1 Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8S 4L8
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ABSTRACT |
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PKC is a critical effector of plasma
membrane dynamics, yet the mechanism and isoform-specific role of PKC
are poorly understood. We recently showed that the phorbol ester PMA
(100 nM) induces prompt activation of the novel isoform PKC followed
by late activation of the conventional isoform PKC
in T84 intestinal
epithelia. PMA also elicited biphasic effects on endocytosis,
characterized by an initial stimulatory phase followed by an inhibitory
phase. Activation of PKC
was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKC
was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKC
on basolateral endocytosis. Inhibition of PKC
by the selective
conventional PKC inhibitor Gö-6976 (5 µM) completely blocked
the late inhibitory phase and markedly prolonged the stimulatory phase
of endocytosis measured by FITC-dextran uptake. The PKC
-selective agonist carbachol (100 µM) induced prolonged stimulation of
endocytosis devoid of an inhibitory phase. Actin disassembly caused by
PMA was completely blocked by Gö-6850 but not by Gö-6976,
implicating PKC
as the key isoform responsible for actin disruption.
The Ca2+ agonist thapsigargin (5 µM) induced early
activation of PKC
when added simultaneously with PMA. This early
activation of PKC
blocked the ability of PMA to remodel basolateral
F-actin and abolished the stimulatory phase of basolateral endocytosis.
Activation of PKC
stabilizes F-actin and thereby opposes the effect
of PKC
on membrane remodeling in T84 cells.
protein kinase C isoforms; cytoskeleton; intestinal mucosa; calcium
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INTRODUCTION |
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PROTEIN KINASE C (PKC) is a family of at least 10 different serine/threonine isoforms (20) that have been implicated in a variety of cellular responses such as membrane trafficking, migration, ion transport, and cell differentiation. Each isoform has distinct enzymological properties, tissue distribution, and subcellular localization (22), implying that each affects a unique complement of biological functions. The basis for this functional diversity and for the selective regulation of individual isoforms is not well understood.
PKC isoforms are usually subdivided into three groups, conventional
(cPKC), novel (nPKC), and atypical (aPKC), on the basis of their
activation requirements, which, in turn, reflect the structure of their
regulatory domains (20, 21). The regulatory domain of cPKC
isoforms (,
I,
II, and
) contains
two common regions, C1 and C2. The C1 region mediates diacylglycerol
(DAG) and phorbol ester binding. The presence of a C2 region makes the cPKC isoforms distinct from other subfamilies in that they require Ca2+ for activation. The nPKC isoforms (
,
,
, and
) contain a C1 region, and, therefore, DAG and phorbol ester
binding activate these isoforms. However, nPKC are Ca2+
independent because they lack the C2 region. The aPKC isoforms (
and
/
) are independent of DAG or Ca2+ and, as a
general rule, cannot be directly activated by phorbol esters such as
PMA. This structural heterogeneity implies that intracellular
Ca2+ is a key determinant of the specific pattern of PKC
isoform activation.
We previously showed that in model polarized T84 human intestinal
epithelia, PMA induces a dramatic increase in the rate of basolateral
fluid-phase endocytosis without affecting endocytosis at the apical
membrane (28). Of the four PKC isoforms identified in T84
cells (,
,
, and
), PMA induced early activation of two
novel isoforms, PKC
and PKC
, followed by late activation of the
conventional isoform PKC
(27). PMA was also shown to induce biphasic effects on basolateral endocytosis characterized by an
early stimulation period (stimulatory phase) followed by a later return
to baseline rates (inhibitory phase) (28). Selective inhibition of PKC
completely abolished the early stimulatory phase,
suggesting that PKC
is the key isoform responsible for PMA-induced
stimulation of basolateral endocytosis by a mechanism that appeared to
involve localized actin disassembly. The basis for the inhibitory phase
was not determined, but preliminary data reported in abstract form
suggested a possible role for PKC
(29). PKC
did not
appear to be involved in either the stimulatory or inhibitory phase, on
the basis of insensitivity to the PKC
-specific inhibitor rottlerin.
There have been several reports describing antagonistic effects of
different PKC isoforms on the regulation of the same biological function (2, 5, 6, 12, 33). For example, in rat
fibroblasts, PKC and PKC
were shown to have opposite effects on
epidermal growth factor receptor-mediated transformation and
phospholipase D activity. PKC
I and PKC
II
had opposite roles in vascular smooth muscle cell proliferation. PKC
and PKC
mediated opposing effects on proximal tubule
Na+/K+-ATPase activity. These considerations
suggested to us the possibility that, in T84 cells, PKC
could play a
counterregulatory role to PKC
in control of basolateral membrane dynamics.
