© 1999 American Heart Association, Inc.
Original Contributions |
Activated Protein Kinase C Isoforms Target to Cardiomyocyte Caveolae
Stimulation of Local Protein Phosphorylation
From the Departments of Pharmacology (V.O.R., X.X., S.F.S.) and Medicine (S.F.S.), College of Physicians and Surgeons, Columbia University, New York, NY.
Correspondence to Susan F. Steinberg, MD, Associate Professor of Pharmacology and Medicine, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 West 168 St, New York, NY 10032. E-mail SFS1{at}columbia.edu
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Abstract |
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Abstract—Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester–sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4
-PMA, which does not
activate PKC) recruited calcium-sensitive PKC and novel
PKC
and PKC
to this compartment. The subcellular localization of
the phorbol ester–insensitive PKC
isoform was not influenced by
PMA. Endothelin also induced the selective translocation of PKC and
PKC (but not PKC or PKC) to caveolae. Multiple components of
the extracellular signal–regulated protein kinase (ERK) cascade,
including A-Raf, c-Raf-1, mitogen-activated protein kinase
kinase, and ERK, were detected in caveolae under resting
conditions. Although levels of these proteins were not altered by PMA,
translocation of phorbol ester–sensitive PKC isoforms to caveolae was
associated with the activation of a local ERK cascade as well as the
phosphorylation of a 
36-kDa substrate protein in
this fraction. Finally, a minor fraction of a protein that has been
designated as a receptor for activated protein kinase C resides
in caveolae and (along with caveolin-3) could represent a
mechanism to target PKC isoforms to cardiomyocyte caveolae.
These studies identify cardiomyocyte caveolae as a meeting
place for activated PKC isoforms and their downstream
target substrates.
Key Words: protein kinase C • caveolae • cardiomyocyte
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Caveolae were first identified as flask-shaped invaginations on the surface of epithelial and endothelial cells >40 years ago. Investigations of the molecular structure and function of caveolae have only recently advanced with the advent of methods to obtain large quantities of this specialized plasma membrane microdomain and the identification of caveolin, the distinct coat protein that decorates the internal membrane surface of caveolae. Caveolin comprises a multigene family of at least 3 immunologically distinct 21- to 24-kDa isoforms that serve as marker proteins for this organelle. Caveolin-1 is most abundantly expressed in adipocytes, endothelial cells, and fibroblasts; caveolin-2 shows a similar pattern of protein expression; caveolin-3 is the muscle-specific isoform.1 2 3 Caveolae are most conspicuous in highly differentiated cell types in which their role in the transport of macromolecules such as albumin and low-density lipoproteins from the blood to the tissue space (transcytosis), in the delivery of small molecules such as folate from the extracellular space to the cytoplasm (potocytosis), and in signal transduction by receptors for growth factors and hormones has been the focus of intense recent investigation in several laboratories.
There is recent growing evidence that caveolae may act as structurally
and biochemically distinct plasma membrane compartments that localize
and regulate the function of a subset of transmembrane signaling events
(reviewed in Reference 44 ). This hypothesis is based on the
identification of multiple components of signaling cascades in caveolae
at steady state or after ligand-induced activation. Further
studies identify caveolin as a plasma membrane docking protein that
sequesters certain cytoplasmically oriented signaling molecules in
their inactive form, thereby serving to promote the efficient and rapid
coupling of agonist-occupied receptors to effector
mechanisms.5 6 7 Caveolin has been shown to directly
interact with G protein subunits,5 Ras,6
Src,7 endothelial nitric oxide
synthase,8 and the epidermal growth factor
receptor.9 In each case, the interaction has been mapped
to the same cytosolic membrane-proximal region of caveolin (termed the
"caveolin-scaffolding domain").7 Using the scaffolding
domain of caveolin as a receptor, 2 related aromatic amino acid–rich
protein sequences that function as caveolin binding motifs have been
identified; these motifs exist in most G protein subunits, in
numerous G protein–coupled receptors, in several peptide growth factor
receptors, and in the kinase domains of many tyrosine and
serine/threonine protein kinases,4 10 including protein
kinase C (PKC) isoforms.10 Indeed, several studies
identify conventional, calcium-sensitive PKC isoforms as constituent
components of caveolar membranes at steady state,11 12 13 14 15
whereas 1 recent study identified regulated (phorbol
12-myristate 13-acetate [PMA]-induced) affiliation of PKC
with the caveolin-enriched fraction of MDCK cells16
(although phorbol ester–induced loss of PKC from caveolae also has
been reported12 ). Although most cells coexpress multiple
PKC isoforms, curiously, analyses of endogenously
expressed PKC isoforms thus far have been restricted largely to the
conventional, calcium-sensitive PKC and PKCß isoforms.
Nevertheless, there is evidence that PKC activators cause
caveolae to flatten out and prevent the caveolaer uptake of small
molecules such as folate via potocytosis,12 17 suggesting
that localization of PKC isoform(s) to caveolae is associated with
functionally important consequences in certain cell types. Accordingly,
the goal of the present study was to determine whether caveolae
form a signaling module for PKC isoforms in
cardiomyocytes.
