From the Toronto Western Research Institute, Cell and
Molecular Biology Division and the Department of Immunology, University
of Toronto, Toronto, Ontario M5T 2S8, Canada and the
§ Herbert Irving Comprehensive Cancer Center, Columbia
University, New York, New York 10032
Received for publication, May 1, 2001
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ABSTRACT |
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Expression of the COOH-terminal residues 179-330
of the LSP1 protein in the LSP1+ B-cell line W10
increases anti-IgM- or ionomycin-induced apoptosis, suggesting that
expression of this LSP1 truncate (B-LSP1) interferes with a
Ca2+-dependent step in anti-IgM signaling. Here
we show that inhibition of Ca2+-dependent
conventional protein kinase C (cPKC) isoforms with Gö6976
increases anti-IgM-induced apoptosis of W10 cells and that expression
of B-LSP1 inhibits translocation of PKC Many mouse and human B-lymphoma cell lines are susceptible to
anti-IgM-induced apoptosis (1-6). Multiple
mIgM1-coupled signal
transduction pathways such as increased
[Ca2+]i, and production of
ceramides and reactive oxygen species mediate the apoptotic effect of
mIgM stimulation (7-9). However, anti-IgM treatment also activates
potentially anti-apoptotic signaling pathways such as activation of
phosphatidylinositol 3-kinase and its downstream target Akt/PKB or
activation of PKC (10-12). Thus, the outcome of anti-IgM signaling
depends on a balance of pro-apoptotic and anti-apoptotic signals.
Activation and translocation of PKC by phorbol ester protects normal
immature and mature mouse B-lymphocytes, the mouse B-lymphoma cell line
WEHI-231, human B-lymphoma cell lines and human B-CLL cells from
anti-IgM-induced apoptosis (1, 4, 13, 14). The precise PKC isoform
involved in protection from anti-IgM-induced apoptosis is not yet
known. Evidence for a role of one or more of the
Ca2+-dependent cPKC isoforms The mouse leukocyte-specific protein 1 (LSP1) is a 330-amino acid
residue intracellular protein expressed in B- and T-lymphocytes and in
macrophages and neutrophils (15-19). Transfection experiments using
the LSP1+ B-lymphoma cell line WEHI-231/89 or a single cell
subclone, W10, showed that expression of an LSP1 truncate containing
residues 179-330 (designated B-LSP1) significantly increased the
extent of apoptosis induced by anti-IgM or by ionomycin but not by
sorbitol, nocodazole, C2-ceramide, or
H2O2 (20). Expression of B-LSP1 had no effect
on anti-IgM-induced growth arrest. Consistent with a role of LSP1 in
the early induction phase of apoptosis, we found that, after anti-IgM
treatment, the number of cells showing loss of mitochondrial membrane
potential ( Cell Culture and Apoptosis Measurements--
Cells were cultured
in RPMI 1640 medium as described (20). The W10 cell line is a single
cell subclone of the B-lymphoma cell line WEHI-231/89, and the TW10.1
cell line is a stable G418 resistant transfectant derived from W10
cells expressing the LSP1 truncate B-LSP1 containing LSP1 residues
179-330 (20). To overexpress PKC Cell Fractionation and Western Blotting--
For PKC
translocation experiments, W10 or TW10.1 cells were washed twice in
Hank's buffered saline solution (HBSS) and resuspended at 25 × 106 cells/ml in HBSS. Three aliquots of 1 ml in
microcentrifuge tubes were then warmed at 37 °C for 10 min in a
water bath. Goat anti-mouse IgM was added to a final concentration of
50 µg/ml to two tubes, and incubation at 37 °C was then continued.
Cells from the third tube served as the unstimulated controls and were
recovered by centrifugation for 30 s in a microcentrifuge, washed
once in cold HBSS, and stored on ice. Anti-IgM-stimulated cells were
recovered after 5 or 20 min of incubation. To prepare plasma membrane
fractions, cells were resuspended for 30 min in cold hypotonic
fractionation buffer (Buffer A: 5 mM Tris-HCl, pH 7.4, 5 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA) containing a mixture of protease inhibitors (22, 23) and then disrupted by 50 strokes in a small Potter homogenizer. The
lysates were cleared by centrifugation for 5 min at 500 × g. Supernatants were layered on top of 1.2 ml of 1.2 M sucrose in buffer A and spun at 10,000 × g for 15 min to remove the majority of mitochondria. The
lysates were then centrifuged at 100,000 × g for 60 min. The high speed pellet was designated as the plasma membrane
fraction and was solubilized in buffer A, while the supernatant was
designated as the cytoplasmic fraction. Protein concentrations were
determined according to the Bradford method using reagents from Bio-Rad
(Oakville, Ontario, Canada).
