From the Departments of Pharmacology and
Medicine, College of Physicians and Surgeons, Columbia
University, New York, New York 10032 and the ¶ Department of
Pediatrics, University of Cincinnati, Children's Hospital Medical
Center, Cincinnati, Ohio 45229
Received for publication, December 11, 2002, and in revised form, January 30, 2003
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ABSTRACT |
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Recent studies identify
conventional protein kinase C (PKC) isoform phosphorylations at
conserved residues in the activation loop and C terminus as
maturational events that influence enzyme activity and targeting but
are not dynamically regulated by second messengers. In contrast, this
study identifies phorbol 12-myristoyl 13-acetate (PMA)- and
norepinephrine-induced phosphorylations of PKC Protein kinase C (PKC)1
constitutes a family of serine/threonine kinases that play key roles in
signal transduction by receptors coupled to phosphoinositide hydrolysis
(1). The basic primary structure of all PKC isozymes consists of a
single polypeptide chain with N-terminal regulatory and C-terminal
catalytic domains. PKC isoforms are subdivided into three distinct
subfamilies based upon structural differences in their N-terminal
regulatory domains. Conventional PKC isoforms (cPKCs) contain an
autoinhibitory pseudosubstrate domain followed by two
membrane-targeting modules, the C1 domain that binds diacylglycerol and
phosphatidylserine (PS) and the C2 domain that binds anionic lipids in
a calcium-dependent manner. Novel PKC (nPKC) isoforms lack
the C2 domain and are maximally activated by diacylglycerol/phorbol
ester in the absence of calcium; atypical PKCs are regulated by lipids
but are not activated by calcium and diacylglycerol. Current models of
PKC isoform activation focus largely on the allosteric changes that are
induced by cofactor binding to N-terminal membrane-targeting modules;
these conformational changes expel the autoinhibitory pseudosubstrate
domain from the substrate binding pocket, relieve autoinhibition, and
allow PKC to phosphorylate target substrates.
In addition to allosteric activation mechanisms, recent studies
identify a series of ordered "priming" phosphorylations that convert nascent PKC isoforms into the mature forms that reside in the
cytosol and can be activated by diacylglycerol and calcium (2). The
focus of most studies has been on cPKC isoform phosphorylations in
heterologous expression systems. Here, the first of three sequential phosphorylations is at a conserved threonine in the activation loop and
is under the control of phosphoinositide-dependent kinase (PDK-1) (3, 4). According to current models, activation loop
phosphorylation introduces a negative charge that critically aligns
residues in the catalytic site and is required for catalytic competence
of the enzyme. This enables two subsequent phosphorylations at
conserved residues in the C-terminal V5 domain (a predicted autophosphorylation in a proline-flanked "turn motif" and an
additional phosphorylation in a hydrophobic
FXXFS/TF/Y motif 19 residues C-terminal to the
turn motif). Phosphorylations at these two C-terminal sites are
believed to lock the enzyme in a closed, catalytically competent,
stabilized, and protease/phosphatase-resistant conformation (2, 5).
Although nPKC isoforms undergo a series of equivalent priming
phosphorylations, there has been only limited progress defining the
order of these phosphorylation events, the identity of the regulatory
kinase(s) that operate on the activation loop and C-terminal sites, and
the consequences of multisite phosphorylations on nPKC isoforms.
Current concepts regarding nPKC isoform phosphorylation mechanisms are
based largely on studies of serum-deprived cells maintained in
suspension culture because nPKC isoforms remain highly phosphorylated
in most adherent cell types grown in the presence of serum (6).
However, in the course of studies on adherent neonatal cardiomyocyte
cultures, we identified dynamically regulated serine/threonine
phosphorylations of PKC Materials--
Antibodies against total and phosphorylated forms
of PKC were from the following sources. Phospho-PKC-Pan,
PKC Cardiomyocyte Culture and Transfection--
Cardiomyocytes were
isolated from the hearts of 2-day-old Wistar rats by a trypsin
dispersion procedure according to a protocol that incorporates a
differential attachment procedure to enrich for cardiomyocytes followed
by irradiation as described previously (7). The yield of cardiomyocytes
typically is 2.5-3 × 106 cells/neonatal ventricle.
Cells were plated on protamine sulfate-coated culture dishes at a
density of 5 × 106 cells/100-mm dish. Experiments
were performed on cultures grown for 5 days in minimum Eagle's medium
(Invitrogen) supplemented with 10% fetal calf serum and then
serum-deprived for the subsequent 24 h. PKC overexpression was
accomplished as described previously (8). Cardiomyocyte infections were
for 2 h with adenoviral vectors that contain nearly the
full-length Ad5 genome (lacking regions E1 and E3) and a
cytomegalovirus promoter driving expression of wild-type or dominant
negative mouse PKC Immunoblot Analysis--
Immunoblot analysis was performed on
whole cell extracts or soluble and particulate fractions prepared
according to methods described previously (9, 10). Briefly, cells were
washed with phosphate-buffered saline and then transferred immediately to ice-cold homogenization buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 6 mM
Immunoprecipitation and Immunocomplex PKC
Immunoprecipitates (from 0.3 mg of protein assay) were used in
immunocomplex kinase assays carried out in 200 µl of a reaction mixture containing buffer (26 mM Tris, pH 7.5, 5 mM MgCl2, 0.6 mM EGTA, 0.6 mM EDTA, 0.5 µM protein kinase
inhibitor, 10 µM PP1, and 0.25 mM
dithiothreitol) in the absence and presence of 80 µg/ml PS plus 160 ng/ml PMA with 0.5 mg/ml histone III-S, 50 µM PMA Stimulates PKC
The most prominent immunoreactive species detected with
anti-phospho-PKC-Pan in resting cardiomyocyte cultures corresponds to
PKC
Anti-phospho-PKC-Pan detects low levels of immunoreactivity that
comigrates with PKC
To examine PKC isoform phosphorylation in the context of
activation-dependent redistribution of the enzyme to
membranes, immunoblot analysis also was performed on cell extracts
partitioned into soluble and particulate fractions. Fig. 2C
shows that PKC
PKC
PKC
Fig. 3 (left panel) examines
the kinetics of PMA-induced phosphorylations and translocation of PKC
isoforms in greater detail. PMA-dependent translocation of