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Materials and Methods |
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Materials
Antibodies and their sources were as follows: mouse monoclonal anti-caveolin-1 and anti-caveolin-3, mouse monoclonal anti-c-Raf-1, rabbit polyclonal affinity-purified anti–mitogen-activated protein kinase kinase (MEK), mouse monoclonal anti-annexin II, and mouse monoclonal anti–receptor for activated protein kinase C (RACK) (Transduction Laboratories); rabbit polyclonal affinity-purified anti-A-Raf and rabbit polyclonal affinity-purified anti–extracellular signal–regulated protein kinase (ERK) (Santa Cruz Biotechnology); mouse monoclonal anti–plasma membrane calcium ATPase (PMCA; Affinity Bioreagents, Inc); and mouse monoclonal anti-Golgi 58-kDa protein IgG1 (Sigma). The mouse monoclonal anti-PKC
antibodies were from Upstate Biotechnology, the mouse monoclonal
anti-PKC was from Transduction Laboratories, the rabbit polyclonal
anti-PKC antibodies were from GIBCO-BRL, and the polyclonal
anti-PKC was a generous gift from Dr Doriano Fabbro (CIBA-Geigy).
125I-labeled goat anti-rabbit IgG
F(ab')2 fragment and
125I-labeled sheep anti-mouse IgG
F(ab')2 fragment were purchased from DuPont NEN.
The enhanced chemiluminescence (ECL) kit was from Amersham Pharmacia
Biotech. All other chemicals were reagent grade.
Cell Culture
Cardiac myocytes were isolated from 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 cardiac
myocytes as described previously.18 The yield of myocytes
typically is 2.5 to 3x106 cells per neonatal
heart. Cells were plated at a density of 0.5x106
cells per milliliter in fibronectin-coated 100-mm culture dishes.
Although the preplating step effectively decreases fibroblast
contamination of the cultures, a small number of cells with
proliferative capability such as cardiac fibroblasts persists in
myocardial cell cultures. Proliferation of these cells was further
curtailed with an irradiation protocol.18 Experiments were
performed after 5 to 6 days of culture in MEM (GIBCO-BRL) with
10% FCS, 5x10–6 mol/L hypoxanthine, and
12 mmol/L NaHCO3.
Purification of Caveolin-Rich Membrane Fractions
Two procedures were used to prepare caveolin-enriched fractions.
The first method followed the detergent-free purification scheme
described by Song et al.6 All steps were carried out at
4°C. Briefly, cells (from 5 dishes, 100 mm-diameter each) were
washed twice with ice-cold PBS and then scraped into 0.5 mol/L sodium
carbonate, pH 11.0 (0.5 mL per dish). Cells from the 5 dishes were
combined (total volume, 2.5 mL) for each preparation. To disrupt
cellular membranes, homogenization was carried out
sequentially with a loose-fitting Dounce homogenizer
(10 strokes), a Polytron tissue grinder (three 10-second bursts), and a
sonicator (three 20-second bursts). The homogenate was then
adjusted to 40% sucrose by adding an equal volume of 80% sucrose
prepared in Mes-buffered saline (MBS; 25 mmol/L Mes, pH 6.5, and
0.15 mol/L NaCl) and placed on the bottom of an ultracentrifuge
tube. A 5% to 35% discontinuous sucrose gradient was formed
above the sample (3 mL of 5% sucrose and 4 mL of 35% sucrose,
both in MBS containing 250 mmol/L sodium carbonate) and
centrifuged at 38 000 rpm for 16 to 18 hours in a rotor (model
SW40, Beckman Instruments). For profiles, 1-mL fractions
starting at the top of each gradient were collected to yield a total of
12 fractions. Alternatively, the light scattering band confined to the
5% to 35% interface (fractions 3 to 4, which contains caveolin but
excludes most other cellular proteins) was diluted 3-fold with MBS and
centrifuged at 40 000g for 1 hour to pellet the
caveolae, which were then solubilized with SDS-PAGE sample buffer.
In some experiments, caveolae also were prepared according to the method that relies on their resistance to solubilization by the nonionic detergent Triton X-100 at low temperatures (essentially as described by Sargiacomo et al19 ). In this case, cells were scraped into MBS containing (in mmol/L) sodium vanadate 0.1, NaF 25, and phenylmethylsulfonyl fluoride 1; 10 µg/mL leupeptin; 10 µg/mL aprotinin; and 1% Triton X-100. The extract was homogenized with 10 strokes of a loose-fitting Dounce homogenizer, adjusted to 40% sucrose, and placed on the bottom of an ultracentrifuge tube. A 5% to 35% discontinuous sucrose gradient was formed above the lysate (in MBS containing protease inhibitors but lacking Triton X-100), and the Triton X-100–insoluble caveolin-enriched complexes that form a flocculent band at the 5% to 35% interface were collected, diluted 3-fold in MBS containing protease inhibitors (but lacking Triton X-100), sedimented by centrifugation (40 000g for 1 hour), and solubilized in SDS-PAGE sample buffer.
Transmission Electron Microscopy
Pellets were fixed with 2.5% glutaraldehyde in
0.1 mol/L sodium cacodylate buffer (pH 7.2) for an hour on ice. Samples
were then rinsed in 0.1 mol/L sodium cacodylate buffer, postfixed with
1% OsO4 in cacodylate buffer, and en bloc
stained with 1% aqueous uranyl acetate. Samples were dehydrated in
graded ethanol and embedded in Ladd Lx112 embedding medium (Ladd
Research Industries, Inc). Thin sections were cut on an ultramicrotome
RMC MT-7000, stained with uranyl acetate and lead citrate, and examined
under a transmission electron microscope (Jeol 1200).
