By
From * BASF Bioresearch Corporation, Worcester, Massachusetts 01605; and Division of Cancer
Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
Recent studies have shown that protein kinase C (PKC) is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC
cleavage to induction of apoptosis is unknown. The
present studies demonstrate that full-length PKC
is cleaved at DMQD330N to a catalytically
active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear
fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of
full-length PKC
or a kinase inactive PKC
fragment had no detectable effect. The findings
suggest that proteolytic activation of PKC
by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.
The protein kinase C (PKC) family consists of multiple
subspecies that possess a conserved catalytic domain.
The classic or group A isoforms ( The nematode Ced-3 cysteine protease is related to the
mammalian interleukin-1 The present results demonstrate that PKC In Vitro Cleavage of PKC Analysis of Peptide Proteolysis.
Peptides were synthesized and
purified to ~95% by standard methods and confirmed by mass
spectrometry. Reaction mixtures (810 µl) contained: 100 mM
Hepes (pH 7.5), 20% (vol/vol) glycerol, 5 µM dithiothreitol,
0.5 mM EDTA, and 380 ng N-His CPP32 (19). Peptide substrates were added to final concentrations of 10 µM. The reaction
mixtures were incubated at 30°C. Aliquots were removed at 10 min intervals for 60 min and added to vials containing 3 M HCl
to stop the reactions. The amount of substrate remaining at each
time was quantitated by reverse phase HPLC. Data were fit to the
equation (St/So) = e Cell Transfections.
Cells were seeded at a density of 1.7 × 105
in each well of 6-well dishes 24 h before transfection. For each
well, 2 µg DNA construct and 0.5 µg pSv To determine whether PKC
Table 1.
CPP32 Proteolysis of Peptides Spanning the PARP,
PKC,
, and
) require Ca2+
for activity and contain cysteine-rich motifs that confer
phospholipid-dependent binding of diacylglycerol (1). The
group A PKCs are cleaved at the third variable region (V3)
by the neutral proteases, calpains I and II, to catalytically
active fragments (2). Recent studies have demonstrated that
the Ca2+-independent
isoform, and not the group A
PKCs, is selectively cleaved at V3 to a catalytically active
fragment in cells induced to undergo apoptosis (3, 4). Inhibition of apoptosis by overexpression of Bcl-2 or Bcl-xL is
associated with a block of PKC
cleavage (3, 4). The finding that PKC
is cleaved at a site (DMQD/N) adjacent to
aspartic acid has supported the potential involvement of aspartate-specific cysteine proteases which are known to be
activated during apoptosis.
converting enzyme (ICE) (5, 6).
The demonstration that overexpression of Ced-3 or ICE
induces apoptosis has provided support for involvement of
these cysteine proteases in cell death pathways (7). ICE/
Ced-3 family members include Nedd2/Ich-1, CPP32/
YAMA/apopain, Tx/Ich-2/ICErelII, ICErelIII, Mch2, Mch3/
ICE-LAP3/CMH-1 (reviewed in reference 8), ICE-LAP6 (9), FLICE/Mch5 (10, 11), and Mch4 (11). ICE cleaves the precursor of IL-1
to the active cytokine (6, 12, 13). Other known substrates of the ICE/Ced-3 family include:
(a) poly (ADP-ribose) polymerase (PARP) which is cleaved
by CPP32, Mch3 and Ced-3, but not ICE (14); and (b)
DNA-dependent protein kinase (DNA-PK), the U1 small
nuclear ribonucleoprotein and D4-GDP dissociation inhibitor for the Rho family GTPases (D4-GDI), which are
cleaved by CPP32 (17, 18). However, the functional role
of these cleavage products in the induction of apoptosis is
unclear.
is cleaved by
CPP32 and not certain other ICE/Ced-3 family members.
We also demonstrate that overexpression of the PKC
catalytic fragment is involved in the induction of phenotypic
changes that are characteristic of apoptosis.
and PARP.
The full-length PKC
cDNA was cloned into the SpeI and BamH1 sites of a modified
pSV
plasmid (Clontech, Palo Alto, CA). PKC
(D327A/D330A) was generated in two steps by overlapping primer extension.
