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INTRODUCTION |
Apoptosis is a physiological process of cell death shared by all
multicellular organisms and is critical for removing unwanted cells
during development. This process is essential for the maintenance of
normal tissue homeostasis. Cell death or survival is dependent on the
receipt of continuous signals from the extracellular environment. These
signals are then transduced through the cell surface to intracellular
molecules that regulate apoptotic cell death. A number of such
regulators have been identified. Among them, the BCL-2 family proteins
play an important role in both induction and suppression of apoptosis
(reviewed in Ref. 1). The anti-apoptotic members include the cellular
proteins such as BCL-2, BCL-xL, MCL-1, BFL1, and BCL-w and viral
proteins such as Epstein-Barr virus BHRF1 and adenovirus E1B-19K
proteins. These cellular and viral proteins suppress apoptosis induced
by diverse stimuli. Certain pro-apoptotic proteins such as BAX and BAK,
despite sharing extensive homology with BCL-2, induce apoptosis when
overexpressed. Most other pro-apoptotic proteins such as BAD, BID, BIK,
BIM, BNIP3, BNIP1, HRK, and NOXA share only a single domain
(BH3)1 with BCL-2 and are
hence designated "BH3-alone" proteins; the human BIK protein is the
founding member of this family. In Caenorhabditis elegans, a
BH3-alone protein Egl-1 has been shown to be essential for
developmentally programmed death of somatic cells (2). An interesting
feature of the BCL-2 family of proteins is the ability of the
pro-apoptotic members to heterodimerize with the anti-apoptotic members
(reviewed in Ref. 1). This suggests that one of the mechanisms by which
anti-apoptotic members of the BCL-2 family suppress apoptosis may be
through heterodimerization with pro-apoptotic members. However, at
least in the case of BIK, heterodimerization with anti-apoptotic
proteins such as BCL-2 and BCL-xL is insufficient for induction of
apoptosis (3), suggesting that other factors may also influence the
apoptotic activity of BIK.
Certain apoptotic stimuli appear to modulate the expression and
activity of the BCL-2 family proteins at the level of transcription as
well as by post-translational modifications. For example, expression of
the BH3-alone pro-apoptotic gene Noxa is activated during
p53-mediated apoptosis (4). Hypoxic conditions have shown to activate
the expression of another pro-apoptotic protein BNIP3 (5). The BH3-alone protein BIM (6) has been shown to be essential for cytokine
withdrawal-induced apoptosis in hemopoietic cells (7). BIM is normally
sequestered to the microtubules in an inactive form and is released
during apoptosis (8). During apoptosis mediated by the death receptors
such as CD95, the BH3-alone protein BID is proteolytically cleaved by
caspase-8 to a pro-apoptotic form (tBID) from an inactive form (9, 10).
Post-translational modifications such as phosphorylation also play
important roles in regulating the activity of both anti-apoptotic and
pro-apoptotic BCL-2 family proteins. BCL-2 contains several potential
serine/threonine phosphorylation sites within the "variable
region," which is located between the BH3 and BH4 domains.
Phosphorylation of a serine residue (Ser-70) within this region of
BCL-2 has been shown to be required for its full anti-apoptotic
activity (11). Deletion of the variable region of BCL-2 has been shown
to relieve a novel proliferation restraining activity of BCL-2 (12).