Immunoblot Analysis
Samples (generally 7 to 10 µg of protein from the caveolae
fraction/lane) were separated by SDS-PAGE (10% acrylamide)
and transferred to nitrocellulose, which was cut longitudinally for
incubations with various primary antibodies. Some experiments used
cardiomyocyte soluble and particulate fractions that were
prepared according to standard methods as described
previously.20 The immunodetection of PKC isoforms was
according to methods described in previous
publications.20 21 All other antibodies were diluted in
50 mmol/L Tris, pH 7.5, 0.2 mol/L NaCl containing 5% nonfat dry
milk, 0.05% Tween 20, and 0.02% NaN3. Primary
antibodies were used at final dilutions of 1:5000 (caveolin-1 and
caveolin-3), 1:2500 (RACK-1), 1:2000 (annexin II), 1:1000 (MEK and
58-kDa protein), 1:500 (ERK), 1:300 (PMCA), 1:250 (c-Raf-1), and 1:100
(A-Raf), and bound primary antibodies were visualized with the
appropriate 125I-labeled secondary antibodies
(see Figures 1
, 2, 3, 5, and 6) or
ECL according to the manufacturer's instructions (Figure 4).
Signals were quantified with a phosphor imager (model 445SI
PhosphorImager, Molecular Dynamics, for
125I-labeled secondary antibodies) or
densitometric analysis (for experiments that used ECL to detect
antigen-antibody complexes). Each of the antibodies was initially
screened with total cell lysates to ensure that they reacted with a
band (or bands) of the appropriate molecular mass in
cardiomyocytes. For the polyclonal antibodies (PKC
isoforms, A-Raf, MEK, and ERK), preliminary experiments established
that the immunoreactive bands were specific (ie, immunoreactivity was
completely blocked by an excess of the respective competing antigen
peptide).
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Assay of ERK Activity in SDS-Polyacrylamide Gels Containing
Myelin Basic Protein (MBP)
Caveolae from control cells and cells exposed to 300 nmol/L PMA
for the indicated intervals were assayed for ERK activity with MBP as
substrate by "in-the-gel"–kinase assays as described
previously.20
Metabolic Labeling of Proteins in Cardiomyocyte
Caveolae
For in vivo phosphorylation assays, the
monolayer was rinsed with Pi-free MEM (4 mL per
100 mm-diameter dish) and then incubated for 4 to 5 hours at
37°C in Pi-free MEM supplemented with
[32P]orthophosphate (0.15 to 0.26 mCi/mL; 4 mL
per dish). To study the effects of PKC activation, PMA (300 nmol/L) was
added during the final 30 minutes of the labeling period as indicated.
At the end of the treatment interval, the radioactive medium was
aspirated, dishes were rinsed with ice-cold PBS (4 mL per dish), and
cells were scraped into MBS containing (in mmol/L) sodium vanadate
0.1, NaF 25, and phenylmethylsulfonyl fluoride 1; 10 µg/mL
leupeptin; 10 µg/mL aprotinin; and 1% Triton X-100. This was
followed by the isolation of caveolae (according to the method
described above), electrophoresis (8% polyacrylamide gels),
and autoradiography to detect PMA-dependent
incorporation of 32P into the resolved caveolar
proteins.
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Results |
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We initially used the sodium carbonate–based detergent-free method of Song et al6 to purify caveolae from quiescent cultured neonatal rat ventricular myocytes. The fractionation scheme is based on the resistance of integral membrane proteins to solubilization in sodium carbonate as well as the low buoyant density of caveolae due to their high cholesterol and glycosphingolipid content. After solubilization in sodium carbonate, cell lysates were adjusted to 40% in sucrose and then overlaid with a 5% to 35% discontinuous sucrose gradient. A small amount of protein moves up the gradient during centrifugation for 16 hours at 285 000g and can be visualized as a sharp light-scattering band at the 5% to 35% interface of the discontinuous sucrose gradient (fractions 3 to 4). Figure 1
illustrates that these fractions
exclude the bulk of the total cell protein that remains in the 40%
sucrose layer (fractions 8 to 12) or sediments to the bottom of the
tube as an insoluble pellet (P). Western blot analysis
demonstrates that the muscle-specific caveolin-3 isoform is recovered
in fractions 3 to 4; small amounts of caveolin-3 also are recovered in
the insoluble pellet, perhaps as a result of incomplete cell lysis
(Figure 1D). Consistent with recent results reported by
others,22 this fraction contains abundant
immunoreactivity for the PMCA but excludes the 58-kDa protein, a marker
for the Golgi23 (Figure 1E). By transmission
electron microscopy, the caveolin-rich membrane domains appear as
50- to 100-nm vesicular structures that resemble the
plasmalemmal vesicles (caveolae) that are seen in
transmission micrographs of whole cells (Figure 1F). There also
are some curved membrane fragments (similar to the morphology observed
for caveolin-enriched domains isolated from other cell
types,13 19 ) but no morphologically recognizable
contaminants. In agreement with a previous study,1
caveolin-1 (the immunologically distinct form of this protein, which is
abundant in adipocytes, endothelial cells, and
fibroblasts) is not detected in this preparation (even when 5 times as
much protein is loaded on the gel; data not shown).