Equal amounts of protein were separated on 12.5% SDS-polyacrylamide
gels, transferred to polyvinylidene difluoride membranes, and analyzed
by using antibodies specific for PKC Activation of ERK2--
Cells (25 × 106
cells/ml) were warmed to 37 °C for 10 min and then stimulated with
50 µg/ml anti-IgM with or without pretreatment with 0.25 µM Gö6976 or 25 µM PD098059 for 15 min. Cells were harvested at different times after addition of
anti-IgM, pelleted for 30 s in a microcentrifuge, lysed in 1×
sample buffer, and immersed in boiling water for 3 min. Phosphorylation
of ERK2 was then analyzed on polyacrylamide gels with an
acrylamide:bisacrylamide ratio of 118:1 (24), followed by Western
analysis with anti-ERK2 antibodies (Santa Cruz Biotechnology Inc.,
catalog no. sc-154).
ERK2 kinase activity was measured in an in vitro immune
kinase assay. Cells were harvested as above and resuspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.2 mM
NaVO3, 1 mM dithiothreitol, 10 µg/ml
aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). After incubation on ice
for 15 min, the insoluble material was pelleted for 15 min at 13,000 rpm in a microcentrifuge and ERK2 was precipitated from the soluble
lysates by addition of 5 µg of anti-ERK2 antibody followed by
incubation at 4 °C. After 1 h, 20 µl of protein G-agarose
slurry (Pierce) was added and incubation was continued for an
additional 30 min. Protein G-agarose beads were recovered by
centrifugation, washed three times in lysis buffer, and washed once in
kinase buffer (20 mM HEPES, pH 7.2, 5 mM
MgCl2, 1 mM EGTA). To measure kinase activity,
beads were resuspended in 30 µl of kinase buffer supplemented with 2 mM sodium vanadate, 5 mM LSP1/PKC Interactions--
To probe for LSP1/PKC interactions in
cell lysates, 4 × 107 W10 cells were lysed in lysis
buffer (20 mM Tris, pH 7.5, 0.15 M NaCl, 5 mM EDTA) containing 0.5% Nonidet P-40 and a mixture of
protease inhibitors as described (22, 23) and incubated with 1-2 µg
of a GST-LSP1 fusion protein containing the intact LSP1 (residues
1-330). After 1 h of incubation at 4 °C, 10 µl of a 1:1
slurry of glutathione-Sepharose beads (Sigma-Aldrich) was added and
incubated for an additional 30 min at 4 °C. The beads were recovered
by brief centrifugation in a microcentrifuge and washed five times with
1 ml of lysis buffer + 0.5% Nonidet P-40, and the recovered proteins
were analyzed by Western blotting. To determine binding of LSP1 with
PKC Inhibition of cPKC Isoforms Increases Anti-IgM-induced
Apoptosis--
Treatment of mature B-cells with a cPKC-specific
inhibitor renders these cells susceptible to anti-IgM-induced
apoptosis, suggesting that anti-IgM-activated cPKC plays a role in the
protection of B-cells from anti-IgM-induced cell death (13). To
determine whether activated cPKC has a similar role in the B-lymphoma
cell line W10, we treated these cells with anti-IgM in the presence or
absence of Gö6976, a PKC inhibitor that preferentially inhibits the cPKC isoforms Expression of LSP1 Residues 179-330 Inhibits Translocation of
PKC
To determine whether the increased anti-IgM-induced apoptosis of
TW10.1 cells is due to the inhibition of PKC Inhibition of PKC
Anti-IgM stimulation activates ERK2 in a PKC-independent manner through
the RAS/Raf-1/MEK1 pathway (12, 32, 33) and in a
PKC-dependent manner through activation of Raf-1 (34-36). Different PKC isoforms can contribute to Raf-1 activation when overexpressed in COS cells (37), but the specific PKC isoform involved
in anti-IgM-induced Raf-1/MEK1/ERK2 activation in B-cells is not yet
known. Fig. 3A shows that
treatment of W10 cells with 0.25 µM Gö6976 or with
25 µM PD098059 significantly inhibits anti-IgM-induced
ERK2 activation as measured by the appearance of phosphorylated ERK2,
which has a slightly lower mobility on SDS-PAGE than the
non-phosphorylated form of ERK2. These results confirm that, in W10
cells, activated cPKC isoforms contribute to the activation of ERK2 and
that activation of ERK2 depends on active MEK1. It is interesting to
note that the effect of Gö6976 is more pronounced after 20 min
than after 2 min of anti-IgM stimulation, whereas the MEK1 inhibitor
PD098059 acts equally well at both time points. Increasing the
pre-incubation time for Gö6976 to 35 min gave a similar result
(data not shown). This suggests that the early activation of MEK1/ERK2
by anti-IgM stimulation is less dependent on activation of cPKC,
whereas at later times activated cPKC contributes significantly to ERK2
activation. The early activation of ERK2 activation may be more
dependent on activated RAS.
