(Received for publication, May 14, 1997)
From the Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
Protein kinase C (PKC
) is a member of the
novel or nPKC family. A functional role for PKC
is unknown. The
present studies demonstrate that PKC
is cleaved in the third
variable region (V3) in apoptosis induced by diverse agents. PKC
cleavage is blocked in cells that overexpress the anti-apoptotic
Bcl-xL or the baculovirus p35 protein. PKC
is
cleaved by Caspase-3 and by apoptotic cell lysates at a
DEVD354/K site. We also show that overexpression of the
cleaved kinase-active PKC
fragment, but not full-length PKC
or a
kinase-inactive fragment, results in induction of sub-G1
phase DNA, nuclear fragmentation, and lethality. These findings
indicate that proteolytic cleavage of PKC
by Caspase-3 induces
events characteristic of apoptosis.
The 11 known isoforms of the protein kinase C
(PKC)1 family have been
divided into the classical (cPKC; ,
,
), novel (nPKC;
,
,
,
, µ), and atypical (aPKC;
,
) groups (1). The
Ca2+-dependent cPKCs contain the conserved
regulatory regions, C1 and C2, while the Ca2+-independent
nPKC and aPKC isoforms lack the C2 domain. Cleavage of cPKCs in the
third variable (V3) region by calpains I and II deletes the C1 and C2
regulatory regions and results in catalytically active fragments (2).
Other studies have shown that the nPKC
isoform is activated by the
Caspase-3 cysteine protease in cells induced to undergo apoptosis
(3-5). Caspase-3-mediated cleavage of PKC
at a DMQD/N site in the
V3 region deletes the C1 regulatory domain (3-5). Overexpression of
the anti-apoptotic Bcl-2 and Bcl-xL proteins blocks PKC
cleavage (3, 4). These findings have suggested that PKC
is
functionally involved in the induction of apoptosis.
The PKC isoform is structurally related to PKC
(6-8), although
the V3 domain of PKC
has no significant homology with that in PKC
or the other PKC isoforms. Few insights are available regarding the
functional roles of PKC
. Whereas PKC
transcripts are found
ubiquitously, PKC
is predominantly expressed in hematopoietic cells
and skeletal muscle (6, 8). Studies in T cells have demonstrated that
PKC
is involved in antigen-specific activation (9). PKC
interacts
with 14-3-3 proteins (10) and is involved in AP-1-mediated
transcription (11). Other work has shown that the human
immunodeficiency virus Nef protein inhibits translocation of PKC
from the cytosolic to membrane fraction after phorbol ester stimulation
(12). Unlike the cPKCs and PKC
, there are no reports of proteolytic
cleavage of the PKC
isoform.
The present studies demonstrate that PKC is cleaved to an activated
form in cells induced to undergo apoptosis. The results indicate that
PKC
is cleaved by the Caspase-3 protease. We also show that
overexpression of the PKC
catalytic fragment induces characteristics
of apoptosis.
Human U-937 myeloid leukemia cells (American
Type Culture Collection, Rockville, MD) were grown in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/liter
L-glutamine. U-937 cells overexpressing bcl-xL,
CrmA, and p35 were prepared as described (13-15). Cells were treated
with 10 µmol/liter 1--D-arabinofuranosylcytosine (ara-C; Sigma), 3 µg/ml etoposide (Bristol-Myers Squibb Co.,
Princeton, NJ), and 100 µmol/liter cisplatinum (Sigma).
Cytoplasmic extracts were prepared and
fractionated through Q-Sepharose columns as described (3, 4). Proteins
were subjected to electrophoresis in 10% SDS-polyacrylamide gels and
then transferred to nitrocellulose paper. The residual binding sites
were blocked by incubating the filters with 5% dry milk in PBST
(phosphate-buffered saline/0.05% Tween 20). The filters were incubated
with anti-PKC polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA). After washing twice with PBST, the blots were incubated with
anti-rabbit IgG peroxidase conjugate (Amersham Corp.). The
antigen-antibody complexes were visualized by chemiluminescence (ECL
detection system; Amersham).
Cells (5 × 106) were harvested, washed, and incubated in 50 µl of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K (Sigma) for 6 h at 50 °C. The samples were incubated with 50 µl of 10 mM EDTA (pH 8.0) containing 2% (w/v) low-melting-point agarose and 40% sucrose for 10 min at 70 °C. The DNA was separated in 2% agarose gels. After treatment with RNase, the gels were visualized by UV illumination.