PKC isoforms, phosphorylation of PKC Kinases That Mediate nPKC Isoform Phosphorylation--
There is
mounting evidence that PKC phosphorylations are mediated by at least
two distinguishable signaling pathways (2, 6). Largely based upon
in vitro studies of isolated PKC isoforms (or studies of PKC
isoforms heterologously expressed in undifferentiated cell types),
activation loop phosphorylation has been attributed to the actions of
PDK-1 (at least for PKC
The acute PMA-dependent translocation of PKC NE Promotes the Phosphorylation of PKC PKC Activity Measurements--
Because activation loop
phosphorylation is viewed as a mechanism to regulate PKC kinase
activity, the catalytic activity of PKC
Fig. 8A shows that PKC
Because PMA-dependent changes in PKC PKC Phosphorylation in Cardiomyocytes That Overexpress Wild-type
and Kinase-inactive Dominant Negative nPKC Isoforms--
The studies
with pharmacologic inhibitors identify a nPKC catalytic activity
requirement for PMA-dependent phosphorylations of PKC
The anti-PKC
A potential role for PKC It is almost a decade since PKC isoforms first emerged as
important regulatory elements in fundamental physiological processes in
cardiomyocytes, including hypertrophic signaling, ion channel regulation, and ischemic preconditioning. The recognition that cardiomyocytes coexpress multiple PKC isoforms with unique modes of
activation, localization, and substrate specificity has provided the
impetus to consider PKC isoforms as promising targets for the therapy
of cardiovascular disorders. Strategies to modulate PKC function have
focused on mechanisms that alter the abundance, activity, and/or
subcellular localization of each isoform. Although ordered
phosphorylations of key serine/threonine residues at the activation
loop and C terminus have been identified as important regulatory events
required for full maturation of catalytically competent cPKCs,
knowledge of the controls and consequences of regulatory
phosphorylations on nPKCs is more limited, and PKC phosphorylation in
cardiomyocytes has never been considered. This study provides novel
evidence that activation loop and hydrophobic domain phosphorylations
constitute highly regulated events that impact on the catalytic
activity, substrate specificity, and perhaps even
trafficking/down-regulation of nPKCs in cardiomyocytes.
Consistent with previous literature that identifies PKC This study demonstrates that PKC There is mounting evidence that hydrophobic motif phosphorylations
promote the release of cPKCs from membranes, allowing for reversible
stimulation by physiologic agonists and/or trafficking to intracellular
membranes for PMA-dependent down-regulation (26). Studies
reported herein are consistent with a similar regulatory role for
PKC These studies indicate that PKC The functional importance of PKC The observation that PKC (at the
C-terminal hydrophobic motif) and PKC
(at the activation loop) as
events that accompany endogenous novel PKC (nPKC) isoform activation in
neonatal rat cardiomyocytes. Agonist-induced nPKC phosphorylations are
prevented (and the kinetics of PMA-dependent PKC
down-regulation are slowed) by pharmacologic inhibitors of nPKC kinase
activity. PKC
is recovered from PMA-treated cultures with increased
in vitro lipid-independent kinase activity (and altered
substrate specificity); the PMA-dependent increase in PKC
kinase activity is attenuated when PKC
activation loop
phosphorylation is prevented. To distinguish roles of individual nPKC
isoforms in nPKC phosphorylations, wild-type (WT) and dominant negative (DN) PKC
and PKC
mutants were introduced into cardiomyocyte cultures using adenovirus-mediated gene transfer. WT-PKC
and WT-PKC
are highly phosphorylated at activation loop and hydrophobic motif sites, even in the absence of allosteric activators. DN-PKC
is
phosphorylated at the activation loop but not the hydrophobic motif;
DN-PKC
is phosphorylated at the hydrophobic motif but not the
activation loop. Collectively, these results identify a role for PKC
in nPKC activation loop phosphorylations and PKC
in nPKC hydrophobic
motif phosphorylations. Agonist-induced nPKC isoform phosphorylations
that accompany activation/translocation of the enzyme contribute
to the regulation of PKC
kinase activity, may influence
nPKC isoform trafficking/down-regulation, and introduce functionally important cross-talk for nPKC signaling pathways in cardiomyocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PKC
which accompany
ligand-dependent PKC activation. Given the paucity of information regarding the regulation of nPKC phosphorylation in differentiated cell types (including cardiomyocytes) and the importance of nPKC isoforms as components of the signaling pathways leading to
hypertrophy, apoptosis, and ischemic preconditioning in the heart, this
study examines the regulation and consequences of nPKC isoform
phosphorylations in cardiomyocytes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-pS643, and PKC
-pT505 were from Cell
Signaling Technology. PKC
and PKC
were from Invitrogen. PKC
was from Santa Cruz Biotechnology. The PKC
substrate peptide
(
-peptide) and the PKC
substrate peptide (
-peptide) were
purchased from Calbiochem and Upstate Biotechnology, respectively. PMA
was from Sigma. All other chemicals were reagent grade.
(K436R substitution at the ATP binding site of
the catalytic domain), wild-type or dominant negative rabbit PKC
(analogous K376R substitution at the ATP binding), or
-galactosidase
(as a control). Protein extracts were prepared 24 h later, in some
experiments after a 15-min treatment with 200 nM PMA or vehicle.
-mercaptoethanol, 50 µg/ml aprotinin, 48 µg/ml leupeptin, 5 µM pepstatin A, 1 mM phenylmethylsulfonyl
fluoride, 0.1 mM sodium vanadate, and 50 mM
NaF); cells were lysed by sonication and centrifuged at 100,000 × g for 1 h. The supernatant was saved as the soluble
fraction. PKC isoforms were extracted from the pellets by incubation on
ice for 10 min in homogenization buffer containing 1% Triton X-100
followed by centrifugation at 100,000 × g for 30 min
at 4 °C. Electrophoresis was on 8% SDS-polyacrylamide gels followed
by transfer to nitrocellulose for immunoblotting with a panel of
antibodies that recognize total PKC
, PKC
, or PKC
protein
expression (identified previously as the phorbol ester-sensitive PKC
isoforms expressed by cardiomyocyte cultures (9, 11)), PKC
phosphorylated at the activation loop (anti-PKC
-pT505),
PKC
phosphorylated at the turn motif
(anti-PKC
-pS643), or an antibody that detects all
phorbol ester-sensitive PKC isoforms phosphorylated at the conserved
hydrophobic motif serine (anti-phospho-PKC-Pan; Fig.