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MATERIALS AND METHODS |
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Cell culture. T84 human intestinal epithelial cells obtained from Dr. Kim Barrett (University of California, San Diego, CA) were grown to confluence as described previously (27).
Fluid-phase endocytosis. Uptake of fluorescein isothiocyanate-dextran (FITC-dextran, MW 12,000, 0.73 mol fluorescein/mol dextran) from the basolateral aspect of confluent T84 monolayers grown on collagen-coated permeable supports (4.7 cm2, 3.0-µm pore size) was measured as described previously (27).
Subcellular fractionation. T84 cells grown to confluence on collagen-coated permeable supports (4.7 cm2) were fractionated into the cytosolic and membrane fractions as described previously (28). Briefly, monolayers were scraped with the cold homogenization buffer (HB) containing 20 mM Tris · HCl, pH 7.5, 250 mM sucrose, 4 mM EDTA, 2 mM EGTA, and Complete protease inhibitor cocktail tablets. The cells were homogenized on ice, and the homogenate was ultracentrifuged at 86,000 g for 50 min at 4°C (TLA 45 rotor, TL-100 Ultracentrifuge; Beckman). The supernatant was designated the cytosolic fraction. The pellet was resuspended in HB containing 0.5% (vol/vol) Triton X-100 and incubated in ice for 30 min. At the end of the incubation period, the samples were centrifuged at 14,000 g for 20 min at 4°C. The resulting supernatant was designated the membrane fraction.
In vitro kinase assay.
Confluent T84 monolayers grown on 4.7-cm2 permeable
supports were treated with various PKC agonists, and proteins were
extracted with the lysis buffer containing 50 mM Tris · HCl, pH
7.5, 150 mM NaCl, 1% (vol/vol) Triton X-100, 2 mM EDTA, 1 mM EGTA, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM
Na3VO4, and Complete protease inhibitor
cocktail tablets (27). Polyclonal antibodies against cPKC (2 µg), nPKC
(4 µg), or nPKC
(2 µg) were added to
each lysate for overnight rotation at 4°C. After incubation, immune complexes were precipitated with the use of protein A-agarose beads,
resuspended in kinase buffer (35 mM Tris · HCl, pH 7.5, 10 mM
MgCl2, 0.5 mM EGTA, 10 µCi [
-32P]ATP, 60 µM cold ATP, and 1 mM Na3VO4), and incubated
with myelin basic protein (MBP) as a substrate at 30°C for 30 min.
After incubation, the reaction was terminated with Laemmli sample and
subjected to SDS-PAGE (15% gels). The gel was then dried and subjected
to autoradiography.
Gel electrophoresis and Western blotting. Equal amounts of protein (~50 µg/sample) were subjected to SDS-PAGE and Western blot as described previously (27). Briefly, proteins were separated on 8% gels, transblotted to nitrocellulose membranes, and incubated with the polyclonal antibodies to different PKC isoforms for 1 h. After brief washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody for 1 h, washed, and visualized with enhanced chemiluminescence (ECL) detection reagent.
Immunofluorescence and microscopy.
Monolayers grown on 0.33-cm2 permeable supports were
treated with various agonists and prepared for confocal microscopy as described previously (27). Briefly, cells were fixed and
permeabilized with 0.1% (vol/vol) Triton X-100. Cells were then
incubated with the blocking buffer (1% normal goat serum, 3% BSA in
PBS) followed by the primary antibody against PKC. After overnight
incubation in a moisture chamber at 4°C, monolayers were incubated in
rhodamine-conjugated goat anti-rabbit polyclonal IgG along with
FITC-phalloidin for F-actin staining. Confocal images were acquired
with a Zeiss inverted microscope equipped with MRC-1024 and Lasersharp
software (Bio-Rad).
Materials.
Tissue culture reagents and protein A-agarose beads were purchased from
Invitrogen. Gel electrophoresis and Western blotting reagents were from
Bio-Rad, with the exception of ECL detection reagent, which was
purchased from Amersham. Complete protease inhibitor cocktail tablets
were from Roche. Anti-PKC was obtained from Sigma and Santa Cruz
Biotechnology for Western blotting and immunostaining, respectively.
Anti-PKC
was purchased from Santa Cruz Biotechnology. Secondary
antibodies were obtained from Bio-Rad and Jackson Laboratories for
Western blotting and immunostaining, respectively. Vectashield mounting
medium was from Vector Laboratories. The PKC inhibitors Gö-6976,
Gö-6850, and rottlerin were obtained from Calbiochem.