Both BCL-2 and BCL-xL have been shown to be phosphorylated in cells
following treatment with microtubule disrupting agents (13). Taken
together, it can be concluded that the activities of BCL-2 and BCL-xL
can be influenced by phosphorylation. Similarly, phosphorylation of the
pro-apoptotic protein BAD on Ser-112, Ser-136, and Ser-155, following
interleukin-3 treatment, has been shown to prevent its
heterodimerization with BCL-2 and BCL-xL, thus rendering it inactive
(14-18). Several protein kinases have been shown to phosphorylate the
BCL-2 family members. The pro-apoptotic protein BAD is a target for
phosphorylation by the survival promoting protein kinase Akt (19) and
mitochondrial protein kinase A (15). Similarly, phosphorylation of
non-BCL-2 members also modulates apoptosis. Phosphorylation of
pro-caspase 9 by Akt appears to inhibit activation of this caspase by
proteolysis (20). Exposure to DNA damaging agents that leads to
p53-mediated apoptosis also induces phosphorylation of p53 at Ser-392
by casein kinase II (CKII) (21). In this article, we report that the
pro-apoptotic protein BIK is a phosphoprotein and that phosphorylation
is required for the full apoptotic activity of BIK.
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EXPERIMENTAL PROCEDURES |
BIK and BIK Mutants--
The HA-tagged BIK expression plasmid
(pcDNA3HA-BIK) has been described (22). Plasmids pET21b-BIK and
mutant BIK were constructed by cloning polymerase chain
reaction-amplified BIK cDNA encoding aa 1-134 in expression vector
pET21b (Novagen) in frame with the His6 tag at the C
terminus. All BIK mutants were constructed by polymerase chain reaction
according to a method described by Taylor et al. (23).
Purification of Recombinant BIK Protein--
His-tagged wt and
mutant BIK proteins were expressed in Escherichia coli
strain BL21 and purified over Ni2+-NTA-agarose resin
(Qiagen). One liter of bacterial culture was centrifuged and
resuspended in 10 ml of sonication buffer (50 mM Tris-Cl,
pH 7.5, 50 mM NaCl, 0.5 mM DTT, 1% Nonidet
P-40). The cell suspension was sonicated four times for 1 min each. The lysates were centrifuged at 12,000 × g for 30 min at
4 °C. The supernatant was diluted with sonication buffer without
Nonidet P-40 to adjust the Nonidet P-40 concentration to 0.5%. The
extracts were loaded onto the Ni2+-NTA columns equilibrated
with sonication buffer containing 0.5% Nonidet P-40. The proteins were
eluted with a linear gradient of 0-0.3 M imidazole.
In Vivo Labeling and Immunoprecipitation of BIK--
The wt and
mutant (T33A/S35A) BIK proteins were expressed in BSC40 cells
using vaccinia/T7 RNA polymerase vector system (24). Briefly, cells
were transfected with 10 µg of pcDNA3-based plasmids expressing
either HA-tagged wt or mutant (T33A/S35A) BIK and infected with 10 pfu/cell vaccinia virus vTF7-3. The cells were labeled either in
phosphate-free DMEM with 100 µCi/ml
[32P]orthophosphoric acid (PerkinElmer Life
Sciences) or in methionine-free DMEM with
[35S]methionine-cysteine mixture (PerkinElmer Life
Sciences) for 4 h. Eighteen to 20 h after infection, cells
were lysed with 50 mM Tris, pH 7.4, 150 mM
NaCl, 0.1% SDS, 1% Nonidet P-40 with the protease inhibitors
leupeptin and aprotinin. Cell lysates were subjected to
immunoprecipitation using HA monoclonal antibody 12CA5 (Roche Molecular
Biochemicals), and the immunoprecipitates were resolved by 12%
SDS-PAGE. The proteins were visualized by autoradiography.
In Vitro Kinase Assay--
Purified wt BIK (1-134 amino acids)
or mutant BIK (T33A/S35A) proteins were phosphorylated in
vitro in 40 mM Hepes, pH 8.0, 2 mM DTT, 10 mM MgCl2, 0.5 mM EGTA, 50 µM ATP, 5 µCi of [
-32P]ATP by 5 µl
of crude HeLa cell cytoplasmic extract or partially purified HeLa cell
extract. The mixture was incubated at 30 °C for 30 min following
removal of unincorporated 32P by precipitation with
trichloroacetic acid, the BIK protein was separated by SDS-PAGE and
examined by autoradiography.