PARP cDNA was generated by PCR cloning. The proteins were
labeled with [35S]methionine by coupled transcription and translation reactions (Promega, Madison, WI). Labeled proteins were
incubated with 5 µg/ml Escherichia coli-derived CPP32
in 50 mM
Hepes (pH 7.5), 10% glycerol, 2.5 mM DTT, and 0.25 mM
EDTA at room temperature for 30 min. The reaction products
were analyzed by electrophoresis in 10-20% SDS-polyacrylamide
gels and then autoradiography. For the kinase assays, full-length
PKC
, PKC
(D327A/D330A), PKC
catalytic fragment (CF),
and PKC
CF(K-R) were prepared by coupled transcription and
translation. PKC
and PKC
(D327A/D330A) were incubated with 5 µg/ml CPP32
at room temperature for 30 min. Protein kinase assays using MBP as a substrate were performed as described
(PKC Assay Kit; GIBCO BRL, Gaithersburg, MD).
kt, where k is the decay rate constant equal
to Vmax/Km. Observed Vmax/Km values were normalized to 1.00 for the PARP peptide.
plasmid containing
-gal were coprecipitated with calcium phosphate. Cells were incubated with the coprecipitate for 30 h at 37°C and then analyzed
by X-gal staining. Cells (1.7 × 105/well) were also transfected
with 2 µg DNA construct for 30 h at 37°C, fixed with 4%
paraformaldehyde, postfixed with 5% acetic acid in ethanol and
then stained with 5 µg/ml Hoechst dye. For sub-G1 DNA content, cells transfected by lipofectamine were stained with propidium iodide and monitored by FACScan®. Chromatin condensation was assessed by staining with acridine orange and ethidium
bromide (20).
is cleaved by one of the
known ICE-like proteases, full-length 78-kD PKC
labeled with [35S]methionine was incubated with purified recombinant proteases. Cleavage of PKC
to a 40-kD fragment was observed with purified CPP32
(14) (Fig. 1 A).
In contrast, ICE failed to cleave PKC
at concentrations up
to 600 U/µl (3). The related Ich-1, Ich-2, Mch2, Mch3,
and ICErelIII proteases also failed to cleave PKC
(data not
shown). Because PKC
is cleaved at DMQD330N in vivo (3, 4), we asked whether this site is responsible for CPP32mediated cleavage in vitro. CPP32 may prefer peptidic
substrates with aspartic acid at the P1 and P4 positions (15).
Consequently, we prepared a PKC
mutant with substitution of D327A and D330A. Incubation with CPP32 resulted in no detectable CPP32-mediated cleavage of this
mutant to the 40-kD catalytic fragment, while there was
partial digestion to a species of ~55 kD (not observed with
wild-type substrate) (Fig. 1 A). Recombinant CPP32 also
cleaved the 116-kD full-length PARP to the predicted
85-kD fragment (14, 15) (Fig. 1 A). Using peptides derived
from the cleavage sites of PARP and PKC
in proteolytic
assays, we found that CPP32 cleaves both substrates and
not a peptide spanning the IL-1
maturation site (Table 1).
These findings confirm that PKC
, like PARP, is a substrate for CPP32.
Fig. 1.
PKC is proteolytically activated by CPP32 in vitro. (A)
PKC
(full-length: FL), PKC
(D327A/D330A) and PARP were labeled
with [35S]methionine and incubated with recombinant CPP32
. The reaction products were analyzed by SDS-PAGE and autoradiography. The
kinase active PKC
catalytic fragment (CF) and the kinase inactive
PKC
CF(K-R) were labeled with [35S]methionine and analyzed under
similar conditions. (B) Recombinant PKC
and PKC
(D327A/D330A)
were incubated with CPP32 and then assayed for protein kinase activity
using MBP as substrate.
[View Larger Version of this Image (20K GIF file)]
, and IL-1
Cleavage Sites
Substrate
Sequence
Relative
Vmax/Km
PARP
Ac-WGDEVD216-GVDEVW-NH2
1.00
PKC
Ac-GEDMQD330NSGTYW-NH2
0.42
IL-1
Ac-NEAYVHD116APVRSLY-NH2
0.00
We also asked whether cleavage of PKC by CPP32 is
associated with activation of the kinase function. Fulllength PKC
exhibited a low level of myelin basic protein
(MBP) phosphorylation, while incubation with CPP32 resulted in a greater than sixfold increase in kinase activity
(Fig. 1 B). In contrast, CPP32 had no detectable effect on kinase function of the PKC
(D327A/D330A) mutant (Fig. 1
B). A recombinant 40-kD CF of PKC
(amino acids 331676) exhibited constitutive kinase activity, while a mutant
of the fragment with K-378 in the ATP binding site mutated to R (K378R; designated K-R) yielded background
levels of MBP phosphorylation found with control bacterial
lysates (Figs. 1, A and B). These findings collectively demonstrate that CPP32-mediated cleavage of the DMQD330N
site activates PKC
.