1). The specificity of the anti-PKC antibodies has been established previously (10, 12); the specificity of
all anti-phospho-PKC antibodies was validated in control experiments showing that anti-phospho-PKC immunoreactivity is stripped by treatment
of samples with alkaline phosphatase (data not shown) and is
down-regulated along with PMA-dependent down-regulation of
the cognate protein (see Fig. 3). Bands were detected by enhanced chemiluminescence, with each panel in each figure from a single gel
exposed for a uniform duration.
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Fig. 1.
The conserved sequences surrounding the
priming phosphorylation sites in PKC ,
PKC
, and PKC
.
The bold type denotes the phosphorylation sites identified
by the anti-PKC
-pT505, anti-PKC
-pS643,
and anti-phospho-PKC-Pan antibodies used in this study.
Assays--
After
incubation with the compounds indicated in the figures, cells were
lysed in homogenization buffer (20 mM Tris-Cl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 0.5 mM
dithiothreitol, 1 µg/ml aprotinin, 5.5 µg/ml leupeptin, 0.25 mM phenylmethylsulfonyl fluoride, 1 µM
pepstatin A, 0.1 mM sodium orthovanadate, 0.2% Triton
X-100). Lysates were centrifuged at 4 °C for 15 min at 1,500 × g. For immunoprecipitation, supernatant from five 100-mm
dishes was incubated with 5 µg of anti-PKC
antibody for 1 h
at 4 °C followed by the addition of 200 µl of protein A/G
PLUS-agarose beads (Santa Cruz Biotechnology) and incubation for 3 h at 4 °C. The immunoprecipitates were washed twice with washing
buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 150 mM NaCl) and suspended in 1.15 ml of storage buffer (18 mM Tris-Cl, pH 7.5, 1.8 mM EDTA, 1.8 mM EGTA, 0.45 mM dithiothreitol, 225 mM NaCl, 10% glycerol).
-peptide, or 50 µM
-peptide as substrate.
-Peptide is a synthetic peptide that corresponds to the
pseudosubstrate domain of PKC
, with a phosphorylatable serine for
alanine substitution (ERMRPRKRQGSVRRRV).
-Peptide is a
highly specific PKC
substrate that corresponds to amino acids
422-443 of murine eEF-1
(RFAVRDMRQTVAVGVIKAVDKK). Reactions were initiated by the addition 13 µCi of 66 µM [
-32P]ATP and were performed in
quadruplicate at 30 °C for 16 min. Assays were terminated by placing
samples on ice followed by centrifugation at 1,500 × g
for 10 min at 4 °C. 40 µl of supernatant was spotted onto
phosphocellulose filter papers (P-81), which were immediately dropped
into water, washed five times for 5 min, and counted for radioactivity.
Pellets were subjected to SDS-PAGE and immunoblotting for PKC
to
normalize for any minor differences in immunoprecipitated enzyme.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and PKC
Phosphorylation in
Cardiomyocytes--
PKC phosphorylation is increasingly recognized as
a mechanism to regulate cPKC targeting and catalytic activity. However, there is still only limited information on the inputs that promote nPKC
phosphorylation or its importance as a mechanism to regulate nPKC
targeting and activity; PKC isoform phosphorylation in cardiomyocytes has never been considered. Therefore, PKC isoform phosphorylation was
examined in cardiomyocyte cultures at rest and after stimulation with
PMA. Fig. 2A shows that
anti-phospho-PKC-Pan (an antibody that recognizes phorbol
ester-sensitive PKC isoforms phosphorylated at the conserved
hydrophobic motif) identifies three major bands that comigrate with
PKC
, PKC
, and PKC
(~78, 82, and 96 kDa, respectively),
corresponding to PKC
phosphorylated at Ser662, PKC
phosphorylated at Ser657, and PKC
phosphorylated at
Ser729. Atypical PKC isoforms contain an acidic Glu in
place of the Ser phosphoacceptor site in the hydrophobic motif and are
not detected by the anti-phospho-PKC-Pan antibody (2).
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Fig. 2.
PMA-dependent phosphorylation and
translocation of phorbol ester-sensitive PKC isoforms in
cardiomyocytes. Day 5 cardiomyocyte cultures were serum starved
for 24 h followed by stimulation for 20 min with vehicle or 100 nM PMA. Whole lysates (A) or soluble and
particulate fractions (C and D) were subjected to
SDS-PAGE and immunoblot (IB) analysis with antibodies that
recognize phosphorylated (A and D) or total
(C) PKC protein. In C, PKC isoforms are presented
according to their molecular masses (with the slowest migrating PKC
at the top and the most rapidly migrating PKC
at the
bottom), to facilitate comparisons of total and
phosphorylated proteins (C and D). Results are
representative of data obtained on four or five separate culture
preparations. B, extracts from cardiomyocyte cultures
stimulated for 20 min with 100 nM PMA were subjected to
immunoprecipitation (IP) with antibodies that recognize RSK2
or PKC
as indicated. The pelleted proteins were probed for RSK,
PKC
, and anti-phospho-PKC-Pan immunoreactivity as indicated. Similar
results were obtained in two experiments on separate culture
preparations.
; PKC
-Ser657 phosphorylation is not detectably
altered by PMA. PKC
phosphorylation at the hydrophobic domain
(Ser662) is also detected in quiescent cardiomyocyte
cultures; treatment with PMA slows the migration of PKC
in SDS-PAGE
(characteristic of a phosphorylation-induced change in electrophoretic
mobility) but induces little to no increase in hydrophobic motif
phosphorylation. This suggests that PMA promotes PKC
phosphorylation
at a site that is distinct from Ser662. Therefore,
immunoblot analysis also was performed with antibodies that recognize
PKC
phosphorylated at Ser643 in the turn motif
(anti-PKC
-pS643) or Thr505 in the activation
loop (anti-PKC
-pT505). Fig. 2A shows that
PKC
-pS643 immunoreactivity is similar in quiescent and
PMA-stimulated cultures (i.e. PKC
phosphorylations at the
C-terminal turn and hydrophobic motifs are relatively stable
modifications that are not influenced by PMA-dependent
activation). In contrast, there is little to no
PKC
-pT505 immunoreactivity in quiescent cultures; PKC
phosphorylation at the Thr505 activation loop site is
increased markedly in association with PMA-dependent PKC
activation.
in quiescent cardiomyocyte cultures; immunoreactivity with PKC
-like mobility is considerably more abundant and (in some experiments) resolved as a doublet (similar to
the appearance of PKC
protein; compare Fig. 2, A and
C) in cultures stimulated with PMA. Because PKC
is
recovered from many cell types as a mixture of species (with
phosphorylation-induced differences in electrophoretic mobility (13)),
the doublet recognized by anti-phospho-PKC-Pan could represent a
mixture of Ser729-phosphorylated PKC
species with
distinct phosphorylation patterns at other residues. Alternatively, the
doublet could represent a phosphorylated form of PKC
and a
phosphorylated form of a distinct gene product that comigrates with
PKC
and also contains a phosphorylatable hydrophobic motif sequence.