Phosphoamino Acid Analysis--
Recombinant BIK (aa 1-134) was
phosphorylated in vitro using HeLa cell extract (pooled
SP-Sepharose fractions). The phosphorylated protein was separated by
SDS-PAGE, and the gel slice containing phosphorylated BIK was excised
and washed sequentially with 25% isopropyl alcohol, 10% methanol, and
50 mM NH4HCO3 (pH 8.8). The protein
was digested with 100 µg of tosylphenylalanyl chloromethyl ketone-treated trypsin in 50 mM
NH4HCO3 for 24 h at 37 °C. The tryptic
peptides were hydrolyzed in 6 N HCl at 110 °C for 3 h under vacuum. Nonradioactive phosphoserine, phosphothreonine, and
phosphotyrosine were added to the sample, which were together applied
onto a TLC plate and separated by electrophoresis at 1000 V for 3 h with pH 1.9 buffer (25). The locations of phosphoamino acids were
determined by ninhydrin staining and autoradiography.
Transient Cell Death Assay--
HeLa cells in 12-well
(105 cells/well) plates were transfected with 0.5-3.0 µg
of HA epitope-tagged wt BIK or mutant BIK (T33A/S35A) expressed from
the pcDNA3 vector, along with 0.1-0.6 µg of the reporter plasmid
pCMV-lacZ by the calcium phosphate method. The cells were fixed at
8-10 h after transfection and stained with X-gal. Briefly, the cells
were washed with PBS and fixed with 0.5% glutaraldehyde for 5 min.
After washing the cells twice with PBS for 5 min, they were stained
with 2 ml of staining solution (2 mM MgCl2 + 5 mM potassium ferricyanate + 5 mM potassium
ferrocyanate + 1 mg/ml X-gal in PBS) at 37 °C overnight. The stained
cells were microscopically examined and counted. Blue color cells
() were scored as round (apoptotic) and flat (viable) cells.
Transformation Assay--
E1A/T24 ras
oncogene cooperative transformation assays were carried out using
primary baby rat kidney (BRK) cells prepared from 4-day-old Fisher rats
as described earlier (26). The use of this transformation assay to
assess the activity of pro-apoptotic proteins has been described
(27).
Preparation of HeLa Cell Extracts--
HeLa cells (4 × 107) were scraped from tissue culture flasks, washed with
PBS. The cell pellet was suspended in buffer A (20 mM
HEPES, pH 7.5, 10% glycerol, 0.1% Nonidet P-40, 0.5 mM
DTT, and 0.2 mM phenylmethylsulfonyl fluoride), lysed at
4 °C for 30 min, and centrifuged at 12,000 × g. The
supernatant was loaded on to a cation exchange column (SP-Sepharose,
Amersham Pharmacia Biotech). The column was washed with 10 ml of buffer
A and eluted with a continuous salt gradient (0.1-0.5 M
NaCl). Two-ml fractions were collected on ice.
In Gel Kinase Assay--
A 12% SDS-polyacrylamide mini-gel
containing 500 µg/ml purified BIK (aa 1-134) was prepared. 25 µg
of partially purified HeLa cell extract (pooled fractions 5-11) was
fractionated (along with prestained Rainbow SDS-PAGE molecular weight
markers) by electrophoresis. The proteins were denatured and renatured
in situ to restore functional activity. The gel was immersed
in 100 ml of a solution containing 50 mM Tris-Cl, pH 8.0, and 20% isopropanol for 1 h to remove SDS. The gels were washed
with 50 mM Tris-Cl, pH 8.0, and 5 mM DTT for
1 h. The proteins were then denatured in a solution containing 50 mM Tris-Cl, pH 8.0, and 6 M guanidine
hydrochloride for 1 h. The concentration of guanidine
hydrochloride was then gradually reduced from 6 M to 0.75 M, thus facilitating slow renaturation of proteins. In gel
phosphorylation was carried out by incubating the gels in 10 ml of
kinase buffer (40 mM HEPES, pH 8.0, 2 mM DTT,
10 mM MgCl2, 0.5 mM EGTA, 50 µM ATP) and 5 µCi/ml [
-32P]ATP
(PerkinElmer Life Sciences) for 1 h. The gels were washed five
times with 250 ml of 5% trichloroacetic acid and 1% sodium pyrophosphate, dried, and subjected to phosphorimager analysis.