[
-32P]ATP with specific activity of 3,000 Ci/mmol was
purchased from NEN. All other chemicals were from Sigma.
Statistical analysis. Data are reported as means ± SE. Data were analyzed by one-way ANOVA with Bonferroni/Dunn's post hoc test for comparison with control.
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RESULTS |
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Activation of PKC and PKC
are temporally associated with
stimulation and inhibition of basolateral endocytosis, respectively.
Our previous results (28) suggested that PMA elicits
biphasic effects on basolateral uptake of FITC-dextran in T84
monolayers. This was confirmed in repeated experiments, shown
in Fig. 1A, that extended the
time course of observation over 150 min. During the initial stimulatory
phase, 100 nM PMA progressively increased basolateral endocytosis,
reaching a peak at ~60 min (Fig. 1A). Subsequently, during
the inhibitory phase, basolateral endocytosis slowly returned to its
basal level. To elucidate the specific PKC isoform that may be involved
in each phase of the endocytosis, we compared this response to the time
course of activation of the conventional isoform PKC
and the novel
isoform PKC
. The results of these isoform translocation and in vitro
kinase assays, shown in Fig. 1, B and C, are
similar to findings in our earlier report (27). For
example, in a new set of experiments, 100 nM PMA initially induced
translocation of PKC
from the cytosolic to the membrane fraction
during the 30- to 60-min window that corresponds to the initial
stimulatory phase of endocytosis (Fig. 1B), whereas
translocation of PKC
lagged ~1 h behind the activation of PKC
and was temporally associated with the inhibitory phase. In vitro
kinase assays for PKC
and PKC
were consistent with these
translocation data (Fig. 1C) and with our earlier reported results (29).
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PMA induces actin rearrangement via activation of PKC, not
PKC
.
We previously showed that PMA increases basolateral endocytosis via
disruption of actin cytoskeleton (28). As shown in Fig. 4, PMA induced significant remodeling of
basolateral F-actin and condensation of staining around the cell
periphery. We examined sensitivity of PMA-elicited cytoskeletal
remodeling to isoform-selective PKC inhibitors. Pretreatment with
Gö-6850 but not Gö-6976 attenuated this actin remodeling,
suggesting that PKC
is the isoform responsible for actin disassembly
caused by PMA. Inhibition of PKC
by Gö-6976 did not attenuate,
and instead appeared to qualitatively exacerbate, the degree of
disruption of the actin cytoskeleton.
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Thapsigargin prevents stimulation of basolateral endocytosis by
PMA.
We wondered whether the combination of PMA plus the
Ca2+-ATPase inhibitor thapsigargin would accelerate
activation of PKC and allow us to more directly address the
potential opposing actions of PKC
and PKC
. Indeed, this proved to
be the case. Thapsigargin alone had no effect on PKC
or PKC
.
However, the combined addition of PMA and thapsigargin accelerated
activation of PKC
. As shown in Fig.
5A, PKC
had already
translocated to the membrane by 30 min. Activation of PKC
by PMA, in
contrast, was not affected by thapsigargin. Moreover, the combined
addition of thapsigargin and PMA failed to stimulate basolateral
endocytosis (Fig. 5B); stated differently, thapsigargin
blocked the ability of PMA to stimulate endocytosis. The
simultaneous activation of PKC
and PKC
by costimulation with
thapsigargin and PMA led to inhibition of the early stimulatory effects
of PMA on basolateral endocytosis. In contrast to thapsigargin, the
Ca2+ agonist CCh, which does not induce early activation of
PKC
either alone or in combination with PMA, did not antagonize the
effects of PMA on basolateral endocytosis. In fact, combined addition of CCh and PMA exaggerated the initial stimulatory phase (data not
shown). Although CCh may have other effects that may interfere with
PMA's ability to induce basolateral endocytosis, these data further
strengthen the possibility that PKC
may be involved in inhibition of
endocytosis.
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PKC rapidly translocates to the basal membrane upon addition of
thapsigargin with PMA.
