Protein Phosphatase 6 Controls
BCR-Induced Apoptosis of WEHI-231 Cells
by Regulating Ubiquitination of Bcl-xL
This information is current as
of June 15, 2017.
Ryutaro Kajihara, Hitomi Sakamoto, Kano Tanabe, Kazuki
Takemoto, Masayoshi Tasaki, Yukio Ando and Seiji Inui
J Immunol 2014; 192:5720-5729; Prepublished online 7 May
2014;
doi: 10.4049/jimmunol.1302643
http://www.jimmunol.org/content/192/12/5720
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Supplementary
Material
The Journal of Immunology
Protein Phosphatase 6 Controls BCR-Induced Apoptosis of
WEHI-231 Cells by Regulating Ubiquitination of Bcl-xL
Ryutaro Kajihara,* Hitomi Sakamoto,* Kano Tanabe,* Kazuki Takemoto,*
Masayoshi Tasaki,† Yukio Ando,† and Seiji Inui*
Crosslinking BCR in the immature B cell line WEHI-231 causes apoptosis. We found that Bcl-xL was degraded by polyubiquitination upon BCR crosslinking and in this study explored the mechanism that controls the degradation of Bcl-xL. Ser62
of Bcl-xL was phosphorylated by JNK to trigger polyubiquitination, and this was opposed by serine/threonine protein phosphatase 6 (PP6) that physically associated with Bcl-xL. We show BCR crosslinking decreased PP6 activity to allow Ser62 phosphorylation of Bcl-xL. CD40 crosslinking rescues BCR-induced apoptosis, and we found PP6 associated with CD40 and PP6 activation
in response to CD40. Our data suggest that PP6 activity is regulated to control apoptosis by modulating Ser62 phosphorylation of
Bcl-xL, which results in its polyubiquitination and degradation. The Journal of Immunology, 2014, 192: 5720–5729.
*Department of Immunology and Hematology, Faculty of Life Sciences, Graduate
School of Health Sciences, Kumamoto University, Kumamoto 862-0976, Japan; and
†
Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 862-0976, Japan
Received for publication October 1, 2013. Accepted for publication April 15, 2014.
This work was supported by a Grant-in-Aid for Scientific Research (24790557) from
the Japan Society for the Promotion of Science and institutional support from the
Kumamoto University.
Address correspondence and reprint requests to Prof. Seiji Inui, Graduate School of
Health Sciences, Kumamoto University, 4-24-1, Kuhonji, Chuoku, Kumamoto 8620976, Japan. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; OA, okadaic acid; PP6, protein phosphatase 6; WCL, whole-cell lysates; WT,
wild-type.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302643
dimerizes with proapoptotic molecules and suppresses their activities. Bcl-xL phosphorylation has been reported in response
to apoptotic signals (16, 17). Several different kinases have been
implicated in the phosphorylation of Bcl-xL including JNK, protein kinase C, and CDK (18, 19). Bcl-xL has been reported to be
degraded by the proteasome after the induction of its ubiquitination
in some cells (20, 21). However, the relationship between the phosphorylation of Bcl-xL and its ubiquitination has not been clearly
elucidated.
The MAPK family consists of three members, ERK, p38 MAPK,
and JNK (22). Generally, ERK promotes cell survival, whereas
JNK and p38 MAPK are associated with apoptosis (22). Prolonged, rather than transient, activation of JNK has been reported
to be involved in apoptosis (23, 24). The induction of apoptosis by
JNK may be conveyed by c-Jun, which results in new gene expression. JNK may also control apoptosis by phosphorylating
Bcl-2 family members without the need for new protein synthesis (25). Stimuli such as osmotic stress and TNF induce activation of the JNK and induce apoptosis (26). JNK deficiency
was reported to result in defects in thymocyte apoptosis (27). In
WEHI-231 cells, BCR crosslinking was shown to activate JNK
to induce apoptosis. These reports demonstrated the relevance of
JNK in the control of apoptosis in immature lymphocytes.
Protein phosphatase 6 (PP6), a serine/threonine phosphatase,
belongs to the protein phosphatase 2A (PP2A) subfamily that
comprises PP2A, PP4, and PP6 (28). These PP2A subfamily
members are sensitive to active site inhibitors, such as okadaic
acid (OA) (29, 30). PP6 consists of a catalytic subunit, PP6c, and
regulatory molecules including SAPS1, -2 and -3. PP6 is mainly
expressed in lymphoid, cardiac, and neuronal cells (31–33). SIT4,
a yeast homolog of PP6, was identified as a molecule required for
the G1 to S transition of the cell cycle (34). Human PP6 was also
reported to be a cell-cycle regulator (32, 35, 36). Kinases and
phosphatases have been identified as essential for cell survival and
apoptosis (37–39). In particular, it was reported that PP6 was an
important regulator of apoptosis (37). In our previous study,
PP6 played an important role in the regulation of apoptosis (40).
Although PP6 regulates the NF-kB pathway (33), the precise
mechanism underlying the regulation of apoptosis by PP6 has
not been fully addressed.
The induction of apoptosis in WEHI-231 cells by anti-IgM can
be rescued by T cell–derived costimulation signals, such as CD40
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B
cell receptor signaling induces opposite results depending on the stage of B cell development (1–3). In mature
B cells, BCR signaling induces proliferation and differentiation. In contrast, the same BCR engagement leads to cell
death by apoptosis in immature B cells. Self-reactive B cells with
BCR, which have high affinity for self-antigens, are thus eliminated in the bone marrow (4, 5). The mechanism underlying this
difference in BCR signaling remains to be determined. However,
Bcl-xL has emerged as one of the molecules that shows different
responses to BCR signaling depending on the stage of B cell
development. Bcl-xL expression is induced by BCR crosslinking
in mature B cells, whereas it decreases in immature B cells following the same stimulation (6, 7). The murine B cell line, WEHI231, has been widely employed as a model to analyze the mechanism underlying the induction of apoptosis by self-antigens in
immature B cells (8, 9). WEHI-231 cells undergo G0/G1 cell-cycle
arrest and apoptosis when stimulated with anti-IgM, which mimics
Ag stimulation (10, 11).
