receptor agonists facilitate isotype switch
to IgE stimulated by CD40 and IL-4 in cultures of human PBMC (84).
PGE2 or agents that elevate cAMP enhance proliferation of
purified tonsillar B cells signaled through CD40 and promote the
secretion of IgM, IgG, and IgA induced by IL-10 in conjunction with
CD40, but inhibit IL-4-induced switching to IgG and IgE (85). Thus,
agonists for adenylate cyclase can either promote or inhibit responses
regulated by CD40 depending on the cellular and cytokine context in
which cAMP synthesis is induced. Whether activation of adenylate
cyclase is a direct result of CD40 ligation is unclear. Synthesis of
cAMP in B cells treated with membrane preparations from activated T
cells has been observed in some laboratories (86, 87), but not in
others (88). Interestingly, inhibitors of PK-A block DNA synthesis
stimulated by membranes from activated T cells (87) and abrogate
NF-B dependent activation of the HIV-1 long terminal repeat induced
by signals through CD40 (89). However, a direct effect of CD40
ligation on the regulation of adenylate cyclase activity has not been
reported. Since ERK activation plays a central role in the transmission
of mitogenic signals to the nucleus (90, 91), regulation of ERK
activity through a cAMP-insensitive pathway enables agonists for
adenylate cyclase to act in concert with signals through CD40 without
inhibiting mitogenic signal transduction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeff Purkerson, Department of Molecular Microbiology and Immunology, L220, Oregon Health Sciences University, Portland, OR 97201.
3 Abbreviations used in this paper: BCR, B cell Ag receptor; mIg, membrane-bound Ig; TAM, tyrosine activation motif; TRAF, tumor necrosis factor receptor-associated factor; NF-B, nuclear factor-B; JNK, Jun N-terminal kinase; ERK, extracellularly regulated kinase; MAPK, mitogen activated protein kinase; CD40L, CD40 ligand; MBP, myelin basic protein; GST, glutathione S-transferase; PK-A, protein kinase A.
Received for publication May 29, 1997. Accepted for publication November 6, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 DNA rearrangement in B cells activated with dextran-anti-IgD antibodies and interleukin 4: a three component model for Ig class switching. J. Exp. Med. 178:1577.2 upon crosslinking of membrane Ig on murine B lymphocytes. J. Immunol. 148:3021.[Abstract]
and Ig-ß with distinct cytoplasmic effectors. Science 258:123.2. J. Exp. Med. 179:673.B activation. Genes Dev. 10:963.B by TNF receptor 2 and CD40. Science 269:1424.B activation prevents cell death. Cell 87:565.[Medline]
B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. Int. Immunol. 7:151.B in B cells. J. Exp. Med. 177:1215. rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages. Proc. Natl. Acad. Sci. USA 92:1614.B induction by TNF, CD95 and IL-1. Nature 385:540.[Medline]
B activation prevents cell death. Cell 87:565.
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