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RESULTS |
BIK Is a Phosphoprotein--
When the human BIK protein expressed
in mammalian cells was examined by Western blot analysis, it generally
migrated as a doublet of 24-25 kDa (Fig.
1A). To determine whether one
of the bands may represent the phosphorylated form of BIK, we
metabolically labeled BIK either with
[35S]methionine-cysteine mixture or with
[32P]orthophosphoric acid. For this purpose,
HA-tagged BIK was expressed in BSC40 cells using the vaccinia virus
expression system (24). The proteins were immunoprecipitated with the
HA antibody and analyzed by SDS-PAGE (Fig. 1B). The BIK
protein that was metabolically labeled with 35S migrated as
a doublet, as in the case of Western blot analysis. Comparison of BIK
labeled with 32P revealed that the slower moving band may
be the phosphorylated form of BIK (Fig. 1B). To determine if
endogenous BIK is also phosphorylated, we metabolically labeled SW480
cells either with 35S or 32P,
immunoprecipitated BIK with a BIK antibody (N-19) and analyzed by
SDS-PAGE (Fig. 1C). This analysis also revealed that
endogenous BIK is phosphorylated. These results indicate that BIK is a
phosphoprotein.

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Fig. 1.
Phosphorylation of BIK. A,
Western blot analysis of BIK. Proteins were immunoprecipitated from
extracts of 293T cells, transfected either with pcDNA3HA vector
(V) or pcDNA3HA-BIK (BIK) with the HA
antibody 12CA5 and detected by Western blot using a polyclonal antibody
raised against a BH3 (BIK) peptide. B, in vivo
phosphorylation of BIK. Extracts of BSC40 cells transfected with
pcDNA3HA-BIK and infected with the vaccinia virus vTF7-3, labeled
with 35S or 32P, immunoprecipitated with the HA
antibody, and analyzed on 15% SDS-PAGE and autoradiography.
C, phosphorylation of endogenous BIK. SW480 (ATCC) cells
were labeled with 35S or 32P,
immunoprecipitated with a BIK-specific peptide antibody (Santa Cruz,
SC-1710), and analyzed on 15% SDS-PAGE.
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In Vitro Phosphorylation of BIK--
To characterize the
phosphorylation of BIK further, we determined whether BIK could be
phosphorylated in vitro. For this purpose, a His-tagged
version of BIK that lacks the C-terminal hydrophophic tail (aa 1-134)
was expressed in bacteria, purified using Ni2+-NTA-agarose
affinity matrix, and used as the substrate for phosphorylation. The BIK
protein that was incubated with a cytoplasmic extract from HeLa cells
was efficiently phosphorylated while BIK incubated without the extract
was not phosphorylated (Fig.
2A). The cytoplasmic extract
was also further fractionated through a cation exchange (SP-Sepharose)
column and the various fractions were assayed to determine their
ability to phosphorylate the recombinant BIK-(1-134) protein. These
results indicated that fractions 5-11 contained the kinase activity
that phosphorylated BIK (Fig. 2B). To identify the
phosphorylated amino acids of BIK, we carried out phosphoamino acid
analysis of in vitro phosphorylated BIK. For this purpose SP-Sepharose fractions 5-11 were pooled and used to phosphorylate BIK.
The phosphoamino acid analysis indicated that Ser and Thr residues were
phosphorylated and no detectable phosphorylation of Tyr was observed
(Fig. 2C). Similar results were also observed using in
vivo phosphorylated BIK (not shown).

