EDITORIAL FOCUS
PKC-
regulates basolateral endocytosis in human
T84 intestinal epithelia: role of F-actin and MARCKS
Jaekyung Cecilia
Song,
Bruce J.
Hrnjez,
Omid C.
Farokhzad, and
Jeffrey B.
Matthews
Department of Surgery, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
Protein kinase C (PKC) and the actin cytoskeleton are critical
effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the
apical or basolateral membrane is poorly defined. In the present study,
phorbol 12-myristate 13-acetate (PMA) and other activators of PKC
selectively enhanced basolateral but not apical fluid-phase endocytosis
in human T84 intestinal epithelia. Stimulation of basolateral
endocytosis was blocked by the conventional and novel PKC inhibitor
Gö-6850, but not the conventional PKC inhibitor Gö-6976,
and correlated with translocation of the novel PKC isoform PKC-
. PMA
treatment induced remodeling of basolateral F-actin. The actin
disassembler cytochalasin D stimulated basolateral endocytosis and
enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and
jasplakinolide. PMA induced membrane-to-cytosol redistribution of the
F-actin cross-linking protein myristoylated alanine-rich C kinase
substrate (MARCKS). Cytochalasin D also induced MARCKS translocation
and enhanced PMA-stimulated translocation of MARCKS. A myristoylated
peptide corresponding to the phosphorylation site domain of MARCKS
inhibited both MARCKS translocation and PMA stimulation of endocytosis.
MARCKS translocation was inhibited by Gö-6850 but not
Gö-6976. The results suggest that a novel PKC isoform, likely
PKC-
, stimulates basolateral endocytosis in model epithelia by a
mechanism that involves F-actin and MARCKS.
cytoskeleton; microfilaments; pinocytosis; protein kinase C
isoforms; cell polarity; carbachol; diacylglycerol
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INTRODUCTION |
PROTEIN KINASE C (PKC) represents a family
of serine-threonine protein kinases that are expressed to varying
degrees in all mammalian cells and that are implicated in numerous
biological events ranging from cell growth and differentiation to
cytoskeletal organization, membrane trafficking, ion transport, and
cell-cell communication (6, 19, 33). To date, at least 11 isoforms of
PKC are known. These isozymes exhibit different patterns of tissue,
cell, and subcellular distribution, suggesting that each subserves
distinct intracellular functions (24). PKC isozymes are subclassified
into three main groups depending on their requirements for
2,3-diacylglycerol (DAG) and Ca2+.
Tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate
(PMA) are well known to translocate, activate, and eventually
downregulate conventional (DAG and
Ca2+ dependent) and novel (DAG
dependent, Ca2+ independent)
isoforms by virtue of their high affinity for the DAG binding site of
these PKC isozymes. The demonstration that PMA experimentally affects a
given cellular property is often taken as evidence for the regulatory
involvement of one or more classical or novel PKC isoforms in that
process. However, because neither the specific isoform nor its
downstream target(s) can be inferred from such observations, such data
can yield only limited mechanistic insight into the precise role of PKC.
PMA complexly influences a number of fundamental properties of
epithelial cells, including vectorial transport. For example, a number
of investigators showed that PMA affects epithelial
Cl
secretion, the
physiological process that accounts for mucosal surface hydration and
the transport event whose disregulation accounts for diseases such as
cystic fibrosis and secretory diarrhea. In several epithelial cell
lines, PMA progressively inhibits cAMP-regulated Cl
secretion (3, 30, 36,
43). The mechanism underlying this observation has not been clearly
established. Initial studies in T84 cells suggested that PMA inhibition
of Cl
secretion might
involve inhibition of apical membrane
Cl
conductance via reduced
gene expression of the cystic fibrosis transmembrane conductance
regulator (CFTR) (43). However, downregulation of CFTR mRNA and channel
function by PMA was shown to lag several hours behind, and thus cannot
account for, the loss of Cl
secretion. A closer temporal correlation with inhibition of two basolateral transport sites, the basolateral
Na+-K+-2Cl
cotransporter (NKCC1) and the basolateral
K+ conductance, was reported by
several groups (3, 29, 30, 36). We showed that PMA induces a loss of
binding sites for the NKCC1 inhibitor bumetanide (29) before any
detectable change in steady-state levels of NKCC1 mRNA or protein (17)
and suggested that PMA may reduce the surface expression of NKCC1.
