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INTRODUCTION |
A growing body of evidence suggests that the commitment to
and execution of apoptosis are mediated through the phosphorylation of
specific proteins by several protein kinases (1). A number of
PKC1 isoforms appear to be
among these protein kinases (2, 3). Apoptogens may activate or
inactivate and cause the translocation of various PKC isoforms from the
cytosol onto cytoskeletal components, cytoplasmic membranes,
mitochondria, and/or the nuclear envelope (4-11); induce their
migration from such subcellular structures to nucleoplasmic and/or
cytosolic fractions (8, 12); or cause the PKC holoenzymes to be cleaved
into N-terminal regulatory and C-terminal catalytic fragments (CFs) by
several proteases, including caspases (11, 13-15). Despite suggestions
that a particular PKC isoform (e.g. the novel PKC-
and/or
its CFs) might play a pivotal role in apoptosis (13, 14),
the available evidence indicates the involvement and cleavage of novel
PKCs (e.g. PKC-
and PKC-
), the classical
Ca2+-stimulable PKCs (e.g. PKC-
), atypical
PKCs (e.g. PKC-
), and PKC-related (e.g. PRK)
kinases in apoptosis (2, 3, 15).
This is the third report from a continuing study of the roles of
protein kinase C isozymes in drug-induced apoptosis using a
polyomavirus-transformed embryo rat fibroblast, the pyF111 cell, as a
model (10, 11). We chose this fibroblast, since it is prone to
apoptosis because it cannot make the antiapoptotic Bcl-2 and
Bcl-XL proteins but can make the proapoptotic Bax protein (10, 11). We have shown that whereas a surge of the activity of PKC-
holoprotein anchored in the cytoplasmic particulate fraction is
a common component of the signal given by diverse apoptogenic drugs to pyF111 cells, only the DNA-damaging topoisomerase-II inhibitors cause a prompt, large, and irreversible fall of PKC-'s specific activity at the nuclear envelope, despite the accumulation and
proteolysis of PKC- holoproteins at the envelope (10, 11). Thus,
according to the apoptogen used, a PKC isoform can be activated, inactivated, or unaffected at the same time in different parts of the
pyF111 cell.
The present experiments focused on the behavior and activities of other
PKC isoforms at the nuclear envelope of apoptosing VP-16-treated pyF111
cells. The results of these experiments indicate that during the
execution phase of apoptosis, catalytically competent PKC-II holoproteins move onto the nuclear envelope,
where they target specific substrates and are cleaved into CFs.
In our search for the nuclear substrates of PKC-II and
PKC-, we focused on the lamins. The nuclear lamina is a network of lamins attached to the inner nuclear membrane (16, 17). Lamins are type
V intermediate filaments with a conserved, central 
-helical rod
domain flanked by non--helical N- and C-terminal domains (16-18).
The nuclear lamina can be reconfigured by phosphorylation while
operating as an important anchoring site for specific chromatin regions
and contributing to the higher order chromatin organization in
interphase (18). The relaxation of the nuclear lamina during the
S-phase (19) and its dissolution during mitosis are caused by several
different protein kinases, such as suPKC1 kinase in interphase sea
urchin eggs (20), cyclin B·CDK-1 kinase (21-23), S6 kinase II (24),
cAMP-dependent protein kinase (25), and PKC- (26) or
PKC-II (27-29). Dissolution of the nuclear lamina is
also needed to package collapsed and fragmented chromatin into apoptotic bodies (30). Thus, nuclear lamins A and B1 are phosphorylated and before DNA fragmentation are cleaved at their -helical rod domains into 45-kDa fragments during the apoptosis caused by
topoisomerase-I and -II inhibitors (30, 31), anti-CD95 antibody (32),
and serum withdrawal (33). Lamins A and B1 are cleaved at amino acids
Asp230 and Asp231, respectively, by caspase-6
but not by caspase-3 (34-37). Overexpression of mutant,
caspase-resistant lamin A or B1 slows the onset of DNA fragmentation,
indicating that lamin proteolysis is part of the execution phase of
apoptosis (34). However, different cells may have different apoptotic
lamin kinases. Thus, the mitotic lamin kinase, CDK-1·cyclin B, is not
also, as might be expected, the apoptotic lamin kinase in thymocytes
(38), PKC- may be the apoptotic lamin kinase in camptothecin-treated
human promyelocytic leukemia HL-60 cells (26), and PKC- may be the
apoptotic lamin kinase in Ara-C-treated HL-60 cells (39). Here we
report the results of experiments indicating that PKC-II
is an apoptotic lamin kinase in VP-16-treated pyF111 rat fibroblasts.
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EXPERIMENTAL PROCEDURES |
Cell Cultures--
Polyomavirus-infected/transformed pyF111
Fischer rat embryo fibroblasts, a gift from Dr. L. Raptis (Department
of Microbiology, Queen's University, Kingston, Ontario, Canada), were
cultured and handled as previously detailed (10, 11).
Apoptosis Induction and Detection--
Experiments were started
by planting 1.2 × 106 cells in each of 20-25 F-160
flasks. Twenty-four hours later (i.e. at the experimental 0 h), the cells in some flasks were sampled (zero time controls), the medium in some other flasks was replaced with the same, but fresh,
medium (parallel or proliferation control group), and the medium in the
remaining flasks was replaced by fresh medium containing 1.0 µg
ml
1 of VP-16 (etoposide or
4N-dimethylepipodophyllotoxin
4-[4,6-O-ethylidene--D-glucopyranoside]; Sigma), a topoisomerase-II inhibitor (30). The medium was not changed
again during observation periods ranging from 6 to 48-72 h.
We considered as "viable" only those cells that excluded trypan
blue or ethidium bromide (EB) and whose nucleic acids were stained
normally with SYTO-10TM or acridine orange (AO) (all from Molecular
Probes Inc., Eugene, OR). Morphological signs of apoptosis were looked
for according to the procedure of Spector et al. in AO-EB
doubly stained samples (40). Loss of high molecular weight DNA and the
generation of oligonucleosomal ladder-type DNA fragments were detected
by gel electrophoresis according to Pellicciari et al. (41).
To determine the mitotic cell cycle fractions and the fractions with
hypodiploid DNA contents, the sub-G1 fractions, cellular
DNA contents were measured with a fluorescence-activated cell sorter
(FACStar PlusTM; Becton Dickinson, Sunnyvale, CA) as previously
described (11).