We determined the involvement of PKC PKC
We do not as yet know why expression of LSP1 residues 179-330 in the
LSP1+ TW10.1 cells inhibits translocation of PKC
The study of PKC isoform-specific functions is hampered by the lack of
isoform-specific activators or inhibitors. Our data suggest that B-LSP1
is a unique reagent that specifically inhibits the anti-IgM-induced
activation of PKCI but not of PKC
II or
PKC
to the plasma membrane. The increased anti-IgM-induced apoptosis
is partially reversed by overexpression of PKC
I. This shows that the
B-LSP1-mediated inhibition of PKC
I leads to increased anti-IgM-induced apoptosis. Expression of constitutively active PKC
I
protein in W10 cells activates the mitogen-activated protein kinase
ERK2, whereas expression of B-LSP1 inhibits anti-IgM-induced activation
of ERK2, suggesting that anti-IgM-activated PKC
I is involved in the
activation of ERK2 and that inhibition of ERK2 activation contributes
to the increased anti-IgM-induced apoptosis. Pull-down assays show that
LSP1 interacts with PKC
I but not with PKC
II or PKC
in W10 cell
lysates, while in vitro LSP1 and B-LSP1 bind directly to
PKC
I. Thus, B-LSP1 is a unique reagent that binds PKC
I and
inhibits anti-IgM-induced PKC
I translocation, leading to inhibition
of ERK2 activation and increased apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
I,
II,
or
in this protection comes from experiments showing that the
protective action of phorbol esters on immature B-lymphocytes (13)
is abrogated by a cPKC-specific inhibitor. This inhibitor also renders
mature B-lymphocytes susceptible to anti-IgM-induced apoptosis (13),
suggesting that anti-IgM-induced activation of cPKC isoforms plays an
important role in regulating susceptibility of different B-lymphocyte
lineage cells to anti-IgM-induced apoptosis.
m) increased faster in the B-LSP1
transfectant than in the parental cells (20). From these experiments we
concluded that LSP1 regulates an early step in the induction of
anti-IgM-mediated apoptosis, downstream of the anti-IgM-induced
increase in [Ca2+]i, but upstream
of the loss of
m. Given the protective role of the
Ca2+-dependent cPKC isoforms in
anti-IgM-induced apoptosis, we asked whether the expression of B-LSP1
inhibits anti-IgM-induced translocation of cPKC isoforms and, if so,
whether this increases anti-IgM-induced apoptosis by inhibiting the
cPKC-regulated activation of the MAP kinase ERK2.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
I, TW10.1 cells were co-transfected
with the pcDNA3 vector containing a rat PKC
I cDNA and with
the pBABE-puro vector, mixed in a 2:1 molar ratio. Cells were selected
in 1 mg/ml G418 and 0.25 µg/ml puromycin. The constitutively active
PKC
I
NPS construct encoding rat PKC
I without the
pseudosubstrate containing residues 1-30 was expressed following
electroporation of W10 cells and selection in 1 mg/ml G418. Parental
and transfected cells were cultured in 24-well plates with or without
the addition of 5 µg/ml goat anti-mouse IgM (Sigma-Aldrich, Oakville,
Ontario, Canada). Apoptosis was assessed using fluorescence-activated
cell sorting analysis to determine the fraction of viable cells
identified by their normal forward scatter/side scatter (FSC/SSC)
profile after 72 h in culture or by determination of the fraction
of cells containing subdiploid DNA 48 h after the initiation of
culture (20, 21). Inhibitors were added either alone or 15 min before the addition of anti-IgM.
(Sigma-Aldrich) or PKC
I or
PKC
II (Santa Cruz Biotechnology Inc., Santa Cruz, CA). Blots were
developed with ECL reagent (Amersham Pharmacia Biotech, Oakville,
Ontario, Canada) followed by exposure to film or with SuperSignal West
Femto reagent (Pierce) followed by imaging in a Bio-Rad
Fluor-Smax. Film exposures were converted to .tif files
using a scanner, and differences in band intensities were quantitated
using Quantity One software from Bio-Rad. Each gel contained a set of
2-fold dilutions of a total cell lysate from W10 or TW10.1 cells to
construct a standard curve used for quantitation of protein signals.
Total lysates were prepared by lysing 107 cells directly in
1 ml of Laemmli sample buffer.
-2-mercaptoethanol,
7 µg of myelin basic protein (MBP), and 5 µCi of
[
-32P]ATP (3000 mCi/mmol) and incubated for 15 min at
30 °C. The reaction was stopped by adding 30 µl of 3× Laemmli
sample buffer prewarmed to 55 °C, followed by immersion in boiling
water for 3 min. Phosphorylation of MBP was then analyzed on 12.5%
SDS-acrylamide gels and quantitated using a phosphoimager (Personal FX,
Bio-Rad).