The most likely candidate is 90-kDa ribosomal S6 kinase (RSK), which is
reported to undergo PMA-dependent phosphorylation at a
consensus FRGFS386F hydrophobic motif sequence (14, 15). To
determine whether RSK contributes to anti-phospho-PKC-Pan
immunoreactivity, RSK and PKC
were immunoprecipitated individually
from PMA-treated cultures and probed for anti-phospho-PKC-Pan
immunoreactivity. Fig. 2B shows that anti-phospho-PKC-Pan
immunoreactivity is recovered from PMA-treated cardiomyocyte cultures
along with PKC
but not with RSK, effectively excluding RSK as the
source of anti-phospho-PKC-Pan immunoreactivity in cardiomyocyte cultures.
protein is recovered largely from the soluble
fraction of quiescent cardiomyocyte cultures and the particulate
fraction of cultures stimulated with PMA. Immunodetection of PKC
with the anti-phospho-PKC-Pan antibody essentially tracks total PKC
protein (Fig. 2D; i.e.
Ser657-phosphorylated PKC
is recovered largely from the
soluble fraction of quiescent cultures and the particulate
fraction of cultures stimulated with PMA). These results are
consistent with the experiments on whole cell lysates (Fig.
2A), which show no PMA-dependent change in
PKC
-Ser657 phosphorylation.
protein is recovered from both soluble and particulate fractions
of resting cardiomyocytes, but the phosphorylated forms of PKC
are
confined to the particulate fraction (Fig. 2, C and D); phosphorylated forms of PKC
are not detected in the
soluble fraction, even with increased protein loading and prolonged
exposures of the gel. PKC
phosphorylation at the C terminus
(Ser662 and Ser643 in the hydrophobic and turn
motifs, respectively) is similar in resting and PMA-stimulated
cultures. In contrast, PKC
phosphorylation at Thr505 in
the activation loop increases during stimulation with PMA, in
association with PMA-dependent translocation of PKC
protein to membranes (and the PMA-induced decrease in its mobility in SDS-PAGE).
also distributes between soluble and particulate fractions of
quiescent cardiomyocytes. PKC
-Ser729 phosphorylation is
detected at trace levels in quiescent cardiomyocyte cultures and at
markedly increased levels in both the soluble and particulate fractions
of cultures treated with PMA (Fig. 2D). A phosphorylated
form of PKC
in the soluble fraction of PMA-treated cultures was not
anticipated because the bulk of PKC
protein is recovered from the
particulate fraction. However, C-terminal phosphorylations have been
described as modifications that promote the release of PKC from
membranes and facilitate its down-regulation (16); the highly
phosphorylated form of PKC
recovered in the soluble fraction of
PMA-treated cardiomyocytes could represent a pool of PKC
newly
released from membranes.
at Ser729, and
phosphorylation of PKC
at Thr505 are rapid events that
are readily detected at 5 min (the first time point measured).
Phosphorylations of PKC
at Ser729 and PKC
at
Thr505 are stable modifications; both decline in parallel
with the agonist-induced down-regulation of these enzymes.
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Fig. 3.
The kinetics of PMA-dependent
translocation and phosphorylation of phorbol ester-sensitive PKC
isoforms. Cardiomyocytes were pretreated for 60 min with vehicle
or 5 µM GF109203X followed by stimulation with vehicle or
100 nM PMA for the indicated intervals. Immunoblot analysis
was on soluble and particulate fractions with antibodies that recognize
total PKC , PKC
, and PKC
as well as anti-phospho-PKC-Pan and
anti-PKC
-pT505 as described in Fig. 2. Similar results
were obtained in three experiments performed on separate culture
preparations.
,
, and
), and the C-terminal
phosphorylations have been characterized as intramolecular autophosphorylations (2, 4, 17, 18). These results predict that certain
PKC phosphorylations would require intact intrinsic PKC activity.
Therefore, initial studies examined the effects of GF109203X (an
inhibitor of cPKCs and nPKCs (19)). Figs. 3 (right panel)
and 4 show that GF109203X prevents PMA-dependent phosphorylation of PKC
at Ser729. Somewhat unexpectedly,
GF109203X also prevents PMA-dependent phosphorylation of
PKC
at Thr505 (as well as the PMA-dependent
shift in PKC
mobility). The inhibitory effects of GF109203X (on
PKC
phosphorylation at Ser729, PKC
phosphorylation at
Thr505, and the PKC
mobility shift) are mimicked by
Go6983 (another bisindolylmaleimide that inhibits cPKCs and nPKCs) but
not by Go6976 (an indolocarbazole which selectively inhibits cPKC
but not nPKCs, Fig. 4).
PMA-dependent phosphorylations of PKC
at Ser729 and PKC
at Thr505 are not inhibited
by the phosphoinositide 3-kinase inhibitor LY294002 or the Src
family kinase inhibitor PP1 (Fig. 5,
under conditions where the efficacy of LY294002 and PP1 was validated, data not shown). These results identify a role for nPKC catalytic activity in the PMA-dependent phosphorylations of native
PKC
(at Ser729) and PKC
(at Thr505) in
cardiomyocytes.
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Fig. 4.
The role of PKC isoforms in
PMA-dependent phosphorylations of PKC
and PKC
. Cardiomyocytes were
pretreated with 5 µM GF109203X, Go6983, or Go6976 (30 min
for each) and then incubated for 30 min with 100 nM PMA.