Moreover, we recently found (32) that PMA decreases the surface
expression of another basolateral membrane transporter not directly
involved in Cl
secretion,
specifically, a facilitated nucleoside transporter. A unifying
explanation for these findings would be that PMA induces a generalized
increase in the rate of basolateral endocytosis by PKC, resulting in
the increased internalization of multiple membrane proteins including
those involved in transmembrane transport.
Plasma membranes are remodeled continually by endocytosis, a tightly
regulated multistep process that has been shown in diverse cell types
to be profoundly influenced by PMA and, hence, presumably, by PKC (2,
11, 27). In epithelial cells, the plasma membrane is subdivided into
distinct apical and basolateral domains and the process of membrane
cycling appears to be regulated in a polarized fashion (9). Recent
studies have shown that changes in fluid-phase endocytosis in
epithelial cells may indeed correlate with changes in the surface
expression of specific transport proteins, although to date, this has
appeared to apply primarily if not exclusively to the apical membrane.
For example, in T84 cells, apical but not basolateral endocytosis is
inhibited by cAMP, an event that parallels a rapid increase in CFTR
Cl
channels (7, 8).
Although PMA was reported to exert no effect on apical membrane
endocytosis in T84 cells (6a), basolateral membrane effects have not
been addressed. However, in Madin-Darby canine kidney
epithelial cells, Mostov and co-workers (11) showed that activation of
PKC increased basolateral endocytosis, transcytosis of membrane markers
from the basolateral to the apical domain, and apical recycling.
Many fundamental questions about the role of PKC and specific PKC
isoforms in the regulation of endocytosis remain unanswered. PMA exerts
profound effects on cell shape and the organization of actin (16, 42).
Although there are numerous reports linking cortical F-actin networks
to various forms of endocytosis, the precise relationship between the
effects of PKC on cytoskeletal organization and membrane traffic has
yet to be established. In the present study, we examine the role of PKC
in fluid-phase endocytosis in polarized T84 cells and address its
isoform selectivity and potential cytoskeletal dependence. In
particular, we examine the potential roles of F-actin and the F-actin
cross-linking protein myristoylated alanine-rich C-kinase substrate
(MARCKS) (21) in the regulation of endocytosis by PKC.
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EXPERIMENTAL PROCEDURES |
Cell culture.
Human T84 intestinal epithelial cells obtained from American Type
Culture Collection (Manassas, VA) and Dr. K. Barrett (University of
California, San Diego) were grown to confluence at pH 7.4 in 162-cm2 flasks (Corning Costar)
with a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12
nutrient mixture (Ham) supplemented with 6% fetal bovine serum (FBS),
15 mM HEPES, 14.3 mM NaHCO3, and antibiotics-antimycotic. Flasks were passaged weekly and fed every 3 days. Cell monolayers for experiments were grown to confluence on
collagen-coated Transwell inserts (Corning Costar, MA). Cells were fed
every 3 days and used after stable transepithelial electrical resistance was achieved, ~7-14 days after plating.
Fluid-phase endocytosis.
Uptake of FITC-dextran (mol wt 12,000, 0.73 mol fluorescein/mol
dextran) from either the apical or the basolateral aspect of confluent
T84 monolayers grown on collagen-coated permeable supports (4.7 cm2, 3.0-µm pore size) was
measured. Monolayers were incubated with 15 mg/ml of FITC-dextran in
either apical or basolateral medium for 6 min at 4 or 37°C.
Monolayers were then washed extensively with ice-cold
HEPES-phosphate-buffered Ringer solution (HPBR) containing (in mM) 135 NaCl, 5 KCl, 3.33 NaHPO4, 1 CaCl2, 1 MgCl2, 10 glucose, and 5 HEPES at
pH 7.4. The membrane was excised from its plastic support, inserted
into 430 µl of distilled water, sonicated for 20 s (setting 3, model
440 Sonic Dismembrator, Fisher Scientific) and spun twice for 5 min at
14,000 g. Fluorescence of the clear
supernatant was measured by using a fluorimeter (Spex DM3000) with
excitation and emission set at 495 and 565 nm, respectively. Nonspecific (temperature insensitive) binding represented at most 50%
of total binding in control monolayers and was not affected by PMA
treatment. Because it would have been impractical and costly to include
parallel experiments at both 4 and 37°C for each experimental condition, the data reported here represent total uptake of
FITC-dextran.