Subcellular Fractionation--
Cells were harvested by scraping
them into cold (4 °C) phosphate-buffered saline and centrifuging the
suspension at 200 × g for 10 min. The sedimented cells
were carefully resuspended in a solution containing 10 mM
Hepes, pH 7.9, 10 mM KCl, 1.0 mM MgCl2, 1.0 mM dithiothreitol, 20 µM sodium orthovanadate, and complete EDTA-free protease
inhibitor mixture (Roche Molecular Biochemicals). The cells were then
chilled on ice for 15 min and gently lysed by adding 0.6% (v/v)
Nonidet P-40. The lysate was centrifuged at 200 × g
for 10 min to produce the whole nuclei fraction pellet, containing
whole nuclei, and the SN1 supernatant. The whole nuclei fraction was
suspended in an excess volume of hypotonic buffer (10 mM
Tris, pH 7.4, 10 mM Na2HPO4, 1.0 mM dithiothreitol, 20 µM sodium
orthovanadate, and complete EDTA-free protease inhibitor mixture (Roche
Molecular Biochemicals) containing DNase I and heparin (0.2 and 5.0 mg/mg of nuclear protein, respectively)). This suspension was incubated
at 4 °C for 45 min and then centrifuged at 9500 × g
for 15 min. The resulting pellet was the nuclear membrane-enriched NM
fraction, and the supernatant was the nucleoplasmic (NP) fraction. The
SN1 supernatant was centrifuged at 100,000 × g for
1 h to yield the SN2 supernatant or cytosolic fraction (CS) and
the P2 pellet or cytoplasmic particulate fraction. The purities of
these fractions were assessed from their contents of the cytosolic
marker lactate dehydrogenase (EC 1.1.1.27) measured by the method of McIntosh and Plummer (42), the plasma membrane marker 5'-nucleotidase (EC 3.1.3.5) assayed by the method of Ipata as modified by McIntosh and
Plummer (42), and the nuclear marker lamin B1 evaluated by Western
immunoblotting (43). In this study, we focused on the nuclear fractions
(the NM and NP fractions), but cytoplasmic fractions (e.g.
CS) were also used whenever required.
Western Immunoblotting--
The protein contents of the
subcellular fractions were assayed according to Bradford (44) using
bovine serum albumin as a standard. Equal amounts (10 or 20 µg) of
proteins from each NM sample were boiled in sample buffer (0.0625 M Tris-HCl, pH 6.8, 2% (w/v) SDS, 5% (w/v)
-mercaptoethanol, 10% (v/v) glycerol, 0.002% (w/v) bromphenol
blue) and electrophoresed in 10% (w/v) SDS-polyacrylamide gel. The
separated proteins were blotted onto a nitrocellulose membrane (0.45 µm; Bio-Rad) (10, 11). To immunodetect PKC-, PKC-I,
PKC-II, and PKC-, the blots were probed with
isotype-specific rabbit IgG polyclonal antibodies (final dilution 1.0 µg ml1), which recognized the
carboxyl-terminal amino acid sequences of each isoform (Sigma (PKC-)
and Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The staining of
the particular band pertaining to each PKC isoform could be suppressed
by preincubating each antibody with the peptide against which it had
been raised (Santa Cruz Biotechnology). Blots were also probed with
antibodies (final dilution 1.0 µg ml1) to
detect cytochrome c, lamin B1, tyrosine 15-phosphorylated (Tyr(P)15, i.e. inactive) CDK-1 kinase or
total ("pan," i.e. both active and
Tyr(P)15-inactive) CDK-1 kinase (Santa Cruz Biotechnology),
and serine-phosphorylated (Ser(P)) proteins (Calbiochem). Blots were
next incubated with alkaline phosphatase-conjugated anti-mouse or
anti-rabbit IgG (Santa Cruz Biotechnology) and stained with
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium liquid
substrate reagent (Sigma). The developed blots were photographed with
an Olympus 3300TM digital camera, and the determination of the
Mr and the densitometric analysis of each
specific band were carried out with SigmagelTM software (Jandel Corp.,
Erkrath, Germany).
Immunoprecipitation of PKC Isoforms, Lamin B1, and Ser(P)
Proteins--
The same amount of protein (300 µg) from each NM
sample was used for the immunoprecipitation experiments. Protein
samples were incubated at 4 °C for 3 h with antibodies directed
against PKC-, PKC-II, and PKC- (anti-PKC- from
Sigma and anti-PKC-II and anti-PKC- from Santa Cruz
Biotechnology), lamin B1 (Chemicon International, Temecula, CA), or
Ser(P)-proteins (Calbiochem) conjugated to Immunopure-immobilized
protein A (Pierce). The immunocomplex-bearing beads were collected by
centrifugation at 2500 rpm for 5 min at 4 °C and washed five times
with Tris-buffered saline (20 mM Tris, pH 7.4, 200 mM NaCl), 1.0 mM dithiothreitol, 20 µM sodium orthovanadate, and complete EDTA-free protease
inhibitor mixture (Roche Molecular Biochemistry). After a final wash,
the immunocomplex-bearing beads were resuspended in Tris-buffered
saline to measure PKC activity or in sample buffer for Western immunoblotting.
Assay of Immunopurified Native PKC-II or PKC-
Specific Activity--
A colorimetric PKC activity assay kit, the
Spinzyme FormatTM (Pierce) was used (10, 11). This kit includes as a
PKC-specific substrate, the -peptide (i.e.
XERMRPRKRQGSVRRRV, where X is lissamine rhodamine
B). To measure the specific activity per µg of protein of an
immunoprecipitated PKC isoform, the assay mixture (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA (or alternatively 0.5 mM calcium), 2.0 mM ATP, 10 mM
MgCl2, 0.002% (v/v) Triton X-100, 0.36 mM
dye-labeled -peptide) without any cofactor was added to the
immunocomplex-bearing beads. All of the assay mixtures were incubated
for 30 min at 30 °C, and the amounts of phosphorylated peptide
were determined as recommended by the supplier. The results were
expressed in arbitrary units calculated for each sample as the ratios
between optical density values and µg of the corresponding
immunoprecipitated protein.
Isolation of Cytoplasms and Nuclei for Cell-free Reconstituted
System Studies--
We basically followed the procedure of Shimizu
et al. (26). Untreated or VP-16-treated (1.0 µg
ml1 for 6, 18, or 24 h) pyF111 cells
were harvested by scraping them into cold (4 °C) phosphate-buffered
saline, washed twice by centrifugation/resuspension cycles in cold
phosphate-buffered saline, and next incubated on ice for 10 min at a
density of 1.0 × 107 cells
ml1 in a lysis buffer (150 mM
NaCl, 1.0 mM KH2PO4, 5.0 mM MgCl2, 1.0 mM EGTA, 1.0 mM dithiothreitol, 20 µM sodium
orthovanadate, complete EDTA-free protease inhibitor mixture (Roche),
5.0 mM Hepes, pH 7.4, 10% glycerol, and 0.3% Triton
X-100). Lysates were next centrifuged (2000 × g for 10 min at 4 °C), and their supernatants were the control cytoplasmic
fractions from untreated cells or apoptotic cytoplasmic fractions from
cells incubated with VP-16 for 6, 18, and 24 h. Nuclei isolated
from the untreated cells were next washed twice by
centrifugation/resuspension in the lysis buffer without Triton X-100
and finally suspended at a density of 1.0-2.0 × 107
nuclei ml1. Control or apoptotic cytoplasms
(4 volumes) were then incubated at 30 °C for 30 min with nuclei (1 volume) from untreated cells either with or without hispidin (5.0 µM) (Calbiochem), a PKC-, but not PKC-, inhibitor
(45-47). Equal amounts of protein (i.e. 300 µg) from the
NM fractions of these reconstituted nucleus-cytoplasm (N-C) mixtures
were immunoprecipitated with an anti-Ser(P) antibody, and such
precipitates were blotted and challenged with
anti-PKC-II or anti-lamin B1 antibody.