The Bcl-2 family proteins play an important role in the regulation of apoptosis (12–15). The antiapoptotic members include
Bcl-2, Bcl-xL, and other proteins. The proapoptotic members are
further divided into two subgroups: Bax subfamily members,
which have a similar structure to Bcl-2 and Bcl-xL with multiple
Bcl-2 homology domains, and the BH3-only proteins, which include Bad and Bim (13–15). Bcl-xL has been reported to play
critical roles in the apoptosis of lymphocytes (7). Bcl-xL hetero-
The Journal of Immunology
ligation (41). CD40 is a membrane molecule that functions in
various ways to activate B cells (42). CD40 is essential for B cell
activation, germinal center formation, class switching, and so on
(43). CD40 is expressed on the surface of both immature and mature
B cells. The mechanism by which CD40 rescues BCR-induced apoptosis remains unclear. The signal transduction of CD40 involves
both the NF-kB and MAPK pathways (44, 45).
In the current study, we found that Bcl-xL was degraded by
polyubiquitination induced by BCR crosslinking in WEHI-231
cells. Ser62 phosphorylation by JNK triggered this ubiquitination.
We investigated whether any phosphatases regulated the apoptosis
by interfering with the JNK-induced phosphorylation of Bcl-xL
and found that the PP6 activity was modulated by BCR crosslinking, and PP6 bound to and protected Bcl-xL by dephosphorylating Ser62. CD40 associated with PP6, and CD40 ligation
suppressed apoptosis by enhancing the PP6 activity. Together, our
results indicate that PP6 plays an important role in the regulation
of apoptosis in WEHI-231 cells.
Cells and reagents
The WEHI-231 cell line was described previously (46), and the HEK293T
(293T) cell line was a kind gift from Dr. Kaisho at Osaka University in
Japan. The anti–Bcl-xL Ab (Abcam, Cambridge, MA), anti–Bcl-xL (pS62)
phosphospecific polyclonal Ab (Millipore, Bedford, MA), anti-K48
linkage-specific polyubiquitin Ab (Millipore), anti-JNK Ab (Cell Signaling Technology, Danvers, MA), anti–p-JNK Ab (Cell Signaling Technology), and anti-PP2Ac Ab (Upstate Biotechnology, Lake Placid, NY) were
purchased for Western blot and/or immunoprecipitation studies. Anti-PP6c
was a kind gift from Dr. Brautigan (University of Virginia). The anti-IgM
mAb M41 was a kind gift from Dr. Rolink at the University of Basel
(Basel, Switzerland). The anti-murine CD40 Ab was purified from LB429
culture supernatant (47). MG-132 was purchased from Merck Millipore.
SP600125 and OA were purchased from Wako Pure Chemical Industries
(Osaka, Japan).
Preparation of immature B cells
BALB/c female mice between 6 and 12 wk old were used for the experiments. Animals were housed at the Center for Animal Resources and
Development. Experiments were approved by the committee on animal
experiments of Kumamoto University. Bone marrow from femurs was
harvested, and single-cell suspensions were prepared. B cells were purified
using a B cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the
manufacturer’s protocol. Purified B cells were stained with the mixture of
biotinylated anti-IgD (Abcam) and biotinylated anti-CD21 (BioLegend)
Ab followed by streptavidin Microbeads. Immature B cells were negatively
selected using MACS LS columns (Miltenyi Biotec) according to the
manufacturer’s protocol to avoid preactivation (48).
Construction of expression plasmids and site-directed
mutagenesis
The PP6c cDNA was described previously (40). The PP4c and PP2Ac
cDNAs were kind gifts from Dr. Maeda at Kumamoto University in Japan.
Human Bcl-xL and murine CD40 cDNAs were amplified from mRNAs of
the human B-lymphoid cell line RPMI1866, and murine B cell line WEHI279, respectively, by PCR and subcloned into pCAGGS or pCMV-HA
vectors. Substitution of Ser62 of Bcl-xL with Ala (S62A) in the pCAGGS–
Bcl-xL plasmid was performed with the PrimeSTAR mutagenesis basal kit
(TaKaRa Bio, Shiga, Japan) using the following specific oligonucleotides
(Hokkaido System Science, Sapporo, Japan): sense, 59-GCAGACGCCCCCGCGGTGAATGGAGCC-39 and antisense, 59-CGCGGGGGCGTCTGCCAGGTGCCAGGA-39. PP6c-D84N was prepared similarly with the following
primers: sense, 59-TTTTGTAAACAGAGGTTACTATAGTT-39 and antisense,
59-CCTCTGTTTACAAAATCACCCATAAA-39 (Hokkaido System Science).
All of the sequences of the cDNAs and mutations in the plasmids were verified
by DNA sequencing with an ABI Prism 310 instrument (Applied Biosystems,
Foster City, CA).
Cell transfection
To establish stably transfected WEHI-231 cells, the cells in exponential
growth were washed once with PBS and resuspended at a concentration of
2 3 107 cells/ml in serum-free medium. Cells (500 ml) were preincubated
on ice for 10 min with linearized plasmid DNA (30 mg) plus 2 mg pSV2neo
vector as a selectable marker. Cells were transfected by electroporation at
250 V and 960 mF using a Gene Pulser (Bio-Rad, Hercules, CA). After
incubation on ice for 5 min, the cell suspension was mixed with complete
medium, and the cells were incubated for 48 h at 37˚C before selection.