Peptide synthesis.
A 25-amino acid tetra-serine (tet-Ser) peptide containing 4 serine
residues and corresponding to the phosphorylation site domain (PSD) of
MARCKS was synthesized by SynPep (Dublin, CA) and was >98% pure
as determined by HPLC and mass spectrographic analysis. A tetra-alanine
(tet-Ala) peptide that shares the same sequence as the tet-Ser,
except that the four serines are replaced by alanines, was also
synthesized as a negative control peptide. The sequences of the
peptides are tet-Ser peptide: KKKKKRFSFKKSFKLSGFSFKKNKK and tet-Ala
peptide: KKKKKRFAFKKAFKLAGFSFKKNKK. To render the peptides cell
permeant, they were myristoylated at the
NH2 terminus.
Fluorescent microscopy.
Fluorescent staining of F-actin was performed as previously described
(22, 39). Polarized T84 cells grown to ~90% confluence on
collagen-coated Anocell inserts (0.33 cm2, 0.2-µm pore size, Whatman)
were rinsed in PBS, fixed in 3.7% formaldehyde, and permeabilized in
20°C acetone. F-actin was detected by staining the cells
with rhodamine-phalloidin for 1 h at room temperature. After being
washed in PBS, filters were mounted on microscope slides in Vectashield
mounting medium. Confocal images were acquired using a Zeiss inverted
microscope equipped with MRC-1024 and Lasersharp software (Bio-Rad).
Subcellular fractionation.
T84 cells grown to confluence on collagen-coated permeable supports
were washed with ice-cold PBS three times and scraped into cold
homogenization buffer (HB) containing (in mM) 20 Tris · HCl (pH 7.5), 250 sucrose, 4 EDTA, and 2 EGTA
and complete protease inhibitor cocktail. The cells were homogenized on
ice with 25 strokes of a glass tissue homogenizer. The resulting
homogenate was ultracentrifuged at 86,000 g for 50 min at 4°C (TLA 45 rotor, TL-100 Ultracentrifuge, Beckman). The supernatant was designated as the
cytosolic fraction. The pellet was resuspended in 800 µl of HB
containing 0.5% (vol/vol) Triton X-100 by brief sonication 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 as the
membrane fraction.
Immunoprecipitation of MARCKS.
Equal amounts of protein (~2.5 mg/sample) extracted in HB were
further solubilized in 1% Triton X-100, 0.5% NP-40, and 60 mM
n-octyl
-D-glucopyranoside. These
samples were first precleared with 10% (vol/vol) protein A agarose for
30 min at 4°C and subjected to immunoprecipitation with 5 µg of
monoclonal anti-human MARCKS overnight at 4°C. After overnight
incubation, 10% (vol/vol) protein A agarose was added to each sample,
incubated for 1.5 h at 4°C, and centrifuged for 2 min at 14,000 g. The pellet with bound MARCKS was
washed three times with HB containing 1% Triton X-100 and 0.5% NP-40
and finally with HB containing no detergents. The pellets were mixed
with 40 µl of Laemmli's sample buffer containing 5% (vol/vol)
-mercaptoethanol and boiled for 5 min. After brief vortexing, the
pellets were centrifuged at 14,000 g
for 2 min and the supernatants were processed by SDS-PAGE as described
in Gel electrophoresis and Western
blotting.
Gel electrophoresis and Western blotting.
Equal amounts (~50 µg/sample) of protein, as determined by the
Bradford assay, were combined with Laemmli's sample buffer containing
5% (vol/vol)
-mercaptoethanol and boiled for 5 min. Proteins were
separated by electrophoresis on 10% SDS-PAGE gels and transblotted to
nitrocellulose membranes. The protein-bound nitrocellulose sheets were
first incubated for overnight at 4°C in a blocking buffer
containing 20 mM Tris (pH 7.5), 500 mM NaCl, and 5% nonfat dry milk.