Particulate fractions (containing the bulk of the PKC isoform
immunoreactivity) were subjected to SDS-PAGE and immunoblot analysis
with anti-phospho-PKC-Pan, anti-PKC
-pT505, and
anti-PKC
(to normalized for total protein). Results are
representative of data obtained in three separate experiments on
separate cultures.
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Fig. 5.
PMA-dependent phosphorylation of
PKC and PKC
requires
intact nPKC kinase activity. Cardiomyocytes were
pretreated for 30 min with vehicle, 10 µM LY294002, 5 µM GF109203X, or 10 µM PP1. Stimulations
were with vehicle or 100 nM PMA for 10 min. Immunoblot
analysis on total cell extracts was according to methods described in
Fig. 4, with similar results obtained in three separate experiments on
separate culture preparations.
to the
particulate fraction is similar in cultures treated with PMA alone or PMA plus GF109203X (Fig. 3). These results indicate that
phosphorylation of PKC
at Ser729 is not required for
PKC
association with membranes. However, Fig. 3 shows that GF109203X
slows the kinetics of PMA-induced PKC isoform down-regulation (compare
levels of PKC
, PKC
, and PKC
immunoreactivity in the
particulate fractions of cultures treated for 24 h with PMA alone
versus PMA plus GF109203X). These results suggest that PKC
kinase activity and/or the resultant PKC isoform phosphorylations
influence the kinetics of down-regulation. To examine more carefully
the effect of PKC inhibitors on the kinetics of PKC isoform
down-regulation, PKC isoform abundance was compared in resting cultures
(as a reference for total PKC isoform immunoreactivity), cultures
treated for 31 h with PMA alone, and cultures treated for 31 h with PMA plus PKC inhibitors. Fig. 6
shows that PMA effectively down-regulates phorbol ester-sensitive PKC
, PKC
, and PKC
and that PMA-dependent
down-regulation of PKC
, PKC
, and (to a lesser extent) PKC
is
impaired by GF109203X and Go6983 (general inhibitors of cPKC and nPKC
kinase activities) but not by Go6976 (a selective cPKC isoform
inhibitor). This identifies a requirement for intact nPKC isoform
kinase activity and suggests two possible mechanisms. Because some
studies identify C-terminal PKC isoform phosphorylations as
prerequisites for the release of PKC from membranes and its subsequent
down-regulation (16), nPKC kinase inhibitors might slow PKC
down-regulation by preventing its C-terminal phosphorylation. However,
the effect of nPKC isoform inhibitors to slow the kinetics of PKC
and PKC
down-regulation (i.e. to regulate PKC
in
trans) suggests a more general role for nPKC kinase activity
in the events that govern PKC trafficking/down-regulation (20, 21).
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Fig. 6.
PMA-dependent down-regulation of
nPKC isoforms is attenuated by GF109203X and Go6983 but not by
Go6976. Cardiomyocytes were pretreated with vehicle or 5 µM GF109203X, Go6983, or Go6976 (for 30 min each)
followed by stimulation with PMA for 30 min (first lane) or
31 h (second through fifth lanes) in the
absence or continued presence of the PKC inhibitors. Immunoblot
analysis of whole extracts was with anti-PKC , anti-PKC
, and
anti-PKC
. Similar results were obtained in three separate
experiments on separate culture preparations.
and PKC
in
Cardiomyocytes--
PMA promotes high affinity irreversible
interactions of PKC isoforms with membranes. Because PMA does not
necessarily mimic the effects of physiologic G protein-coupled receptor
agonists, the studies next examined the effects of
1-adrenergic receptor activation with norepinephrine
(NE). Fig. 7 shows that NE promotes the
phosphorylation of PKC
at Ser729 and PKC
at
Thr505. PKC
phosphorylation at Ser729 is
detected in both soluble and particulate fractions of NE-stimulated cultures but only after a lag of ~10 min; the intensity of PKC
phosphorylation at Ser729 is similar in cultures treated
for 10 min with either NE or PMA. In contrast, PKC
phosphorylation
at Thr505 is confined to the particulate fraction, is
detected at a much earlier time point (0.5 min), and is sustained for
at least 20 min. PKC
phosphorylation at Thr505 is more
prominent in cultures treated with PMA than cultures treated with NE.
The NE-dependent increases in PKC
phosphorylation at
Ser729 and PKC
phosphorylation at Thr505 are
inhibited by the
1-adrenergic receptor antagonist
prazosin or GF109203X (and not by the
-adrenergic receptor
antagonist propranolol or Go6976; data not shown).
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Fig. 7.
NE promotes nPKC isoform
phosphorylation. Cardiomyocytes were stimulated with vehicle,
10 5 M NE for the indicated intervals, or 100 nM PMA for 10 min, as a positive control. Immunoblot
analysis was of soluble and particulate fractions (for
anti-phospho-PKC-Pan) or whole cell lysates (for
anti-PKC
-pT505). Similar results were obtained in four
separate experiments on separate culture preparations.
recovered from quiescent
cultures (with little to no PKC
-Thr505 phosphorylation)
and cultures treated for 10 min with PMA (with demonstrable
PKC
-Thr505 phosphorylation) was compared; assays were
performed under basal conditions and with lipid (PS plus PMA) added to
the in vitro incubations. To determine whether
PMA-dependent activation/phosphorylation leads to a change
in PKC
substrate specificity, we measured the incorporation of
32Pi from [
-32P]ATP into a
panel of substrates, including
-peptide and
-peptide (model
substrates for PKC
and PKC
, respectively) as well as histone
(generally considered to be a better substrate for cPKC isoforms than
for nPKC isoforms).
immunopurified from quiescent cardiomyocyte cultures exhibits little to
no lipid-independent kinase activity toward any substrate; histone,
-peptide, and
-peptide kinase activities are increased markedly
by the addition of lipid (PS/PMA) to the in vitro kinase
assays (although surprisingly, 32Pi
incorporation into
-peptide is consistently relatively modest compared with the activity measured with
-peptide or histone as
substrates). PKC
is recovered from PMA-treated cultures with markedly different cofactor requirements, substrate specificity, and
activity. Here, lipid-independent kinase activity toward all substrates is increased significantly. However, the increment in
lipid-independent histone kinase activity is relatively modest in
magnitude (~40%); the addition of PS/PMA to the in vitro
kinase assay results in ~3-fold further increase in histone
phosphorylation, whereas the level of histone phosphorylation by PKC
recovered from PMA-stimulated cultures is relatively low level compared with the robust histone phosphorylation induced by PKC
recovered from quiescent cultures (i.e. histone is a particularly poor
substrate for PKC
recovered from PMA-stimulated cultures). In
contrast, PKC
is recovered from PMA-activated cultures as a markedly
activated (largely lipid-independent)
-peptide kinase. The pattern
for PKC
phosphorylation of
-peptide is intermediate.