Assay of Caspase-3 and -6 Activity--
The activities of
caspase-3 and -6 were measured in various subcellular fractions and in
immunoprecipitates from the NM fractions with the specific fluorometric
substrates Ac-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC)
and Ac-Val-Glu-Ile-Asp-7-Amido-4-methylcoumarin (Ac-VEID-AMC),
respectively (both from Alexis Corp., San Diego, CA). Base-line
activities were determined in samples from untreated pyF111 cultures at
0 h (48). The results pertaining to immunoprecipitates were
expressed in arbitrary units calculated for each sample as the ratios
between fluorescence values and µg of the corresponding immunoprecipitated protein.
Statistical Analysis--
Statistical significance was assessed
with Student's t test for unpaired samples; only
differences with p < 0.05 were regarded as significant.
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RESULTS |
Apoptosis Induction by VP-16 in pyF111 Cells--
The number of
substrate-adhering cells in untreated pyF111 cultures increased by
9.8-fold on the average between 0 h (i.e. 24 h
after planting in F-160 flasks and a fresh medium change) and 72 h, when more than 99.7% of the cells were viable as indicated by their
exclusion of trypan blue or EB fluorochrome (Fig.
1A; see also Refs. 10 and
11).
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Fig. 1.
The G2-M block and increased size
of the apoptotic sub-G1 fraction caused by VP-16 in pyF111
cells. A, pyF111 fibroblasts (1.2 × 106/flask) were either untreated (0 h; left) or
treated with VP-16 (1.0 µg ml1)
(right). The cells were doubly stained with AO-EB
(46) and observed under the fluorescence microscope. In untreated
actively proliferating cells, only the membrane-permeable AO
stains the nuclei yellow-green and the cytoplasms pale
green. After a 72-h exposure to VP-16, most of the cells have
died, and the remaining AO-stained ones appear to be shrinking or have
just been converted into apoptotic bodies (solid
arrow), whose clumps of chromatin emit a dazzling
yellow fluorescence. Conversely, aged apoptotic bodies have
lost their membrane integrity and emit a red fluorescence
due to penetration of the otherwise membrane-impermeable EB
(dashed arrow). Magnification, ×120.
B, pyF111 cells (1.2 × 106/flask) were
either untreated (0 and 48 h) or treated (48 h) with VP-16 (1.0 µg ml1). Cellular DNA contents and sizes of
the several cell cycle fractions and of the sub-G1
(hypodiploid) fraction were assessed with a fluorescence-activated cell
sorter as previously detailed (11). Bars represent mean
values from three independent experiments. S.E. values (not shown)
were within 11% of the mean values. *, p < 0.05.
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Both at 0 and 48 h later (i.e. when they were
exponentially growing), the fractions of untreated pyF111 cells in the
G1, S, and G2-M phases of the cell cycle did
not significantly change, and the apoptotic hypodiploid
(sub-G1) cell population remained negligible (Fig.
1B).
As expected and as previously shown (10, 11), VP-16 (1.0 µg
ml1 of culture medium) caused cells to stop
and accumulate in the G2-M phase (i.e. from
around 15-50% by 48 h) with a corresponding drop in the
G1 and S fractions (Fig. 1B) and triggered
apoptosis, the execution of which started 24 h after the drug was
added to the cultures and had killed 50% of the cells by 48 h and
75% by 72 h (Fig. 1A; see also Refs. 10 and 11). The
size of the hypodiploid or sub-G1 fraction had also risen
greatly by 48 h (Fig. 1B). Between 24 and 72 h,
the cells lost their high molecular weight DNA, which had been cleaved
into a characteristic ladder of oligonucleosomal DNA fragments (data
not shown; see Refs. 10 and 11 for identical results). As previously
shown (10, 11), PKC- specific activity at the nuclear envelope soon
began dropping dramatically, while cytochrome c escaped from
the mitochondria into the cytosol, where its level peaked by 24 h
and stayed at that level for the next 48 h (data not plotted).
Effects of VP-16 on the Translocation, Specific Activity, and
Cleavage of Activated PKC-II Holoproteins at the
NM--
As revealed by immunoblotting, at both 0 and 48 h there
were nearly undetectable amounts of 80-kDa PKC-II
holoproteins in the NM fraction of untreated, actively proliferating
pyF111 cells (Fig. 2, A and
B). And no PKC-II CFs were detected in such
NM samples even when the amount of protein loaded into each lane was 30 µg instead of the usual 10 µg. The specific activity of the trace
amounts of Ca2+-stimulable PKC-II
immunoprecipitated by anti-PKC-II-specific antibody from
the NM of untreated cells was unchanged between 0 and 48 h (Fig.
2C) and could not be increased by adding 0.5 mM
Ca2+ to the assay mixtures (Fig. 2C).
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Fig. 2.
The translocation, levels of specific
activity, and proteolysis of
PKC-II in the NM fractions of
VP-16-treated pyF111 cells. A, NM fractions isolated
from untreated (0 h and 48-h proliferating (48p)) and VP-16
(1.0 µg ml1)-exposed cells containing equal
amounts of protein were subjected to SDS-PAGE, transferred onto
nitrocellulose membranes, and challenged with an
anti-PKC-II antibody reacting with an isotype-specific
C-terminal amino acid sequence, and the bands were revealed as
indicated. Molecular masses are designated on the right. The
immunoblots are typical of five separate experiments. B,
densitometric analysis of the bands pertaining to the
PKC-II holoprotein and its CFs in the NM fractions
isolated and processed as indicated. C,
PKC-II was immunoprecipitated from equal aliquots (300 µg of protein) of the NM fractions from untreated (controls, 0 and
48 h) and VP-16 (1.0 µg ml1)-treated
pyF111 cells, and both their native activities and activities in the
presence of added calcium (0.5 mM) were measured using the
dye-labeled -peptide as substrate as described under "Experimental
Procedures." Points on the curves in B and C
represent means ± S.E. from five independent experiments.
W.B., Western blot; I.P.,
immunoprecipitation.
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During the first 18 h of exposure to VP-16 (1.0 µg
ml1), the amount of PKC-II
holoproteins in the NM changed only slightly (Fig. 2, A and
B), but the specific activity of the PKC-II
immunoprecipitated from the NM rose by 44% (p < 0.05 by 18 h) (Fig. 2C). Between 18 and 72 h, the
amount of PKC-II holoproteins increased 4 times (p < 0.001) in the NM fraction, where it also
underwent a massive proteolysis that resulted in large accumulations of
47- and 40-kDa PKC-II CFs (Fig. 2, A and
B).