Nitrocellulose sheets were then incubated with monoclonal anti-human
MARCKS (1 g/ml) in the blocking buffer for 1 h at room temperature and
rinsed for 30 min with a wash buffer containing 20 mM Tris, pH 7.5, 500 mM NaCl, and 0.2% Tween-20. Finally, the membranes were incubated with
horseradish peroxidase-conjugated goat anti-rabbit IgG antibody
(1:3,000 dilution) for 1 h at room temperature and washed for 30 min
with agitation, with the wash buffer changed every 5 min. MARCKS bands
were visualized with enhanced chemiluminescence (ECL) detection reagents.
Materials.
Tissue culture reagents were purchased from Life Technologies. 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 was from Boehringer
Mannheim, and rhodamine-phalloidin was from Molecular Probes.
Monoclonal antibodies against human MARCKS were purchased from Upstate
Biotechnology. Antibodies against PKC-
and
-
1 were obtained from Sigma,
anti-PKC-
and -
were obtained from Santa Cruz Biotechnology, and
anti-PKC-
was obtained from Alexis Biochemicals. Protein A agarose
was from Life Technologies, and Vectashield mounting medium was
purchased from Vector Laboratories. Calphostin C, myristoylated PKC
pseudosubstrate, Gö-6976, rottlerin, and myristoylated coenzyme A
were obtained from Calbiochem. Gö-6850 was purchased from Alexis
Biochemicals, and staurosporine was from Sigma. All other chemicals
were from Sigma.
Statistical analysis.
Data are reported as means ± SE. Data were analyzed by Student's
t-test for unpaired variates and by
two-way ANOVA, where appropriate, with
P < 0.05 considered statistically significant.
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RESULTS |
PKC selectively stimulates basolateral membrane fluid-phase
endocytosis.
Uptake of FITC-dextran from either the apical or the basolateral aspect
of confluent T84 monolayers was time dependent at 37°C and
completely blocked at 4°C (Fig. 1); by
fluorescent microscopy, FITC-dextran was visualized in punctate
distribution consistent with vesicular compartmentalization (not
shown). On the basis of these experiments, a 6-min uptake period at
37°C was chosen. Treatment of monolayers with PMA increased the
rate of basolateral but not apical uptake of FITC-dextran in a time-
and dose-dependent fashion. At 10 nM PMA, a time-dependent increase in
basolateral endocytosis is seen (Fig.
2A) that
reaches a peak at ~60 min, with the earliest increase detectable at
15 min after exposure. At higher concentrations of PMA, substantial and
dose-dependent increases in basolateral endocytosis were detectable
within 10 min (Fig. 2B). The
increase in endocytosis elicited by PMA was not uniformly sustained, an
effect that was particularly evident at higher concentrations of PMA.
For example, the peak stimulatory effect of 100 nM PMA was observed at
30 min (202 ± 24% control) and then declined thereafter
such that after 120 min the basolateral uptake was 128 ± 26%
control. We then examined whether the effect of PMA on basolateral
endocytosis was exerted via PKC. As shown in Fig.
3, two additional phorbol esters also
stimulated basolateral FITC-dextran uptake, whereas the PKC-inactive
-isomer of PMA exerted no effect. Additionally, the DAG analog
1-oleoyl-2-acetylglycerol as well as the nonphorbol PKC agonist
bryostatin 1 also markedly enhanced basolateral endocytosis.

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Fig. 1.
Uptake of FITC-dextran (FD) across apical and basolateral aspect of
confluent T84 monolayers. Time course of FITC-dextran uptake measured
at 4 and 37°C from either apical or basolateral aspect of confluent
T84 monolayers grown on 4.7-cm2
permeable supports is shown. Monolayers were incubated with 15 mg/ml
FITC-dextran in either apical (A) or
basolateral (B) medium for time
indicated (n = 3 at each time point)
at either 4 or 37°C.
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Fig. 2.
Phorbol 12-myristate 13-acetate (PMA) selectively stimulates
basolateral membrane fluid-phase endocytosis of T84 cells in time- and
dose-dependent fashion. Confluent T84 monolayers grown on
4.7-cm2 permeable supports were
treated with PMA, and the rate of FITC-dextran uptake was measured from
either apical or basolateral aspect of confluent monolayers as
described in EXPERIMENTAL PROCEDURES.