After 48 h, 1 mg/ml G418 (Life Technologies, Grand Island, NY) was
added to the cells, and cultures were maintained under antibiotic selection
for 2 wk. Individual clones were obtained by limiting dilution. For the
transient transfection of 293T cells, transfection was performed using the
HilyMax transfection reagent (Dojindo, Kumamoto, Japan) according to
the manufacturer’s instructions.
Real-time PCR
Total RNA was isolated from WEHI-231 cells with RNAiso Plus (TaKaRa
Bio). First-strand cDNA was synthesized using PrimeScript II 1st strand
cDNA Synthesis Kit (TaKaRa Bio) according to the manufacturer’s instructions. The real-time PCR was performed using SYBR Green Realtime PCR
Master Mix (Toyobo, Osaka, Japan) with the primers for Bcl-xL: sense, 59GCTGGGACACTTTTGTGGAT-39 and antisense, 59-TGTCTGGTCACTTCCGACTG-39. The PCR was performed with an ABI Prism 7500 Real-Time
PCR System (Applied Biosystems). The specificity was verified by a melting
curve analysis. The results were calculated using the DD threshold cycle
method relative to the expression of GAPDH and are presented as the foldinduction relative to unstimulated samples.
Cell-viability assay
Cells were cultured at 1 3 104 cells/well in 96-well microtiter plates and
treated with anti-IgM or OA for 24 or 48 h. The relative cell viabilities
were analyzed using the Cell Counting kit-8 (Dojindo). In brief, the cells
were pulsed with WST-8 for the last 3 h of culture. The absorbance was
then measured with an ELISA plate reader at a wavelength of 450 nm. The
viability of untreated cells was defined as 100%.
Cell-cycle analysis
Cells were cultured in 24-well culture dishes at a density of 1 3 105 cells/ml
and treated with anti-IgM for 24 h. After treatment, the cells were fixed in
70% ethanol for 3 h and stained with Guava Cell Cycle Reagent (Millipore), assayed in Guava easyCyte 6HT/2L (Millipore), and analyzed using
the guavaSoft 2.6 software program (Millipore), all according to the
manufacturer’s instructions.
Immunoprecipitation and the Western blot analysis
For the immunoprecipitation of polyubiquitinated Bcl-xL, whole-cell lysates
(WCL) were prepared in RIPA buffer (50 mM Tris-HCl [pH 8], 150 mM
NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Nonidet P-40)
containing 50 mM MG-132 and 10 mM N-ethylmaleimide with freshly
added protease inhibitors and phosphatase inhibitors. For coimmunoprecipitation, cells were lysed in buffer containing 25 mM Tris-HCl (pH 7.6),
0.5% Triton X-100, 100 mM NaCl, 1 mM MgCl2, and 2 mM DTT with
freshly added protease inhibitors and phosphatase inhibitors. Each cell
lysate was precleared by incubation at 4˚C with protein G–coupled
Sepharose beads for 1 h. These precleared lysates were immunoprecipitated at 4˚C by adding specific Abs overnight, followed by 1.5 h of
incubation with protein G–coupled Sepharose beads. Immunoprecipitates
were washed five times with the buffer used to prepare the cell lysates and
then dissolved in SDS sample buffer. The Western blot analysis was performed as described previously (40). Briefly, samples were separated by
SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The
membranes were incubated for 1 h in blocking buffer (TBS containing
0.05% Tween 20 and 5% nonfat dry milk) followed by a 1-h or overnight
incubation with the primary Ab in blocking buffer. After extensive washing,
the blot was incubated with a relevant secondary Ab for 1 h and processed
using the ECL reagents (GE Healthcare, Buckinghamshire, U.K.) according
to the manufacturer’s instructions.
In vitro phosphatase assay
To detect the phosphatase activity, anti-FLAG immunoprecipitates from
FLAG-PP6c–expressing WEHI-231 cells or WCL were prepared as described in the previous paragraph. After washing the precipitates with the
reaction buffer (50 mM imidazole and 0.1% 2-ME) the last two times,
90 ml reaction buffer was added to the immunoprecipitates. To assay the
WCL, 85 ml reaction buffer was added to 5 ml WCL. A 10-ml aliquot of
6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP; Invitrogen, Carlsbad, CA; dissolved at 100 mM) was added to the precipitates or WCL to
start the reaction. After 10 min of incubation at 30˚C, 40 ml reaction
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Materials and Methods
5721
5722
mixture was transferred to a 96-well microtiter plate, and the fluorescence
(emission 450 nm and excitation 360 nm) was measured in a fluorescence
plate reader (MTP-800; Corona Electric, Ibaraki-ken, Japan). The fluorescence of untreated cells was defined as 100%.
Statistical analyses
The Student t test was used to calculate the p values. The differences were
considered to be significant at a 5% level.
Results
BCR crosslinking in WEHI-231 cells induces Bcl-xL
degradation by ubiquitination
Bcl-xL UBIQUITINATION REGULATED BY PP6
ence of 10 mg/ml of anti-IgM, and expression of the antiapoptotic
factor Bcl-xL was assessed by Western blot. BCR crosslinking
induced a near-complete loss of Bcl-xL protein levels between
2 and 6 h, which preceded induction of apoptosis in WEHI-231
cells (Fig. 1A). Real-time PCR was performed to see whether there
was any reduction in mRNA of Bcl-xL after anti-IgM stimulation;
as shown in Fig. 1B, there was no change, at least up to 6 h. There
were some previous reports that showed that Bcl-xL degraded
by the ubiquitin–proteasome pathway in other systems (20, 21).