Lipid-independent
-peptide kinase activity approximately doubles
when cultures are treated with PMA; the addition of PS/PMA to the
in vitro kinase assay results in ~2-fold further increase
in
-peptide phosphorylation. Importantly, control experiments
established that the phosphorylations of
- and
-peptide (which do
not contain tyrosines) and histone are mediated by PKC rather than
another kinase that coimmunoprecipitates with PKC
; all
phosphorylations are inhibited completely by the addition of 5 µM GF109203X to the in vitro kinase assay
buffer.
View larger version (25K):
[in a new window]
Fig. 8.
PKC is recovered
from PMA-activated cardiomyocytes with altered substrate-specificity,
cofactor requirements, and activity. A, cultures were
treated with vehicle or 300 nM PMA and then subjected to
extraction, immunoprecipitation, and immunocomplex PKC
kinase assays
with histone,
-peptide, or
-peptide as substrate, assayed in the
absence or presence of PS/PMA, as described under "Experimental
Procedures." B, cultures were treated with vehicle, 300 nM PMA, or PMA + GF109203X (each for 20 min). Kinase
activity was assayed with
-peptide as substrate in the absence or
presence of PS/PMA. The levels of lipid-independent PKC
kinase
activity recovered from unstimulated cultures, cultures stimulated with
PMA alone, and cultures stimulated with PMA + GF109203X differed
significantly from each other (p < 0.05). In each
case, results are the mean ± S.E. of quadruplicate measurements,
with similar results obtained in three separate experiments on separate
culture preparations.
kinase activity
could be caused by phosphorylation at Thr505 in the
activation loop or other PMA-induced regulatory modifications of the
enzyme (such as tyrosine phosphorylation or the release of chelated
zinc ions from the cysteine-rich zinc finger domains (22, 23)), we
compared PKC
kinase activity in cultures treated with PMA alone and
cultures treated with PMA plus GF109203X (to prevent Thr505
activation loop phosphorylation). The effect of PMA to reduced basal
and PS/PMA-stimulated histone phosphorylation was not prevented when
PMA stimulation was in the presence of GF109203X (data not shown).
However, Fig. 8B shows that the effect of PMA to render PKC
highly lipid-independent toward
-peptide as substrate is significantly attenuated when cultures are treated with PMA plus GF109203X. Nevertheless, lipid-independent
-peptide phosphorylation by PKC
recovered from PMA/GF109203X-treated cultures remains significantly higher than the base line values recovered from quiescent
cultures. Control experiments excluded the trivial explanation that
GF109203X inhibition results from carryover of drug from the culture
medium into the in vitro kinase reaction; PKC
kinase activity is not reduced when cultures are treated with GF109203X alone
(data not shown). Collectively, these results indicate that PMA
profoundly alters the cofactor requirements and substrate specificity
of PKC
and that the effects of PMA result from Thr505
phosphorylation as well as other mechanisms.
at
Thr505 in the activation loop and PKC
at
Ser729 in the hydrophobic motif. Adenovirus-mediated gene
transfer, with constructs that drive expression of wild-type and
dominant negative mutants of PKC
and PKC
(full-length proteins
with single Lys
Arg substitutions in their ATP binding sites), were
used to resolve the relative roles of individual nPKC isoforms in this process. The integrity of these PKC-encoding adenoviruses was established in a previous study (8). Fig.
9A shows that protocols were
designed to achieve comparable levels of WT and DN nPKC isoform overexpression, with exogenous proteins overexpressed at levels that
are 5-7 times higher than the cognate endogenous PKC isoforms (as
reported previously (8)). Overexpression of WT- or DN-PKC
has no
obvious effect on expression of the endogenous PKC
protein; WT-PKC
overexpression also does not alter endogenous PKC
expression. However, DN-PKC
overexpression promotes the accumulation
of endogenous PKC
.
View larger version (53K):
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Fig. 9.
Phosphorylation of wild-type and
kinase-inactive nPKC isoforms overexpressed in cardiomyocytes.
Adenovirus-mediated gene transfer was used to overexpress WT-PKC ,
DN-PKC
, WT-PKC
, DN-PKC
, and
-galactosidase
(
-gal) as a control. A, immunoblot analysis of
total cell lysates was used to compare total PKC
and PKC
expression, PKC
activation loop phosphorylation (with
anti-PKC
-pT505), and hydrophobic domain phosphorylation
(anti-phospho-PKC-Pan). B-D, cultures were serum starved
for 24 h, treated with vehicle or 100 nM PMA, and then
partitioned into soluble and particulate fractions. Immunoblot analysis
was used to detect PKC
activation loop phosphorylation (with
anti-PKC
-pT505; B), hydrophobic domain
phosphorylation (anti-phospho-PKC-Pan; C), and turn motif
phosphorylation (anti-PKC
-pS643; D). Similar
results were obtained in two separate experiments on separate culture
preparations.
-pT505 antibody was used to explore the role
of nPKC kinase activity in activation loop phosphorylation. Fig.
9A shows that the anti-PKC
-pT505 antibody
readily detects WT and DN-PKC
. Although native PKC
is
phosphorylated at Thr505 only in the particulate fraction
of PMA-stimulated cardiomyocytes (Fig. 2), overexpressed WT-PKC
is
highly Thr505-phosphorylated in both the soluble and
particulate fractions (and in both resting and PMA-stimulated cultures,
Fig. 9B). These results indicate that the tight regulation
of PKC
phosphorylation at Thr505 in the activation loop
is lost during PKC
overexpression. Because DN-PKC
is
phosphorylated at the activation loop, PKC
cannot be required for
Thr505 phosphorylation. Of note, the
anti-PKC
-pT505 antibody detects a prominent band that
comigrates with PKC
in cultures that overexpress WT-PKC
(Fig.