In Vitro Translation and Protease Cleavage AssaysThe
full-length (FL) PKC cDNA (provided by J. Anthony Ware, Beth
Israel Hospital, Boston) was cloned into BamHI sites of a
modified pSV
plasmid (CLONTECH). A PKC
(D351A
and D354A) mutant was generated in two steps by overlapping primer
extension. PARP cDNA was generated by polymerase chain reaction
cloning (5). [35S]methionine-labeled proteins (PKC
FL,
PKC
FL(D-A), PARP) were synthesized by coupled transcription and
translation reactions (Promega, Madison, WI). Labeled proteins were
incubated with 5 µg/ml Escherichia coli-derived
Caspase-3
, Caspase-1, Caspase-2, Caspase-4, Caspase-6, or Caspase-7
in 50 mM Hepes (pH 7.5), 10% glycerol, 2.5 mM
dithiothreitol, and 0.25 mM EDTA at room temperature for 30 min (16). Cleavage reactions were also performed in the presence of 5 µg of cytoplasmic extract from untreated or ara-C-treated cells and
in the presence of recombinant CrmA or p35 (14, 15). The reaction
products were analyzed by electrophoresis in 10 or 12%
SDS-polyacrylamide gels and then autoradiography.
Recombinant PKC proteins
were prepared by coupled transcription and translation. A vector
expressing a PKC
fragment (CF; amino acids 355-706) was generated
by polymerase chain reaction cloning from the full-length PKC
cDNA. A mutant PKC
CF with Lys-409 substituted by Arg (K-R) was
generated by overlapping primer extension. Protein kinase assays were
performed as described (PKC assay kit; Life Technologies, Inc.).
PKCFL, PKC
CF, or PKC
CF(K-R)
were cloned into the pEGFP-C1 vector (CLONTECH).
HeLa cells were suspended at a density of 1 × 107
cells/ml and transfected by electroporation (Gene Pulsar, Bio-Rad; 0.22 V, 960 µF). At 40 h post-transfection, cells were sorted by
FACScan (Becton Dickinson, Mansfield, MA), and viability was checked by
trypan blue exclusion. Transfected cells were stained with propidium
iodide. FACScan was used to determine sub-G1 content in
cells positive for green fluorescence. Chromatin fragmentation was
determined by staining methanol-fixed cells with 0.5 µg/ml DAPI
(Molecular Probes, Eugene, OR).
Treatment of U-937 cells with ara-C and other DNA-damaging agents
results in the induction of apoptosis (17, 18). Whereas the V3 region
of PKC has a DEVD/K site similar to that cleaved in PARP during
apoptosis (19, 20), we asked whether PKC
is also susceptible to
cleavage. PKC
was detectable as a 78-kDa band in control cells (Fig.
1A). By contrast,
ara-C-induced apoptosis was associated with cleavage of PKC
to a
40-kDa fragment (Fig. 1A). Similar results were obtained
during apoptosis induced by cisplatinum, etoposide, and 20-gray
ionizing radiation (Fig. 1B and data not shown).
To determine whether PKC cleavage is associated with induction of
apoptosis, we studied cells that overexpress the anti-apoptotic Bcl-xL protein and exhibit resistance to induction of
apoptosis (13). Exposure of control U-937/neo cells to ara-C resulted in cleavage of PKC
, while there was no apparent effect of this agent
on PKC
in the U-937/Bcl-xL transfectant (Fig.
2A). The cowpox protein CrmA
(21) and the baculovirus protein p35 (16) block apoptosis in diverse
models by directly inhibiting members of the ICE/Ced-3 family of
cysteine proteases. Overexpression of CrmA had no effect on
ara-C-induced PKC
cleavage or apoptosis (Fig. 2B). By
contrast, ara-C treatment of cells that overexpress p35 resulted in no
detectable cleavage of PKC
or induction of apoptosis (Fig.
2B). These findings indicated that PKC
is cleaved by an
ICE/Ced-3-like protease.