The PKC-II specific activity immunoprecipitated from the
NM reached twice its starting level (p < 0.001)
between 18 and 48 h but then dropped to only 57% of the starting
activity (p < 0.01) during the next 24 h in the
VP-16-treated cells (Fig. 2C). Adding 0.5 mM
Ca2+ to the immunoprecipitated test mixtures from the NM
fractions of the VP-16-exposed cells between 18 and 72 h did not
further increase the PKC-II specific activity,
indicating that all of the PKC-II holoproteins loaded
into the NM fraction were active (Fig. 2C).
It should be noted that the PKC-II holoproteins and
their C-terminal fragments were held in the NM fraction. At no time
after VP-16 addition did any detectable immunoprecipitable
PKC-II activity appear in the NP fractions (not shown).
These results demonstrate that, during the execution phase of
VP-16-induced apoptosis, active PKC-II holoproteins were
massively loaded onto the nuclear envelope and cleaved into 47- and
40-kDa PKC-II CFs, which remained attached to the envelope.
PKC-II Associates with Lamin B1 in the NM of pyF111
Cells during VP-16-induced Apoptosis but Not during the G2
Build-up to Mitosis--
These observations prompted us to search for
possible substrates of the PKC-II collecting at the NM
of apoptosing pyF111 cells.
Lamin B1 is a PKC-II substrate during the G2
build-up to mitosis in human promyelocytic leukemia HL-60 cells (49,
50) and human erythroleukemia K562 cells (51). To find out whether this
also applies to the pyF111 fibroblasts, we treated NM fractions from
untreated 0 h and from logarithmically proliferating 48-h cells
with anti-lamin B1 or anti-PKC-II antibodies, but
neither PKC-II holoproteins nor PKC-II
CFs coimmunoprecipitated with lamin B1 from the normal NM samples (Fig.
3, A-D). Therefore, PKC-II is probably not a significant mitotic lamin
kinase in the untreated fibroblasts, because if it were, the more than
15% of the cell population that was in G2-M (Fig.
1B) should have had at least a detectable amount of
coimmunoprecipitable lamin B1, PKC-II holoprotein, and
PKC-II CFs. And, as noted above, there was still no
measurable PKC-II in triple the amount of NM protein
from the proliferating cells.
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Fig. 3.
PKC-II
coimmunoprecipitates with lamin B1 only from the NM fractions of
VP-16-treated cells. A, NM fractions from untreated (0 h and 48-h proliferating (48p)) and VP-16 (1.0 µg
ml1)-exposed cells were isolated as
indicated. PKC-II was immunoprecipitated from equal
aliquots (300 µg of protein) of the NM fractions, and the whole
immunoprecipitate samples were subjected to SDS-PAGE, transferred onto
nitrocellulose membranes, and challenged with an anti-lamin B1
antibody. The immunoblot is typical of those from five separate
experiments. Molecular mass is indicated on the right.
B, densitometric analysis of the 69-kDa lamin B1 bands
coimmunoprecipitated with PKC-II from the NM fractions.
C, lamin B1 was immunoprecipitated from the same NM
fractions as in A, and the whole immunoprecipitated samples
were subjected to SDS-PAGE, transferred onto nitrocellulose membranes,
and challenged with an anti-PKC-II antibody. The
immunoblot shown is a representative one out of five separate
experiments. Molecular masses are shown on the right.
D, densitometric analysis of the bands pertaining to
PKC-II holoprotein and its catalytic fragments
coimmunoprecipitated with lamin B1 from NM fractions. Points on the
curves in B and D represent means ± S.E. of
the values from five separate experiments. I.P.,
immunoprecipitation.
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In contrast, a substantial amount of lamin B1 could be
immunoprecipitated from the NM fractions with the
anti-PKC-II antibody by 6 h after adding VP-16, and
it continued rising up to 24 h (Fig. 3, A and
B) but then began falling very slowly (Fig. 3B). Conversely, no PKC-I could be immunoprecipitated with
the anti-PKC-II antibody from the NM of either untreated
or VP-16-treated cells (not shown). PKC-II holoproteins
and their 47- and 40-kDa CFs were also strongly immunoprecipitated
along with lamin B1 by the anti-lamin B1 antibody from the NM fractions
of the VP-16-treated pyF111 cells (Fig. 3, C and
D). There were both 80- and 77-kDa, PKC-II
holoprotein bands in the anti-lamin B1 antibody-precipitated fractions
after both 24 and 48 h of treatment. The faster holoprotein band
might have been a dephosphorylated form of the catalytically competent
enzyme (52). Immunoprecipitated PKC-II
holoprotein·lamin B1 complexes predominated during the first 24-48
h, but by 72 h nearly equal amounts of PKC-II
holoprotein·lamin B1, 47-kDa PKC-II CF·lamin B1, and
40-kDa PKC-II CF·lamin B1 complexes had appeared (Fig.
3, C and D). These results suggest that a part of
the VP-16-triggered apoptogenic mechanism is the formation at the NM of
PKC-II·lamin B1 complexes, in which the lamin B1 is
marked by the PKC for cleavage by caspase-6 (53, 54-57). Moreover, it
appears that, unlike human cells (49-51, 54, 55), VP-16-exposed pyF111
rat cells use PKC-II as an apoptotic rather than a
mitotic lamin kinase.
Active Caspase-3 and -6 Associate with Lamin
B1·PKC-II Complexes in the NM Fraction of
VP-16-treated Cells--
The VP-16-induced rapid surge of cytosolic
cytochrome c (see Ref. 10) triggered a cascade of events
including a relatively early activation of the cytoplasmic
"executioner" or "effector" caspase, caspase-3, the activity of
which, as detected by using the selective fluorogenic substrate
Ac-DEVD-AMC, increased 3.25-fold (p < 0.001) to a peak
between 6 and 18 h and then fell back to the starting level by
72 h (Fig. 4A). The
nuclear caspase-3 activity rose gradually during the first 24 h
but then sharply rose 3.2-fold (p < 0.02) to a
peak at 48 h and then fell (Fig. 4B). It was this burst
of nuclear caspase-3-like activity that probably started the execution
phase of apoptosis (10, 11). It has been reported that the nuclear
lamina is broken down during the execution phase of apoptosis by
caspase-6 activated by caspase-9 and/or caspase-3 (48, 53, 56-60).
Using the caspase-6-specific substrate, Ac-VEID-AMC (48), we found that
this protease was only marginally active in both the whole cytoplasmic
(SN1) and NP fractions of untreated (0-h) pyF111 cells (Fig. 4,
A and B), but the caspase-6 specific activity in
the cytoplasm (the SN1 fractions) had increased 2.4 times
(p < 0.001) by 6 h after adding VP-16, although
it took 18 h for this activity to double (p < 0.001) in the NP fractions (Fig. 4, A and B). By
48 h, the cytoplasmic caspase-6 activity was 19.7 times higher
than the starting value (p < 0.001) and then slowly
dropped (Fig 4A). At the same time, the nucleoplasmic activity was 20.3 times higher than the starting value
(p < 0.001), and then it too began dropping (Fig.