9A). Given the high degree of primary sequence homology
surrounding the activation loop phosphorylation site in PKC isoforms
(see Fig. 1), this band is presumed to represent cross-reactivity with
PKC
overexpressed at high levels and phosphorylated at the
activation loop. Precedent for this type of cross-reactivity (with an
antibody raised to Thr497 in the activation loop of PKC
recognizing phosphorylated forms of PKC
I and PKC
II) has been
published. In this context, it is noteworthy that DN-PKC
, which is
overexpressed at similarly high levels, is not detected by the antibody
that recognizes activation loop phosphorylation. This suggests that the
activation loop site of DN-PKC
is not phosphorylated (and that the
nPKC kinase activity requirement for PMA-dependent PKC
activation loop phosphorylation reflects a requirement for PKC
catalytic activity).
or PKC
kinase activity in nPKC isoform
C-terminal phosphorylations was explored in a similar fashion. Fig. 9,
A and C, show that WT-PKC
and WT-PKC
are
highly phosphorylated at their hydrophobic motifs; WT-PKC
also is
highly phosphorylated at Ser643 in the turn motif (Fig.
9D). Although C-terminal phosphorylations of endogenous
PKC
are confined to the particulate fraction, overexpressed WT-PKC
is phosphorylated at the C-terminal regulatory sites in both
the soluble and particulate fractions (identifying deregulated C-terminal phosphorylation of overexpressed PKC
). Fig. 9,
A and C, shows that DN-PKC
is highly
phosphorylated at the hydrophobic motif, effectively excluding an
intramolecular autophosphorylation mechanism. In contrast, DN-PKC
is
not phosphorylated at the hydrophobic motif. In the context of the
pharmacologic data that identifies a requirement for nPKC kinase
activity for hydrophobic motif phosphorylation, these results are most
consistent with a model that attributes PKC
hydrophobic motif to an
intramolecular autophosphorylation and characterizes PKC
hydrophobic
motif phosphorylation as a reaction that requires intact PKC
kinase
activity. Collectively, these results are most consistent with a model
that incorporates a high level of nPKC isoform cross-regulation, with
PKC
catalytic activity required for nPKC activation loop
phosphorylations and PKC
catalytic activity required for nPKC
hydrophobic motif phosphorylations, as schematized in Fig.
10.
View larger version (16K):
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Fig. 10.
Model for the cross-regulation of nPKC
isoform phosphorylation in cardiomyocytes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phosphorylation as a priming event completed during maturation of the
enzyme and not dynamically regulated by second messengers (2), PKC
hydrophobic motif phosphorylation was detected in resting
cardiomyocytes and was not increased by PMA. In contrast, certain
PKC
and PKC
phosphorylations were dynamically regulated by PMA
and NE. The rapid kinetics of ligand-induced nPKC isoform phosphorylations in cardiomyocytes (measured in minutes) contrasts with
the much slower kinetics for phosphorylation of heterologously expressed nPKC isoforms in suspension and/or serum-deprived HEK293 cell
cultures (6, 24). The most obvious potential explanation for these
discrepant results is that phosphorylation events on native and
overexpressed nPKC isoforms (and in differentiated cardiomyocytes
versus undifferentiated cell lines) are regulated differently. The excessive/dysregulated phosphorylation patterns identified for nPKC isoforms overexpressed in cardiomyocytes (which differ strikingly from the phosphorylation pattern of the native cardiomyocyte enzymes) lend support to this formulation. It is tempting
to speculate that dynamically regulated nPKC phosphorylations are
widespread components of the nPKC isoform activation mechanism (and
that these events are best appreciated in studies of native enzymes in
highly differentiated cell types; they are obscured in studies of
overexpressed enzymes).
is phosphorylated at
hydrophobic and turn motifs in resting cardiomyocytes. C-terminal
phosphorylations on PKC
appear to be stable modifications that are
not regulated during PMA-dependent activation or
down-regulation. In contrast, PKC
retains little hydrophobic motif
phosphorylation in resting cardiomyocytes; the hydrophobic motif of
PKC
becomes phosphorylated during allosteric activation by agonist.
cPKC C-terminal phosphorylations are described as intramolecular
autophosphorylations (25); the mechanism(s) for PKC
hydrophobic
motif phosphorylation has been disputed. Cenni et al. (24)
characterized this event as an autophosphorylation reaction based upon
evidence that WT-PKC
overexpressed in HEK293 cells is phosphorylated
at its hydrophobic motif (Ser729) but kinase-inactive
PKC
is not, PKC
-Ser729 phosphorylation is reduced in
cells incubated with GF109203X, and PKC
autophosphorylates at
Ser729 in vitro. However, others attribute
PKC
hydrophobic motif phosphorylation to a heterologous kinase
complex (not inhibited by GF109203X (6)). This study identifies
PMA-dependent phosphorylation of native PKC
at the
hydrophobic motif as an event that requires nPKC kinase activity in
cardiomyocytes. An autophosphorylation reaction is highly unlikely
because kinase-inactive PKC
is phosphorylated at its hydrophobic
motif when overexpressed in cardiomyocytes (a result that differs from
the previous findings in HEK293 cells (24) and could suggest
differences related to the cell types harboring the kinase-inactive PKC
mutant). Rather, the observation that DN-PKC
is not phosphorylated
at its hydrophobic motif when overexpressed in cardiomyocytes suggests
that PKC
hydrophobic motif phosphorylation results from an
autophosphorylation reaction and that PKC
kinase activity
contributes to PKC
hydrophobic motif phosphorylation in
trans. However, it is worth noting that this model assumes a
similar pharmacology for the phosphorylation of native and
overexpressed nPKC isoforms. Although this assumption seems reasonable
(and underlies much of the literature that has used a molecular
strategy to characterize nPKC isoform phosphorylation mechanisms), it
might deserve more careful scrutiny given the distinct phosphorylation
patterns for native and overexpressed nPKCs in cardiomyocytes
identified in this study.