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Fig. 2.
In vitro
phosphorylation of BIK. A, phosphorylation by
total cytoplasmic extract. One hundred ng of BIK (aa 1-134) was
incubated in the presence (+) or absence ( ) of 5 µl of crude HeLa
cell extract and 5 µCi of [ -32P]ATP in a 20-µl
reaction volume. The phosphorylated protein was precipitated with
trichloroacetic acid, run on 12% SDS-PAGE, and analyzed by
autoradiography. B, phosphorylation by partially purified
extract. Five-µl aliquots of various SP-Sepharose fractions of the
cytoplasmic extract were assayed for the kinase activity using
BIK-(1-134) as in A. The reactions were carried out in the
presence of 100 ng of BIK (indicated by +) or absence of BIK (indicated
by ). C, phosphoamino analysis. One µg of BIK was
phosphorylated by 10 µl of pooled HeLa cell fractions 5-11 as in
A and hydrolyzed, and phosphoamino acids were separated by
TLC. Circled areas represent the location of phosphoserine
(Ser), phosphothreonine (Thr), and
phosphotyrosine (Tyr) markers indicated by ninhydrin
staining.
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Mapping of the Phosphorylation Site--
To identify the
phosphorylation site(s), we performed site-directed mutagenesis by
converting all serine, threonine, and tyrosines residues contained
within the N-terminal 134-amino acid region of BIK to alanine residues
(Table I). The various mutants were tagged with the HA epitope and transiently expressed in 293T cells. The
protein extracts were analyzed by Western blot and probed with the HA
antibody. All the mutant proteins except mutant
S2A/S8A/T15A/Y18A were expressed as stable proteins. The various
mutant proteins, except mutant T33A/S35A were expressed as doublets,
suggesting that they are not defective in phosphorylation (summarized
in Table I). On the other hand, mutant T33A/S35A had only the fast migrating band, suggesting that this mutant may be defective in phosphorylation. To confirm that mutant T33A/S35A is defective in
phosphorylation, we performed metabolic labeling of 293T cells transiently transfected with wt BIK or mutant with 32P and
35S. Analysis of the proteins labeled with 35S
indicated that both wt and mutant proteins were expressed at comparable
levels, while there was no significant incorporation of 32P
in the mutant protein (Fig.
3A, rightmost
lane). These results suggest that mutant T33A/S35A is
strongly defective in phosphorylation under conditions where the wt BIK
protein is phosphorylated efficiently. The T33A/S35A mutation was also
found to abolish phosphorylation of purified BIK (aa 1-134) protein
in vitro (Fig. 3B). These results suggest that
residues Thr-33 or/and Ser-35 of BIK are required for phosphorylation.
To determine which of the two residues constitute the phosphorylation
site of BIK, we constructed two different single amino acid
substitution mutants of BIK in which either Thr-33 or Ser-35 was
mutated to an Ala residue (mutants T33A and S35A). The mutant proteins
were metabolically labeled with 32P and 35S and
analyzed (Fig. 3C). Both mutants (T33A and S35A) were found to be defective in phosphorylation, suggesting that both residues are
required for phosphorylation of BIK. However, these results do not
distinguish between the possibility that both residues may be
phosphorylated perhaps in a cooperative manner or only one is
phosphorylated and the other residue plays a structural role. Since the
phosphoamino acid analysis indicates that both Thr and Ser residues are
phosphorylated, it is possible that both Thr-33 and Ser-35 may be
target for phosphorylation.
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Table I
BIK mutants and phosphorylation
Various BIK mutants were analyzed by Western blot. A number of
different analyses are summarized in Table I. Mutants that expressed
both the slow and fast migrating forms of BIK were considered positive
for phosphorylation, and the mutant that expressed only the fast
migrating form was considered negative for phosphorylation.
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Fig. 3.
Phosphorylation of BIK mutants.