I in vitro, 1 µg of GST fusion protein containing
LSP1 residues 1-330, 1-178, or 179-330 was mixed with 50 ng of
recombinant human PKC
I (PanVera Corp., Madison, WI) in 250 µl of
1× PKC buffer (20 mM Tris, pH 7.5, 5 mM
MgCl2). After incubation for 1 h at 4 °C,
glutathione-Sepharose beads were added and the incubation was continued
for an additional 30 min. Beads were recovered by centrifugation and
washed four times in 1 ml of 1× PKC buffer, and the amounts of bound
PKC
I and recovered GST and GST fusion protein were analyzed by
Western blotting.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
I, and
II (25, 26). The extent of
apoptosis was determined by measuring changes in the FSC/SSC profile of the cells at 72 h (20, 21). Treatment of W10 cells with 0.25 µM Gö6976 or with 5 µg/ml anti-IgM results in
only a slight reduction of viable cells; 70.6% of anti-IgM-treated
cells and 76.4% of Gö6976-treated cells display a normal FSC/SSC
profile after 72 h of culture (Fig.
1). In contrast, the addition of both
agents reduces the number of cells with a normal FSC/SSC profile to
10.4%. The addition of Gö6976 could be delayed for at least 20 min after addition of anti-IgM, which shows that the protective action
of cPKC depends on a relatively late event and that activation of cPKC
immediately following anti-IgM stimulation is not sufficient to protect
W10 cells from anti-IgM-induced apoptosis (data not shown). The
combined effect of Gö6976 and anti-IgM on apoptosis of W10 cells
is similar to the effect of expressing B-LSP1 as only 16.8% of the
anti-IgM-treated TW10.1 cells display a normal FSC/SSC profile after
72 h. To confirm that cell death occurs by apoptosis, we
determined the number of cells containing subdiploid DNA after 48 h of culture. W10 cells treated with anti-IgM or with Gö6976
contain 20.8% or 20.7% cells with subdiploid DNA, respectively,
whereas W10 cells treated with both agents contain 50.1% cells with
subdiploid DNA. Again, the effect of expressing B-LSP1 is similar to
the effect of treatment with Gö6976, as 55.2% of
anti-IgM-treated TW10.1 cells contain subdiploid DNA (data not shown).
Since the cPKC
isoform is not expressed in these cells (27), these
results suggest that the anti-apoptotic effect of activated cPKC is due
to activation of one or more of the PKC
, -
I, or -
II
isoforms.
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Fig. 1.
Inhibition of cPKC or MEK1 increases
anti-IgM-induced apoptosis. W10 cells, TW10.1 cells (10.1), and
PKC I-overexpressing TW10.1 transfectants (10.1/
I) were cultured
in 24-well plates as described with or without anti-IgM (5 µg/ml) or
the PKC inhibitor Gö6976 (0.25 µM) or the MEK1
inhibitor PD098059 (25 µM) as indicated. The inhibitors
were added 15 min before addition of anti-IgM, and the percentage of
viable cells was determined by their normal FSC/SSC profile after
72 h of culture.
I to the Plasma Membrane--
Increased anti-IgM-induced
apoptosis was evident after treatment of W10 cells with Gö6976 or
after transfection with B-LSP1 containing LSP1 residues 179-330. To
determine whether expression of B-LSP1 inhibits the activation of cPKC,
we measured the extent of anti-IgM-induced translocation of PKC
,
-
I, and -
II to the plasma membrane fractions of W10 cells and of
the transfectant TW10.1. Translocation of PKC is often used as a
measure of activation (28). Plasma membrane fractions were prepared
from unstimulated cells and from cells stimulated at 37 °C with
anti-IgM for 5 or 20 min. Equal amounts of plasma membrane protein were
analyzed for the presence of PKC
, -
I, and -
II by Western
blotting. All three PKC isoforms tested translocated to the plasma
membrane after treatment of W10 cells with anti-IgM (Fig.
2). Translocation was evident after 5 min
of stimulation and did not change significantly for the next 15 min.
Translocation of PKC
and PKC
II in TW10.1 cells did not differ
significantly from that measured in W10 cells. Interestingly,
translocation of PKC
I was significantly inhibited in TW10.1 cells,
when measured 5 or 20 min after addition of anti-IgM. We conclude from
these data that expression of B-LSP1 specifically inhibits
anti-IgM-induced PKC
I translocation to the plasma membrane.
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Fig. 2.
Expression of LSP1 truncate 179-330 inhibits
anti-IgM-induced translocation of PKC I.
A, plasma membrane fractions were prepared from unstimulated
W10 and TW10.1 cells and from cells stimulated for 5 or 20 min with 50 µg/ml anti-IgM. Equal amounts of plasma membrane protein were then
separated on 12.5% SDS-acrylamide gels and analyzed for PKC
I or
PKC
II. Lanes 1-4 are 2-fold dilutions of a
total lysate from W10 cells to construct a standard curve used to
quantitate the amount of PKC
I or PKC
II in the different plasma
membrane fractions (lanes 5-10). A 79-kDa
molecular mass marker is indicated at the left. The results
shown are of one experiment typical of four experiments performed.
B, results are presented as average -fold increase over
unstimulated cells calculated from three or four experiments performed.
, W10 cells.
