Published online before print June 7, 2001, doi: 10.1161/hh1201.092994
© 2001 American Heart Association, Inc.
Integrative Physiology |
Protein Kinase C 
–Src Modules Direct Signal Transduction in Nitric Oxide–Induced Cardioprotection
Complex Formation as a Means for Cardioprotective Signaling
From the Department of Physiology and Biophysics (T.M.V., J.Z., C.S., X.C., C.P.B., J.M.P., R.B., P.P.) and Division of Cardiology (T.M.V., J.Z., C.S., X.-L.T., X.C., C.P.B., J.M.P., S.W., R.B., P.P.), Department of Medicine, University of Louisville, Louisville, Ky.
Correspondence to Peipei Ping, PhD, Departments of Physiology and Biophysics, Medicine, Division of Cardiology, Suite 122 Baxter Bldg, 570 S Preston St, Louisville, KY 40202. E-mail peipeiping{at}hotmail.com
Abstract
Abstract—An
essential role for protein kinase C 
(PKC) has been shown in
multiple forms of cardioprotection; however, there is a distinct
paucity of information concerning the signaling architecture that is
responsible for the manifestation of a protective phenotype. We
and others have recently shown that signal transduction may proceed via
the formation of signaling complexes (Circ
Res. 2001;88:59–62). In order to understand if the assembly
of multiprotein complexes is the manner by which signaling is conducted
in cardioprotection, we designed a series of experiments to
characterize the associations of Src tyrosine kinase with PKC in a
conscious rabbit model of nitric oxide (NO)-induced late
preconditioning. Our data demonstrate that PKC and Src can form
functional signaling modules in vitro: PKC interacts with Src; the
association with PKC activates Src; and adult cardiac cells
receiving recombinant adenoviruses encoding PKC exhibit increased
Src activity. Furthermore, our results show that NO-induced late
preconditioning involved PKC-Src module formation and enhanced the
enzymatic activity of PKC-associated Src. Inhibition of PKC blocked
cardioprotection, module formation, and PKC-associated Src activity,
providing direct evidence for a functional role of the PKC-Src
module in the orchestration of NO-induced cardioprotection in conscious
rabbits.
Key Words: proteomics • ischemic injury • preconditioning • protein-protein interactions
The protein kinase
C (PKC) signaling system in cardioprotection has two conceptual
components: (1) the molecules that are involved in PKC signaling and
(2) the manner in which these molecules interact with PKC. The first
of these components has been extensively
studied,1 2 3 4 5 6 7 8 9 10 11 12
which has resulted in the identification of numerous signaling elements
that may participate in PKC-mediated preconditioning (PC). However,
the latter component, that is, the specific infrastructure established
by these signaling elements to engender cardioprotection, remains
undefined. Evidence from recent studies in numerous cellular theaters
suggests that signal transduction may proceed via the formation of
multiprotein signaling
complexes.13 14 15 16 17
Our laboratory has shown that PKC forms signaling complexes with at
least 36 proteins in the heart, and that PKC-mediated
cardioprotection is associated with dynamic modulation of these
complexes.17 Despite this,
the specific manner in which PKC might interact with a member of its
complex in order to perform functional signaling tasks is at
present unknown.
Previous studies pertaining to the components of the
signaling system in cardioprotection have implicated a role for
tyrosine
kinases.8 12 18 19 20 21 22
Our laboratory has reported that ischemic PC induces activation
of Src in a PKC-dependent manner in conscious
rabbits.8 Mounting evidence
has demonstrated that the administration of a nitric oxide (NO) donor
fully mimics the delayed cardioprotective effect afforded by
ischemic
PC.1 7 Furthermore,
this pharmacological PC also utilizes a PKC-dependent signaling
mechanism.1 7
However, the ability of, and the mechanism used by, NO to
activate Src and to modulate the involvement of Src in
NO-induced late PC in the myocardium remains to be
elucidated. It was recently found in murine fibroblasts that Src could
be induced to autophosphorylate and become active by
NO,23 which raises the
possibility that NO may activate Src via separate,
non-PKC-dependent mechanisms in the heart. In rat
cardiomyocytes, oxidative stress is effective in activating
extracellular signal–regulated kinases (ERKs) via an Src-dependent
pathway, a phenomenon which is not blocked by inhibitors of
PKC and/or PKA.23 Therefore,
it is unclear whether NO can activate Src in the
myocardium, and whether this occurs via activation of
PKC.
Analyses of PKC signaling complexes in the murine
myocardium revealed that Src, a member of the Src family of
nonreceptor tyrosine kinases, resides in the PKC
complex.17 Although the
individual roles of Src and PKC have been extensively
studied,2 24 25 26 27 28 29
it remains unknown if PKC interacts with Src, and if so, whether
their direct interaction promotes signal transduction, ie, whether
association with PKC activates Src. The fulfillment of these
criteria would indicate that PKC and Src constitute a signaling
module.
Accordingly, we designed a study to comprehensively
analyze the interactions of PKC and Src in NO-induced late
PC. We reasoned that if signaling in NO-induced PC proceeds by the
assembly of a module containing PKC and Src, then the association of
Src with PKC in this fashion might lead to increased enzymatic
activity of Src. To test this, we analyzed the enzymatic
activity and subcellular localization of Src and the association of Src
with the PKC signaling complex. The PKC inhibitor
chelerythrine (CHE) was given, at a dose shown previously to block late
NO-induced PC, to determine whether the formation of a PKC-Src
module is an essential part of the signaling architecture that protects
the heart against injury. We found that NO-induced cardioprotection
involves the formation of a module containing PKC and Src in the
particulate fraction. Association of Src with PKC in this module
dynamically enhances Src kinase activity during the late phase of
protection.