A, effect of T33A/S35A mutation on in vivo
phosphorylation. 32P- or 35S-labeled proteins
were immunoprecipitated with the HA antibody from extracts of 293T
cells transfected either with pcDNA3HA-BIK (WT) or
pcDNA3HA-BIK (T33S35A). Proteins were fractionated by
SDS-PAGE (15%) and analyzed by autoradiography. B, in
vitro phosphorylation of BIK-(1-134) and BIK-T33A/S35A mutant
proteins. One hundred ng of either wt BIK-(1-134) or BIK mutant
(T33A/S35A) proteins were phosphorylated using 5 µl of pooled HeLa
cell fractions 5-11 and analyzed as in Fig. 2. Upper
panel shows the autoradiogram of a 15% SDS-PAGE. The
Western blot shown in the lower panel indicates
the relative amounts of the BIK proteins. C, effect of T33A
and S35A mutations on in vivo phosphorylation. Proteins
labeled with 32P or 35S were immunoprecipitated
with the HA antibody and analyzed as in A.
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Effect of Phosphorylation on BIK Activities--
To investigate
the role of phosphorylation on BIK activities, we first determined the
apoptotic activities of wt BIK and mutant T33A/S35A in a transient
apoptosis assay. HeLa cells were transfected with plasmids expressing
wt BIK or mutant T33A/S35A along with a plasmid that expresses the
E. coli lacZ reporter gene. In cells transfected with the
empty vector (pcDNA3HA) less than 10% of the cells (that expressed
the lacZ gene) exhibited apoptotic features at all DNA
concentrations examined (0.5-3.0 µg), whereas, in cells transfected
with wt BIK or mutant BIK, apoptosis increased as a function of DNA
concentration (Fig. 4A). In
cells transfected with wt BIK, about 30-50% of the transfected cells
exhibited apoptosis, depending on the DNA concentration. In cells
transfected with the mutant BIK, there was significantly lower cell
death at all DNA concentrations, suggesting that the mutation reduces
the apoptotic activity of BIK.

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Fig. 4.
Effect of phosphorylation on BIK-mediated
apoptosis. A, transient apoptosis assay. HeLa cells
were cotransfected with the reporter plasmid
( -galactosidase) along with the empty vector
(pcDNA3HA), pcDNA3HA-BIK (wt), or pcDNA3HA-BIK (T33A/S35A).
Ten hr after transfection, cells were fixed and stained with X-gal and
apoptotic cells were quantitated. The empty vector is indicated by
filled square, wt BIK is indicated by
open square, and the mutant BIK is indicated by
filled diamond. The levels of BIK expression
(Western blot analysis with the HA antibody) in cells transfected with
BIK wt and BIK mutant T33A/S35A are shown in the bottom
panel. B, cell survival assay. Primary BRK cells
were transformed with the E1A hyper-transforming mutant 177-9 (26) and
T24 ras in the presence of wt BIK or mutant BIK or empty
vector. Transfected cells were selected with 50 µg/ml G418 for 10 days, stained with Giemsa, and photographed. DNA
conc., DNA concentration; V, vector (pcDNA3);
WT, wild type.
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In addition to the transient apoptosis assay, the effects of wt BIK and
the mutant BIK were also examined in a cell survival assay (27). This
survival assay is based on the principle that coexpression of a
pro-apoptotic gene along with the transforming oncogenes (E1A and T24
ras) in primary BRK cells reduces the formation of
transformed foci. In BRK cells transfected with the transforming oncogenes and wt BIK, formation of transformed colony formation was
severely reduced as compared with cells transfected with the oncogenes
and the empty vector (Fig. 4B). In cells transfected with
the oncogenes and the mutant, there was consistent increase in the
number of foci formation compared with cells expressing wt BIK,
suggesting that the mutant induced less efficient cell death. Both the
transient apoptosis assay (Fig. 4A) and the cell survival
assay (Fig. 4B) suggest that phosphorylation of BIK is required for the manifestation of efficient cell death activity.
