From the Laboratory of Molecular Pharmacology, Division of Basic
Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892-4255
Protein phosphorylation plays an important role
in signal transduction, but its involvement in apoptosis still remains
unclear. In this report, the p53-null human leukemia HL60 cells were
used to investigate phosphorylation and degradation of lamin B during apoptosis. We found that lamin B was phosphorylated within 1 h after addition of the DNA topoisomerase I inhibitor, camptothecin, and
that lamin B phosphorylation preceded lamin B degradation and DNA
fragmentation. Using a cell-free system we also found that cytosol from
camptothecin-treated cells induced lamin B phosphorylation and
degradation in isolated nuclei from untreated HL60 cells. Lamin B
phosphorylation was prevented by the protein kinase C (PKC) inhibitor
7-hydroxystaurosporine (UCN-01) but not by the Cdc2
inhibitor, flavopiridol. Phosphorylation of lamin B was inhibited by immunodepletion of PKC
from activated cytosol and was restored by
addition of purified PKC
. PKC
activity also increased rapidly as
lamin B was phosphorylated after initiation of the apoptotic response
in HL60 cells. These data suggest that lamin B is phosphorylated by
PKC
and proteolyzed before DNA fragmentation in HL60 cells undergoing apoptosis.
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INTRODUCTION |
The nuclear lamins are karyophilic proteins located at the
nucleoplasmic surface of the inner nuclear membrane where they assemble
in a polymeric structure referred to as the nuclear lamina (for review,
see Refs. 1-3). Lamins belong to the family of intermediate filaments,
which share a tripartite organization consisting of a central
-helical rod domain of conserved size, flanked by N- and C-terminal
non-
-helical end domains of variable size and sequence (see Fig. 9).
The lamina has been suggested to serve as a major chromatin anchoring
site of nuclear scaffold-associated regions during interphase and
possibly to be involved in organizing higher order chromatin domains.
The lamina is a dynamic structure regulated by phosphorylation.
Phosphorylation by p34cdc2 kinase is key to the dissolution of
the nuclear lamina during mitosis. Other lamin kinases include
mitogen-associated protein kinases, c-AMP-dependent protein
kinase (PKA)1 and protein
kinase C (PKC) (1-3). Major PKC phosphorylation sites have been mapped
to serine residues located in close proximity to the nuclear
localization signal in the C-terminal non-
-helical region, and
phosphorylation of these residues interferes with the nuclear transport
of lamin B (2). The p34cdc2 phosphorylation sites are on both
sides of the central
-helical rod domain. While many mammalian cells
contains three distinct lamins (lamins A, B, and C), human leukemia
HL60 cells express primarily lamin B (4).
Lamin proteolysis during apoptosis has been reported in various cell
lines treated with different stimuli. In human leukemia HL60 cells
treated with etoposide (VP-16) (5) or camptothecin (CPT) (6), apoptosis
is accompanied by diminished levels of lamin B. Etoposide is a
topoisomerase II inhibitor (7) and CPT a topoisomerase I inhibitor (8).
Both drugs are effective anti-cancer agents. Lamin B1 degradation was
also reported to precede DNA fragmentation in apoptotic thymocytes (9)
and in HeLa cells treated with anti-CD95 antibody (10). Lamin A and B
proteolysis into 45-kDa fragments is also observed in apoptosis induced
by serum starvation of ras transformed primary rat embryo cells (11) and in reconstituted cell-free systems (6, 12). The site of
lamin A and B cleavage yielding the 45-kDa fragment has recently been
mapped to a conserved aspartate residue at position 230 (13, 14)
corresponding to a consensus sequence for caspases. Furthermore, lamin
A has been shown to be cleaved by caspase 6 (Mch-2
) but not caspase
3 (CPP32/YAMA) (13, 15).
The death-related cysteine proteases of the caspase family play a
central role in the execution phase of apoptosis (16-19). Each caspase
cleaves selectively a subset of cellular proteins. For instance,
poly(ADP-ribose)polymerase is preferentially cleaved by caspase 3 (CPP32/Yama) (20, 21), and lamin A can be cleaved by caspase 6 (Mch-2)
(13, 15). Interestingly, recent observations demonstrated that
overexpression of mutant lamins A or B resistant to caspase
cleavage delayed DNA fragmentation, suggesting that lamin cleavage
participates in the activation of DNA fragmentation and nuclear
apoptosis (14). Thus, lamins are presently the only known caspase
substrates known to be directly involved in the execution phase of
apoptosis.