A: monolayers were exposed to 10 nM
PMA for time indicated (total n > 100), and 6-min uptake of FITC-dextran was measured and expressed as
percentage of control. PMA increased basolateral but not apical uptake
of FITC-dextran in time-dependent fashion
(P < 0.001 by ANOVA). Peak effect
was shown after 60-min exposure to 10 nM PMA.
B: monolayers were exposed to
indicated concentrations of PMA for 10 min. PMA dose-dependently
increased basolateral uptake of FITC-dextran.
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Fig. 3.
Activators of protein kinase C (PKC) enhance basolateral fluid-phase
endocytosis. Monolayers were treated with 2 phorbol esters known to
activate PKC as well as nonphorbol PKC agonists bryostatin 1 (bryo-1)
and 2,3-diacylglycerol (DAG) analog 1-oleoyl-2-acetylglycerol (OAG),
and uptake of basolateral FITC-dextran was measured. A 60-min exposure
to 100 nM phorbol dibutyrate (PdBu) and phorbol didecanoate (PdDc)
enhanced basolateral uptake (n = 5 and
n = 12, respectively), whereas
PKC-inactive -isomer of PMA exerted no effect
(n = 3). Additionally, 60-min exposure
to 100 nM bryo-1 (n = 5) and 20 µM
OAG (n = 3) markedly stimulated
basolateral endocytosis. * P < 0.0001.
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PMA-stimulated basolateral endocytosis occurs via activation of
PKC-
.
PMA-stimulated endocytosis was attenuated by the general PKC inhibitors
staurosporine, calphostin C, and a myristoylated pseudosubstrate inhibitor (Fig. 4). Isoform selectivity to
this effect was suggested by the finding that the conventional PKC
inhibitor Gö-6976 (26) and the PKC-
-selective inhibitor
rottlerin (27) did not block endocytosis at concentrations up to 10 µM. At 5 µM, Gö-6976 should completely inhibit PKC-
(IC50
2-6 nM for
conventional isoforms) but exerts no effect against novel PKC isozymes
(47). At 10 µM, rottlerin is rather specific for the novel isoform
PKC-
(IC50
3-6 µM),
weakly active against conventional isoforms
(IC50 ~ 30-40 µM), and
inactive against PKC-
(IC50 > 80 µM) (20a). Moreover, the bisindoylmaleinimide Gö-6850, which
inhibits both conventional and novel PKC isoforms at nanomolar
concentrations (47), nearly completely inhibited PMA-stimulated
endocytosis. Because Gö-6976 was completely ineffective at
blocking PMA-induced endocytosis, it is unlikely that a conventional
PKC isoform is responsible for the PMA effect. Similarly, PKC-
is
unlikely to be involved, because rottlerin failed to inhibit the PMA
effect. In contrast, the ability of Gö-6850 to block the PMA
effect strongly implicates PKC-
as the isoform underlying
PMA-stimulated endocytosis.

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Fig. 4.
General PKC inhibitors and the conventional and novel PKC inhibitor
Gö-6850 attenuate PMA-stimulated endocytosis. Monolayers were
treated with various PKC putative inhibitors for 1 h followed by 10 nM
PMA for 1 h in presence of inhibitors. Basolateral uptake of
FITC-dextran was subsequently measured and compared with control.
Nonselective PKC inhibitors including 1 µM calphostin C, 100 µM
staurosporine, and 100 µM myristoylated PKC pseudosubstrate peptide
significantly attenuated PMA-stimulated basolateral endocytosis (each
n > 3, P < 0.005). Conventional and novel
PKC isoform inhibitor Gö-6850 attenuated PMA-elicited endocytosis
at a concentration of 5 µM (n = 3, P < 0.01). However, conventional
(but not novel) PKC inhibitor Gö-6976 enhanced PMA-stimulated
endocytosis at a concentration of 5 µM
(n = 5, P < 0.01). PKC- inhibitor
rottlerin and atypical PKC inhibitor myristoylated coenzyme A (myr-coA)
did not attenuate PMA-stimulated endocytosis at concentrations up to 10 µM. None of the agents that inhibited PMA-elicited endocytosis
altered FITC-dextran uptake in absence of PMA (not shown).