We previously showed that inactive PKC
is localized in the
basal cytoplasm of T84 cells (27). Vertical images
captured from confocal microscopy revealed that PKC
is found in
basal zone of the cell in a diffuse cytoplasmic pattern in control
monolayers (Fig. 7A). When PMA
was added alone, PKC
moved apically and was found in the apical
domain (Fig. 7B). In striking contrast, with PMA plus
thapsigargin, PKC
rapidly cleared from the basal cytoplasm and
relocated to the basal membrane (Fig. 7D), suggesting that Ca2+ influx in the basal region could redirect the
subcellular localization of PKC
from the apical domain (without
thapsigargin) to the basal membrane (with thapsigargin). Thapsigargin
alone without PMA-induced elevation of DAG had no effect on PKC
distribution (Fig. 7C).
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PMA-mediated actin rearrangement is prevented by thapsigargin.
Because thapsigargin prevented the PMA-elicited increase in
basolateral endocytosis, we anticipated that thapsigargin would also
inhibit PMA-induced actin disassembly. Indeed, this was the case, as
shown in Fig. 8D. Pretreatment
with the PKC inhibitor Gö-6976 abolished this effect of
thapsigargin (Fig. 8E). This finding strongly suggests that
thapsigargin-induced translocation and/or activation of PKC
to the
basal membrane (in the context of PMA treatment) inhibits
PKC
-mediated actin disassembly as well as stimulation of basolateral
endocytosis.
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DISCUSSION |
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In our earlier report, we found that a variety of
physiological and pharmacological activators of the novel
Ca2+-independent PKC isoform rapidly induced actin
remodeling and enhanced endocytosis at the basolateral aspect of
polarized T84 monolayers (27). In the present study, we
developed evidence to suggest that activation of the conventional
Ca2+-dependent PKC
isoform opposes this action of
PKC
. Treatment of T84 monolayers with PMA was observed to
sequentially activate PKC
and PKC
in temporal association with an
early stimulatory and a later inhibitory phase of basolateral
endocytosis. The inhibitory phase was eliminated by concurrent
treatment with Gö-6976, a selective Ca2+-dependent
PKC inhibitor that has been validated in our system, at the
concentration used, to be largely selective for PKC
. Cytoskeletal remodeling induced by PMA was prevented by the PKC
/PKC
inhibitor Gö-6850 but was exacerbated by Gö-6976. Although the
Ca2+-ATPase inhibitor thapsigargin did not by itself affect
PKC isoform activity, addition of thapsigargin plus PMA induced
simultaneous rather than sequential activation of PKC
and PKC
.
Moreover, thapsigargin prevented PMA-elicited stimulation of
basolateral endocytosis and actin remodeling. Interestingly, this
action of thapsigargin depended on the presence of Ca2+ in
the basolateral bath and was abrogated by the SOC inhibitor La3+, suggesting that basolaterally restricted capacitive
Ca2+ entry may play a key strategic role in activation of
PKC
and in regulation of dynamic basolateral membrane/cytoskeletal
remodeling. This concept was further supported by the observation that
CCh, in contrast to PMA, activates only PKC
and not PKC
. The
different pattern of isoform activation between PMA and CCh appears to
account for the different time courses of their effects on basolateral endocytosis. CCh induced an extended stimulation of fluid-phase tracer
uptake without the subsequent Gö-6976-sensitive inhibitory phase
observed with PMA attributable to PKC
.
The subcellular localization of inactive PKC isoforms and their dynamic
translocation to other subcellular compartments upon activation likely
accounts for specificity in regulation of various biological processes
(3, 8, 15). Spatial constraints may therefore determine
the relative availability or accessibility of key activating cofactors
to the PKC isozymes. Thus the location of inactive cPKC or nPKC
isoforms in reference to sites of DAG generation in response to
phospholipase-coupled membrane receptors could determine their degree
of activation. In our initial report (28), we found that
the DAG mimetic PMA sequentially activated PKC and then PKC
. CCh,
an acetylcholine analog that induces DAG generation via basolaterally
located M3 muscarinic receptors, was also shown to activate
PKC
; however, there was no evidence of activation of PKC
by CCh
(27). We were puzzled by this observation, given the known
ability of CCh to generate DAG and to increase cytosolic
Ca2+ from phosphoinositol-sensitive stores. One possible
explanation is that DAG production in response to CCh occurs only in
the limited vicinity of the basolateral M3 receptor and is
available only to PKC
within this microdomain, whereas PMA from the
bulk solution can diffuse throughout the cytoplasm in essentially
unlimited capacity and can reach both PKC
and PKC
. Alternatively,
the increase in cytosolic Ca2+ elicited by CCh could be
spatially restricted to a subcellular localization in which either DAG
is not available or inactive PKC
is scarce.