Protein phosphorylation is probably important to regulate apoptosis
(22). For instance, unscheduled activation of p34cdc2 kinase,
one of the lamin kinases (2), is associated with cytotoxic T
lymphocyte-mediated apoptosis (23) and precedes CPT- and DNA damage-induced apoptosis in HL60 cells (24). In the present study, we
investigated lamin B phosphorylation and degradation during apoptosis
in response to camptothecin in HL60 cells and in a previously described
cell-free system (6, 25-27). The identity of the lamin B protease is
not known. Both caspases and the nuclear scaffold-associated serine
protease have been suggested as candidate proteases (28, 29).
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MATERIALS AND METHODS |
Chemicals, Drugs, and Antibodies--
CPT,
7-hydroxystaurosporine (UCN-01), and flavopiridol were obtained from
the NCI Drug Chemistry and Synthesis Branch. Drugs were freshly
dissolved in dimethyl sulfoxide at 10 mM and further diluted in water prior to each experiment. Radiolabeled precursor [14C]thymidine (53.6 mCi/mmol) was purchased from NEN
Life Science Products and [32P]orthophosphate was
obtained from Amersham Pharmacia Biotech. 3,4-Dichloroisocoumarin (DCI)
was purchased from Boehringer Mannheim. All other chemicals were of
reagent grade and purchased from Sigma or other local sources.
Anti-lamin B monoclonal antibody from mouse (101-B7) was purchased from
Oncogene Research Products (Cambridge, MA) and anti-protein kinase C
(anti-PKC
) polyclonal antibodies from rabbit was purchased from
Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-PKC monoclonal
antibody 1.9 and recombinant PKC
from baculovirus were purchased
from Life Technologies, Inc. The horseradish peroxidase-conjugated anti-mouse immunogloblin secondary antibody was purchased from Amersham
Pharmacia Biotech.
Cell Culture, Drug Treatment, DNA, and Protein
Labeling--
Human promyelocytic leukemia HL60 cells were grown in
suspension culture in RPMI 1640 medium supplemented with 10% fetal
calf serum (Life Technologies, Inc.), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in an
atmosphere of 95% air and 5% CO2. For filter elution
assays, HL60 cells were incubated with [14C]thymidine for
1-doubling time (about 24 h). Cell cultures were then washed with
fresh medium twice and chased in isotope-free medium overnight before
drug treatment. Unless otherwise indicated, camptothecin treatments
were with 5 µM. For in vivo phosphorylation, HL60 cells were washed twice in phosphate-free RPMI 1640 medium containing 10% dialyzed fetal calf serum, resuspended in the
same medium and incubated with 250 µCi of
[32P]orthophosphate/107 cells. Following 1-h
incubation, the 32P-labeled cells were washed twice and
resuspended in phosphate-free RPMI 1640 with dialyzed serum for 30 min
prior to drug treatment.
Isolation of Nuclei and Cytosol for Reconstituted Cell-free
System Studies--
We followed our previously published procedure
(25, 26). Briefly, untreated and treated cells (5 µM
camptothecin for 3 h) were spun down, rinsed three times in cold
PBS (phosphate-buffered saline), and resuspended at a density of
approximately 107 cells/ml in nucleus buffer (1 mM KH2PO4, 150 mM NaCl,
5 mM MgCl2, 1 mM EGTA, 0.1 mM AEBSF, 0.15 unit/ml aprotinin, 1.0 mM
Na3VO4, 5 mM HEPES, pH 7.4, 10%
glycerol), including 0.3% Triton X-100. After incubation at 4 °C
for 10 min and gentle agitation, cellular mixes were centrifuged at
2,000 × g for 10 min, rinsed once by centrifugation/resuspension in nucleus buffer without Triton X-100, and
used as nuclei suspensions at a density of 1-2 × 107
nuclei/ml. Supernatants were centrifuged at 10,000 × g
for 10 min and used as cytosol. [14C]Thymidine-labeled
cells were used to prepare nuclei for filter elution assay.