* P < 0.0001 for difference from control.
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Subsequent Western blot experiments identified four PKC isoforms in T84
cell homogenates (
,
,
, and
). We examined each PKC isoform
for evidence of activation (defined as translocation from cytosol to
membrane fraction) in response to PMA within a time period that could
account for stimulation of endocytosis. In response to 100 nM PMA,
PKC-
translocated within 10 min, consistent with the earliest
detectable effect on endocytosis (Fig. 5).
PKC-
also showed evidence of translocation, but this was not evident until 60 min after treatment (data not shown). Thus the temporal correlation of PKC-
movement in response to PMA with the stimulation of endocytosis, combined with the sensitivity of endocytosis to Gö-6850, strongly implicates the PKC-
isoform in this event.

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Fig. 5.
PMA induces membrane translocation of PKC- . Cytosolic (C) and
membrane (M) fractions of T84 homogenates were obtained as described in
EXPERIMENTAL PROCEDURES. Fractions
were subjected to SDS-PAGE and blotted with PKC isoform-specific
antibodies to examine redistribution of different PKC isoforms in
response to PMA. Four different PKC isoforms, , , , and ,
were detected in T84 cells and were found to be dominantly associated
with cytosolic fraction in inactive forms.
PKC- 1 was not found in these
cells by Western blot. In response to 100 nM PMA, PKC- translocated
to membrane fraction within 10 min, a time consistent with earliest
detectable effect of PMA on endocytosis. Translocation of other PKC
isoforms was not evident. Representative experiment shown was repeated
3 times.
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PMA-stimulated endocytosis is associated with remodeling of F-actin
cytoskeleton.
Fluorescent imaging of rhodamine-phalloidin-labeled actin filaments in
PMA-treated cells revealed attenuation of stress fibers and
condensation of staining around the periphery of the cells, changes
that were confined to the region below the perijunctional actin ring in
monolayers viewed en face (Fig. 6). No
changes were evident at the level of F-actin within the core of
microvilli. Because this pattern resembled that previously reported for
the microfilament disassembler cytochalasin D (28), we examined whether
cytochalasin D also affected fluid-phase endocytosis. Treatment of T84
monolayers with 20 µM cytochalasin D (but not the F-actin-inactive
analog chaetoglobosin in equimolar concentrations; not shown) enhanced
basolateral uptake of FITC-dextran. The enhancement of endocytosis by
PMA and cytochalasin D together was no greater than when maximal
stimulatory concentrations of either agent were used (Fig.
7); at lower concentrations, however, there
appeared to be additivity (not shown), observations that suggest a
common target of action (e.g., the actin cytoskeleton). To further
address whether PMA-stimulated endocytosis was associated with
reorganization of the actin cytoskeleton, we used two structurally
distinct F-actin stabilizers, phalloidin and jasplakinolide. In
monolayers loaded by overnight incubation in medium containing 10 µM
phalloidin and in monolayers treated with 1 µM jasplakinolide for 1 h, stimulation of FITC-dextran uptake by PMA was markedly attenuated
(Fig. 7). Neither jasplakinolide nor phalloidin prevented actin
reorganization induced by cytochalasin D, and neither agent blocked
cytochalasin D-induced endocytosis (not shown).

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Fig. 6.
PMA remodels basolateral F-actin. Confocal images of
rhodamine-phalloidin labeled actin filaments were acquired from T84
monolayers as described in EXPERIMENTAL
PROCEDURES. Three fields from each slide were chosen at
random, and vertical (z) sections
were collected at a step size of 1 µm. Cell heights of each monolayer
were measured from tip of microvilli to basal membrane on the basis of
staining of rhodamine-phalloidin. A:
mean cell height of control monolayers was 31.9 ± 3.2 µm
(n = 9). Basal stress fibers appeared
as randomly dispersed homogeneous filaments at 6.0 ± 0.7 µm above
basal membrane. B: after 1-h exposure
to 100 nM PMA, mean cell height remained unchanged at 34.2 ± 3.4 µm (n = 8). At optical section of
6.6 ± 0.9 µm above basal membrane, F-actin was displaced toward
periphery of cells and basal stress fibers were significantly
attenuated. Representative images are shown. PMA-treated cells could be
distinguished from control monolayers on the basis of these features in
100% of monolayers examined.