We also examined the effect of phosphorylation of BIK on its ability to
heterodimerize with the cellular anti-apoptosis protein BCL-2. For this
purpose, 293T cells were transfected with HA-tagged wt BIK or mutant
(T33A/S35A) BIK or empty vector (pcDNA3-HA) and BCL-2. Protein
extracts were immunoprecipitated either with the HA antibody or with
the BCL-2 antibody. The immunoprecipitates were subjected to Western
blot analysis using the BCL-2 antibody (Fig.
5). This analysis revealed that the
phosphorylation-deficient mutant T33A/S35A complexed with BCL-2 at
ratios similar to that of wt BIK, suggesting that phosphorylation of
BIK at residues Thr-33 and Ser-35 apparently does not significantly
influence its affinity for BCL-2. In addition, we have also observed
that the T33A/S35A mutant had neither an altered half-life nor an
abnormal subcellular localization pattern compared with wt BIK (data
not shown).

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Fig. 5.
Interaction of phosphorylation-defective
mutant of BIK with BCL-2. Lysates from 293T cells coexpressing
HA-BIK (WT) or HA-BIK T33A/S35A (indicated by M)
and BCL-2 were immunoprecipitated either with a polyclonal anti-BCL-2
antibody (lanes marked IP:BCL-2) or anti-HA
monoclonal antibody 12CA5 (lanes marked IP:HA).
The blots were probed with anti-BCL-2 antibody. V, vector
(pcDNA3-HA).
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Characterization of the Kinase Activity--
To determine the
identity of the protein kinase responsible for phosphorylation of BIK,
we analyzed the primary amino acid sequences of BIK for the putative
consensus phosphorylation sites for known Ser/Thr protein kinases.
Computer-assisted analysis revealed that BIK contains four putative
CKII sites and two PKC sites (Fig.
6A). Interestingly, two of the
predicted CKII sites (Thr-33 and Ser-35) correspond to the
phosphorylation sites determined by mutational analysis of BIK, raising
the possibility that CKII may be a candidate kinase for phosphorylation
of BIK. It should be noted that mutations of other potential CKII and
PKC phosphorylation sites did not affect phosphorylation of BIK (Table
I).

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Fig. 6.
In vitro
characterization of BIK-specific kinase(s). A,
schematic representation of putative CKII and PKC phosphorylation
sites. B, size estimation. In gel kinase was performed using
300 µg/ml BIK-(1-134) as the substrate for phosphorylation by the
HeLa cell extract (pooled fractions 5-11). C, Western blot
analysis of HeLa cell extract. Blots of purified CKII and pooled
SP-Sepharose fractions 5-11 were probed with anti-CKII antibody.
D, phosphorylation of BIK with CKII. One hundred ng of wt or
T33A/S35A mutant of BIK-(1-134) was phosphorylated with 0.5 units of
CKII (New England Biolabs). The phosphorylated proteins were analyzed
by 12% SDS-PAGE and autoradiography. E, effect of DRB on
BIK phosphorylation. BIK-(1-134) protein was phosphorylated in the
presence of indicated concentrations of DRB either with purified CKII
or with the HeLa cell extract (pooled fractions 5-11). The
phosphorylated proteins were analyzed by 12% SDS-PAGE and
phosphorimager analysis. Level of phosphorylation in the absence of DRB
was set at 100%.