4B).
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Fig. 4.
The activation of caspase-3 and -6 and the
coimmunoprecipitation of active caspase-3 and -6 with lamin
B1·PKC-II complexes from the NM
fractions of VP-16-treated pyF111 cells. A and
B, NP and SN1 fractions were obtained from untreated (0-h)
and VP-16 (1.0 µg ml1)-treated cells, and
the specific activity of caspase-3 and -6 was assayed in equal
aliquots (50 µg of protein) using Ac-DEVD-AMC and Ac-VEID-AMC as the
respective specific fluorogenic substrates as indicated. C
and D, lamin B1 and PKC-II were
immunoprecipitated from equal aliquots (300 µg of protein) of the NM
fractions isolated from untreated (0-h) and VP-16 (1.0 µg
ml1)-treated cells using anti-lamin B1 or
anti-PKC-II antibody, and the activities of the caspases
were determined in each immunoprecipitated sample. Points on the curves
in A-D represent means ± S.E. for five separate
experiments. E, lamin B1 proteolysis in the NP fractions of
VP-16-treated pyF111 cells. NP fractions were isolated from untreated
(0-h) and VP-16 (1.0 µg ml1)-treated cells
and immunoprecipitated with anti-lamin B1 antibody from equal aliquots
(300 µg of protein) of the fractions, and the whole
immunoprecipitates were subjected to SDS-PAGE, transferred onto
nitrocellulose membranes, and challenged with an anti-lamin B1
antibody. Molecular masses are shown on the right. The
figure is typical of the immunoblots from five separate
experiments. W.B., Western blot; I.P.,
immunoprecipitation.
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The caspase-6 activity immunoprecipitated from the NM fractions by
either the anti-lamin B1 or anti-PKC-II antibody was
about 5-10 times higher than in the NP fractions (Fig. 4, compare
B with C), because of the enzyme's accumulation
in lamin B1·PKC-II complexes in the nuclear envelope.
The caspase-6 activity was relatively low in both the anti-lamin B1 and
anti-PKC-II immunoprecipitates from the NM fractions of
untreated 0-h cells. The immunoprecipitable activity increased only
1.3-1.5-fold (p > 0.05) during the first 24 h
after adding VP-16, but between 24 and 48 h the activity shot up
4.9-fold in the anti-lamin B1 antibody precipitates (p < 0.001) and 5.6-fold in the anti-PKC-II antibody
precipitates (p < 0.001). Between 48 and 72 h,
the caspase-6 activity rose another 2.9 times (p < 0.001) in the anti-lamin B1 antibody precipitates, but by then
anti-PKC-II antibody precipitates with caspase-6 activity had begun disappearing (Fig. 4C). Thus, since up to
48 h the caspase could be increasingly precipitated with either
anti-lamin B1 or anti-PKC-II and since lamin B1 could be
precipitated with anti-PKC-II antibody and since
PKC-II could be precipitated with anti-lamin B1
antibody, active caspase-6 was probably contained in increasing numbers
of caspase-6·lamin B1·PKC-II complexes during the
execution phase of apoptosis.
Trace amounts of caspase-3 activity were also immunoprecipitated by
anti-PKC-II antibody from the NM fraction of untreated cells (Fig. 4D), and PKC-II-bound caspase-3
activity changed relatively little during the first 24 h of VP-16
exposure but surged 5-fold between 24 and 48 h only to drop
thereafter (Fig. 4D). Thus, active caspase-3 was also a part
of a large complex containing PKC-II,
PKC-II CFs, active caspase-6, and lamin B1.
While PKC-II holoprotein or any of its CFs were never
found in the NP of untreated or VP-16-treated pyF111 cells (not shown), the surge of caspase-6 activity in the NP (Fig. 4B)
was associated, especially between 24 and 48 h, with the
appearance in the NP of 45-kDa fragments of lamin B1, which is
normally restricted to the NM fraction (17) (Fig. 4E).
VP-16 Causes PKC-II to Rapidly Act as an Apoptotic
Lamin Kinase in a Cell-free Reconstituted N-C System--
After having
identified PKC-II as a nucleus-seeking apoptotic lamin
kinase, we set out to show that this active, hence
serine-phosphorylated (49, 61), PKC-II originated
in the cytoplasm in response to VP-16's apoptogenic signal. To do
this, we used a cell-free N-C model consisting of normal, intact nuclei
from untreated (0-h) cells mixed with either untreated normal
cytoplasms from 0-h cells or apoptotic cytoplasms from cells
that had been exposed to VP-16 for 6, 18, or 24 h. These N-C
mixtures were incubated at 37 °C for only 30 min before isolating
their NM fractions. And the experiments were carried out either in the
presence or in the absence of hispidin, a PKC- (but not PKC-)
inhibitor (45-47). Hispidin was used at a concentration of 5.0 µM, which inhibited by 90% the peak specific activity of
the PKC-II immunoprecipitated from the NM fractions of
48-h VP-16-treated cells, but did not at all affect the peak specific
activity of PKC- immunoprecipitated from the NM fractions of
untreated cells (10) (Fig. 5). Moreover,
hispidin (up to 10 µM) must not have affected the cells'
other PKCs because it did not affect the total PKC activity
assayable in NM fractions isolated from untreated cells, which
contained very small amounts of -isoforms (not shown). The NM
fractions isolated from the cell-free N-C mixtures were
immunoprecipitated with an anti-Ser(P) antibody, and Western
immunoblots of the components of these immunoprecipitates were probed
with either the anti-lamin B1 antibody or the
anti-PKC-II antibody.
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Fig. 5.
Hispidin inhibits the maximal specific
activity of PKC-II but not PKC-
immunoprecipitated from the NM fractions of pyF111
cells. (i) NM fractions were isolated from pyF111 cells
that had been exposed for 48 h to VP-16 (1.0 µg
ml1), and the PKC-II was
immunoprecipitated by an anti-PKC-II antibody from
aliquots (300 µg of protein) of the fractions; or (ii) NM fractions
were isolated from untreated (0-h) pyF111 cells, and PKC- was
immunoprecipitated by anti-PKC- antibody from aliquots (300 µg of
protein) of the fractions. Each sample was incubated for 30 min at
30 °C with the dye-labeled -peptide (i.e. the PKC
substrate) either in the presence or in the absence of increasing
concentrations of the PKC- inhibitor hispidin. PKC activity was then
assayed as described under "Experimental Procedures." Points are
means ± S.E. for three separate experiments.
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Only trace amounts of phosphorylated PKC-II could be
precipitated by the anti-Ser(P) antibody from the NM fractions of
untreated nuclei mixed with control cytoplasms for 30 min (Fig.