Materials and Methods
Recombinant Proteins and Reagents
In vitro studies used cDNAs of wild-type PKC
(rabbit), wild-type Src (mouse), and a mutant of Src, which were cloned
into the pAcGHLT vector, expressed in the baculovirus system, and
purified to generate glutathione
S-transferase (GST) fusion
proteins (Pharmingen). Cold or
[35S]-methionine–labeled recombinant
proteins of wild-type PKC, wild-type Src, and a mutant of Src
(Y529F) were also made through in vitro transcription and translation
using the TNT Quick-Coupled reticulocyte system
(Promega). The GST-PKC, GST-Src, and the in
vitro–translated PKC and Src were all verified to retain kinase
activity (data not shown). Inhibitor sources were as
follows: PKC inhibitors chelerythrine
(Sigma) and GF109203X
(Calbiochem); Src inhibitor PP2
(Calbiochem).
Assessing Protein-Protein Interactions Via GST
Affinity Pull-Down Assays
Briefly, GST-recombinant proteins were
immobilized on GST beads, mixed with either recombinant
proteins of interest or rabbit myocardial homogenates, and
incubated in binding buffer with various concentrations of NaCl, as
previously
reported.17 30
The GST-protein complexes were washed, resolved by SDS-PAGE, and
analyzed via immunoblotting with antibodies
against corresponding proteins. Parallel reactions were conducted using
equal molar amounts of GST-null proteins in place of the tested
GST-fusion proteins (negative control).
The Conscious Rabbit Model of DETA/NO-Induced
Late PC
The present study was performed in accordance with
the guidelines of the Animal Care and Use Committee of the University
of Louisville School of Medicine and with the Guide for the Care
and Use of Laboratory Animals (Department of Health and Human
Services, publication No. [NIH] 86-23).
A well-established conscious rabbit model of NO-induced PC
was used as previously
described.7 31 32
Briefly, male New Zealand White rabbits (Myrtle’s Rabbitry Inc,
Thompson Station, Tenn; 2.0 to 2.5 kg, age 3 to 4 months) were
intravenously administered the NO donor
diethylenetriamine/NO (DETA/NO, 0.1 mg/kg once every 25 minutes for 75
minutes; total dose of 0.4 mg/kg). This dose of DETA/NO has been shown
to be sufficient to trigger activation of PKC on day 1 and to
protect against myocardial infarction and stunning on day
2.7 31 32
To determine whether inhibition of PKC, which has been demonstrated to
block NO-induced PC, would affect PKC-Src signaling module
formation, the PKC inhibitor chelerythrine (CHE; dose 5
mg/kg) was given 5 minutes before the first DETA/NO
injection.7 10
This dose of CHE has been shown to abolish NO-induced activation of
PKC and to abrogate NO-induced late
cardioprotection.7 10
Rabbits in control groups received vehicle. To characterize PKC-Src
module formation during the temporal development of NO-induced PC,
cardiac tissues were taken at two time points: (1) at 30 minutes after
the last DETA/NO injection (day 1) and (2) at 24 hours after DETA/NO
(day 2). Hearts were frozen and stored at -80°C. Before biochemical
analysis, tissue samples were fractionated as previously
reported.6
Recombinant Adenoviral-Mediated PKC
Transfection in Adult Rabbit Cardiomyocytes
Recombinant adenoviruses encoding active PKC were
used to produce activation of PKC in cardiomyocytes.
Generation of recombinant viruses and isolation of adult
cardiomyocytes were performed as detailed
previously.5 9
Immunoprecipitation and Western
Blotting
PKC and Src antibody-based immunoprecipitations
were performed as previously
documented.7 8 17 33
IgG (Sigma) was substituted to determine
nonspecific binding. Immunoblotting was performed for
PKC and Src as previously
described.6 17 33
PKC and Src Activity Assays
The phosphorylation activity of
PKC and Src was determined as previously
described.7 8
Briefly, cytosolic or particulate samples were immunoprecipitated
overnight with either PKC (Pharmingen) or Src (Santa Cruz)
antibodies. The PKC phosphorylation activity was
determined by incubating immunoprecipitates with the PKC-selective
substrate (ERMRPRKRQGSVRRRV) in the PKC
phosphorylation
cocktail.7 Src
phosphorylation activity was assessed by incubating the
immunoprecipitates with either the Src-selective peptide
(KVEKIGEGTYGVVYK) (Upstate) or enolase (Sigma)
as substrates in the Src phosphorylation
cocktail.8
The PKC-associated Src phosphorylation
activity was determined by subjecting the PKC-immunoprecipitated
complex to the Src phosphorylation assay using the
peptide as a substrate.
Statistical Analysis
For ease of comparison, measurements of kinase
activity or protein level in each individual experiment were expressed
as a percentage of the average value for the respective control group.
Differences among the experimental groups were analyzed using
ANOVA. When necessary, post hoc contrasts and Student’s
t tests for unpaired data using
Bonferroni correction were
performed.34 Data are
reported as mean±SEM.