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To gain further insight into the nature of the protein kinase, we
determined the size of the kinase(s) present in the HeLa cell
cytoplasmic extract (pooled SP-Sepharose fractions 5-11) by the in
gel kinase assay using recombinant BIK (aa 1-134) protein as the
substrate. This analysis revealed that BIK-(1-134) was phosphorylated
by three proteins of about 36, 40, and 44 kDa (Fig. 6B). It
is interesting to note that molecular sizes of two of these proteins
(40 and 44 kDa) are similar to the size of the CKII subunits. Western
blot analysis indicate that the pooled HeLa cell cytoplasmic fractions
contain the 40- and 44-kDa subunits (
and
') of protein kinase
CKII (Fig. 6C, lane 2). We also
determined the effect of purified CKII on phosphorylation of BIK. As
shown in Fig. 6D, wt BIK (aa 1-134) was efficiently
phosphorylated by CKII while the T33A/S35A mutant was defective in
phosphorylation. These results raise the possibility that CKII may be
the candidate kinase for phosphorylation of BIK. We then tested the
effect of a CKII inhibitor, DRB, on phosphorylation of BIK-(1-134) by
purified CKII and HeLa cell extract. DRB inhibited BIK phosphorylation by CKII in a dose-dependent manner (Fig. 6E).
However, phosphorylation of BIK by HeLa cell extracts (SP-Sepharose
fractions 5-11) was not significantly affected by DRB, although the
extracts contain CKII (see Fig. 6C). Taken together, the
above result implicate that a kinase related to CKII, but insensitive
to DRB, may be the candidate kinase for phosphorylation of BIK.
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DISCUSSION |
We have demonstrated that the human pro-apoptotic protein BIK is a
phosphoprotein and that it is phosphorylated on consensus CKII
phosphorylation sites (Thr-33 and/or Ser-35). We have observed that
phosphorylation is required for efficient BIK-mediated cell death. This
is opposite of the role of phosphorylation on another BH3-alone
pro-apoptotic protein BAD. In the case of BAD, survival factors induce
phosphorylation via survival-promoting kinases Akt and protein kinase A
(19) and negatively influence its apoptotic activity. The precise
mechanism by which phosphorylation plays a role in the apoptotic
activity of BIK is not known. It has been proposed that BH3 proteins
mediate apoptosis primarily by interaction with BCL-2 family
anti-apoptosis proteins (see Ref. 28). In the case of BAD,
phosphorylation of a Ser residue within the BH3 domain affects its
ability to interact with BCL-2 (17, 29), which may contribute at least
partially toward inactivation of BAD activity. Here, we have observed
that the phosphorylation defective mutant of BIK interacts with BCL-2
at levels similar to that of wt BIK, despite its lower apoptotic
activity. We have also observed similar results in interaction studies
between the phosphorylation-defective mutant (T33A/S35A) and BCL-xL
(data not shown). These observations lend support to our earlier
observation (3) that heterodimerization of BIK with cellular
anti-apoptosis proteins is alone insufficient for its pro-apoptotic
activity. It is possible that phosphorylation of BIK may play role in
interaction with other potential cellular targets. In case of
pro-apoptotic proteins BAK and BID, the BH3 domain becomes exposed in
response to apoptotic stimuli (9, 10, 30). In the case of BID,
N-terminal protein processing appears to exposes the BH3 domain. It is
possible that phosphorylation of BIK confers certain conformational
changes that expose the BH3 domain.
Our results suggest that BIK may be a target for phosphorylation by a
kinase related to CKII. Although CKII-related kinases have shown to
phosphorylate apoptosis modulators such as p53 and c-Myc (33-36), BIK
would be the first BCL-2 family protein target for CKII. Previously,
BIK-induced apoptosis has been shown to be suppressed by
expression of the survival-promoting kinase, Akt (37). It appears that
the effect of Akt on BIK activity may be indirect as Akt suppresses the
activity of a number of other pro-apoptotic stimuli. Recently, it has
been reported that Akt may inhibit BAD and BIK-induced apoptosis
indirectly by inducing expression of BCL-xL (31).
BIK is constitutively expressed in several different human tissues and
appears to be expressed at elevated levels in skeletal and cardiac
muscles (32). It is possible that the activity of BIK may be activated
by phosphorylation in response to different apoptotic stimuli in
various human tissues. The effect of various apoptotic stimuli on
phosphorylation of BIK remains to be investigated.