Filter Elution Assays for Measurement of DNA
Fragmentation--
DNA fragmentation related to apoptosis was measured
by filter elution as described previously (25, 30). Briefly, reaction mixtures were deposited onto protein-adsorbing filters (Metricel, Gelman Science, Ann Harbor, MI) and washed with 3 ml of nucleus buffer.
This fraction (W) was collected. Lysis was performed with 5 ml of LS10
(2 M NaCl, 0.04 M Na2EDTA,
0.2% Sarkosyl, pH 10) followed by washing with 5 ml of 0.02 mM Na2EDTA, pH 10. The lysis (L) and EDTA (E)
fractions were collected. All fractions (W, L, and E) and filters (F)
were counted by liquid scintillation. DNA fragmentation was calculated
as the percent of DNA eluting from the filter as: percent DNA
fragmentation = 100 × (W + L + E)/(W + L + E + F). All
experiments were repeated at least two or three times.
Immunoblotting for Lamin B--
After drug treatment, cells were
washed in PBS and resuspended in reducing loading buffer (62.5 mM Tris-HCl, pH 6.8, 6 M urea, 10% glycerol,
2% SDS, 0.003% bromphenol blue, 5% 2-mercaptoethanol) and sonicated
for 20 s. The lysates containing 2.5 × 105 cells
were heated at 65 °C for 15 min and then loaded in 12% SDS-polyacrylamide gels (precast gel from NOVEX, San Diego, CA). After
electrophoresis, proteins were electrophoretically transferred from the
gels to polyvinylidene difluoride membranes (Immobilon-P from
Millipore Co., Bedford, MA) according to the manufacturer's protocol.
Membranes were incubated at room temperature for 1 h in the
primary antibody solutions after blocking in 5% non-fat dry milk
solution for 1 h, followed by 1-h incubation with secondary antibody. Bands were visualized by enhanced chemiluminescence (SuperSignal, Pierce).
In Vivo Lamin B Phosphorylation--
After drug treatment,
32P-labeled HL60 cells (107 cells/sample) were
washed in phosphate-free RPMI 1640 medium without serum once, and the
cell pellets were lysed in buffer A (PBS containing 1% Nonidet P-40, 1 µg/ml leupeptin, 5 mM NaF, 1 mM
Na3VO4, 2 mM AEBSF, 4 units/ml
aprotinin, and 1% bovine serum albumin) with 0.4% SDS before
sonication. The cell lysates were centrifuged at 14,500 rpm for 15 min,
and the supernatants were mixed with 1.5 µg of anti-human lamin B
antibody and 20% protein G-Sepharose suspensions in lysis buffer
followed by overnight mixing at 4 °C. At the end of incubation, the
immune complex was washed in buffer A and buffer A without bovine serum
albumin. The immune complex was boiled for 10 min after adding 3 × SDS loading buffer. Samples were analyzed in 12% SDS-PAGE. Protein
gels were dried up and subjected to autoradiography after being dried
up.
In Vitro Lamin B Phosphorylation in Cell-free System--
The
nuclei from untreated HL60 cells (1.5 × 106 nuclei)
were incubated with cytosol in the presence of
[
-32P]ATP. After incubation, buffer A supplemented
with 0.4% SDS was added to the samples before brief sonication.
Afterward the procedures were the same as for in vivo lamin
B phosphorylation.