, TW10.1 cells. Differences were tested for
statistical significance using Student's t test. **,
p < 0.01. *, p < 0.05.
I activation, we
transfected TW10.1 cells with an expression vector encoding intact rat
PKC
I and selected four colonies in which the expression levels of
PKC
I were 2-3 times higher than in the untransfected TW10.1 cells.
We also isolated four control colonies that express the puromycin
resistance gene but have no increased levels of PKC
I. Stimulation
with anti-IgM showed that overexpression of PKC
I rendered the TW10.1
cells less susceptible to anti-IgM-induced apoptosis (Fig. 1). Although
only 16.8% of TW10.1 cells or 17.1% of the puromycin resistant
control colonies (data not shown) were viable after 72 h of
anti-IgM stimulation, 35.4% of PKC
I-overexpressing TW10.1
transfectants remained viable. This shows that the B-LSP1-mediated inhibition of PKC
I contributes significantly to the B-LSP1-mediated increase in anti-IgM-induced apoptosis of TW10.1 cells. The partial reversal may be related to the level of overexpression of PKC
I. Alternatively, it may indicate that expression of B-LSP1 also affects
anti-IgM-induced apoptosis through mechanisms that do not involve
PKC
I activation.
I Translocation Inhibits Anti-IgM-induced ERK2
Activation--
To determine whether the specific inhibition of
anti-IgM-induced translocation of PKC
I affects a known
cPKC-regulated, anti-IgM-stimulated signaling pathway, we measured the
anti-IgM-induced activation of the MAP kinase ERK2 in W10 and TW10.1
cells. Activation of ERK2 is associated with survival in many cell
types (29, 30), and results in Fig. 1 show that this is the case in W10
cells as well. Treatment of W10 cells with 25 µM
PD098059, an inhibitor specific for MEK1 (31), the direct activator of
ERK2, does not significantly affect cell viability as 89.1% of W10
cells displayed a normal FSC/SSC profile after 72 h of culture.
However, in cultures treated with PD098059 and 5 µg/ml anti-IgM, only
34.3% of cells were viable, showing that anti-IgM-induced activation
of ERK2 protects W10 cells from anti-IgM-induced apoptosis.
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Fig. 3.
Inhibition of PKC I
leads to inhibition of ERK2 activation. A, W10 cells
were stimulated with anti-IgM alone (top row) or
with anti-IgM and the cPKC inhibitor Gö6976 (middle
row) or with anti-IgM and the MEK1 inhibitor PD098059
(bottom row). Cells were harvested just before or
2 or 20 min after addition of anti-IgM and lysed in sample buffer. ERK2
phosphorylation was determined by Western blotting. Phosphorylated ERK2
is indicated with an asterisk (*) and has a slightly lower
mobility during PAGE than the non-phosphorylated ERK2. B,
W10 were transfected with the constitutively active PKC
I
NPS
construct. Two control transfectants and two transfectants
overexpressing PKC
I
NPS (~3-fold compared with the endogenous
PKC
I, data not shown) were assayed for ERK2 activity using an
in vitro immune kinase assay with MBP as substrate. Both
PKC
I
NPS expressing transfectants contained 4-5-fold more ERK2
activity than the control cells. C, anti-IgM-induced ERK2
activation of W10 and TW10.1 cells. *, phosphorylated ERK2.
D, ERK2 activity was measured using an immune kinase assay.
The results shown in A-D are of one experiment typical for
three experiments performed. E, quantitative representation
of anti-IgM-induced ERK2 activation as measured by immune kinase assay.
Results are expressed as -fold increase ± S.E. of MBP
phosphorylation in anti-IgM stimulated samples over unstimulated
controls. Solid bars, W10 cells. Open
bars, TW10.1 cells.