Results
PKC and Src Form a Functional Module
Recombinant PKC and GST-Src proteins were used to
determine whether PKC and Src were binding partners. GST-Src was
found to interact with PKC in vitro
(Figure 1
). The intensity of interaction between PKC and
Src varied with NaCl concentration, indicating that the binding between
these two molecules is ionic in nature
(Figure 1A, lanes 1 through 3). Next, we determined whether
Src colocalizes with PKC in the rabbit myocardium.
Western immunoblots for Src revealed that
endogenous Src associated with the GST-PKC complex
(Figure 1B). Interestingly, the association of Src with the
GST-PKC complexes was found to be subcellular location-specific, as
only Src from the particulate fraction, and not that from the
cytosolic, interacted with the GST-PKC complex
(Figure 1B, lanes 8 and 9 and 3 and 4, respectively). To our
knowledge, this is the first demonstration that PKC can bind
directly to Src.
|
To determine whether the conformation of Src affects its
binding affinity for PKC, we performed GST pull-down assays using
GST-PKC and in vitro–translated wild-type Src and the Y529F mutant
Src, which exists in an open conformation
(Figure 1C
). GST-PKC exhibited stronger binding with the
mutant Src than with wild-type Src, a phenomenon that appeared to be
concentration-dependent
(Figure 1C). These data show that when Src is in its open
configuration, it displays higher binding affinity for
PKC.
We then proceeded to assess whether the in vitro association
of PKC and Src results in the transduction of a signal, that is,
whether activation of PKC enhances Src activity. Active PKC was
incubated with recombinant Src and the Src-specific substrate protein
enolase. Activity of Src, as assessed by its
phosphorylation of enolase, was significantly higher in
the presence of PKC compared with that in the absence of PKC
(Figure 1D), indicating that PKC activates Src. To
verify this result, we also performed Src
phosphorylation activity assay using the Src-selective
substrate peptide.8 These
data confirmed the finding that PKC induced activation of Src in
vitro
(Figure 1D).
To further characterize the interaction of PKC with Src
in cardiomyocytes, we performed transfection experiments
with adenoviruses encoding active
PKC.5 9 Adult
rabbit cardiomyocytes receiving PKC adenoviruses
exhibited activation of
PKC5 9 and
displayed a marked increase in Src kinase activity (235.8±8.8% of
null vector; P<0.05)
(Figure 1E). Treatment with the PKC inhibitor
GF109203X blocked activation of
PKC5 as well as
PKC-induced activation of Src (80.4±7% of null vector). As
expected, Src activation was also blocked by the Src
inhibitor PP2 (91.9±9.0% of null vector). These data
indicate that, in adult cardiomyocytes, PKC activation
is sufficient to stimulate increased Src kinase
activity.
Increased Association of Src With the PKC
Complex During the Development of NO-Induced
Cardioprotection
We next examined whether the association of Src with
the PKC complex was concomitant with the development of
cardioprotection in vivo. It has been well documented that NO-induced
PC requires activation of
PKC.1 7 Although
tyrosine kinases have been implicated in
PC,8 12 20 35
the manner in which Src participates in PKC-dependent signaling
during NO-induced PC is unknown. To address this issue, we determined
whether NO-induced cardioprotective signaling involves the
colocalization of Src and PKC, and whether the interactions of
PKC and Src correspond with the temporal development of NO-induced
PC. Rabbits were administered DETA/NO at a dose that has been
previously demonstrated to generate a late phase of cardioprotection
against infarction and
stunning.1 7 31 32
We found that this dose of DETA/NO promoted recruitment of Src to the
PKC complex. The amount of Src protein associated with the PKC
complex was significantly increased at 30 minutes (187.0±1.1% of
control; P<0.05) and at 24
hours (286.0±3.5% of control;
P<0.05) after DETA/NO
(Figure 2A). These data suggest that PKC-mediated signal
transduction during the genesis (30 minutes) and the manifestation (24
hours) of NO-induced late PC involves an increased formation of
PKC-Src modules.
|
Assembly of PKC-Src Modules Increases Src
Enzymatic Activity In Vivo
In order to understand if, and the manner by which,
complex formation might facilitate signal transduction in vivo, we
determined the PKC-associated Src kinase activity in hearts from
conscious rabbits. Cardiac tissues were immunoprecipitated with PKC
antibodies and subjected to Src activity assay. In hearts obtained 30
minutes after DETA/NO administration, we observed a significant
increase in PKC-associated Src kinase activity (181.0±0.4% of
control; P<0.05)
(Figure 2B). Of particular importance, in hearts obtained 24
hours after DETA/NO administration, we found a marked increase in the
PKC-associated Src kinase activity (1082±319.1% of control;
P<0.05)
(Figure 2B), a magnitude of increase far exceeding that
observed at 30 minutes. Importantly, when analysis of Src
activity included both the non–PKC-associated Src and the
PKC-associated Src (30 minutes: 141.5±2.6% of control; 24 hours:
277.0±5.4% of control;
P<0.05)
(Figure 2C), the increase in Src activity induced by NO was
significantly less compared with the activity of PKC-associated Src
alone. The basal Src activity measurements in control animals (data not
shown) were comparable to those previously
reported.8 Taken with the
data shown in
Figure 2B, these findings indicate that the
PKC-associated Src accounted for the increased activity attributed
to the particulate Src and exhibited higher activity than that of the
non–PKC-associated Src.
These observations indicate that within the PKC complex,
a module containing PKC and Src is formed, and that assembly of this
module facilitates the transduction of a signal by increasing the
enzymatic activity of Src.