Intracellular Ca2+ concentration ([Ca2+]i) plays a central signaling role for a variety of cellular functions. In polarized epithelial cells such as pancreatic acinar cells, Ca2+ signaling has been shown to occur in a highly compartmentalized fashion (35). Inositol 1,4,5-trisphosphate (IP3)-elicited [Ca2+]i release begins in an apical "trigger zone" (9, 10, 32), where the vast majority of IP3 receptors are localized (13, 19, 36). Spreading of Ca2+ through the cytoplasm is modulated by activation of ryanodine receptors localized at the basal pole of acinar cells (30). Ca2+ spreading is also dependent on agonist concentration (31). When cells are stimulated to low levels, Ca2+ spikes remain restricted to the apical pole. Greater levels of stimulation cause Ca2+ waves to propagate toward the basal pole. These findings indicate that agonist-specific regulation of Ca2+ signaling and compartmentalization are likely to exist in polarized cells and that activation of Ca2+-dependent PKC isoforms could be precisely regulated on the basis of agonist type.
Thapsigargin prevents refilling of Ca2+ stores by
inhibiting Ca2+-sequestering ATPase pumps in the
endoplasmic reticulum membrane. This slowly depletes Ca2+
stores and thereby activates SOCs. We have found that, in T84 cells,
the SOCs activated by thapsigargin are functionally restricted to the
basolateral membrane domain (26). Although CCh is known to
activate SOCs in other cell systems, we have found no evidence of
sustained activation of basolateral membrane SOCs by CCh in T84 cells
(Ref. 26 and unpublished data). Thus, whereas both thapsigargin and CCh induce an increase in
[Ca2+]i, only thapsigargin is associated with
sustained activation of basolateral SOCs. In the present study, we were
able to use the Ca2+-ATPase inhibitor thapsigargin to
selectively manipulate the timing of activation of PKC after PMA
stimulation. We found that thapsigargin prevented PMA-stimulated
endocytosis, an effect that was inhibited by the PKC
-selective
inhibitor Gö-6976 and that appeared to require basolateral
Ca2+ entry via a La3+-sensitive pathway.
Translocation of specific PKC isoforms to distinct intracellular
structures following activation likely represents one mechanism for
determining their unique physiological roles (17).
Targeting of activated PKC isoforms involves association with anchoring proteins such as receptors for activated C-kinase (RACKs). RACKs are
thought to increase PKC phosphorylating efficiency by stabilizing the
active kinase near its target substrate (25). RACKs
specific for different PKC isoforms have been identified (4,
18). Both PMA and CCh appear to induce PKC translocation
specifically to the basolateral domain. Although the specific RACK(s)
governing this response remains uncertain, F-actin has been shown to
represent a PKC
-specific RACK (23); in addition,
'-COP, which has been associated with caveolae, may also be a RACK
for PKC
(4).
We previously showed that stimulation of basolateral endocytosis by PMA
and CCh involved remodeling of the F-actin cytoskeleton via PKC and
the actin cross-linker MARCKS (myristoylated alanine-rich C kinase
substrate) (28). In the present study, we found that thapsigargin prevents PMA-induced actin remodeling and stimulation of
endocytosis in association with activation of PKC
, an effect blocked
by Gö-6976. The detailed basis for PKC
antagonism of PKC
-induced actin disassembly is unclear. The small GTPase protein RhoA, a well-known regulator of stress fiber formation in many cell
types, is a candidate target for PKC
(7, 16). One
possible scenario, therefore, is that thapsigargin, by inducing
basolateral Ca2+ influx via SOCs, allows PMA to activate
PKC
and, in turn, RhoA, which then prevents remodeling of F-actin
cytoskeleton by PKC
.
In summary, we have found that activation of PKC, whether by PMA
alone or in conjunction with thapsigargin, antagonizes the ability of
PKC
to disassemble basolateral F-actin and stimulate basolateral
membrane endocytosis in a model intestinal epithelium. Our data suggest
that cytoskeletal and membrane structure may be dynamically modulated
by a balance between nPKC
and cPKC
that appears to depend on
subtleties in agonist-regulated subcellular redistribution of the
isoenzymes and the microorganization of Ca2+ signaling.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. B. Matthews, Dept. of Surgery, Univ. of Cincinnati College of Medicine, 231 Albert B. Sabin Way, PO Box 670558, Cincinnati, OH 45267-0558 (E-mail: Jeffrey.Matthews{at}uc.edu).
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.
July 24, 2002;10.1152/ajpcell.00105.2002
Received 7 March 2002; accepted in final form 17 July 2002.
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