Cardiomyocytes are known to contain at least 2 proteins that at least
theoretically could serve to anchor or regulate PKC isoforms in
caveolin-enriched domains. The first is the muscle-specific caveolin-3
isoform, the scaffolding domain sequence of which was recently shown to
directly interact in in vitro binding assays with the phorbol
ester–sensitive PKC isoform.15 A second class of
proteins that potentially could anchor PKC isoforms in caveolae is the
RACK proteins. According to this formulation, individual PKC isoforms
translocate to distinct intracellular loci as a result of specific
high-affinity interactions between unique sequences in individual PKC
isoforms and their respective anchoring RACK proteins.24
Because RACK proteins were first identified in the Triton X-100
insoluble fraction of neonatal rat hearts, and RACK immunoreactivity
was recently reported to be detectable in the caveolae fraction of
kidney epithelial cells,12 we next examined whether RACK
proteins could constitute a class of proteins that bind PKC in
caveolae. Figure 1C shows that the bulk of the RACK protein is
recovered as a 36-kDa protein in the heavy fractions. Nevertheless, a
minor component of total cellular RACK immunoreactivity (0.05%)
also is detectable in cardiomyocyte caveolae.
Having identified at least 2 potential PKC anchoring proteins in
cardiomyocyte caveolae, caveolin-enriched complexes were
isolated from basal and PMA-stimulated (300 nmol/L for 30 minutes)
cultured neonatal rat ventricular myocytes and probed for
PKC isoform immunoreactivity. Previous studies established that
neonatal rat ventricular myocytes coexpress multiple PKC
isoforms, including the phorbol ester–/calcium-sensitive PKC, the
phorbol ester–sensitive novel PKC and PKC, and the atypical
PKC isoforms.21 25 26 These isozymes differ in their
cofactor requirements for enzymatic activation, their substrate
specificity, and their subcellular localization in
cardiomyocytes and other cell types.27 Figure 2 shows that no PKC
immunoreactivity could be detected in the caveolae fraction from
control cells when 125I-labeled secondary
antibodies were used as the method to detect antigen-antibody complexes
(although it was possible to visualize small amounts of
immunoreactivity for each PKC isoform when blots were developed with
the more sensitive ECL method; see Figure 4). Immunoreactivity
for all 4 PKC isoforms was recovered in great abundance in the heavy
gradient fractions (data not shown). Immunoreactivity for each of the
phorbol ester–sensitive PKC isoforms increased dramatically in
caveolae in response to treatment with PMA (but not 4-PMA, which
does not activate PKC). Incubation with PMA did not lead to the
recruitment of the atypical PKC to the caveolin-enriched domain
(data not shown). These results indicate that the effects of PMA are
due to the specific activation of phorbol ester–sensitive PKC isoforms
(rather than to a nonspecific effect on membrane structure).
The time course for the PMA-induced translocation of PKC isoforms to
the caveolin-enriched fraction is shown in Figures 2B and 3. For each PKC isoform, immunoreactivity
was detected at 5 minutes, became further elevated by 15 minutes, and
remained relatively constant for at least the subsequent 2 hours of
stimulation with PMA. The PMA-induced recruitment of PKC isoforms to
the caveolae fraction occurred in the absence of any changes in the
yield of caveolin-enriched vesicles or caveolin-3 recovery (Figure 2B, bottom). It should be noted that the caveolin-enriched
fraction of cells treated with PMA for 30 minutes contained between
10% and 20% of total cellular PKC immunoreactivity for each phorbol
ester–sensitive isoform. Although this represents an enormous
enrichment in phorbol ester–sensitive PKC isoforms in the caveolae
compartment (which excludes >99% of the cellular proteins), the heavy
fractions retain the vast majority of each PKC isoform. Figure 3
shows that prolonged treatment with PMA ultimately leads to the
disappearance of PKC isoforms from the caveolin-enriched domain. For
each isoform, the time course of this process parallels the kinetics of
downregulation in the particulate fraction of the cell. Thus, there is
a substantial downregulation of PKC and PKC by 8 hours, and this
process is complete by 24 hours. Consistent with previous
observations in cardiomyocyte
preparations,20 26 the downregulation of PKC is a
slower process, with the abundance of PKC immunoreactivity reduced
to 20% of the initial level only at the 24-hour time point.
Collectively, these results indicate that stimulation with PMA leads to
the temporary stable association of phorbol ester–sensitive PKC
isoforms with the caveolae fraction of neonatal rat
ventricular myocytes.
Further experiments using endothelin provide evidence that the
translocation of PKC isoforms to caveolae is a component of the
physiological activation process. Endothelin is
known to stimulate phosphoinositide hydrolysis and
induce the diacylglycerol-dependent translocation of novel PKC
isoforms (PKC and PKC) from the soluble to the particulate
fraction of cardiomyocytes.20 26 Figure 4 demonstrates that endothelin promotes
the rapid association of PKC and PKC with the caveolae fraction
(70% and 100% over basal, respectively). Although these responses to
endothelin are relatively modest compared with the 10-fold increase in
PKC isoform immunoreactivity in the caveolae fraction elicited by PMA,
this result is consistent with previous studies demonstrating
that direct pharmacological activation of PKC isoforms with PMA is more
robust than physiological activation through G
protein–coupled receptors.20 Endothelin does not alter
the level of immunoreactivity for the phorbol ester–sensitive PKC
isoform in caveolae (data not shown). This result was somewhat
surprising, but there is a precedent for a translocation/activation
process in cardiomyocytes that is selective for PKC and
PKC (and does not involve PKC).28 Consistent
with previous reports that the subcellular distribution of PKC is
not altered by endothelin,20 26 endothelin does not
promote the translocation of the phorbol ester–insensitive PKC
isoform to caveolae.