NO-Induced Late PC Involves Increased
Particulate Expression of PKC But Not Src
The subcellular alterations that could lead to
increased module formation are undoubtedly extensive. However, four
fundamental scenarios, designed to incorporate a range of conceivable
biochemical changes that could result in increased module formation,
are discussed in
Figure 3. In the present study, we measured the level of
expression and activity of PKC and Src during NO-induced PC to
determine which, if any, of these mechanisms might contribute to
increased module formation between PKC and Src.
|
In agreement with our previous
report,7 we found that
DETA/NO induced PKC translocation at 30 minutes after NO-induced PC
(Figure 4A). Moreover, we also observed significant
translocation of PKC at 24 hours after NO-induced PC
(Figure 4A). The increased particulate localization of PKC
at 24 hours appeared to be partially supported by an increase of total
PKC protein (196.3±20.0% of control;
P<0.05), whereas total PKC
expression was not altered at 30 minutes after DETA/NO (data not
shown). Furthermore, inhibition of PKC with CHE before DETA/NO
treatment abolished NO-induced translocation of PKC both at 30
minutes and at 24 hours (data not shown). CHE also abrogated the
increased expression of PKC protein that was seen 24 hours later
(127.1±5.0% of control).
|
As shown in
Figure 2B, DETA/NO induced a significant increase in the
PKC-associated Src activity. We next examined the effect of NO on
Src protein level at 30 minutes and 24 hours after DETA/NO. Our data
show that Src expression remained unaltered at either 30 minutes or 24
hours after DETA/NO administration
(Figure 4B). Furthermore, DETA/NO did not cause a subcellular
redistribution of Src kinase
(Figure 4B), in that the expression of Src was unchanged when
either the total or the particulate fraction alone was considered at
both 30 minutes (total Src: 8 3.6±6.0% of control; particulate
Src: 105.0±0.9% of control) and at 24 hours (total Src: 121.4±21.7%
of control; particulate Src: 102.4±0.4% of control). Treatment with
CHE before DETA/NO treatment did not alter the expression of Src in
either the total or particulate pool both at 30 minutes and 24 hours
after DETA/NO administration (data not shown).
CHE Inhibits PKC-Src Module Formation and
Blocks Cardioprotection
Previous studies have demonstrated that CHE, given at a
dose that inhibits PKC activation, is sufficient to block the
salubrious effects afforded by ischemic and NO-induced
PC.7 10 In the
present investigation, our data demonstrate that pretreatment with
this same dose of CHE abolished DETA/NO-induced formation of PKC-Src
modules both at 30 minutes (123.4±3.9% of control) and at 24 hours
(75.4±0.6% of control)
(Figure 5). In accordance with the disruption of module
formation by CHE was the observation that CHE also attenuated the
NO-induced increase in PKC-associated Src activity at 30 minutes
(132.3±18.9% of control) and at 24 hours (95.8±10.3% of control)
(Figure 2B). These data demonstrate that the activation of
PKC by NO is a prerequisite for PKC-Src module formation and
function 24 hours later. Furthermore, the assembly of PKC-Src
modules was disrupted by the same mechanism that blocked
cardioprotection, ie, inhibition of
PKC.7 10
Together, these findings suggest that the formation of PKC-Src
modules in response to NO is a mechanism by which the heart carries out
cardioprotective signaling.
|
Discussion
To our knowledge, this is the first study to
demonstrate that the assembly of a signaling module is a means for
cardiac cell signaling, and that the formation of signaling modules
containing PKC and Src is an essential step in NO-induced
cardioprotection. There are a number of salient observations in this
study. In the in vitro setting, our data demonstrate that PKC
interacts with Src, and that this interaction promotes the activation
of Src. In cardiac cells and in the normal myocardium, our
results show that formation of PKC-Src modules serves to accomplish
signal transduction. In addition, the formation of PKC-Src modules
is subcellular compartment-dependent, in that it only occurs in the
particulate fraction. Finally, and of particular importance, we found
that administration of an NO-donor promotes PKC-Src module formation
during the development of cardioprotection in conscious rabbits. In
NO-induced PC, the configuration of this module facilitates signal
transduction by increasing PKC-associated Src kinase activity.
Inhibition of PKC with CHE, at a dose that has previously been
demonstrated to block NO-induced
cardioprotection,7 10
was sufficient to abolish PKC-Src module formation and
PKC-associated Src activity.
Module Formation as a Means for Signal
Transduction
Recent advances in molecular technologies have allowed
for the identification of molecules that participate in biochemical
responses. However, the precise manner in which these molecules
interact to transmit subcellular signals is unclear. Studies regarding
signaling paradigms in noncardiac tissues have identified the assembly
of signaling complexes as a means for signal
transduction.13 14 15 16
Moreover, our laboratory has conducted an extensive characterization of
the PKC signaling complex in the murine myocardium and
found that Src (along with 
35 other proteins) is a member of this
complex.17 In concert with
these findings, we have proposed the "signaling module hypothesis"
of PKC,36 in which we
suggest that the formation of stimulus- and subcellular
location–specific signaling modules may be a mechanism whereby a
multiprotein complex coordinates signal
transduction.36
In the present study, we undertook the characterization
of a module containing a serine/threonine kinase, PKC, and a
tyrosine kinase, Src. Our data show that the formation of this module
enhanced PKC-associated Src activity, indicating that the
interaction of these two kinases promotes signal transduction.