The Caspase-3 protease is insensitive to CrmA and inhibited by p35 (14,
16, 22). Whereas Caspase-3 cleaves PARP at DEVD216/G (22,
23), we asked whether Caspase-3 induces cleavage at a DEVD/K site in
the V3 region of PKC. Full-length PKC
labeled with
[35S]methionine was cleaved by purified Caspase-3 to a
40-kDa fragment (Fig. 3A). By
contrast, there was no apparent cleavage of PKC
with purified ICE
(Fig. 3A). Cleavage of PKC
at the DEVD/K site predicts
the formation of a catalytic domain of 40 kDa that corresponds physically with the PKC
fragment identified in apoptotic U-937 cells. Nonetheless, to confirm the Caspase-3-mediated cleavage site in
PKC
, we mutated the essential P1 and P4 Asp residues with
substitution by Ala (D351A and D354A). The finding that Caspase-3 fails
to cleave the PKC
(D-A) mutant provided support for involvement of
the DEVD354/K site (Fig. 3A). Caspase-3,
Caspase-7, and Ced-3 cleave PARP at the DEVD216/G site (22,
23). The finding that PKC
is cleaved by Caspase-3, and not
Caspase-7, provided support for selectivity of the PKC
cleavage site
(Fig. 3B). There was also no detectable cleavage of PKC
with Caspase-2, Caspase-9, or Caspase-6 (Fig. 3B). Other
studies have demonstrated that Caspase-3 is activated in cells treated
with ara-C (14, 15). Addition of labeled PKC
to lysates from
ara-C-treated, but not control, cells was associated with cleavage of
PKC
(Fig. 3C). Preincubation of the active lysate with
CrmA had no effect, while p35 blocked PKC
cleavage (Fig.
3C). Taken together, these results indicate that PKC
is
cleaved by Caspase-3 in cells induced to undergo apoptosis.
To assess whether cleavage of PKC at the DEVD/K site is associated
with activation of kinase function, we assayed recombinant PKC
proteins for phosphorylation of the pseudosubstrate region of PKC
(amino acids 19-31) with replacement of Ala-25 by Ser. The
[A25S]PKC(19-31) peptide serves as a substrate for PKC
(24). The
PKC
-cleaved fragment (CF; amino acids 355-706) was over 6-fold more
active than PKC
full-length (FL) and the PKC
FL(D-A) mutant (Table
I). Moreover, a mutant of the cleaved
fragment with Lys-409 in the ATP binding site mutated to Arg (K409R;
designated K-R) had little if any activity above that found for control
bacterial lysates (Table I). These findings demonstrate that cleavage
at the DEVD/K site results in PKC
activation.
|
To determine if PKC contributes to apoptosis, we transfected HeLa
cells with PKC
FL, PKC
CF, or PKC
CF(K-R) cloned into vectors expressing the green fluorescence gene. Positive transfectants were
selected by flow cytometry, reseeded in medium, and assayed for
viability by trypan blue exclusion. Over 90% of the PKC
FL transfectants were viable, while only 10-15% of the
PKC
CF-transfected cells survived (Fig.
4A). The finding that over
90% of the PKC
CF(K-R) transfectants were viable provided further
support for the selective effects of PKC
CF expression (Fig.
4A). To assess whether transfection of PKC
CF induces
apoptosis, we monitored the appearance of green fluorescence-positive
cells with sub-G1 DNA content. Transfection of PKC
FL or
PKC
CF(K-R) was associated with 7-10% of cells with sub-G1 DNA (Fig. 4B). Significantly,
transfection of kinase-active PKC
CF resulted in 48% of cells with
sub-G1 DNA (Fig. 4B). Cells were also stained
with DAPI to assess nuclear morphology (25). Transfection of PKC
CF,
but not PKC
FL or PKC
CF(K-R), was associated with nuclear
fragmentation (Fig. 4C).
Recent studies have demonstrated that the aPKCs (PKC and
)
interact with Par-4 and abrogate the ability of Par-4 to induce apoptosis (26). These findings have suggested that the aPKCs exhibit an
anti-apoptotic function. By contrast, the present results and previous
work on PKC
(3, 4) support a potential role for at least certain
nPKCs in promoting apoptosis. The absence of detectable cleavage of
PKC
, -
, -
, and -
further supports the selective involvement
of PKC
and -
in apoptosis (3, 4). Previous studies have
demonstrated that Caspase-3 cleaves PARP (22, 23), DNA-PK (27, 28),
D4-GDI (29), U1 small nuclear riboprotein (27), and PKC
(5). We show
that PKC
is also cleaved by Caspase-3 and that Bcl-xL
functions upstream to this event. The finding that the cleaved fragment
of PKC
induces characteristics typical of apoptosis further supports
a role for PKC
in mediating apoptotic events and not simply a
bystander effect of Caspase-3 activation.
We are grateful to Robert Talanian (BASF Bioresearch Corp., Worcester, MA) for providing recombinant proteases and protease inhibitors. We thank Tariq Ghayur (BASF Bioresearch Corp.) for helpful discussions.