The recruitment of activated PKC isoforms to caveolae could
lead to the activation of downstream targets at this site. A key
signaling cascade that might be activated by PKC isoforms in
caveolae is the ERK subfamily of mitogen-activated protein
kinases; this pathway involves the sequential
phosphorylation and activation of Raf, MEK, and the 42-
and 44-kDa isoforms of ERK. Only a few sporadic reports have
investigated the presence of components of the ERK cascade in caveolae,
and the results have been inconsistent. Thus, there is evidence
that epidermal growth factor induces the transient appearance and
activation of c-Raf-1 in a detergent-free caveolae fraction from
Rat-1 fibroblasts.29 However, MEK and the 44-kDa
isoform of ERK are reported to be excluded from caveolin-enriched
fractions (when detergent is used in the preparation of the
complex13 ). Although the 42-kDa isoform of ERK has been
detected in caveolae prepared from resting human fibroblasts and mouse
lung tissue13 30 (and there is very recent evidence that
it becomes activated in response to stimulation with PDGF in
human fibroblasts),31 a recent study reports the release
of ERK from the caveolin-enriched fraction of MDCK cells on activation
with PMA.16 Accordingly, studies illustrated in Figure 5 examined the extent to which components
of the ERK signaling cascade partition to the caveolin-enriched domain
of neonatal ventricular myocytes. Since certain signaling
molecules may be recruited to caveolae (or conversely may disappear
from caveolae) after agonist stimulation, samples were prepared under
resting conditions and after stimulation with PMA.
Previous studies established that neonatal myocytes express 2 isoforms
of Raf, A-Raf and c-Raf-1, and that both are activated (albeit
with distinct kinetics) by PKC activators.32
Figure 5 shows that A-Raf and c-Raf-1, as well as MEK and the
42- and 44-kDa isoforms of ERK, cosegregate to caveolae and that these
species are present in equal abundance in the caveolin-enriched
fractions of resting and PMA-treated cardiomyocytes. To
determine whether the colocalization of these signaling molecules
represents a mechanism to facilitate signaling through an ERK
phosphorylation cascade localized to the caveolae
membrane domain, we measured in-the-gel MBP kinase activity in caveolae
from control and PMA-stimulated cardiomyocytes. Figure 5 shows that ERK is activated in response to PMA
(2.2±0.08-fold over basal, n=3, P<0.05) and that the
kinetics of ERK activation parallels the time course for PKC
translocation to caveolae (ie, the increase in ERK activity is
detectable by 5 minutes and reaches a maximal level at 15 minutes,
which persists for an additional 15 minutes of stimulation with
PMA).
The detection of MEK and both isoforms of ERK in caveolae contrasts
with the previous inability to detect these species in this
compartment.13 To determine whether technical issues
related to differences in the method used to prepare caveolae could
explain these discrepant results, we performed immunoblot
analysis on caveolae prepared according to both sodium
carbonate– and Triton X-100–based purification schemes. Figure 6 shows that the presence of detergent
does not alter the abundance of MEK in caveolae. In contrast, detergent
modestly reduces the abundance of ERK, dramatically reduces the
abundance of PKC, and totally eliminates PKC and PKC from this
compartment. These results indicate that the association of PKC
isoforms and (to a lesser extent) ERK with caveolae is weak (or lipid
dependent) such that these species are removed by the Triton
X-100–based extraction protocol.
Finally, to determine whether activation of PKC leads to the
phosphorylation of resident caveolae proteins, neonatal
cardiomyocytes were metabolically labeled with
[32P]orthophosphate and then left untreated or
stimulated with PMA for 30 minutes before the isolation of
caveolin-enriched complexes. Figure 7
shows that stimulation with PMA enhances the
phosphorylation state of proteins in the 80- to 90-kDa
range (which could at least in part represent
phosphorylation of the MARCKS protein, an 80-kDa
protein that is a known PKC substrate and is abundant in the immature
rat heart26 33 ) and several 42- to 50-kDa species, and
leads to the appearance of a smaller 36-kDa phosphoprotein in the
caveolae fraction. With respect to the 36-kDa phosphoprotein, the
heavy chain of annexin II is a known PKC substrate with a similar
molecular mass (36 to 39 kDa) that has been reported to
compartmentalize to caveolae13 19 ; we performed further
studies to determine whether this lower molecular mass phosphoprotein
species in caveolae fractions from PMA-treated
cardiomyocytes is the heavy chain of annexin II. Although
Figure 7 (far right lane) shows that annexin II immunoreactivity
is detectable in cardiomyocyte caveolae, its mobility is
considerably slower than the lower molecular mass phosphoprotein
species in caveolae fractions from PMA-treated
cardiomyocytes. These results argue that the 36-kDa
protein substrate for PKC in cardiomyocyte caveolae is not
the heavy chain of annexin II.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although several laboratories have initiated a systematic analysis of the signaling molecules resident in caveolae, the literature on PKC isoform localization to caveolin-enriched domains is largely limited to an analysis of PKC
, the data are
inconsistent (with phorbol esters variably recruiting PKC to
caveolae or inducing a loss of PKC from this
compartment12 16 ), and there is very limited information
on the molecular composition of caveolae in cardiomyocytes.