Molecular analyses of Src reveal that both the SH2 and the SH3
domains contain multiple PKC phosphorylation motifs
(a.a. S/T-X-K/R). Several investigations have shown that the SH2 and
SH3 domains are the preferred regions for interaction of Src tyrosine
kinase with other proteins, including the epidermal growth factor
receptors,37 the
ß3-adrenergic
receptors,14 and the
receptors for activated C
kinases.27 To our knowledge,
the present study represents the first demonstration that
PKC can directly interact with Src and that this interaction is
sufficient to lead to the activation of Src.
NO-Induced Cardioprotection Requires PKC-Src
Module Formation
To determine whether the assembly of multiprotein
complexes serves as a means for cardioprotective signal transduction,
we characterized the formation of PKC-Src modules during the genesis
of NO-induced PC. We found that NO-induced late PC involves an increase
in module formation between PKC and Src. This increase in
association was accompanied by a dramatic (10-fold) enhancement of
PKC-associated Src kinase activity only in the particulate fraction
of the cell, indicating that the association of Src with the PKC
signaling complex is a subcellular location-specific occurrence. It was
also found that the PKC inhibitor CHE, given at a dose
shown previously to abrogate the infarct- and stunning-sparing effects
of NO-induced PC, was sufficient to block the increased PKC-Src
module formation afforded by NO. In addition, this dose of CHE also
attenuated the increase in PKC-associated Src activity that was seen
30 minutes and 24 hours after NO treatment. To our knowledge, no
previous study has identified the formation of signaling modules as a
mechanism of accomplishing signal transduction in vivo. Furthermore,
the findings suggest that the formation of signaling complexes is not a
random association of proteins or an artifact of immunoprecipitation
procedures. Rather, the fact that association with PKC enhances Src
enzymatic activity suggests that the formation of these modules is a
highly organized event that facilitates the transmission of a
subcellular signal. In view of the fact that both PKC and Src have
been shown to play necessary roles in NO-induced
PC,7 34 the data
herein indicate that PKC-Src modules direct signal transduction in
NO-induced cardioprotection.
While it has been shown that PKC associates with a large
number of other proteins within a signaling complex, it is rational to
hypothesize that not all of the proteins within the PKC complex
participate in all of the physiological processes
that involve PKC. Accordingly, we propose that signaling complexes,
like the PKC complex characterized by our
laboratory,17 which contain
within them stimulus-responsive and subcellular location–specific
modules, like the PKC-Src module that is described herein, may
represent a means by which the cell uses multifunctional
signaling elements to perform distinct subcellular
tasks.36 For example, the
PKC-Src signaling module is cardioprotective, whereas a different
subset of proteins within the PKC complex containing connexin43,
that would constitute a distinct module, may interact to coordinate
excitation-contraction
coupling.38
Molecular Mechanisms Underlying Module
Formation
To elucidate the molecular mechanisms responsible
for increased module formation, we characterized the expression levels
of PKC and Src, as well as the nature of the affinity of these two
molecules for each other. In these studies, we found that NO induced an
increase in the particulate PKC protein at 30 minutes and at 24
hours, with no change in particulate Src protein at either of these
time points
(Figure 2). These data indicate that one of the mechanisms
leading to increased complex formation between PKC and Src is an
increase in the ratio of PKC protein to Src protein relative to
basal conditions
(Figure 3). Furthermore, we found that PKC and Src
interactions only existed in the particulate fraction where Src has
been reported to be in its open
configuration.26 This
evidence, combined with the in vitro finding that the open conformation
of Src favors its interaction with PKC
(Figure 1C), suggests that an altered affinity of Src for
PKC may also contribute to the formation of PKC-Src modules
(Figure 3).
Conclusion
A challenge for the development of pharmacological
strategies that target specific signaling events is the
physiological ubiquity of many of the proteins
involved in protective phenomena (ie, preconditioning). In addition to
participating in cardioprotection, both PKC and Src tyrosine kinase
are also involved in numerous other signaling events across a variety
of cell types. An essential step toward addressing this challenge is
the characterization of the distinct manner in which proteins interact
to define a module. This information would provide a level of
specificity that could not be achieved solely from the biochemical
properties of the individual molecules. Thus, rather than to
activate a single molecule, which would impact multiple
signaling events, future pharmacological and/or genetic interventions
may be tailored to specifically recapitulate the endogenous
interactions that constitute a protective
module.
Acknowledgments
This study was supported by AHA EIG-40167N (P.P.), NIH HL-63901 (P.P.), NIH HL-65431 (P.P.), NIH HL-43151 (R.B.), NIH HL-55757 (R.B.), the University of Louisville Research Foundation, the Commonwealth of Kentucky Research Challenge Trust Fund, and Jewish Hospital Research Foundation.
Footnotes
Original received March 21, 2001; revision received May 9, 2001; accepted May 14, 2001.
1 These authors contributed equally to this work. 
This manuscript was sent to Eugene Braunwald, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
References
1. Bolli R. The late phase of preconditioning. Circ Res. 2000;87:972–983.
2.
Dorn GW II,
Souroujon MC, Liron T, Chen CH, Gray MO, Zhou HZ, Csukai M, Wu G,
Lorenz JN, Mochly-Rosen D. Sustained in vivo cardiac protection by a
rationally designed peptide that causes protein kinase C
translocation. Proc Natl Acad Sci
U S A. 1999;96:12798–12803.
3.