In Vitro Histone H1 Kinase Assay for Protein Kinase C
Activity--
After drug treatment, cells (107
cells/sample) were harvested, washed in cold PBS once, and resuspended
in 340 µl of lysis buffer (150 mM NaCl, 50 mM
Tris-HCl, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, 5 mM EGTA, 10 mM NaF, 5 µg/ml leupeptin,
1 mM Na3VO4, 1 mM
AEBSF) followed by 30-min incubation on ice. The supernatant was
collected after centrifugation at 14,500 rpm for 30 min and incubated
with protein G-Sepharose and 1 µg of anti-PKC
at 4 °C for
2 h to make PKC immunoprecipitates. The Sepharose beads associated
with immunoprecipitates were washed in lysis buffer three times and
twice in kinase buffer (50 mM Tris-HCl, pH 7.4, 10 mM NaF, 5 µg/ml leupeptin, 1 mM
Na3VO4, 1 mM AEBSF, 2 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA). The washed beads were incubated with reaction
buffer (20 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 10 µM ATP, 0.4 µg/ml histone H1, 5 µCi of [
-32P]ATP) in the presence of 1.2 mM CaCl2, 40 µg/ml phosphatidylserine, and
3.3 µM dioleylglycerol at 30 °C for 10 min. After the
incubation, 3 × SDS loading buffer was added to stop the kinase
reaction and samples were boiled for 10 min. Samples were run on 12%
SDS-PAGE, and the phosphorylated histone H1 bands were analyzed by
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Immunodepletion of PKC
--
Anti-PKC
antibody was
incubated with protein G-Sepharose beads at 4 °C for 3 h. The
beads were collected by centrifugation. After removal of the
supernatant, the beads were washed once with nucleus buffer and
incubated with cytosol from CPT-treated cells (CPT-cytosol:antibody = 5:1 (v/v)) overnight in a rotator at
4 °C. The beads were subsequently pelleted by centrifugation at
10,000 × g. The supernatant was subjected to
immunoblotting for PKC
and was used as CPT-cytosol immunodepleted of
PKC
. Mock-depleted CPT-cytosol was made just using nuclei buffer to
replace antibody.
 |
RESULTS |
Lamin B Degradation and Phosphorylation in Apoptotic HL60
Cells--
HL60 cells are remarkably sensitive to various apoptotic
stimuli, including chemotherapeutic DNA-damaging agents (5, 31) such as the topoisomerase I inhibitor camptothecin, protein kinase inhibitors (25, 32), and the Golgi poison, brefeldin A (33). Consistent
with previous studies (31), Fig.
1A shows that camptothecin induces apoptotic DNA fragmentation in HL60 cells with rapid kinetics. Lamin B protein was cleaved with similar kinetics as the DNA
fragmentation, yielding two cleavage bands (Fig. 1B)
corresponding to 45- and 32-kDa polypeptides that were detected 3 h after the beginning of drug treatment.

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Fig. 1.
DNA fragmentation and lamin B cleavage in
apoptotic HL60 cells after treatment with camptothecin. A,
[14C]thymidine-labeled cells were treated without
(Control) or with camptothecin (CPT) for
the indicated times. DNA fragmentation was assayed by filter elution.
B, at the indicated times, cell lysates from
camptothecin-treated cells were separated by SDS-PAGE. Lamin B was
detected by Immunoblotting. Numbers to the left
correspond to migration position of protein markers (in kilodaltons).
Lamin B-specific bands are indicated to the right.
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Since phosphorylation is critical for modulating lamin stability and
the nuclear and chromatin structure both in mitosis and interphase (2),
we studied lamin B phosphorylation during apoptosis in HL60 cells. As
shown in Fig. 2, phosphorylation of the
69-kDa lamin B polypeptide increased rapidly during camptothecin
treatment. Three hours after the beginning of treatment, the 32-kDa
lamin B cleavage product was also phosphorylated (Fig. 2A).
These results indicate that lamin B phosphorylation occurs early during
apoptosis and is associated with its degradation.

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Fig. 2.
Lamin B phosphorylation in apoptotic HL60
cells. [32P]Orthophosphate-labeled HL60 cells were
treated with camptothecin. Lamin B protein from cell extracts
was immunoprecipitated with anti-lamin B antibody. A,
samples were separated by 12% SDS-PAGE and processed by
PhosphorImager. Lanes 1-4 are samples taken 0.5, 1, 2, and
3 h after addition of camptothecin. B, quantification
of lamin B phosphorylation in camptothecin-treated (CPT) and
control HL60 cells.
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Cytosol from Apoptotic HL60 Cells Also Induced Lamin B Cleavage and
Phosphorylation in Vitro--
We next used a cell-free system that we
previously established to demonstrate the role of serine proteases in
triggering apoptotic DNA fragmentation (25, 26, 34). Consistent with
our previous results, the cytosol from apoptotic HL60 cells induced DNA
fragmentation in nuclei from untreated HL60 cells (Fig.
3A). Cytosol from apoptotic cells also cleaved lamin B from naive nuclei to a 45-kDa product (Fig.