I in anti-IgM-induced ERK2
activation by two approaches. First, W10 cells were transfected with a
pcDNA3-based expression vector encoding a constitutively active
PKC
I protein (PKC
I
NPS, lacking the NH2-terminal
pseudosubstrate region) and two colonies expressing the PKC
I
NPS
protein were selected. Two G418-resistant colonies that do not express
PKC
I
NPS were selected as control cells. Fig. 3B shows
that ERK2 activity is 4-5 times higher in the PKC
I
NPS-expressing
transfectants than in the control cells, showing that activation of
PKC
I leads to activation of the ERK2 pathway. Second, we analyzed
ERK2 activation in W10 and TW10.1 cells in which the anti-IgM-induced
translocation of PKC
I is inhibited. ERK2 activation was measured 2, 10, and 20 min after anti-IgM treatment, using the decreased mobility of phosphorylated ERK2 on SDS-PAGE as a read-out (Fig. 3C)
or by measuring ERK2 activity in an immune kinase assay (Fig. 3, D and E). In W10 cells, ERK2 is activated
efficiently after 2 min of anti-IgM stimulation and does not change
significantly over the next 15 min. The extent of activation of ERK2 in
TW10.1 cells is similar to that found in W10 cells when measured 2 or 10 min after stimulation but after 20 min is significantly less than in
W10 cells. These data show that inhibition of cPKC with Gö6976 or
inhibition of PKC
I by expression of B-LSP1 both inhibit only the
late but not the early activation of ERK2 by anti-IgM. This is strong
evidence that anti-IgM-induced activation of PKC
I contributes
significantly to the late activation of ERK2. Given that inhibition of
ERK2 by PD098059 increases the extent of anti-IgM-induced apoptosis, we
suggest that the inhibition of ERK2 activation in TW10.1 cells
contributes to the increase in anti-IgM-induced apoptosis. The
designation of ERK2 as an anti-apoptotic protein does not agree with a
report showing that inhibition of anti-IgM-induced activation of ERK2
in WEHI-231 cells protects from anti-IgM-induced apoptosis (38). These
discordant findings may be related to the different methods used to
inhibit ERK2 activation. Whereas we established a protective role for
ERK2 using the MEK1 inhibitor PD098059 to inhibit ERK2, the
pro-apoptotic role of ERK2 was established using expression of the
phosphatase MKP-1 to inhibit ERK2. However, this phosphatase is not
specific for ERK2 and inhibits other members of the MAP kinase family,
p38 and stress-activated protein kinase/c-Jun NH2-terminal
kinase 1 as well (39).
I Interacts with LSP1 Residues 179-330--
We used
pull-down experiments to determine whether LSP1 interacts with PKC
I.
A GST fusion protein containing the intact LSP1 or the GST protein was
mixed with Nonidet P-40-soluble lysates from W10 cells. After
incubation for 1 h, the GST protein and the LSP1/GST fusion
proteins were recovered using glutathione-Sepharose beads and analyzed
by Western blotting. Using this protocol PKC
I but not PKC
or
PKC
II were recovered from lysates when mixed with the LSP1/GST
fusion protein. No detectable amounts of PKC
, -
I, or -
II were
recovered using the GST protein, indicating a specific interaction of
LSP1 with PKC
I (Fig. 4A).
To determine whether PKC
I and LSP1 interact directly, we performed
in vitro binding assays using human recombinant PKC
I and
GST fusion proteins containing different LSP1 sequences. One µg of
GST or GST fusion protein was incubated with 50 ng of recombinant human
PKC
I. Fig. 4B shows that PKC
I interacts preferentially
with intact LSP1 or with the COOH-terminal domain residues 179-330. In
replicate experiments, binding to the NH2-terminal residues
1-178 is not significantly higher than binding to the GST protein
alone, indicating that the preferential binding site or sites for
PKC
I are located between LSP1 residues 179 and 330.
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Fig. 4.
LSP1 interacts with
PKC I. A, pull-down experiments
were performed by incubating GST or a GST fusion protein containing
LSP1 residues 1-330 with Nonidet P-40-soluble lysates from W10 cells
as described, and proteins associated with GST or the GST-LSP1 fusion
protein were analyzed for the presence of PKC
, -
I, and -
II by
Western blotting. TL, total lysate prepared by lysis of W10
cells directly into SDS-PAGE sample buffer. The position of a 73-kDa
molecular mass marker is indicated at the left.
B, recombinant human PKC
I (50 ng) was incubated with 1 µg of GST or GST fusion proteins containing LSP1 residues 1-330,
1-178, or 179-330. GST and GST fusion proteins were recovered on
glutathione-Sepharose beads and analyzed for the presence of PKC
I
(top panels) and GST or GST fusion proteins
(bottom panels) by Western blotting. In the
bottom panels, only that portion of the Western
blots containing the GST or GST fusion protein is shown.
I, but
we propose that the endogenous, intact LSP1 sequesters inactive PKC
I
to a cytosolic localization and that, in response to anti-IgM-generated signals, the LSP1/PKC
I complex dissociates, allowing for the translocation of PKC
I. The release of PKC
I from B-LSP1 in
response to anti-IgM stimulation may be less efficient, leading to
inhibition of PKC
I translocation. LSP1 is a Ca2+-binding
protein and contains two putative Ca2+-binding EF-hand
motifs near the NH2 terminus (16). Thus, binding of
Ca2+ to the NH2-terminal domain may result in a
structural change in the COOH-terminal domain, leading to dissociation
of PKC
I. Since both EF-hand motifs are absent from B-LSP1,
suggesting that B-LSP1 does not bind Ca2+, the
B-LSP1/PKC
I complex may not dissociate after the anti-IgM-induced increase in [Ca2+]i thereby
inhibiting the movement of PKC
I to the plasma membrane.
Alternatively, since the NH2-terminal domain of LSP1 contains a phosphorylation site for casein kinase II (40) and several
putative PKC phosphorylation sites, it is possible that the binding of
PKC
I to LSP1 residues 179-330 is regulated by phosphorylation of
specific sites in the LSP1 NH2-terminal domain. Thus, LSP1
may protect from apoptosis only in response to certain signals such as
increased [Ca2+]i. Apoptosis
induced by sorbitol, nocodazole, C2-ceramide, or
H2O2 is not regulated by LSP1 (20), possibly
because these signals do not lead to dissociation of PKC
I from
LSP1.