Liu GS, Cohen MV,
Mochly-Rosen D, Downey JM. Protein kinase C is responsible for the
protection of preconditioning in rabbit cardiomyocytes.
J Mol Cell Cardiol. 1999;31:1937–1948.
4. Tong H, Chen W, Steenbergen C, Murphy E. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res. 2000;87:309–315.
5.
Li RC, Ping P,
Zhang J, Wead WB, Cao X, Gao J, Zheng Y, Huang S, Han J, Bolli R.
PKC modulates NF-
B and AP-1 via mitogen-activated protein
kinases in adult rabbit cardiomyocytes.
Am J Physiol. 2000;279:H1679–H1689.
6.
Ping P, Zhang J,
Qiu Y, Tang XL, Manchikalapudi S, Cao X, Bolli R. Ischemic
preconditioning induces selective translocation of protein kinase C
isoforms and 
in the heart of conscious rabbits without a
subcellular redistribution of total protein kinase C activity.
Circ Res. 1997;81:404–414.
7. Ping P, Takano H, Zhang J, Tang XL, Qiu Y, Li RC, Banerjee S, Dawn B, Balafanova Z, Bolli R. Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning. Circ Res. 1999;84:587–604.
8. Ping P, Zhang J, Zheng YT, Li RC, Dawn B, Tang XL, Takano H, Balafanova Z, Bolli R. Demonstration of selective protein kinase C-dependent activation of Src and Lck tyrosine kinases during ischemic preconditioning in conscious rabbits. Circ Res. 1999;85:542–550.
9. Ping P, Zhang J, Cao X, Li RC, Kong D, Tang XL, Qiu Y, Manchikalapudi S, Auchampach JA, Black RG, Bolli R. PKC-dependent activation of p44/p42 MAPKs during myocardial ischemia-reperfusion in conscious rabbits. Am J Physiol. 1999;276:H1468–H1481.
10.
Qiu Y, Ping P,
Tang XL, Manchikalapudi S, Rizvi A, Zhang J, Takano H, Wu WJ, Teschner
S, Bolli R. Direct evidence that protein kinase C plays an essential
role in the development of late preconditioning against myocardial
stunning in conscious rabbits and that is the isoform involved.
J Clin Invest. 1998;101:2182–2198.
11. Xuan YT, Tang XL, Qiu Y, Banerjee S, Takano H, Han H, Bolli R. Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits. Am J Physiol. 2000;279:H2360–H2371.
12. Nakano A, Cohen MV, Downey JM. Ischemic preconditioning: from basic mechanisms to clinical applications. Pharmacol Ther. 2000;86:263–275.
13. Husi H, Ward MA, Choudhary JS, Blackstock WP, Grant SG. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat Neurosci. 2000;3:661–669.
14. Cao W, Luttrell LM, Medvedev AV, Pierce KL, Daniel KW, Dixon TM, Lefkowitz RJ, Collins S. Direct binding of activated c-Src to the ß3-adrenergic receptor is required for MAP kinase activation. J Biol Chem. 2000;275:38131–38134.
15. Fraser ID, Cong M, Kim J, Rollins EN, Daaka Y, Lefkowitz RJ, Scott JD. Assembly of an A-kinase-anchoring protein-ß2-adrenergic receptor complex facilitates receptor phosphorylation and signaling. Curr Biol. 2000;10:409–412.
16. Yasuda J, Whitmarsh AJ, Cavanagh J, Sharma M, Davis RJ. The JIP group of mitogen-activated protein kinase scaffold proteins. Mol Cell Biol. 1999;19:7245–7254.
17.
Ping P, Zhang J,
Pierce WM Jr, Bolli R. Functional proteomic analysis of protein
kinase C signaling complexes in the normal heart and during
cardioprotection. Circ Res. 2001;88:59–62.
18. Vahlhaus C, Schulz R, Post H, Rose J, Heusch G. Prevention of ischemic preconditioning only by combined inhibition of protein kinase C and protein tyrosine kinases in pigs. J Mol Cell Cardiol. 1998;30:197–209.
19. Baines CP, Wang L, Cohen MV, Downey JM. Protein tyrosine kinase is downstream of protein kinase C for ischemic preconditioning’s anti-infarct effect in the rabbit heart. J Mol Cell Cardiol. 1998;30:383–392.
20. Maulik N, Yoshida T, Zu YL, Sato M, Banerjee A, Das DK. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am J Physiol. 1998;275:H1857–H1864.
21. Fryer RM, Schultz JE, Hsu AK, Gross GJ. Importance of PKC and tyrosine kinase in single or multiple cycles of preconditioning in rat hearts. Am J Physiol. 1999;276:H1229–H1235.
22. Dana A, Skarli M, Papakrivopoulou J, Yellon DM. Adenosine A1 receptor induced delayed preconditioning in rabbits: induction of p38 MAPK activation and Hsp 27 phosphorylation via a tyrosine kinase- and protein kinase C-dependent mechanism. Circ Res. 2000;86:989–997.
23. Akhand AA, Pu M, Senga T, Kato M, Suzuki H, Miyata T, Hamaguchi M, Nakashima I. Nitric oxide controls Src kinase activity through a sulfhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. J Biol Chem. 1999;274:25821–25826.
24. Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Tanaka M, Shiojima I, Hiroi Y, Yazaki Y. Oxidative stress activates extracellular signal-regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest. 1997;100:1813–1821.