3B).

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Fig. 3.
DNA fragmentation and lamin B degradation
induced in untreated isolated nuclei by cytosol from apoptotic HL60
cells. A, nuclei from untreated
[14C]thymidine-labeled HL60 cells were incubated at
37 °C for the indicated times with cytosol from untreated HL60 cells
(Control Cytosol) or cells treated with camptothecin
(CPT Cytosol). DNA fragmentation was measured by filter
elution. B, naive nuclei were incubated at 37 °C with
CPT-cytosol or control-cytosol for the indicated times. At the
indicated times samples were analyzed by SDS-PAGE and lamin B
immunoblotting.
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Lamin B phosphorylation was then studied in the cell-free system after
incubation of nuclei suspensions with cytosols from apoptotic or
control cells in the presence of [
-32P]ATP. After
immunoprecipitation with anti-lamin B antibody, samples were run on
SDS-PAGE, and phosphorylated lamin B was analyzed by autoradiography
and PhosphorImager (Molecular Dynamics). Fig. 4 shows that cytosol from apoptotic cells
enhanced lamin B phosphorylation.

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Fig. 4.
Apoptosis-associated lamin B phosphorylation
in a cell-free system. A, nuclei from untreated HL60 cells
were incubated at 37 °C for the indicated times with cytosol from
HL60 cells treated with camptothecin for 3 h (CPT
Cytosol) or from untreated HL60 cells (Control Cytosol)
in the presence of [ -32P]ATP. After incubation, lamin
B was immunoprecipitated from the reaction mixtures with anti-lamin B
monoclonal antibody. Phosphorylated lamin B (32P
Lamin B) was analyzed by a PhosphorImager after SDS-PAGE. A
representative experiment is shown. B, lamin B
phosphorylation induced by CPT-apoptotic cytosol was quantitated
relative to untreated (control) cytosol.
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Lamin B Cleavage, but Not Phosphorylation, in the Cell-free
System Can Be Inhibited by the Serine Protease Inhibitor DCI--
We
observed previously that DNA fragmentation induced by apoptotic cytosol
could be inhibited by the serine protease inhibitor DCI (6). Fig.
5 shows while DCI blocked DNA
fragmentation induced by CPT, lamin B cleavage also was abolished. The
result suggested that serine protease activation was required for
both DNA fragmentation and lamin B degradation. We next asked whether
lamin B phosphorylation could be inhibited by DCI. Fig. 5C
shows that lamin B phosphorylation was not affected by DCI. This
finding suggests that protease activation does not affect lamin B
phosphorylation.

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Fig. 5.
Effects of the serine protease inhibitor DCI
on DNA fragmentation, lamin B degradation, and phosphorylation in a
cell-free system. Naive nuclei were incubated at 37 °C for 30 min with cytosol from apoptotic HL60 cells (CPT cytosol) or
from untreated HL60 cells (Control cytosol) in the absence
or presence of DCI. A, DNA fragmentation was measured by
filter elution. B, Western blotting for lamin B protein.
C, lamin B phosphorylation measured after
immunoprecipitation with anti-lamin B antibody.
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Investigation of the Lamin B Kinase during Apoptosis in HL60
Cells--
Cyclin B/Cdc2 (p34cdc2) kinase is critical for
lamin depolymerization during mitosis (2). We found that flavopiridol,
a potent Cdk inhibitor (35, 36) could not inhibit lamin B
phosphorylation induced by cytosol from apoptotic cells even at high
concentrations (100 µM) (Fig.
6A). We also found that
flavopiridol had not effect on either DNA fragmentation or lamin B
cleavage in the cell-free system (Fig. 6, B and
C). These observations suggested that p34cdc2 kinase
was not responsible for phosphorylation of lamin B by the apoptotic
cytosol.

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Fig. 6.
Effects of the cyclin-dependent
protein kinase inhibitor flavopiridol on DNA fragmentation, lamin B
degradation, and phosphorylation in vitro. Nuclei from
untreated HL60 cells were incubated at 37 °C for 30 min with cytosol
from apoptotic HL60 cells (CPT cytosol) or from untreated
HL60 cells (Control cytosol) in the absence or presence of
flavopiridol. A, lamin B phosphorylation. B, DNA
fragmentation. C, Western blotting for lamin B
protein.