I. Experiments to determine whether B-LSP1 also
inhibits PKC
I translocation in response to other
Ca2+-generating signals such as ionomycin or anti-CD20
stimulation are currently under way. We have used expression of B-LSP1
to inhibit anti-IgM-induced translocation of PKC
I in a transformed B-cell line, which significantly impacted on the susceptibility of
these cells to anti-IgM-induced apoptosis. Many B-lymphoma cell lines
are susceptible to anti-IgM-induced apoptosis, a characteristic that
forms the basis for using anti-Ig antibodies as therapy for B-lymphoma
in vivo (41, 42). Our results suggest that the efficiency of
anti-Ig therapy may be enhanced by the concomitant inhibition of
PKC
I.
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ACKNOWLEDGEMENTS |
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We thank Atri Persad for technical help and Dr. C. Whiteside (University of Toronto, Toronto, Ontario, Canada) for a kind gift of anti-PKC antibodies.
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FOOTNOTES |
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* This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society and from the Cancer Research Society, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Toronto Western Hospital, Rm. 13-419; 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.: 416-603-6481; Fax: 416-603-5745; E-mail: jongstra@uhnres.utoronto.ca.
Published, JBC Papers in Press, May 1, 2001, DOI 10.1074/jbc.M103883200
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ABBREVIATIONS |
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The abbreviations used are: mIgM, membrane immunoglobulin M; LSP1, leukocyte-specific protein 1; PKC, protein kinase C; cPKC, conventional protein kinase C; GST, glutathione S-transferase; MAP, mitogen-activated protein; MBP, myelin basic protein; HBSS, Hanks' buffered saline solution; FSC, forward scatter; SSC, side scatter.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Benhamou, L. E., Cazenave, P. A., and Sarthou, P. (1990) Eur. J. Immunol. 20, 1405-1407[Medline] [Order article via Infotrieve] |
2. | Hasbold, J., and Klaus, G. G. B. (1990) Eur. J. Immunol. 20, 1685-1690[Medline] [Order article via Infotrieve] |
3. | Andjelic, S., and Liou, H. C. (1998) Eur. J. Immunol. 28, 570-581[CrossRef][Medline] [Order article via Infotrieve] |
4. | Knox, K. A., Finney, M., Milner, A. E., Gregory, C. D., Wakelam, M. J., Michell, R. H, and Gordon, J. (1992) Int. J. Cancer 52, 959-966[Medline] [Order article via Infotrieve] |
5. |
Kaptein, J. S.,
Lin, C.-K. E.,
Wang, C. L.,
Nguyen, T. T.,
Kalunta, C. I.,
Park, E.,
Chen, F.-S.,
and Lad, P. M.
(1996)
J. Biol. Chem.
271,
18875-18884 |
6. |
Graves, J. D.,
Draves, K. E.,
Craxton, A.,
Krebs, E. G.,
and Clark, E. A.
(1998)
J. Immunol.
161,
168-174 |
7. | Genestier, L., Bonnefoy-Berard, N., Rouault, J.-P., Flacher, M., and Revillard, J.-P. (1995) Int. Immunol. 7, 533-540[Abstract] |
8. | Fang, W., Rivard, J. A., Ganster, J. A., LeBien, T. W., Nath, K. A., Mueller, D. L., and Behrens, T. W. (1995) J. Immunol. 155, 66-75[Abstract] |
9. |
Wiesner, D. A.,
Kilkus, J. P.,
Gottschalk, A. R.,
Quintans, J.,
and Dawson, G.
(1997)
J. Biol. Chem.
272,
9868-9876 |
10. |
Gold, M. R.,
Scheid, M. P.,
Santos, L.,
Dang-Lawson, M.,
Roth, R. A.,
Matsuuchi, L.,
Duronio, V.,
and Krebs, D. L.
(1999)
J. Immunol.
163,
1894-905 |
11. | DeFranco, A. L. (1997) Curr. Opin. Immunol. 9, 296-308[CrossRef][Medline] [Order article via Infotrieve] |
12. | Campbell, K. S. (1999) Curr. Opin. Immunol. 11, 256-264[CrossRef][Medline] [Order article via Infotrieve] |
13. |
King, L. B.,
Norvell, A.,
and Monroe, J. G.
(1999)
J. Immunol.
162,
2655-2662 |
14. |
McConkey, D. J,
Aguilar-Santelises, M.,
Hartzell, P.,
Eriksson, I.,
Mellstedt, H.,
Orrenius, S.,
and Jondal, M.
(1991)
J. Immunol.
146,
1072-1076 |
15. |
Jongstra, J.,
Tidmarsh, G. F.,
Jongstra-Bilen, J.,
and Davis, M. M.