Studies reported herein establish that under basal conditions the level
of immunoreactivity for various PKC isoforms in
cardiomyocyte caveolae is at the limits of detection using
the conventional visualization methods and that these molecules are
selectively recruited to caveolae after pharmacological and
physiological stimuli (PMA and endothelin). Several
factors may reconcile our findings and the discrepant data already
published in the literature. First, the observations that the
association of PKC isoforms with caveolae does not withstand treatment
with Triton X-100 and that PKC isoforms are detected in unstimulated
cardiomyocytes only when a sensitive ECL method is applied
to the analysis identify 2 technical factors (the nature of the
extraction scheme used before the isolation of caveolae and the method
for detection) that may explain at least some of the prior
inconsistent results. As demonstrated in this study and
recently suggested by others, the precise nature of the agonist
stimulation protocol, which if sufficiently intense would lead to a
loss of PKC isoform immunoreactivity due to the downregulation of the
enzyme, also may influence the recovery of PKC isoforms in the caveolae
fraction.16 Finally, it is possible that caveolae from
different cell types act in a specialized fashion to concentrate and
functionally regulate cytoplasmically oriented signaling molecules.
There are at least 3 mechanisms that potentially could mediate the
recruitment of PKC isoforms to caveolin-enriched domains. First, on the
basis of recent studies demonstrating that PKC interacts with the
scaffolding domain of caveolin-3 (and caveolin-1, but not the
structurally distinct homologous region of caveolin-215 ),
it is reasonable to speculate that activation of PKC isoforms might
lead to a conformational change that exposes an aromatic amino
acid–rich consensus caveolin-binding domain that facilitates targeting
to caveolae. Second, despite the partitioning of only a minor component
of total cellular RACK immunoreactivity to caveolae, RACK proteins
still could constitute a second class of proteins that bind PKC only in
the presence of PKC activators in this compartment. Third,
proteins such as the annexins have been reported to bind negatively
charged phospholipids such as phosphatidylserine
and act as PKC binding proteins.34 35 Available evidence
suggests that this process involves protein-protein interactions that
are facilitated or stabilized by lipid cofactors (such as
phosphatidylserine). This final mechanism would be
particularly susceptible to treatment with 1% Triton X-100 (which
removes resident lipid-modified signaling proteins such as G protein
ß
complexes and Ras from caveolae6 ) and therefore
might be particularly pertinent to the results reported herein.
Finally, it should be noted that these mechanisms may not be mutually
exclusive. Pertinent to this point is the observation that PMA recruits
all phorbol ester–sensitive PKC isoforms (, , and ) to the
caveolae fraction, whereas endothelin induces a rapid increase in the
association of PKC and PKC (but not PKC) with the caveolae
fraction. This observation is consistent with the notion that
activated PKC isoforms are recruited to caveolae via mechanisms
that are distinct for isozymes.
These studies demonstrate that PMA induces the phosphorylation of several proteins in the caveolin-enriched domain. Although this further establishes caveolae as a target for PKC isoform actions, there are several issues that require further study. First, we have yet to determine whether PKC activators lead to the phosphorylation of proteins already associated with caveolae in their unphosphorylated form in the absence of PKC activators or whether PKC activation leads to the de novo association of proteins that are susceptible to PMA-dependent phosphorylation within the caveolae compartment. Second, the identity of the phosphorylating kinase was not determined. In this regard, the in vivo phosphorylation experiments cannot discriminate a direct effect of PKC isoforms to phosphorylate resident substrate proteins from an indirect effect secondary to PKC isoform–dependent activation of downstream kinases. In particular, a casein kinase II–like enzyme, which is activated by PKC, has been reported to be the dominant serine kinase in the caveolae fraction36 (although this conclusion was based on an analysis of kinase activity in caveolae prepared from resting cells and does not entirely rule out a dominant function for PKC isoforms after their recruitment to caveolae by PMA). The evidence that stimulation with PMA leads to the local activation of ERK establishes ERK as yet an additional candidate kinase in this compartment. Third, the substrates phosphorylated in cardiomyocyte caveolae have yet to be identified. This study considered the possibility that 1 of the phosphoproteins in caveolae from PMA-stimulated cardiomyocytes is the heavy chain of annexin II. Indeed, annexin II immunoreactivity was detected in cardiomyocyte caveolae. However, the electrophoretic mobilities of annexin II and the phosphoprotein species in the caveolae fraction differ substantially, suggesting that annexin II is not a major substrate for the PKC-induced phosphorylation in caveolae. The identification of the proteins phosphorylated in cardiomyocyte caveolae should provide insights into the functional consequences of PKC-activated signaling events in this compartment.