6, A and B). But
the bands of Ser(P)-PKC-II were more than 11-fold
thicker (p < 0.001) when the NM fractions were from
untreated nuclei that had been mixed for 30 min with the 6- or 18-h
apoptotic cytoplasms (Fig. 6, A and B). Thus,
VP-16-pretreated cytoplasms can load the NM fractions of untreated
nuclei with Ser(P)-PKC-II. The apparently more rapid
rise in the amount of holoenzyme at the NMs of the N-Cs as compared
with that observed in the NMs from the whole cells (cf. Fig.
2A) is probably due to the immunoprecipitation by the
anti-Ser(P) antibody and, possibly, to the arbitrarily set N/C
volumetric ratio (i.e. 1:4) used for the N-Cs. Moreover, two
bands of the Ser(P)-PKC-II holoprotein instead of one
were clearly visible in the NM fraction when the 24-h apoptotic
cytoplasms were added to the nuclei from untreated cells (Fig. 6,
A and B), which was the same as when
PKC-II was immunoprecipitated in complexes with lamin B1
from whole cells (Fig. 3C). Moreover, in both kinds of
specimens (cf. Figs. 3C and 6A), no
PKC-II-CFs could be detected when the exposure to VP-16
did not exceed 24 h. Thus, the apoptotic signal from VP-16
produced active, serine-phosphorylated, PKC-II holoproteins in the cytoplasms that massively translocated to the
nuclear envelopes of the nuclei from untreated cells.
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Fig. 6.
The rapid translocation of Ser(P)
(pSer)-PKC-II
holoproteins onto the NM of nuclei from untreated cells mixed with
apoptotic cytoplasms in a reconstituted cell-free system. Intact
nuclei from untreated (0 h) cells were mixed (1:4, v/v) with either
control cytoplasms (from untreated (0-h) cells) or apoptotic cytoplasms
from cells exposed to VP-16 (1.0 µg ml1)
for 6, 18, or 24 h. The various N-C mixtures were then incubated
for 30 min at 30 °C as indicated. NM fractions were next extracted,
and Ser(P) proteins were immunoprecipitated from equal aliquots (300 µg of protein) of each sample. Samples were subjected to SDS-PAGE,
transferred onto nitrocellulose membranes, and challenged with an
anti-PKC-II antibody. A, a representative
immunoblot out of three distinct experiments. Molecular mass is shown
on the right. Similar doublets can also be seen in Fig.
3C showing immunoprecipitates from whole cells.
B, densitometric analysis of the bands of
Ser(P)-PKC-II translocated onto NM fractions of nuclei
from untreated cells mixed with either control or apoptotic (6, 18, or
24 h of exposure to VP-16) cytoplasms. The data have been
normalized, taking 1 as the value obtained from the nuclei from
untreated cells mixed with control cytoplasms. Bar
heights are the means ± S.E. of the values from three
separate experiments. The bar values of samples incubated
with 24-h apoptotic cytoplasms are the sums of the densitometric values
of the two specific bands on the gels.
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A sizable amount of 69-kDa Ser(P)-lamin B1 could be immunoprecipitated
from the NM fractions of the nuclei from untreated cells mixed with
untreated control cytoplasms (Fig. 7,
A and C). The amount of immunoprecipitable
Ser(P)-lamin B1 nearly doubled when 18- or 24-h apoptotic cytoplasms
were used (Fig. 7, A and C). Importantly, a
45-kDa fragment of Ser(P)-lamin B1 also appeared in the NM fractions
when untreated nuclei were mixed with the 18- or 24-h apoptotic
cytoplasms (Fig. 7, A and D). This was
accompanied by a significant increase in caspase-6 activity in the
nuclei from untreated cells that had been mixed with the 18- or 24-h apoptotic cytoplasms (Fig. 7E). These data are consistent
with there having been a prompt (i.e. within 30 min)
phosphorylation of lamin B1 induced in the normal nuclei by contact
with the 18- or 24-h apoptotic cytoplasms followed by lamin B1 cleavage
by an active caspase-6 from these cytoplasms.
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Fig. 7.
When added to apoptotic cytoplasms, the
PKC- inhibitor hispidin reduces lamin B1
phosphorylation and totally prevents lamin B1 proteolysis. N-C
mixtures were prepared as indicated and incubated for 30 min at
30 °C either with or without the PKC- inhibitor hispidin (5.0 µM). NM fractions were next extracted, and Ser(P)
(pSer)-proteins were immunoprecipitated from equal aliquots
(i.e. 300 µg of protein) of the fractions. Each sample was
processed as in Fig. 6 and challenged with an anti-lamin B1 antibody,
and the antigen-antibody complexes were visualized as indicated.
A and B, representative immunoblots out of three
distinct experiments. Molecular masses are indicated between
the panels. C and D, densitometric
analyses of the bands pertaining to Ser(P)-lamin B1 holoprotein
(69-kDa) (C) and its fragments (45-kDa) (D)
obtained either in the absence or the presence of hispidin
(H). Absolute densitometric values pertaining to the changes
in protein amounts are reported in C and in D. Bars represent mean values ± S.E. for three distinct
experiments. E, the activity of caspase-6 in whole nuclei
from untreated cells mixed with either control or apoptotic cytoplasms
was not significantly changed by adding hispidin (5.0 µM)
to the cytoplasmic fractions. Samples of whole nuclei were taken from
the various N-C mixtures to which hispidin had or had not been
previously added. Caspase-6 activity associated with such nuclei was
assessed as described in the legend to Fig. 4. Bars
represent mean values ± S.E. from three separate
experiments.
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To better define the relevance of PKC-II action in this
lamin phosphorylation and proteolysis, we added the PKC- inhibitor hispidin (45-47) to the apoptotic cytoplasms. The hispidin totally prevented any increase above basal (0-h) values in the amount of 69-kDa
Ser(P)-lamin B1 at the NM when the 6-h apoptotic cytoplasms were used
(Fig. 7, B and C), but another protein kinase may
have phosphorylated the lamin at later times because the level of
69-kDa serine-phosphorylated lamin B1 was reduced by only 30% when the normal nuclei were mixed with hispidin-treated 18- or 24-h apoptotic cytoplasms (Fig. 7, B and C). Most importantly,
however, hispidin, and therefore inhibition of PKC- activity,
totally suppressed the generation of 45-kDa Ser(P)-lamin B1 fragments
at the NM when the 18- or 24-h apoptotic cytoplasms were tested (Fig.
7, B and D). However, hispidin did not stop
caspase-6 activity from increasing in the nuclei of the N-C systems
(Fig. 7E). Thus, inhibiting PKC-II activity
with hispidin only partially reduces the serine phosphorylation of
69-kDa lamin B1, but it completely prevents the cleavage of the lamin
into 45-kDa fragments despite the presence of an increased nuclear
caspase-6 activity. Therefore, caspase-6 specifically needs
PKC-II activity to depolymerize lamin B1 in
VP-16-treated fibroblasts.