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Protein kinase C has also been shown to phosphorylate lamin B in
interphase cells (37). We first used the PKC inhibitor UCN-01 (38) to
test whether lamin B phosphorylation during apoptosis is related to
PKC. Fig. 7A shows that lamin
B phosphorylation was inhibited by UCN-01 in a
dose-dependent manner. An anti-PKC monoclonal antibody,
which acts near the active site of PKC and inhibits PKC activity by
more than 80% (39), was used next. This antibody strongly suppressed
lamin B phosphorylation induced by apoptotic cytosol. Recombinant
PKC
restored lamin B phosphorylation (Fig. 7B). A third
type of experiment was performed to test whether lamin B
phosphorylation in apoptotic cells extracts could be linked to PKC
.
Fig. 7C shows that after immunodepletion of PKC
from apoptotic cytosol, lamin B phosphorylation was reduced by
about 90%. The efficiency of the immunodepletion was tested (Fig.
7C, lower panel) and showed that under these condition
PKC
protein levels were almost undetectable. Together, the results
shown in Fig. 7 suggested that PKC
was critical for lamin B
phosphorylation by cytosol from apoptotic HL60 cells.

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Fig. 7.
Lamin B phosphorylation by apoptotic cytosol
is prevented by the PKC inhibitor UCN-01, by anti-PKC antibody, and by
immunodepletion of cytosolic PKC and is restored by purified
PKC . Nuclei from untreated HL60 cells were incubated with
cytosol from apoptotic cells (CPT cytosol) and
[ -32P]ATP in the absence or presence of various
concentration of UCN-01 (A) or anti-PKC antibody 1.9 and/or
PKC (B) for 5 min at 37 °C. C, inhibition
of lamin B phosphorylation by immunodepletion of PKC . Cytosol from
apoptotic HL60 cells (CPT cytosol) was incubated with or
without anti-PKC antibody (2 µg/100 µl), including protein
G-Sepharose at 4 °C overnight. Upper panel, lamin B
phosphorylation in cell-free system was measured using supernatants.
Lower panel, supernatants were subjected to Immunoblotting
for PKC .
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Protein Kinase C
Is Activated with a Similar Kinetics as
Lamin B Phosphorylation during Camptothecin-induced Apoptosis in HL60
Cells--
A recent study showed that PKC
is activated in cytosol
from apoptotic HL60 cells (40). Now we tested whether camptothecin also
induces PKC
activation in whole HL60 cells. As shown in Fig.
8, PKC
activity increased rapidly
during the first hour after the beginning of camptothecin
treatment.

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Fig. 8.
Induction of PKC activity in apoptotic
HL60 cells after treatment with camptothecin. PKC
immunoprecipitates were prepared from camptothecin
(CPT)-treated and untreated (Control) HL60 cells
and tested for PKC activity using histone H1 as substrate.
A, representative PhosphorImager picture.
32P-H1, radiolabeled histone H1. B,
quantification of PKC activity as percent of untreated
control.
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 |
DISCUSSION |
The present study is the first report of lamin B
phosphorylation during apoptosis. We found that lamin B is
phosphorylated within 1 h after the addition of the apoptotic
inducer (camptothecin) and that lamin B phosphorylation persists for
several hours as lamin B is being cleaved, and DNA fragmentation and
complete apoptosis take place (31). Various kinases are involved in
lamin phosphorylation, including p34cdc2 kinase,
mitogen-associated kinase, PKC, PKA (2). In particular, in mitosis,
Cdc2 kinase is believed to play a critical role in lamin
phosphorylation and thus disassembly of lamin polymers (2, 41). The
present data suggest that PKC is critical for lamin B phosphorylation
in HL60 cell during apoptosis for the following reasons. First, the PKC
inhibitor, UCN-01 (38, 42) effectively blocked the lamin kinase
in vitro, while the cyclin kinase inhibitor flavopiridol
(35) was inactive. Second, lamin B phosphorylation was inhibited by a
monoclonal antibody directed against the active site of PKC. Third,
immunodepletion of apoptotic cell extracts with anti-PKC antibody
inhibited lamin B phosphorylation, while addition of excess PKC
restored lamin B phosphorylation. Fourth and finally, total PKC
activity increased at the time of lamin B phosphorylation (40).