Therefore, we stimulated WEHI-231 cells with anti-IgM in the
We studied the apoptosis-induction system of WEHI-231 cells that
undergo growth arrest and apoptosis following crosslinking of IgM
on the cell surface, which mimics the deletion of self-reactive
B cells in the bone marrow (8–11). Anti-IgM stimulation induces
cell death in 12–24 h. WEHI-231 cells were cultured in the pres-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 1. BCR stimulation induces Bcl-xL polyubiquitination and
proteasomal degradation. (A) WEHI-231 cells were stimulated with
10 mg/ml anti-IgM for various times, and the cell lysates were subjected
to Western blot with anti–Bcl-xL (total) or anti–p-Ser62–specific Bcl-xL
(pSer62). (B) These cells were also assessed for the expression of Bcl-xL
mRNA by real-time PCR. The levels of Bcl-xL mRNA expression were
normalized to those of GAPDH and expressed as a fold of the Bcl-xL level
from unstimulated cells. Bars represent the means 6 SDs from triplicate
wells of three independent experiments. (C) WEHI-231 cells were treated
with 10 mg/ml anti-IgM for the indicated time periods, with or without 10
mM MG-132, for 12 h. The expression levels of the Bcl-xL protein were
determined by Western blot. (D) The lysates of the cells treated with antiIgM and MG-132 in (C) were immunoprecipitated with anti–Bcl-xL. The
samples were analyzed by Western blot for Bcl-xL polyubiquitination.
Similar results were obtained in three independent experiments in (A)–(D).
FIGURE 2. The phosphorylation of Ser62 of Bcl-xL facilitates its
polyubiquitination. WEHI-231 cells were stably transfected with the
pCAGGS vector harboring WT FLAG-Bcl-xL (WEHI-Bcl-xL) or phospho-deficient FLAG-Bcl-xL (WEHI-Bcl-xL-S62A). (A) The cell lysates of
WEHI-Bcl-xL or WEHI-Bcl-xL-S62A cells were assessed for exogenous
expression of FLAG-Bcl-xL. (B) WEHI-Bcl-xL or WEHI-Bcl-xL-S62A
cells were stimulated with 10 mg/ml anti-IgM for the indicated times, and
then the cell lysates were subjected to Western blot with anti-FLAG. (C)
WEHI-Bcl-xL or WEHI-Bcl-xL-S62A cells were treated with 10 mg/ml
anti-IgM and 10 mM MG-132 for 12 h. The cell lysates were immunoprecipitated (IP) with anti-FLAG. The samples were analyzed by Western
blot (WB) with anti-ubiquitin (Ub). (D) WEHI-231, WEHI-Bcl-xL, or
WEHI-Bcl-xL-S62A cells were induced to undergo apoptosis by treatment
with 10 mg/ml anti-IgM for 48 h, and the cell-viability levels were measured as described in Materials and Methods. The viability of untreated
cells was defined as 100%. The bars represent the means 6 SDs from three
independent experiments. The results of (A) are representative of two independent experiments, and those in (B)–(D) are representative of three
independent experiments. **p , 0.01.
The Journal of Immunology
presence or absence of a proteasome inhibitor, MG-132. The
degradation of Bcl-xL was almost completely inhibited by the
addition of 10 mM of MG-132, and the protein level of Bcl-xL
was sustained even after 12 h of anti-IgM stimulation (Fig. 1C).
This result suggested that the ubiquitin–proteasome pathway was
involved in the regulation of Bcl-xL protein level in BCRstimulated WEHI-231 cells. To confirm this, endogenous BclxL was immunoprecipitated with anti–Bcl-xL and blotted with
anti-ubiquitin before and at various times after anti-IgM stimulation. BCR crosslinking induced the ubiquitination of Bcl-xL in
WEHI-231 cells, and this ubiquitination was readily detectable
after 6 h of stimulation (Fig.1D).
Phosphorylation of Ser62 of Bcl-xL is important for its
ubiquitination
stimulation, but significantly less degradation of the S62A form of
Bcl-xL. Next, WEHI-231 cells transfected with Bcl-xL cDNAs
were stimulated with BCR for 12 h. The polyubiquitination of
transfected S62A Bcl-xL was greatly diminished compared with
that of WT Bcl-xL (Fig. 2C). Apoptosis resistance was compared
between WEHI-231 cells expressing either WT or S62A Bcl-xL.
The overexpression of WT Bcl-xL in WEHI-231 cells conferred
resistance to apoptosis induced by BCR crosslinking (Fig. 2D) as
reported previously (9, 51). Although the expression level of
S62A mutant protein was comparable with that of WT Bcl-xL
(Fig. 2A), the S62A mutant protein conferred more resistance to
apoptosis in WEHI-231 cells (Fig. 2D). It was previously reported
that BCR crosslinking induced G0/G1 arrest of WEHI-231 cells
before the induction of apoptosis (10, 11). Anti-IgM stimulation
induced a similar level of G0/G1 arrest in transfectants with WT
and S62A mutant Bcl-xL (Supplemental Fig. 1).
JNK is responsible for the Ser62 phosphorylation of Bcl-xL
A variety of kinases, including JNK, can phosphorylate Ser62 of BclxL (18, 19). We tested the effect of a pharmacological JNK inhibitor on the phosphorylation of Bcl-xL in this system. As shown
in Fig. 3A, Ser62 phosphorylation induced by BCR crosslinking
was effectively blocked by a JNK inhibitor SP600125 (used at 10
mM). To investigate the relevance of JNK in the phosphorylation of
Bcl-xL during the induction of apoptosis in WEHI-231 cells, the
activation of JNK was monitored after BCR crosslinking in WEHI231 cells. The phosphorylation of JNK was detected 15 min after
BCR stimulation (Fig. 3B). Treatment with the JNK inhibitor
SP600125 inhibited the degradation of Bcl-xL induced by BCR
crosslinking (Fig. 3C). Subsequently, WEHI-231 cells transfected
with Bcl-xL cDNA were stimulated by BCR crosslinking for 12 h.