hydrophobic motif phosphorylation in cardiomyocytes. We
demonstrate that PMA induces a stable increase in PKC
hydrophobic motif phosphorylation (which declines in parallel with
PMA-dependent down-regulation of the protein) and that the
kinetics of PMA-dependent PKC
down-regulation is
markedly slowed when PKC
hydrophobic motif phosphorylation is
prevented by nPKC isoform inhibitors. In the context of the
pharmacologic and molecular evidence implicating PKC
kinase activity
in the pathway leading to PKC
hydrophobic motif phosphorylation,
these results suggest a plausible explanation for the accumulation of
PKC
in cardiomyocytes that overexpress DN-PKC
; any intervention
that impairs PKC
kinase activity would reduce PKC
hydrophobic
motif phosphorylation and lead to defective PKC
processing. However,
the observation that PKC
and PKC
accumulate in cells treated with
nPKC kinase inhibitors argues that nPKC kinases play an additional
(more general) role in the processing/down-regulation of all PKC
isoforms. A general requirement for nPKC kinase activity in PKC
processing events is noteworthy, given the growing interest in nPKC
isoform inhibitors for the treatment of cardiac disorders. An effect of
nPKC kinase inhibitors to promote PKC isoform accumulation (even in
kinase-inhibited forms) could lead to important physiologic consequences, given recent evidence that kinase-inactive PKC
induces
apoptosis (27).
is not phosphorylated detectably at
Thr505 (the activation loop) in quiescent cardiomyocyte
cultures; PKC
-Thr505 phosphorylation is increased
dynamically in the particulate fraction of ligand-stimulated cultures.
Concepts regarding the requirements for PKC
-Thr505
phosphorylation have recently evolved. Initial studies identified a
requirement for allosteric activators (diacylglycerol/PMA) and membranes that contain PDK-1 and phosphatidylinositol
(3,4,5)P3 (17). More recent studies indicate that complex
formation between PDK-1 and the C terminus of membrane-localized
unphosphorylated PKC (in an open conformation to expose the activation
loop residue) is sufficient (28, 29). The lack of any requirement for
phosphoinositide 3-kinase or 3'-phosphoinositide binding to the
pleckstrin homology domain of PDK-1 is consistent with the pharmacology
of PKC
-Thr505 phosphorylation in cardiomyocytes, which
is not blocked by a phosphoinositide 3-kinase inhibitor.
However, the observation that PKC
-Thr505 phosphorylation
is inhibited by GF109203X and that kinase-inactive PKC
displays
defective activation loop phosphorylation (whereas kinase-inactive
PKC
does not) suggests that PKC
kinase activity is required for
nPKC activation loop phosphorylations in cardiomyocytes. This result
was unanticipated, based upon previous studies of in vitro
phosphorylation events or phosphorylation of heterologously expressed
enzymes in undifferentiated cell types. However, the result is
consistent with recent evidence that PKC
phosphorylates the
activation loop of PKCµ (and that in vitro and in
vivo requirements for activation loop phosphorylation can
differ (30)).
-Thr505 phosphorylation
remains disputed. Although activation loop phosphorylation is required for the maturation of catalytically competent cPKCs, PKC
expressed in bacteria is not phosphorylated at Thr505 and is
enzymatically active (presumably because an acidic glutamic acid at
position 500 assumes the role of the phosphorylated threonine in cPKC
isoforms (31)). Although wild-type and the PKC
-T505A mutant exhibit
similar Km values for substrates and inhibition by staurosporine-related blockers (and both undergo autophosphorylation and Src-dependent tyrosine phosphorylation (32)),
Thr505 phosphorylation has been identified as a regulated
event that enhances PKC
activity (17). Our studies used a
pharmacologic strategy to assess the functional significance of PKC
activation loop phosphorylation and reached a similar conclusion.
PKC
is recovered from PMA-stimulated cardiomyocytes as a
lipid-independent enzyme with altered substrate specificity; the
PMA-dependent modulation of PKC
kinase activity is
attenuated when activation loop phosphorylation is prevented. However,
some regulatory effects of PMA persist in this context. This is not
entirely surprising because PMA also modulates PKC
kinase activity
via other mechanisms (including by releasing tetrahedrically
coordinated zinc ions from regulatory cysteine-rich motifs and
promoting Src-dependent tyrosine phosphorylation of
PKC
(22, 23)).
activation loop phosphorylation accompanies
(and is required for optimal) PKC
activation deserves emphasis for
two major reasons. First, PKC isoform translocation generally is
considered a hallmark of PKC activation; this study demonstrates that
PKC
activation may be incomplete unless accompanied by
phosphorylation (i.e. translocation may be an imperfect
surrogate marker for PKC activation). The converse scenario, PKC
activation without translocation, also may pertain to certain clinical
conditions; our recent studies identify
H2O2-dependent activation of PKC
in association with its release from membranes in
cardiomyocytes.2 The
imperfect relationship between PKC translocation and activation raises
questions as to whether the pharmaceuticals that have been designed to
block PKC phosphorylations by inhibiting PKC translocation are complete
inhibitors of PKC cardiac actions. Second, our results suggest that
nPKC isoform phosphorylations are functionally interdependent. PKC
activation loop phosphorylation (which is required for optimal PKC
activation) requires intact PKC
kinase activity, whereas PKC
hydrophobic motif phosphorylation (which influences its
down-regulation) requires intact PKC
kinase activity. These results
identify a serious theoretical obstacle to the design of in
vivo nPKC isoform selective inhibitors. Pharmaceuticals with even
high levels of in vitro PKC isoform specificity may not
retain in vivo selectively for a single isoform. This
considerably more elaborate paradigm for nPKC function in
cardiomyocytes, which incorporates phosphorylations as a component of
nPKC activation, suggests new challenges and opportunities for the
development of PKC targeted therapeutic agents.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Ema Stasko for preparing the myocyte cultures.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grant HL-64639 through the NHLBI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: University of Alabama at Birmingham.
** To whom correspondence should be addressed: Dept. of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168 St., New York, NY 10032. Tel.: 212-305-4297; Fax: 212-305-8780; E-mail: sfs1@columbia.edu.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M212644200
2 V. O. Rybin and S. F. Steinberg, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; cPKC, conventional protein kinase C; DN, dominant negative; HEK, human embryonic kidney; NE, norepinephrine; nPKC, novel protein kinase C; PDK, phosphoinositide-dependent kinase; PMA, phorbol 12-myristate 13- acetate; pS, phosphoserine; PS, phosphatidylserine; pT, phosphothreonine; RSK, ribosomal S6 kinase; WT, wild type. PP1, 4- amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo[3,4-d]pyramidine.
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