The lamin B protein kinase C phosphorylation sites have been mapped to
serines 395 and 405 in HL60 cells following PKC activation by
bryostatin (43) (Fig. 9). These sites are
adjacent to the highly conserved central
-helical rod domain, which
is thought to be responsible for the formation of a highly stable
coiled-coil dimer between two lamin molecules. It is also next to the
nuclear translocation signal sequence (NLS) (Fig. 9). In the
case of chicken lamin B2, phosphorylation by PKC in this region has
been shown to alter recognition of this sequence and block nuclear
import of newly synthesized lamin polypeptides (2). Recently,
PKC-mediated lamin B phosphorylation during interphase has been shown
to promote lamin B solubilization and nuclear lamina disassembly (37). Thus, PKC-mediated lamin B phosphorylation during apoptosis is likely
to affect nuclear and chromatin structure.

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Fig. 9.
Schematic diagram of cleavage and PKC
phosphorylation sites of human lamin B. Hypothetical cleavage
sites of caspases at aspartate 231 (14) and putative nuclear scaffold
protease at tyrosine 377 are indicated above the domain organization of
lamin B. PKC phosphorylation sites (43) (solid arrows) and a
p34cdc2 kinase phosphorylation site (open arrowhead)
(2) are shown at the bottom. The nuclear localization signal
(NLS) is represented as an open box and the
C-terminal CaaX motif as the black box. The C-terminal
tetrapeptide referred to as the CaaX box (C = Cys;
a = aliphatic amino acid; X = any amino acid) is
subject to three successive posttranslational modifications
(farnesylation, proteolytic trimming, and carboxymethylation), which
are required for association of nuclear lamins with the nuclear
membrane (2).
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Proteolytic lamin degradation is a common and probably functionally
important biochemical feature of apoptosis. It has been observed in all
the apoptotic cell systems described to date, including HL60 cells
treated with chemotherapeutic agents (5, 6, 34), activation-driven cell
death of T cells (44), thymocyte apoptosis (9), and serum starvation in
ras transformed embryo cells (11). Lamin degradation has
also been reported in apoptosis induced by drICE in insect cells (45).
Interestingly, a recent study of Rao et al. (14)
demonstrated that overexpression of lamin A or B delayed nuclear
apoptosis and DNA fragmentation in the case of
p53-dependent apoptosis in rodent cells. These observations suggest that lamin cleavage plays an active role in the execution phase
of apoptosis. Caspase 6 (Mch-2) has been shown to cleave lamin A at the
conserved aspartic residue at position 230 (13, 14), and the
corresponding lamin B cleavage site has been mapped to
Asp231 (14). This site is located in the conserved
-helical rod domain (Fig. 9), and its cleavage would produce two
polypeptides of 40.3 and 25.9 kDa, respectively. The observed 45-kDa
fragment has been shown to correspond to the predicted 40.3 fragment
(13, 14). Another candidate lamin protease is the nuclear scaffold
protease (46), which would be expected to cleave lamin B at tyrosine 377 and to yield two polypeptides of 43.2 and 23 kDa, respectively (Fig. 9). Therefore, it is possible that serine proteases (6) might
also be involved in lamin B cleavage during apoptosis to produce the
32-kDa lamin B fragment that we observed in addition to the 45-kDa
polypeptide in apoptotic HL60 cells. Since the observed sizes indicate
that cleavage of lamin B during apoptosis occurs in the conserved
-helical rod domain, which is essential for lamin dimerization (Fig.
9), it is likely that lamin B cleavage should promote the dissolution
of the nuclear lamina and affect nuclear condensation. This recent
conclusion is consistent with the works of Rao et al. (14)
and Lazebnik et al. (12), demonstrating an impairment of
apoptotic chromatin condensation upon inhibition of lamin
proteolysis. Chromatin condensation and DNA fragmentation might be
related to the important function of the nuclear lamina as an anchorage
structure for the chromatin scaffold-associated regions, which would
organize the chromatin loop structures. Thus, it is possible that
chromatin release from the nuclear lamina might facilitate the activity
of nucleases and the cleavage and release of chromatin loops during
apoptosis.