The polyubiquitination of transfected Bcl-xL induced by BCR
crosslinking was inhibited by the JNK inhibitor SP600125, again
indicating the importance of phosphorylation in the regulation of
Bcl-xL ubiquitination (Fig. 3D). We further showed JNK was
FIGURE 3. The BCR-triggered phosphorylation and ubiquitination of Bcl-xL are dependent on JNK. (A) The phosphorylation status of Bcl-xL was
assessed by Western blot after anti-IgM (10 mg/ml) and SP600125 (10 mM) treatment for 1 h in WEHI-231 cells. (B) WEHI-231 cells were stimulated with
10 mg/ml anti-IgM for the indicated times, and the cell lysates were subjected to Western blot with anti–p-JNK or anti-JNK. (C) The expression level of BclxL was analyzed by Western blot after anti-IgM (10 mg/ml) and SP600125 (10 mM) treatment for 12 h in WEHI-231 cells. (D) WEHI-Bcl-xL cells were
incubated with 10 mg/ml anti-IgM and 10 mM MG-132, with or without SP600125 (10 mM), for 12 h. The cell lysates were immunoprecipitated (IP) with
anti-FLAG. Samples were analyzed by Western blot (WB) with anti-ubiquitin (Ub). (E) WEHI-Bcl-xL cells were treated with 10 mg/ml anti-IgM for 1 h
and subjected to IP with anti–Bcl-xL. Samples were analyzed by Western blot with anti-JNK. Results in (A)–(C) and (E) are representative of two independent experiments, and those of (D) are representative of four independent experiments.
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In many cases, ubiquitination of a protein is preceded by phosphorylation (49), and the phosphosites are recognized by SCF
family E3 ligases (50). We observed phosphorylation of Ser62 in
Bcl-xL under some apoptotic conditions in the previous studies,
although the significance of this phenomenon was not fully
addressed (16, 17). We investigated whether Ser62 was phosphorylated after BCR stimulation and found that it was indeed
phosphorylated within 15 min of stimulation, and the phosphorylation was elevated substantially up to 2 h, after which time, the
protein levels dropped (Fig.1A). The phosphorylation preceded
the degradation of Bcl-xL. To more clearly demonstrate that
phosphorylation of Ser62 is important for the ubiquitination and
degradation of Bcl-xL, wild-type (WT) and Ser62 to Ala (S62A)
mutant cDNAs of Bcl-xL were prepared as described in the
Materials and Methods. The proteins were expressed in WEHI231 cells, and Western blot analysis showed the expression levels
of WT and S62A mutant Bcl-xL were comparable (Fig. 2A).
However, S62A mutant Bcl-xL was more stable than WT Bcl-xL
after BCR stimulation (Fig. 2B). There was a reduction in the
levels of both WT and S62A proteins after 6 and 12 h of BCR
5723
5724
Bcl-xL UBIQUITINATION REGULATED BY PP6
coprecipitated with Bcl-xL in WEHI-231 cells, and this binding
was enhanced after anti-IgM stimulation (Fig. 3E).
PP6 associates with Bcl-xL
PP6 controls the phosphorylation and ubiquitination of Bcl-xL
To determine whether PP6 regulates the phosphorylation and
degradation of Bcl-xL, PP6c cDNA was transfected into WEHI-231
cells. The overexpression of PP6c in WEHI-231 cells diminished the apoptosis induced by BCR crosslinking (Fig. 5A). This
effect was due to the activity of PP6 because this difference was
not observed in the presence of OA, a potent inhibitor of PP6 (30).
The phosphorylation of Ser62 of Bcl-xL after BCR crosslinking
was also decreased in these cells (Fig. 5B). To confirm this result,
an inactive form of PP6c, PP6c-D84N (52), was prepared and
transfected into WEHI-231 cells. Overexpression of inactive form
PP6c had no effect on the resistance to apoptosis or the phosphorylation of serine 62 of Bcl-xL induced by BCR crosslinking
(Fig. 5A, 5B). Of note, the degradation of Bcl-xL was inhibited
in WEHI-231 cells overexpressing PP6c (Fig. 5C). The polyubiquitination of Bcl-xL induced by BCR crosslinking was also
diminished by PP6c overexpression (Fig. 5D). To elucidate the
mechanism by which Bcl-xL is regulated by PP6, we studied
whether the JNK activation was compromised in PP6c-overexpressing WEHI-231 cells. The JNK activation, as monitored
by the phosphorylation of JNK, was not significantly different
between the parental cells and the transfectant (Fig. 5E). Therefore,
we next tested whether PP6c associated with JNK before or after antiIgM stimulation. No association between JNK and PP6c was observed under either condition (Fig. 5F). In contrast, PP6c associated
with Bcl-xL in WEHI-231 cells, and this association was decreased
by BCR crosslinking (Fig. 5G). These results suggest that PP6
controls the phosphorylation of Bcl-xL.