Other Possible Lamin Kinases in VP-16-treated pyF111 Cells--
To
further gauge how crucial the activity of PKC-II at the
NM is for the execution of apoptosis in the VP-16-treated pyF111 cells,
we determined the possible apoptogenic contributions of other lamin
kinases. For this we immunoprecipitated the NM fractions from both
untreated (proliferating) and VP-16-exposed (apoptosing) pyF111 cells
with the anti-lamin B1 antibody and then probed the immunoblots of the
precipitated components with specific anti-kinase antibodies.
PKC- could have phosphorylated lamin B1 (26), because there were
substantial amounts of the holoprotein and its 40-kDa CF in the NM
fractions of both proliferating and apoptosing cells (not shown). But
neither PKC- nor its CF coimmunoprecipitated with lamin B1 the NM
fractions of either the proliferating or apoptosing cells (not shown).
CDK-1 is the principal prophase-triggering protein kinase in eukaryotic
cells (reviewed in Refs. 50, 54, 55). The results in Fig.
8 (A and B)
demonstrate that CDK-1 kinase coimmunoprecipitated with lamin B1 from
the NM fractions both of proliferating and apoptosing cells as revealed
by a pan-antibody that did not distinguish between CDK-1's active
dephosphorylated form and its inactive phosphorylated form. Clearly,
this pan-antibody revealed that the amounts of CDK-1·lamin B1
(presumably cyclin B·CDK-1·lamin B1) complexes increased both with
the entry of the untreated cultures into their logarithmic growth phase
(i.e. by 48 h) and after adding the
G2/M-blocking VP-16 (Fig. 8, A and
B). Using an anti-Tyr(P)15-CDK-1 antibody, which
bound to the inactive form of the enzyme, we found, as would be
expected, that the CDK-1 immunoprecipitated by anti-lamin B1 antibodies
from the NM fractions of the untreated, proliferating (both 0 and
48 h) cells did not bind the anti-Tyr(P)15-CDK-1
antibody and was therefore unphosphorylated and active (Fig. 8,
C and D). In contrast, the Tyr15
residues of the increasing amounts of CDK-1 that coimmunoprecipitated with lamin B1 from the NM fractions of the apoptosing and
G2-blocked (Fig. 1B) VP-16-treated cells did
bind the anti-Tyr(P)15-CDK-1 antibody and were therefore
phosphorylated and inactive (Fig. 8, C and D).
Thus, the CDK-1 accumulating in the VP-16-treated cells was inactive
and could not have phosphorylated lamin B1. To find out whether the
CDK-1·lamin B1 complexes were or were not different from the
caspase-3·caspase-6·lamin B1·PKC-II complexes, we
looked for the presence or absence of CDK-1 in NM immunoprecipitates obtained with the anti-PKC-II antibody. Our results show
that 48 h after the addition of VP-16, mainly inactive
Tyr(P)15-CDK-1 is significantly associated with and hence
yet another part of the large caspase-3·caspase-6·lamin
B1·PKC-II complex (Fig. 8E).
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Fig. 8.
CDK-1 is a mitotic lamin kinase in pyF111
cells. Lamin B1 was immunoprecipitated from equal aliquots (300 µg of protein) of the NM fractions isolated from untreated (0 h and
48-h proliferating (48p)) and VP-16 (1.0 µg
ml1)-exposed cells. Each sample was subjected
to SDS-PAGE, transferred onto nitrocellulose membranes, and challenged
with either a pan-CDK-1 antibody that binds both active and inactive
CDK-1 (A and E) or an anti-Tyr(P)15
(pTyr15)-CDK-1 antibody that binds inactive CDK-1
(C and E). These immunoblots are representative
of those of four distinct experiments. Molecular mass is indicated on
the left. B and D, densitometric
analyses of the bands of CDK-1 coimmunoprecipitated with lamin B1 from
the NM fractions of cells untreated or treated as above and revealed
with pan-CDK-1 antibody (B) or an
anti-Tyr(P)15-CDK-1 antibody (D). The
bars in B and D are the means ± S.E. from four separate experiments. E, inactive CDK-1
coimmunoprecipitates with PKC-II from the NM fractions
of pyF111 cells treated with VP-16 for 48 h. Experimental
procedures used were as in A and C. I.P., immunoprecipitation.
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Finally, PKC- could also be a lamin kinase in the pyF111 cells,
because the 80-kDa PKC- holoprotein and its 40-kDa CF
coimmunoprecipitated with lamin B1 from the NM fractions of 0-h
proliferating cells (Fig. 9, A
and C). The amount of holoprotein-·lamin B1 complexes had doubled by 48 h when the cells were in their logarithmic
growth phase, while the 40-kDa PKC- CF·lamin B1 complexes had
dropped by 38% (Fig. 10, A
and C). PKC-, like CDK-1 kinase, was also
immunoprecipitated from the NM fractions of the apoptosing cells
by anti-lamin B1 antibody, but, instead of increasing like the CDK-1 in
CDK-1·lamin B1·PKC-II complexes, the PKC-
holoprotein·lamin B1 complexes dropped by 38-43% between 24 and
48 h after the drug was added (Fig. 9, B and
D). The 40-kDa PKC- CF·lamin B1 complexes dropped slightly by 24 h but then rose 40% by 48 h after VP-16 was
added (Fig. 9, B and D). It must be stressed
here that the fall of PKC--holoprotein·lamin B1 complexes and the
rise in 40-kDa PKC- CF·lamin B1 complexes coincided with the
persistent 77-83% drop in the PKC- specific activity at the NM
that we have previously shown to happen soon after adding VP-16 (10).
It must be noted that unlike CDK-1, but like PKC-I,
PKC- was not immunoprecipitated by
anti-PKC-II antibody from the NM fractions of
VP-16-treated cells (data not shown). This preliminary finding suggests
that there is a PKC-·lamin B1 complex that is separate from the
caspase-3·caspase-6·lamin B1·PKC-II·CDK-1
complex. In conclusion, while PKC- could have served along with
CDK-1 kinase for the phosphorylation of lamin B1 at the
G2-M transition in the proliferating cells, in no way could
it have approached the lamin-phosphorylating action of the massively
surging PKC-II in the NM fractions of the VP-16-treated cells. Therefore, PKC-II is an important, if not the
major, apoptotic lamin kinase in etoposide-treated pyF111 cells.
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Fig. 9.
PKC- is mainly a mitotic lamin kinase in
pyF111 cells. Lamin B1 was immunoprecipitated from 300-µg
protein aliquots of the NM fractions isolated from untreated (0 and
48 h) or VP-16 (1.0 µg ml1)-exposed
cells. Each sample was subjected to SDS-PAGE, transferred onto
nitrocellulose membranes, and probed with an anti-PKC- antibody as
indicated. Shown are the bands of PKC- coimmunoprecipitated with
lamin B1 from the NM fractions of untreated, proliferating cells
(A) and of VP-16 (1.0 µg
ml1)-incubated cells (B). The
immunoblots are representative of the blots from three distinct
experiments. Molecular masses are indicated between the
panels. C and D, densitometric
analyses of the bands of PKC- holoprotein and its CFs
immunoprecipitated by anti-lamin B1 antibody from the same NM fractions
and immunoblotted. Bars represent the means ± S.E.
values of three separate experiments.