These studies are the first to identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their targets, including the phosphorylation cascade that results in the activation of ERK. As such, this report lends further credence to the notion that caveolae represent "signaling processing centers," which orchestrate signaling events at the cell surface that influence cell function. Nevertheless, the full functional significance of this event remains to be determined. Because PKC activators have been reported to dramatically alter the morphology and functional activity of caveolae,12 17 and caveolae act as a processing center for multiple receptor-dependent signaling events (which likely include adrenergic receptors, cholinergic receptors, and their downstream targets),37 38 it is tempting to speculate that activation of PKC can influence subsequent signal output from other receptors. Results reported herein also are consistent with recent evidence that components of the ERK cascade are concentrated in caveolae as a mechanism to facilitate agonist-dependent activation of this pathway.30 31 Indeed, the identification of all of the biochemical machinery for the ERK phosphorylation cascade in caveolae raises the possibility that the ERK signaling pathway performs specific functions in this location that cannot be accomplished elsewhere in the cell.
There is evidence that caveolin-1 acts as a general "kinase inhibitor" and a negative regulator of mitogenic growth at least in part through an effect to inhibit cellular activation through the Ras/ERK pathway.39 Thus, it will be interesting to determine whether caveolae (and caveolin-3, which is structurally homologous to caveolin-1) also influences hypertrophic growth responses in cardiomyocytes. Apart from a potential role in the control of cardiomyocyte growth, PKC isoforms also phosphorylate an array of substrate proteins that fulfill functions that are unique to cardiomyocytes, including modulation of the rate of spontaneous contractile activity, regulation of intracellular calcium ion concentration, ischemic preconditioning of the heart, and the genesis of arrhythmias. The recent identification of a "loss-of-function" mutation in the caveolin-3 gene as the molecular basis for some forms of autosomal dominant limb-girdle muscular dystrophy40 emphasizes the importance of caveolae in muscle cell biology. Studies of patients in this kindred (which are likely to be accompanied by studies in genetically altered mice with specific mutations or targeted disruption of the caveolin-3 gene) will delineate the importance of PKC (and other second-messenger molecules) targeting to caveolae in the contractile or growth response of the heart.
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Acknowledgments |
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This work was supported by US Public Health Service-National Heart, Blood, and Lung Institute grants HL-28958 (to S.F.S.) and HL-49537 (to S.F.S.); an American Heart Association Grant-in-Aid (to S.F.S.); and training grant 2T32 DK07328-18 in Hormones: Biochemistry & Molecular Biology from the National Institute of Diabetes and Digestive and Kidney Diseases (to X.X.). We thank HongLu Zhang for performing the ERK activity assays, Kristy Brown and Dr Michael Shelanski for help with transmission electron microscopy, and Ema Stasko for preparing myocyte cultures.
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Footnotes |
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This manuscript was originally submitted on December 8, 1997, resubmitted on June 10, 1998, and accepted on February 5, 1999.
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References |
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V. O. Rybin, P. W. Grabham, H. Elouardighi, and S. F. Steinberg Caveolae-associated proteins in cardiomyocytes: caveolin-2 expression and interactions with caveolin-3 Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H325 - H332. [Abstract] [Full Text] [PDF]
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 V. O. Rybin, E. Pak, S. Alcott, and S. F. Steinberg Developmental Changes in {beta}2-Adrenergic Receptor Signaling in Ventricular Myocytes: the Role of Gi proteins and Caveolae Microdomains Mol. Pharmacol., June 1, 2003; 63(6): 1338 - 1348. [Abstract] [Full Text] [PDF]
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L. Liu, K. Mohammadi, B. Aynafshar, H. Wang, D. Li, J. Liu, A. V. Ivanov, Z. Xie, and A. Askari Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1550 - C1560. [Abstract] [Full Text] [PDF]
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K. Y. Krotova, S. I. Zharikov, and E. R. Block Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L1037 - L1044. [Abstract] [Full Text] [PDF]
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Y. Shimoni and X.-F. Liu Role of PKC in autocrine regulation of rat ventricular K+ currents by angiotensin and endothelin Am J Physiol Heart Circ Physiol, April 1, 2003; 284 (4): H1168 - H1181. [Abstract] [Full Text] [PDF]
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 H. Hua, S. Munk, and C. I. Whiteside Endothelin-1 activates mesangial cell ERK1/2 via EGF-receptor transactivation and caveolin-1 interaction Am J Physiol Renal Physiol, February 1, 2003; 284(2): F303 - F312. [Abstract] [Full Text] [PDF]
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S. K. Sukumaran, M. J. Quon, and N. V. Prasadarao Escherichia coli K1 Internalization via Caveolae Requires Caveolin-1 and Protein Kinase Calpha Interaction in Human Brain Microvascular Endothelial Cells J. Biol. Chem., December 20, 2002; 277(52): 50716 - 50724. [Abstract] [Full Text] [PDF]