CD40 associates with PP6c and controls its activity
We then studied whether the PP6 activity was altered after BCR
crosslinking in WEHI-231 cells. An in vitro phosphatase assay
was performed using DiFMUP as a substrate, as described in the
Materials and Methods. The phosphatase activity in the anti-FLAG
precipitate decreased after BCR crosslinking (Fig. 6A). It is well
established that CD40 crosslinking rescues BCR-induced apoptosis
(41). CD40 ligation was reported to activate the NF-kB pathway
and increase the transcription of the Bcl-xL gene, which has an
NF-kB binding site (53). We therefore investigated whether PP6
associated with CD40, because PP2Ac was reported to bind to CD28,
a T cell activation molecule (54). First, CD40 cDNA was trans-
FIGURE 4. An OA-sensitive phosphatase is involved in regulating the
phosphorylation status of Bcl-xL. (A) WEHI-Bcl-xL cells were stimulated
with 10 mg/ml anti-IgM in the presence or absence of OA (50 nM) for the
indicated time periods. MG-132 was also added to the media to prevent
Bcl-xL from being degraded. WCL were prepared and tested for the
presence of p–Bcl-xL and total Bcl-xL. (B) WEHI-Bcl-xL cells were incubated with 10 mg/ml anti-IgM and 10 mM MG-132, with or without OA
(50 nM) for 12 h. The cell lysates were immunoprecipitated (IP) with antiFLAG. Samples were analyzed by Western blot (WB) with anti-ubiquitin
(Ub). (C) WEHI-231 cells were induced to undergo apoptosis by treating
them with 10 mg/ml anti-IgM in the presence or absence of OA (50 nM)
for 24 h, and the cell viability was measured as described in Materials and
Methods. The viability of untreated cells was defined as 100%. The bars
represent the means 6 SDs from three independent experiments. (D) The
293T cells were transfected with FLAG-PP1c, PP2Ac, PP4c, or PP6c in
combination with Bcl-xL cDNA. After a 48-h incubation, the cells were
harvested and subjected to IP with anti–Bcl-xL. Then, immunoblotting was
performed with anti-FLAG or anti–Bcl-xL. The results shown in (A) and (D)
are representative of two independent experiments. The results shown in (B)
and (C) are representative of three independent experiments. **p , 0.01.
fected into 293T cells together with PP6c cDNA, and PP6c was
coprecipitated with CD40 (Fig. 6B). The association was specific
to PP6c, because PP2Ac did not bind to CD40 nor did control Ab
coprecipitate PP6c (Fig. 6B). The association between endogenous
PP6c and Bcl-xL was then tested in greater detail. As shown in
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WEHI-231 cells were stimulated with anti-IgM in the absence or
presence of OA, a serine threonine PP family phosphatase inhibitor. MG-132 was added to the culture to prevent the degradation
of Bcl-xL in this experiment. Ser62 phosphorylation induced by
BCR crosslinking was enhanced by the addition of 50 nM of OA
(Fig. 4A). MG-132 alone had no effect on the Ser62 phosphorylation level of Bcl-xL even after 12 h of incubation (Supplemental
Fig. 2). OA also enhanced the polyubiquitination of Bcl-xL as
shown in Fig. 4B. Next, the effect of OA on the induction of apoptosis in WEHI-231 cells was studied. OA itself induced slight
apoptosis in WEHI-231 cells and enhanced the apoptosis induced
by the BCR crosslinking (Fig. 4C). These results indicate the involvement of phosphatase(s) in the regulation of Bcl-xL ubiquitination and the apoptosis of WEHI-231 cells. Because PP2A
subfamily members are similarly affected by OA treatment (29,
30), we tested if any of the type 2A phosphatases were directly
associated with Bcl-xL. As shown in Fig. 4D, PP6c specifically
associated with Bcl-xL when cotransfected into 293T cells.
The Journal of Immunology
5725
Fig. 6C, we found that endogenous CD40 also associated with
PPP6c. Next, we tested if CD40 ligation increased the PP6 activity
in WEHI-231 cells. Cells expressing FLAG-PP6c were stimulated
with anti-CD40 for 1 h, and the lysate was precipitated with antiFLAG. The PP6 activity increased after CD40 ligation, as shown in
Fig. 6A, and CD40 ligation reversed the decrease in PP6 activity
in BCR-stimulated WEHI-231 cells. CD40 stimulation also diminished the phosphorylation of Bcl-xL induced by BCR crosslinking (Fig. 6D). Based on these results, we propose a model in
which the stability of Bcl-xL is controlled by the phosphorylation
of Ser62 (Fig. 7). This phosphorylation status is regulated by JNK
and PP6, both of which are under the control of BCR crosslinking.