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Fig. 10.
The possible interactions of
PKC-II, lamin B1, and caspase-6 at
the NM of VP-16-exposed transformed fibroblasts are
crucial for the execution of apoptosis. Exposure to VP-16 renders
cytosolic PKC-II catalytically competent, first via the
PDK-1 kinase and then via autophosphorylation. The now functionally
competent phospho-PKC-II molecules migrate to the NM,
where they bind to the inner membrane, become active, and
serine-phosphorylate lamin B1. Concurrently, caspase-6 is activated
downstream from the release of cytochrome c from the
mitochondria into the cytosol, the formation of the apoptosome, and the
activation of executioner caspase-9. Active caspase-6 travels to the NM
and the nucleoplasm (NP). At the NM, active caspase-6 binds
to phosphorylated lamin B1·active PKC-II complexes and
cleaves the lamin into 45-kDa fragments. PKC-II
holoproteins and its CFs of PKC-II are never found in
the NP, but active caspase-6 complexes do get into the NP, where they
cleave phosphorylated lamin B1. (It should be noted that caspase-3 also
reaches the nucleus and joins lamin B1 and PKC-II in a
complex on the nuclear envelope, but it would not cleave lamin B1
(34-37).) The interactions of PKC-II, lamin B1, and
caspase-6 in a large complex lead to the dissolution of the nuclear
lamina and to the structural and functional disruption of the
chromatin, events that are required for the fragmentation of the
nucleus (karyorexis) and the formation of apoptotic bodies.
p, phosphorylated form of the molecule; *, activated form of
the enzyme; NM, nuclear envelope; NP,
nucleoplasm.
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DISCUSSION |
The present results are consistent with the hypothesis that at the
onset of the execution of the VP-16-triggered apoptosis another PKC
isoform, PKC-II, is converted into an activable form by
phosphorylation and induced to move from the cytosol to the nuclear
envelope. There, the phospho-PKC-II holoproteins become anchored to the inner membrane where they are activated and
phosphorylate lamin B1 to which active caspase-6 then binds and
cleaves. These findings are the first evidence of PKC-II
being the apoptotic lamin kinase in VP-16-treated transformed
fibroblasts, although it appears to be the mitotic lamin kinase in
erythroleukemia K562 cells and human promyelocytic leukemia HL-60 cells
(49, 51, 62, 63). But other PKC isoforms, PKC- and PKC-, seem,
respectively, to be the main lamin kinases in camptothecin-induced and
Ara C-induced apoptosis in HL-60 cells (26, 39).
In various cell models (i.e. COS-7, HL-60, NIH 3T3, Sf-9,
etc.), when the cells near the end of the G2 build-up to
mitosis, the PKC-II holoproteins are sequentially
phosphorylated at three positions (i.e. first at
Thr500 on the activation loop by the PDK-1 kinase and then
at Thr641 and Ser660 on the turn and
hydrophobic motifs near the C terminus by autophosphorylation) (52, 61,
64, 65). Thus locked in a catalytically competent, but still inactive,
conformation, they move from the cytosol and accumulate at the nuclear
envelope, where they are selectively captured when their C-terminal V5
regions bind to a phosphatidylglycerol in the membrane (49, 51, 62,
66). The phosphatidylglycerol-tethered kinases are then activated by
perinuclear Ca2+ transients and diacylglycerols released
from the nuclear envelope by phosphatidylinositol-specific
phospholipase C (2, 62). The active PKC-II then triggers
the disassembly of the nuclear lamina that is needed for the breakdown
of the nuclear envelope and entry into prophase by phosphorylating
lamin B's Ser395 and Ser405 residues (49).
Thus, PKC-II activity has been considered essential for
cell proliferation (29, 54) and consequently would be expected to be
the lamin B1 phosphorylator in untreated proliferating pyF111 cells.
But it is not. In logarithmically growing pyF111 cultures, when at
least 15% of the cells are in the G2-M phase at 48 h
(Fig. 1B), there were only tiny amounts of activated
PKC-II holoproteins in the nuclear envelope fraction,
and their specific activities were very low when immunoprecipitated
from this fraction. Furthermore, there were no detectable
PKC-II CFs in the nuclear envelope fractions of the
untreated cells, and no lamin B1·PKC-II complexes
could be immunoprecipitated from the nuclear envelope fractions from actively proliferating (both 0- and 48-h) pyF111 cells. Instead of
PKC-II, we found two other active protein kinases,
PKC- and CDK-1, complexed with lamin B1 in the proliferating cells.
Furthermore, although, PKC- is constitutively hyperexpressed and has
the highest immunoprecipitable specific activity at the NM of the
untreated pyF111 cells (10), it seems likely from the large amount of evidence from other cells, that it is only a minor mitotic lamin kinase
compared with active CDK-1 kinase.
On the other hand, although PKC-II did not collect in
the nuclear membrane of the proliferating pyF111 fibroblasts, VP-16 caused it to become functionally competent and move from the cytosol to
the nuclear envelope at the onset of the execution phase of apoptosis
(i.e. between 24 and 48 h of exposure). Although lamin B1·PKC-II complexes could be immunoprecipitated from
the NM fraction even at early time points (e.g. 6 h of
VP-16 exposure), they peaked around 48 h when strong caspase-3 and
-6 activities could be immunoprecipitated with them.
The assumption that the apoptogenic signal from VP-16 induced
PKC-II to become the apoptotic lamin kinase was
validated by the results obtained with reconstituted
nucleus-cytoplasmic mixtures. As in the whole fibroblasts, only nearly
undetectable or trace amounts of PKC-II holoproteins
were in the NM fraction from untreated nuclei mixed with
control cytoplasms from untreated cells. On the other hand,
apoptotic cytoplasms from treated cells had catalytically competent
Ser(P)-PKC-II molecules that rapidly (within 30 min) migrated to the NM.
Moreover, our data suggest that, as in the whole cells, the
serine-phosphorylated lamin B1 immunoprecipitated from the NM fractions
of untreated nuclei mixed with control cytoplasms was the substrate of
PKC- and CDK-1 kinases as well as PKC-II. However, since adding the PKC-II (but not PKC-) inhibitor
hispidin (45-47) to the apoptotic cytoplasms only partially reduced
the serine phosphorylation of lamin B1 but totally suppressed the
lamin's proteolysis, PKC-II activity is essential for
the apoptotic dissolution of
II Is an Apoptotic Lamin Kinase
in Polyomavirus-transformed, Etoposide-treated pyF111 Rat
Fibroblasts*
,
TOP
ABSTRACT
INTRODUCTION