25. Haendeler J, Berk BC. Angiotensin II mediated signal transduction: important role of tyrosine kinases. Regul Pept. 2000;95:1–7.
26. Sicheri F, Kuriyan J. Structures of Src-family tyrosine kinases. Curr Opin Struct Biol. 1997;7:777–785.
27. Chang BY, Conroy KB, Machleder EM, Cartwright CA. RACK1, a receptor for activated C kinase and a homolog of the ß subunit of G proteins, inhibits activity of Src tyrosine kinases and growth of NIH 3T3 cells. Mol Cell Biol. 1998;18:3245–3256.
28. Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, Messing RO. Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol. 2000;279:L429—L438.
29.
Takeishi Y, Ping
P, Bolli R, Kirkpatrick DL, Hoit BD, Walsh RA. Transgenic
overexpression of constitutively active protein kinase C causes
concentric cardiac hypertrophy.
Circ Res. 2000;86:1218–1223.
30. Phizicky EM, Fields S. Protein-protein interactions: methods for detection and analysis. Microbiol Rev. 1995;59:94–123.
31. Shinmura K, Tang XL, Takano H, Hill M, Bolli R. Nitric oxide donors attenuate myocardial stunning in conscious rabbits. Am J Physiol. 1999;277:H2495–H2503.
32. Takano H, Tang XL, Qiu Y, Guo Y, French BA, Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res. 1998;83:73–84.
33.
Pass JM, Zheng
YT, Wead WB, Zhang J, Li RC, Bolli R, Ping P. PKC activation induces
dichotomous cardiac phenotypes and modulates PKC-RACK
interactions and RACK expression. Am
J Physiol. 2001;280:H946–H955.
34. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res. 1980;47:1–9.
35. Tang XL, Kodani E, Takano H, Hill M, Ping P, Bolli R. Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning in conscious rabbits. J Mol Cell Cardiol. Abstract. In press.
36.
Vondriska TM,
Klein JB, Ping P. Use of functional proteomics to investigate
PKC-mediated cardioprotection: the signaling module hypothesis.
Am J Physiol. 2001;280:H1434–H1441.
37. Luttrell DK, Lee A, Lansing TJ, Crosby RM, Jung KD, Willard D, Luther M, Rodriquez M, Berman J, Gilmer TM. Involvement of pp60c-src with two major signaling pathways in human breast cancer. Proc Natl Acad Sci U S A. 1994;91:83–87.
38.
Doble BW, Ping P,
Kardami E. The subtype of protein kinase C is required for
cardiomyocyte connexin-43 phosphorylation.
Circ Res. 2000;86:293–301.
This article has been cited by other articles:
 |
|
 |
|
  I. Moench, H. Prentice, Z. Rickaway, and H. Weissbach Sulindac confers high level ischemic protection to the heart through late preconditioning mechanisms PNAS, November 17, 2009; 106(46): 19611 - 19616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ferdinandy, R. Schulz, and G. F. Baxter Interaction of Cardiovascular Risk Factors with Myocardial Ischemia/Reperfusion Injury, Preconditioning, and Postconditioning Pharmacol. Rev., December 1, 2007; 59(4): 418 - 458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-X. Zhang, X.-M. Lu, S. Kimura, and A. Nishiyama Role of mitochondria in angiotensin II-induced reactive oxygen species and mitogen-activated protein kinase activation Cardiovasc Res, November 1, 2007; 76(2): 204 - 212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Pasdois, C. L. Quinlan, A. Rissa, L. Tariosse, B. Vinassa, A. D. T. Costa, S. V. Pierre, P. Dos Santos, and K. D. Garlid Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mitoKATP, and ROS Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1470 - H1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Slupsky, A. S. Kamiguti, R. J. Harris, J. C. Cawley, and M. Zuzel Central Role of Protein Kinase C{epsilon} in Constitutive Activation of ERK1/2 and Rac1 in the Malignant Cells of Hairy Cell Leukemia Am. J. Pathol., February 1, 2007; 170(2): 745 - 754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Inagaki, E. Churchill, and D. Mochly-Rosen Epsilon protein kinase C as a potential therapeutic target for the ischemic heart Cardiovasc Res, May 1, 2006; 70(2): 222 - 230. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. McGregor and M. J. Dunn Proteomics of the Heart: Unraveling Disease Circ. Res., February 17, 2006; 98(3): 309 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Samarel Costameres, focal adhesions, and cardiomyocyte mechanotransduction Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2291 - H2301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Robia, M. Kang, and J. W. Walker Novel determinant of PKC-{epsilon} anchoring at cardiac Z-lines Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1941 - H1950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Okada, H. Otani, Y. Wu, T. Uchiyama, S. Kyoi, R. Hattori, T. Sumida, H. Fujiwara, and H. Imamura Integrated pharmacological preconditioning and memory of cardioprotection: role of protein kinase C and phosphatidylinositol 3-kinase Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H761 - H767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, D. E. Handy, and J. Loscalzo Adenosine-Dependent Induction of Glutathione Peroxidase 1 in Human Primary Endothelial Cells and Protection Against Oxidative Stress Circ. Res., April 29, 2005; 96(8): 831 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Wang, D. A. Liem, T. M. Vondriska, H. M. Honda, P. Korge, D. M. Pantaleon, X. Qiao, Y. Wang, J. N. Weiss, and P. Ping Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1290 - H1295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, C. P. Baines, C. Zong, E. M. Cardwell, G. Wang, T. M. Vondriska, and P. Ping Functional proteomic analysis of a three-tier PKC{varepsilon}-Akt-eNOS signaling module in cardiac protection Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H954 - H961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, P. Ping, G.