The degradation of Bcl-xL is regulated by JNK and PP6 in
normal immature B cells
We next investigated whether the phenomena observed in WEHI231 cells were applicable to normal immature B cells. We prepared
immature B cells from the bone marrow of BALB/c mice. BCR
crosslinking induced a decrease of Bcl-xL in immature B cells, as
indicated in Fig. 8A. MG-132 treatment inhibited this decrease,
suggesting a role of ubiquitination in this process. BCR crosslinking also induced the phosphorylation of Ser62 of Bcl-xL in
immature B cells (Fig. 8B). The addition of a JNK inhibitor,
SP600125, reduced the Ser62 phosphorylation of Bcl-xL, just
as it did in WEHI-231 cells (Fig. 8B). Anti-IgM treatment induced the phosphorylation of JNK in normal immature B cells
(Fig. 8C). We also found that the Ser62 phosphorylation of BclxL was enhanced by the addition of OA (Fig. 8D). We next
examined whether BCR crosslinking induced a decrease in PP6
activity in normal immature B cells. Because a sufficient amount
of endogenous PP6 was not available for immunoprecipitation
from normal immature B cells, we used WCL to monitor the
phosphatase activity. As shown in Fig. 8E, the phosphatase activity
was reduced after BCR crosslinking and enhanced after CD40
ligation (Fig. 8E). The reduced phosphatase activity induced
by BCR crosslinking was reversed by CD40 ligation (Fig. 8E),
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FIGURE 5. PP6 dephosphorylates Ser62 of Bcl-xL and confers apoptosis resistance to WEHI-231 cells. WEHI-231 cells were stably transfected with
a pCMV-tag2 vector harboring PP6c (WEHI-PP6c) or inactive PP6c (WEHI-PP6c-D84N). (A) WEHI-231, WEHI-PP6c, and WEHI-PP6c-D84N cells were
induced to undergo apoptosis by treatment with 10 mg/ml anti-IgM in the presence or absence of OA (50 nM) for 24 h, and the cell-viability assay was
performed. The viability of untreated cells was defined as 100%. The bars represent the means 6 SDs from three independent experiments. (B) The
phosphorylation status of Bcl-xL was assessed by Western blot after anti-IgM (10 mg/ml) treatment for 1 h in WEHI-231, WEHI-PP6c, or WEHI-PP6cD84N cells. (C) WEHI-231 or WEHI-PP6c cells were incubated with anti-IgM (10 mg/ml) for 12 h, and then expression levels of Bcl-xL were analyzed by
Western blot. (D) WEHI-231 and WEHI-PP6c cells were treated with 10 mg/ml anti-IgM and 10 mM MG-132 for 12 h. The cell lysates were immunoprecipitated (IP) with anti–Bcl-xL. Samples were assessed by Western blot (WB) with anti-ubiquitin (Ub). (E) WEHI-231 and WEHI-PP6c cells were
stimulated with 10 mg/ml anti-IgM for the indicated times, and the cell lysates were subjected to Western blot with anti–p-JNK or anti-JNK. (F) WEHIPP6c cells were incubated with anti-IgM (10 mg/ml) for 1 h, and the cell lysates were IP by FLAG-PP6c. Samples were assessed for the interaction between
PP6 and JNK. (G) WEHI-231 cells were treated with 10 mg/ml anti-IgM for 1 h, and cell lysates were subjected to IP with anti–Bcl-xL. Coimmunoprecipitates were analyzed by Western blot with anti-PP6c or anti-PP2Ac. The results shown in (A) are representative of three independent experiments, and
those shown in (B)–(G) are representative of two independent experiments. **p , 0.01.
5726
Bcl-xL UBIQUITINATION REGULATED BY PP6
FIGURE 6. CD40 ligation upregulates the PP6 activity and rescues the
WEHI-231 cells from BCR-induced apoptosis. (A) WEHI-231 cells expressing FLAG-PP6c were stimulated with 10 mg/ml anti-IgM and/or anti-CD40
(10 mg/ml) for 1 h. The cell lysates were immunoprecipitated with antiFLAG. The phosphatase activities in the complex were assayed using
DiFMUP as a substrate, as described in Materials and Methods. The value
of untreated cells was defined as 100%. The bars represent the means 6
SDs from three independent experiments. (B) FLAG-PP2Ac or PP6c, in
combination with hemagglutinin (HA)-CD40 cDNA, was transfected into
293T cells, and WCL were immunoprecipitated (IP) with anti-HA or
control Ab (rabbit IgG). Samples were subjected to Western blot with
anti-FLAG or anti-HA. (C) The lysates of WEHI-231 cells were IP with
anti-CD40 or control Ab (rabbit IgG). Then, the coimmunoprecipitation of
endogenous PP2Ac or PP6c was tested by Western blot. (D) WEHI-Bcl-xL
cells were stimulated with 10 mg/ml anti-IgM in the presence or absence of
anti-CD40 (10 mg/ml) for 1 h. WCL were prepared and tested for the
presence of p–Bcl-xL and total Bcl-xL. The results in (A) are representative
of three independent experiments, and those shown in (B)–(D) are representative of two independent experiments. *p , 0.05.
which was in agreement with the observation in WEHI-231
cells (Fig. 6A).
Discussion
The WEHI-231 cell line has served as an excellent model to study
apoptosis in immature B cells (8, 9). It was reported that Bcl-xL
played an important role in the life and death decision-making
process in mature versus immature B cells (6, 7). Cellular apoptosis is controlled by Bcl-2 family proteins (12–15). Bcl-xL and
Bcl-2 seem to function in the same apoptotic pathway, and they
are especially important in the control of apoptosis in lymphocytes,
in which both proteins are abundantly expressed (7, 9). The
overexpression of either molecule conferred resistance to BCRinduced apoptosis in WEHI-231 cells (9, 51). Although there
was a report that demonstrated the existence of heterogeneity in
the WEHI-231 cell line (55), our study confirmed the expression
and importance of Bcl-xL in the resistance of WEHI-231 cells to
apoptosis. It was recently reported that Ser62 phosphorylation was
involved in cell-cycle blockade (56), and anti-IgM stimulation of
WEHI-231 was also shown to cause cell-cycle blockade (10, 11).
Therefore, we assessed the effects of overexpressing WT and S62A
mutant Bcl-xL on the cell-cycle blockade induced by BCR crosslinking. Our results showed that there was no apparent effect on
the cell cycle associated with the expression of either molecule
(Supplemental Fig. 1). The differences in the results may be
explained by the differences in the cell types examined and the
employed stimulus between the previous paper (56) and our
present study.
BCR crosslinking induced the polyubiquitination and degradation of Bcl-xL in WEHI-231 cells. This ubiquitination was preceded by the phosphorylation of Ser62 of Bcl-xL. Previous findings
showed that Bcl-xL was degraded following its ubiquitination in
response to apoptosis-inducing signals (20, 21). UV irradiation,
oxidative stress, and antimitotic drugs were all demonstrated to
induce the degradation of Bcl-xL by the proteasome. Phosphorylation was also implicated in the regulation of functions of Bcl-2
family members, although the impact of phosphorylation on resistance to apoptosis was inconsistent (16, 25). The phosphorylation of Ser62 of Bcl-xL abolished its antiapoptotic activity (16).