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Fig. 7.
Effect of chemical manipulation of F-actin on basal and PMA-stimulated
basolateral endocytosis. Monolayers were treated with 20 µM
cytochalasin D (cyto D) for 1 h, and basolateral uptake of FITC-dextran
was compared with control and PMA (100 nM, 1 h)-treated monolayers.
Cytochalasin D alone enhanced basolateral uptake of FITC-dextran to a
degree comparable to effect of PMA. In contrast, F-actin stabilizers
phalloidin (phall) and jasplakinolide (jas) blocked PMA stimulation of
endocytosis. Overnight treatment with 10 µM phalloidin and 1-h
treatment with 1 µM jasplakinolide markedly attenuated PMA
stimulation of FITC-dextran uptake (P < 0.0005 compared with PMA treatment). * P < 0.0001 for
difference from control.
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PMA and cytochalasin D elicit biochemical redistribution of MARCKS.
MARCKS is a widely distributed PKC substrate that has been implicated
in secretion and membrane trafficking in a number of cell types (1). It
is a membrane-associated actin cross-linking protein (21) that is known
to translocate from a membrane to a cytosolic location in response to
PKC-dependent phosphorylation, an event associated with cytoskeletal
remodeling (1). We therefore wondered whether stimulation of
endocytosis by PMA and cytochalasin D was associated with effects on
MARCKS dynamics in T84 cells. Immunoprecipitation and Western blotting
of T84 cell lysates using a monoclonal antibody raised against
recombinant human MARCKS detected a single band corresponding to a
molecular mass of ~65 kDa after SDS-PAGE (Fig.
8). In control monolayers,
MARCKS was found approximately equally distributed between the membrane
and the cytosolic fraction of homogenized T84 cells. In response to PMA, however, MARCKS was observed to translocate from the membrane to
the cytosolic fraction. Over this same time period, PKC-
translocated from the cytosolic to the membrane fraction. Translocation
of MARCKS was blocked by Gö-6850, whereas the conventional
isoform inhibitor Gö-6796 and PKC-
-selective inhibitor
rottlerin failed to do so (Fig. 9).
Cytochalasin D also induced translocation of MARCKS in response to PMA
(Fig. 10), consistent with its ability to
enhance basolateral endocytosis as shown in Fig. 7.

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Fig. 8.
PMA releases membrane-bound myristoylated alanine-rich C kinase
substrate (MARCKS) into cytosol. A:
immunoprecipitation of MARCKS from cell lysates using a monoclonal
antibody against recombinant human MARCKS identified a single protein
of mol wt ~ 65,000. B: T84
monolayers were exposed to 10 nM PMA for time indicated (min), and
subcellular fractions were obtained as described in
EXPERIMENTAL PROCEDURES. Both
cytosolic (C) and membrane (M) fractions were subjected to SDS-PAGE and
probed with antibodies to PKC- and MARCKS. In control monolayers,
MARCKS was equally distributed between the membrane and the cytosolic
fraction of homogenized T84 cells. In response to 100 nM PMA, however,
MARCKS translocated from membrane to cytosolic fraction and by 60 min,
MARCKS completely associated with cytosolic fraction. Over this same
time period, PKC- migrated to membrane fraction.
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Fig. 9.
Gö-6850, but not Gö-6976 or rottlerin, blocks PMA-induced
translocation of MARCKS. Monolayers were pretreated with a panel of PKC
isoform-specific inhibitors, and translocation of MARCKS was then
determined in response to 100 nM PMA.
A: MARCKS was immunoprecipitated from
subcellular fractions of T84 homogenates and blot obtained from
SDS-PAGE was probed with anti-human MARCKS. A 1-h pretreatment with 5 µM Gö-6850 blocked PMA-induced translocation of MARCKS from
membrane (M) to cytosolic (C) fraction. However, conventional PKC
inhibitor Gö-6976 and PKC- inhibitor rottlerin failed to block
MARCKS translocation. B: densitometric
analysis of data from A, expressing
percentage of total MARCKS found in cytosolic fractions (cyto MARCKS).
Similar data were obtained in duplicate experiment.