To study the role of PKC in apoptosis, we used the
transient HeLa cell transfection system previously found to
demonstrate induction of apoptosis by ICE-like proteases
(7). Cotransfection of the kinase inactive PKC
CF(K-R)
mutant with the
-galactosidase (
-gal) marker gene had
little effect on HeLa cell morphology (Fig. 2 A). Most of
the blue X-gal positive cells remained flat and attached to
the dish (Fig. 2 A). Cotransfection of the kinase active
PKC
CF and
-gal resulted in condensed, small blue cells (Fig. 2 B), consistent with the induction of apoptosis (7). Similar findings were obtained with NIH3T3 cells (Figs. 2,
C and D). Overexpression of PKC
in both cell types also
resulted in detachment of non-viable cells into the culture
medium.
Hoechst staining of HeLa cells transfected with a vector
that expresses full-length PKC had no detectable changes
in nuclear morphology (Fig. 3 A), but overexpression of
PKC
CF resulted in fragmented nuclei (Fig. 3 B). Transfection of kinase inactive PKC
CF(K-R) was associated
with a normal nuclear morphology (Fig. 3 C). The changes
observed with expression of the PKC
CF were also compared to those found upon exposure to 1-
-D-arabinofuranosylcytosine (ara-C), a DNA-damaging agent that induces
proteolytic cleavage of PKC
and apoptosis (4). Treatment
of HeLa cells with ara-C resulted in a similar pattern of nuclear fragmentation (Fig. 3 D).
To confirm that the nuclear changes induced by PKCCF
are associated with induction of apoptosis, we assessed the
effects of transfection on the appearance of HeLa cells with
sub-G1 DNA content. Transfection of the empty vector,
full-length PKC
or PKC
CF(K-R) resulted in 10-15% of
cells with sub-G1 DNA (Fig. 4 A and data not shown). By
contrast, transfection of PKC
CF was associated with 30-
35% of cells with sub-G1 DNA (Fig. 4 A). Cells were also
stained with acridine orange and ethidium bromide to assess
chromatin condensation (20). Transfection of PKC
CF, but not PKC
CF(K-R), resulted in the appearance of bright
yellow-green nuclear staining of condensed chromatin
(Fig. 4 B).
To quantify the effects of PKCCF expression on cell
viability, we cotransfected PKC
CF or PKC
CF(K-R) and
the green fluorescence gene (Clontech) into HeLa cells.
Positive transfectants were selected by flow cytometry, reseeded in culture medium and assayed at 24 h for viability
by trypan blue exclusion. Less than 5% of the PKC
CF
transfectants were viable, while over 90% of the kinase inactive PKC
CF(K-R) transfectants were viable and attached to the dish. Viability of 90-95% was observed after
transfection of the null vector and sorting. We conclude
that the kinase active catalytic domain of PKC
induces
characteristics typical of cells undergoing apoptosis: (a) size
reduction and round morphology; (b) nuclear fragmentation; (c) chromatin condensation; (d) sub-G1 DNA content; and (e) detachment and loss of viability.
Multiple events that lead to destruction of nuclear and
cytoplasmic integrity are probably required for apoptosis.
Activation of ICE-family proteases may be a central trigger,
resulting in the cleavage of substrates such as PARP (21),
lamin B1 (22, 23), topoisomerase 1 (23), D4-GDI (18),
DNA-PK, and the U1 small nuclear ribonucleoprotein
(17). PKC, but not PKC
,
,
, or
, is also cleaved at the
onset of apoptosis (3, 4). Little is known about the physiological function of PKC
(24, 25). We demonstrate that
PKC
is cleaved by CPP32 and not other ICE/Ced-3 family members in vitro. The results also demonstrate that expression of the PKC
catalytic fragment induces morphologic changes characteristic of apoptosis. We propose that
the proteolytic cleavage of PKC
is a key mediator of nuclear fragmentation and cell death, and not a bystander effect of protease activation. Moreover, the finding that proteolytic activation of PKC
is blocked by Bcl-2 and Bcl-xL
suggests that these anti-apoptotic proteins act upstream to
this event (3). Elucidation of the substrates phosphorylated
as a consequence of PKC
cleavage should provide insights
into the pathways activated by the catalytic fragment.
Address correspondence to Donald W. Kufe, Division of Cancer Pharmacology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115.
Received for publication 13 August 1996
This investigation was supported by Public Health Service grants CA66996, CA55241, and CA29431 awarded by the National Cancer Institute, DHHS.1. | Nishizuka, Y.. 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science (Wash. DC). 258: 607-614 [Medline] . |
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