Several kinases, including JNK, protein kinase C, and CDK, have
been reported to phosphorylate this site (18, 19). Although JNK
may mediate a transcription-dependent apoptotic pathway, it was
also shown that the effects of UV did not require new gene expression to induce apoptosis (25). However, the relevant kinase
involved in the BCR-induced apoptosis had been unclear, and the
relationship between the phosphorylation of Bcl-xL at Ser62 and
its ubiquitination has not been clearly demonstrated. In this study,
we showed that JNK was responsible for the Ser62 phosphorylation and demonstrated that this phosphorylation was essential for
Bcl-xL ubiquitination.
Phosphatases and kinases are like two sides of a coin, and when
phosphorylation regulates a specific signal transduction pathway,
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FIGURE 7. A proposed model for the regulation of Bcl-xL. Bcl-xL is
degraded by the ubiquitin (Ub)–proteasome pathway upon the phosphorylation of the Ser62 residue. This site is phosphorylated by JNK and
dephosphorylated by PP6. BCR induces the apoptosis of immature B cells
by controlling the activities of both JNK and PP6.
The Journal of Immunology
5727
dephosphorylation must also play a role in the same pathway to
counteract the effect (57). PP2A, which is a ubiquitously expressed
serine/threonine phosphatase, plays an important role in the regulation of cellular functions, including cell-cycle control and survival (28). Previous studies showed that PP2A dephosphorylated
Bcl-2 family members, including Bcl-2 and Bcl-xL, and controlled
its antiapoptotic activity (58, 59). In this study, we showed that
PP6, which belongs to the PP2A subfamily, played an important
role in the regulation of Bcl-xL. This finding was not surprising,
because it was also previously reported that PP6 was important in
the regulation of apoptosis (37, 40), and PP6 is highly expressed in
lymphoid cells (32, 33). In some cases, phosphatases directly associate with the kinases that they regulate (60). However, an association between PP6 and JNK was not observed in either the
nonphosphorylated or phosphorylated forms of JNK. In contrast,
PP6 associated with Bcl-xL, and this association decreased after
BCR crosslinking. Therefore, we propose that PP6 controls the
phosphorylation of Bcl-xL by working on Bcl-xL, rather than JNK
(Fig. 7).
It is well established that activation signals, such as IL-4, LPS,
and CD40, can rescue WEHI-231 cells from BCR-induced apoptosis (41). CD40 induces NF-kB activation and upregulates the
transcription of Bcl-xL, which has an NF-kB binding site in its
promoter region (53). PP2A was shown to associate with CD28,
a T cell activation marker, but not with CD40 (54). Our results
confirmed that PP2A did not bind to CD40. However, CD40 associated with PP6. We then tested if the PP6 activity was involved
in the regulation of Bcl-xL phosphorylation following BCR activation. BCR crosslinking decreased the phosphatase activity, and
CD40 ligation increased the PP6 activity. Furthermore, the BCRinduced decrease of PP6 activity was diminished by CD40 ligation. Therefore, as an additional mechanism to rescue WEHI-231
cells from BCR-induced apoptosis, CD40 may stabilize Bcl-xL by
controlling its phosphorylation status.
BCR crosslinking induces apoptosis in immature B cells (1–5) and
transitional B cells in the neonatal spleen (61). Transitional B cells
are subdivided into T1 and T2 cell subpopulations, and the adult
spleen also contains these transitional T1 cells (62). We prepared
immature B cells from bone marrow and showed that BCR crosslinking induced the degradation of Bcl-xL by the proteasome. It
was reported in a previous study that BCR crosslinking induced a
much lower amount of Bcl-xL in T1 cells compared with T2 cells
(62). Our present study demonstrated that Bcl-xL degradation was
controlled by the induction of polyubiquitination resulting from the
Ser62 phosphorylation by JNK. We further showed that a protein
phosphatase was involved in the control of Bcl-xL degradation.
These results confirmed that the mechanism observed in WEHI-231
cells was applicable to normal immature B cells.
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FIGURE 8. The mechanism observed in WEHI-231 cells is relevant to normal immature B cells in the bone marrow. Normal immature B cells were
prepared from the bone marrow of BALB/c mice as described in Materials and Methods. (A) IgM+IgD2 immature B cells from bone marrow were
stimulated with 10 mg/ml of anti-IgM for 24 h with or without 10 mM MG-132 for 12 h. The cell lysates were subjected to Western blot with anti–Bcl-xL.
The blot with anti-tubulin was performed to show equal loading. Immature B cells were treated with 10 mg/ml of anti-IgM for 2 h, and the cell lysates
were subjected to Western blot with anti–Bcl-xL or anti–p-Ser62–specific Bcl-xL or anti-JNK (B) or anti–p-JNK (C). (D) Immature B cells were
stimulated with 10 mg/ml of anti-IgM for 2 h with or without 50 mM OA. WCL were prepared and tested for the presence of p–Bcl-xL and total Bcl-xL.
(E) Immature B cells were stimulated with 10 mg/ml anti-IgM and/or anti-CD40 (10 mg/ml) for 1 h. The phosphatase activities of the cell lysates were
assayed using DiFMUP as a substrate, as described in Materials and Methods. The value of untreated cells was defined as 100%. The results in (A) are
representative of three independent experiments, and those shown in (B)–(D) are representative of two independent experiments. Five mice were used for
each experiment.
5728
Bcl-xL UBIQUITINATION REGULATED BY PP6
Acknowledgments
We thank Dr. David Brautigan (University of Virginia) for kind advice
regarding the protein phosphatase assay using DiFMUP as a substrate
and the gift of anti-PP6 Ab. We also thank Dr. Brautigan for critical reading of the manuscript.
28.
29.
30.
Disclosures
The authors have no financial conflicts of interest.
31.
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