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J. Zhong, G.-X. Wang, W. J. Hatton, I. A. Yamboliev, M. P. Walsh, and J. R. Hume Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC Am J Physiol Cell Physiol, December 1, 2002; 283(6): C1627 - C1636. [Abstract] [Full Text] [PDF]
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S. E. Woodman, D. S. Park, A. W. Cohen, M. W.-C. Cheung, M. Chandra, J. Shirani, B. Tang, L. A. Jelicks, R. N. Kitsis, G. J. Christ, et al. Caveolin-3 Knock-out Mice Develop a Progressive Cardiomyopathy and Show Hyperactivation of the p42/44 MAPK Cascade J. Biol. Chem., October 4, 2002; 277(41): 38988 - 38997. [Abstract] [Full Text] [PDF]
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 B. Razani, S. E. Woodman, and M. P. Lisanti Caveolae: From Cell Biology to Animal Physiology Pharmacol. Rev., September 1, 2002; 54(3): 431 - 467. [Abstract] [Full Text] [PDF]
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 B. Junoy, H. Maccario, J.-L. Mas, A. Enjalbert, and S. V. Drouva Proteasome Implication in Phorbol Ester- and GnRH-Induced Selective Down-Regulation of PKC ({alpha}, {epsilon}, {zeta}) in {alpha}T3-1 and L{beta}T2 Gonadotrope Cell Lines Endocrinology, April 1, 2002; 143(4): 1386 - 1403. [Abstract] [Full Text] [PDF]
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 D. Wu, T. L. Foreman, C. W. Gregory, M. A. McJilton, G. G. Wescott, O. H. Ford, R. F. Alvey, J. L. Mohler, and D. M. Terrian Protein Kinase C{epsilon} Has the Potential to Advance the Recurrence of Human Prostate Cancer Cancer Res., April 1, 2002; 62(8): 2423 - 2429. [Abstract] [Full Text] [PDF]
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T. L. Yarbrough, T. Lu, H.-C. Lee, and E. F. Shibata Localization of Cardiac Sodium Channels in Caveolin-Rich Membrane Domains: Regulation of Sodium Current Amplitude Circ. Res., March 8, 2002; 90(4): 443 - 449. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, C. Gregorian, R. M. Drenan, Y. Xiang, J. W. Regan, and P. A. Insel Receptor Number and Caveolar Co-localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase J. Biol. Chem., November 2, 2001; 276(45): 42063 - 42069. [Abstract] [Full Text] [PDF]
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J. C. Song, C. M. Hanson, V. Tsai, O. C. Farokhzad, M. Lotz, and J. B. Matthews Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms Am J Physiol Cell Physiol, August 1, 2001; 281(2): C649 - C661. [Abstract] [Full Text] [PDF]
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T. M. Vondriska, J. B. Klein, and P. Ping Use of functional proteomics to investigate PKC{epsilon}-mediated cardioprotection: the signaling module hypothesis Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1434 - H1441. [Abstract] [Full Text] [PDF]
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 G. van Meer Caveolin, Cholesterol, and Lipid Droplets? J. Cell Biol., March 5, 2001; 152(5): 29 - 34. [Full Text] [PDF]
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 J. Szalay, P. Bruno, R. Bhati, J. Adjodha, D. Schueler, V. Summerville, and R. Vazeos Associations of PKC Isoforms with the Cytoskeleton of B16F10 Melanoma Cells J. Histochem. Cytochem., January 1, 2001; 49(1): 49 - 66. [Abstract] [Full Text]
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D. D. Doyle, G. Goings, J. Upshaw-Earley, S. K. Ambler, A. Mondul, H. C. Palfrey, and E. Page Dystrophin Associates With Caveolae of Rat Cardiac Myocytes : Relationship to Dystroglycan Circ. Res., September 15, 2000; 87(6): 480 - 488. [Abstract] [Full Text] [PDF]
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J. M. Hare, R. A. Lofthouse, G. J. Juang, L. Colman, K. M. Ricker, B. Kim, H. Senzaki, S. Cao, R. S. Tunin, and D. A. Kass Contribution of Caveolin Protein Abundance to Augmented Nitric Oxide Signaling in Conscious Dogs With Pacing-Induced Heart Failure Circ. Res., May 26, 2000; 86(10): 1085 - 1092. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, J. D. Violin, S. Coleman, and P. A. Insel Selective Enhancement of beta -Adrenergic Receptor Signaling by Overexpression of Adenylyl Cyclase Type 6: Colocalization of Receptor and Adenylyl Cyclase in Caveolae of Cardiac Myocytes Mol. Pharmacol., May 1, 2000; 57(5): 1075 - 1079. [Abstract] [Full Text]
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R. D. Lasley, P. Narayan, A. Uittenbogaard, and E. J. Smart Activated Cardiac Adenosine A1 Receptors Translocate Out of Caveolae J. Biol. Chem., February 11, 2000; 275(6): 4417 - 4421. [Abstract] [Full Text] [PDF]
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R. S. Ostrom and P. A. Insel Caveolar Microdomains of the Sarcolemma : Compartmentation of Signaling Molecules Comes of Age Circ. Res., May 14, 1999; 84(9): 1110 - 1112. [Full Text] [PDF]
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V. O. Rybin, X. Xu, M. P. Lisanti, and S. F. Steinberg Differential Targeting of beta -Adrenergic Receptor Subtypes and Adenylyl Cyclase to Cardiomyocyte Caveolae. A MECHANISM TO FUNCTIONALLY REGULATE THE cAMP SIGNALING PATHWAY J. Biol. Chem., December 22, 2000; 275(52): 41447 - 41457. [Abstract] [Full Text] [PDF]
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T. L. Yarbrough, T. Lu, H.-C. Lee, and E. F. Shibata Localization of Cardiac Sodium Channels in Caveolin-Rich Membrane Domains: Regulation of Sodium Current Amplitude Circ. Res., March 8, 2002; 90(4): 443 - 449. [Abstract] [Full Text] [PDF]
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