-W. Wang, M. Lu, D. Pantaleon, X.-L. Tang, R. Bolli, and T. M. Vondriska Bmx, a member of the Tec family of nonreceptor tyrosine kinases, is a novel participant in pharmacological cardioprotection Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2364 - H2366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ping Identification of Novel Signaling Complexes by Functional Proteomics Circ. Res., October 3, 2003; 93(7): 595 - 603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Heidkamp, A. L. Bayer, B. T. Scully, D. M. Eble, and A. M. Samarel Activation of focal adhesion kinase by protein kinase C{epsilon} in neonatal rat ventricular myocytes Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1684 - H1696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, P. Ping, T. M. Vondriska, X.-L. Tang, G.-W. Wang, E. M. Cardwell, and R. Bolli Cardioprotection involves activation of NF-{kappa}B via PKC-dependent tyrosine and serine phosphorylation of I{kappa}B-{alpha} Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1753 - H1758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Weiss, P. Korge, H. M. Honda, and P. Ping Role of the Mitochondrial Permeability Transition in Myocardial Disease Circ. Res., August 22, 2003; 93(4): 292 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xu, B. Jian, R. Chu, Z. Lu, Q. Li, J. Dunlop, S. Rosenzweig-Lipson, P. McGonigle, R. J. Levy, and B. Liang Serotonin Mechanisms in Heart Valve Disease II: The 5-HT2 Receptor and Its Signaling Pathway in Aortic Valve Interstitial Cells Am. J. Pathol., December 1, 2002; 161(6): 2209 - 2218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Schwanke, I. Konietzka, A. Duschin, X. Li, R. Schulz, and G. Heusch No ischemic preconditioning in heterozygous connexin43-deficient mice Am J Physiol Heart Circ Physiol, October 1, 2002; 283 (4): H1740 - H1742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Garcia-Dorado, M. Ruiz-Meana, F. Padilla, A. Rodriguez-Sinovas, and M. Mirabet Gap junction-mediated intercellular communication in ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 456 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.F Baxter Role of adenosine in delayed preconditioning of myocardium Cardiovasc Res, August 15, 2002; 55(3): 483 - 494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Oldenburg, Q. Qin, A. R Sharma, M. V Cohen, J. M Downey, and J. N Benoit Acetylcholine leads to free radical production dependent on KATP channels, Gi proteins, phosphatidylinositol 3-kinase and tyrosine kinase Cardiovasc Res, August 15, 2002; 55(3): 544 - 552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Dawn, H. Takano, X.-L. Tang, E. Kodani, S. Banerjee, A. Rezazadeh, Y. Qiu, and R. Bolli Role of Src protein tyrosine kinases in late preconditioning against myocardial infarction Am J Physiol Heart Circ Physiol, August 1, 2002; 283(2): H549 - H556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Robin, I. Boulven, C. Desmyter, S. Harbon, and D. Leiber ET-1 stimulates ERK signaling pathway through sequential activation of PKC and Src in rat myometrial cells Am J Physiol Cell Physiol, July 1, 2002; 283(1): C251 - C260. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. D. Edmondson, T. M. Vondriska, K. J. Biederman, J. Zhang, R. C. Jones, Y. Zheng, D. L. Allen, J. X. Xiu, E. M. Cardwell, M. R. Pisano, et al. Protein Kinase C {epsilon} Signaling Complexes Include Metabolism- and Transcription/Translation-related Proteins: Complimentary Separation Techniques With LC/MS/MS Mol. Cell. Proteomics, June 1, 2002; 1(6): 421 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Balafanova, R. Bolli, J. Zhang, Y. Zheng, J. M. Pass, A. Bhatnagar, X.-L. Tang, O. Wang, E. Cardwell, and P. Ping Nitric Oxide (NO) Induces Nitration of Protein Kinase Cepsilon (PKCepsilon ), Facilitating PKCepsilon Translocation via Enhanced PKCepsilon -RACK2 Interactions. A NOVEL MECHANISM OF NO-TRIGGERED ACTIVATION OF PKCepsilon J. Biol. Chem., April 19, 2002; 277(17): 15021 - 15027. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Baines, J. Zhang, G.-W. Wang, Y.-T. Zheng, J. X. Xiu, E. M. Cardwell, R. Bolli, and P. Ping Mitochondrial PKC{epsilon} and MAPK Form Signaling Modules in the Murine Heart: Enhanced Mitochondrial PKC{epsilon}-MAPK Interactions and Differential MAPK Activation in PKC{epsilon}-Induced Cardioprotection Circ. Res., March 8, 2002; 90(4): 390 - 397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Song, T. M. Vondriska, G.-W. Wang, J. B. Klein, X. Cao, J. Zhang, Y. J. Kang, S. D'Souza, and P. Ping Molecular conformation dictates signaling module formation: example of PKCepsilon and Src tyrosine kinase Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H1166 - H1171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Ninomiya, H. Otani, K. Lu, T. Uchiyama, M. Kido, and H. Imamura Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1933 - H1943. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.L. Mehta, H.J. Chen, and D.Y. Li Protection of Myocytes From Hypoxia-Reoxygenation Injury by Nitric Oxide Is Mediated by Modulation of Transforming Growth Factor-{beta}1 Circulation, May 7, 2002; 105(18): 2206 - 2211. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||