(1988)
J. Immunol.
141,
3999-4004 |
16. | Klein, D. P., Jongstra-Bilen, J., Ogryzlo, K., Chong, R., and Jongstra, J. (1989) Mol. Cell. Biol. 9, 3043-3048[Medline] [Order article via Infotrieve] |
17. | Jongstra, J., Ittel, M.-E., Iscove, N., and Brady, G. (1994) Mol. Immunol. 31, 1125-1131[CrossRef][Medline] [Order article via Infotrieve] |
18. | Li, Y., Guerrero, A., and Howard, T. H. (1995) J. Immunol. 155, 3563-3569[Abstract] |
19. | Pulford, K., Jones, M., Banham, A. H., Haralambieva, E., and Mason, D. Y. (1999) Immunology 96, 262-271[CrossRef][Medline] [Order article via Infotrieve] |
20. | Jongstra-Bilen, J., Wielowieyski, A., Misener, V., and Jongstra, J. (1999) Mol. Immunol. 36, 349-359[CrossRef][Medline] [Order article via Infotrieve] |
21. | Nicoletti, I., Migliorati, M. C., Grignani, F., and Riccardi, C. (1991) J. Immunol. Methods 139, 271-279[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Klein, D. P.,
Galea, S.,
and Jongstra, J.
(1990)
J. Immunol.
145,
2967-2973 |
23. | Jongstra-Bilen, J., Janmey, P. A., Hartwig, J. H., Galea, S., and Jongstra, J. (1992) J. Cell Biol. 118, 1443-1453[Abstract] |
24. |
Scheid, M. P.,
and Duronio, V.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7439-7444 |
25. | Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H. J., and Johannes, F. J. (1996) FEBS Lett. 392, 77-80[CrossRef][Medline] [Order article via Infotrieve] |
26. | Wenzel-Seifert, K., Schachtele, C., and Seifert, R. (1994) Biochem. Biophys. Res. Commun. 200, 1536-43[CrossRef][Medline] [Order article via Infotrieve] |
27. | Tsutsumi, A., Freire-Moar, J., and Ransom, J. T. (1992) Cell. Immunol. 142, 303-312[Medline] [Order article via Infotrieve] |
28. | Liu, W. S., and Heckman, C. A. (1998) Cell. Signal. 10, 529-542[CrossRef][Medline] [Order article via Infotrieve] |
29. | Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract] |
30. | Cross, T. G., Scheel-Toellner, D., Henriquez, N. V., Deacon, E., Salmon, M., and Lord, J. M. (2000) Exp. Cell Res. 256, 34-41[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Alessi, D. R.,
Cuenda, A.,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494 |
32. |
Tordai, A.,
Franklin, R. A.,
Patel, H.,
Gardner, A. M.,
Johnson, G. L.,
and Gelfand, E. W.
(1994)
J. Biol. Chem.
269,
7538-7543 |
33. | Derijard, B., Raingeaud, J., Barrett, T., Wu, I. H., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) Science 267, 682-685[Medline] [Order article via Infotrieve] |
34. |
Hashimoto, A.,
Okada, H.,
Jiang, A.,
Kurosaki, M.,
Greenberg, S.,
Clark, E. A.,
and Kurosaki, T.
(1998)
J. Exp. Med.
188,
1287-1295 |
35. |
Jiang, A.,
Craxton, A.,
Kurosaki, T.,
and Clark, E. A.
(1998)
J. Exp. Med.
188,
1297-1306 |
36. |
Carroll, M. P.,
and May, W. S.
(1994)
J. Biol. Chem.
269,
1249-1256 |
37. |
Schoenwasser, D. C.,
Marais, R. M.,
Marshall, C. J.,
and Parker, P. J.
(1998)
Mol. Cell. Biol.
18,
790-798 |
38. |
Lee, J. R.,
and Koretzky, G. A.
(1998)
J. Immunol.
161,
1637-1644 |
39. |
Chu, Y.,
Solski, P. A.,
Khosravi-Far, R.,
Der, C. J.,
and Kelly, K.
(1996)
J. Biol. Chem.
271,
6497-6501 |
40. |
Gimble, J. M.,
Dorheim, M. A.,
Youkhana, K.,
Hudson, J.,
Nead, M.,
Gilly, M.,
Wood, W. J., Jr.,
Hermanson, G. G.,
Kuehl, M.,
Wall, R.,
and Kincade, P. W.
(1993)
J. Immunol.
150,
115-121 |
41. |
Hsu, F. J.,
Caspar, C. B.,
Czerwinski, D.,
Kwak, L. W.,
Liles, T. M.,
Syrengelas, A.,
Taidi-Laskowski, B.,
and Levy, R.
(1997)
Blood
89,
3129-3135 |
42. |
Davis, T. A.,
Maloney, D. G.,
Czerwinski, D. K.,
Liles, T. M.,
and Levy, R.
(1998)
Blood
92,
1184-1190 |