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Fig. 10.
F-actin disassembler cytochalasin D enhances PMA-induced translocation
of MARCKS. Monolayers were pretreated with 20 µM cytochalasin D for 1 h before subsequent exposure to PMA. MARCKS was immunoprecipitated from
cytosolic (C) and membrane (M) fractions of cell homogenates and
detected by SDS-PAGE and Western blotting with anti-human MARCKS
antibody. A: in response to 20-min
exposure to 100 nM PMA, MARCKS translocated from membrane to cytosol as
shown in previous figures. A 1-h treatment with 20 µM cytochalasin D
alone also showed effects similar to those of PMA on MARCKS
translocation. B: distribution of
MARCKS between cell fractions was determined by densitometric analysis,
and amount of cytosolic MARCKS after each treatment is expressed in
percentage of total. Similar results were confirmed in duplicate
experiment.
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|
A myristoylated peptide corresponding to PSD of MARCKS inhibits
PMA-stimulated basolateral endocytosis.
To determine whether the effects of PMA on MARCKS could be related to
its effect on basolateral endocytosis, we used a synthetic 25-amino
acid peptide containing 4 serine residues (tet-Ser) corresponding to
the PSD of MARCKS. tet-Ser has been shown to inhibit PKC-dependent phosphorylation of MARCKS as well as a number of other PKC targets in
vitro (20). Exposure of T84 monolayers to this peptide did not alter
PMA-stimulated endocytosis (not shown). However, myristoylation of this
peptide, which was hypothesized not only to enhance its membrane
permeability but also to concentrate it at hydrophobic sites similar to
MARCKS, enabled it to markedly inhibit PMA-stimulated endocytosis (Fig.
11). A similar 25-amino acid peptide in
which the 4 serine residues were replaced by 4 alanine residues
(tet-Ala) fails to block PKC-dependent MARCKS phosphorylation in vitro, although it functions as an effective inhibitor of phosphorylation of
other PKC targets (20). This cognate tet-Ala peptide, modified in
similar fashion by NH2-terminal
myristoylation, did not inhibit PMA-elicited endocytosis. The tet-Ser
PSD peptide was also found to block translocation of MARCKS from the
membrane to the cytosolic fraction in response to PMA, whereas tet-Ala
did not (Fig. 12).

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Fig. 11.
A myristoylated peptide corresponding to phosphorylation site domain
(PSD) of MARCKS inhibits PMA-stimulated basolateral endocytosis. A
myristoylated 25-amino acid peptide containing 4 serine residues and
corresponding to PSD of MARCKS (tet-Ser) and a similar myristoylated
25-amino acid peptide in which the 4 serine residues are replaced by 4 alanine residues (tet-Ala) were synthesized. Monolayers were
treated with 25 µM tet-Ser peptide for 1 h, and basolateral uptake of
FITC-dextran was measured in response to PMA. A 1-h exposure to 10 nM
PMA in absence of tet-Ser peptide increased basolateral endocytosis
(n = 6). However, preincubation with
tet-Ser peptide inhibited PMA-stimulated endocytosis by ~80%
(n = 6, P < 0.005). Treatment with the
tet-Ala peptide, however, did not alter PMA-stimulated endocytosis
(n = 3). * P < 0.0001 for
difference from control.
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Fig. 12.
The tet-Ser peptide corresponding to PSD site of MARCKS inhibits
PMA-induced translocation of MARCKS.
A: monolayers were treated with either
25 µM tet-Ser peptide or 25 µM tet-Ala peptide for 1 h followed by
20-min exposure to 100 nM PMA. PMA-induced translocation of MARCKS from
membrane (M) to cytosolic (C) fraction was blocked by tet-Ser peptide
but not by tet-Ala peptide. B: MARCKS
distribution was quantified by densitometric analysis and expressed as
percent cytosolic MARCKS of total. Similar data were obtained in
duplicate experiment.
|
|
Acetylcholine analog carbachol enhances basolateral endocytosis by a
mechanism similar to PMA.
Carbachol (CCH) acts via muscarinic receptors to stimulate
Cl
secretion in T84
monolayers and native tissue, an effect that involves an increase in
intracellular Ca2+ (13). CCH is
known to induce phospholipid turnover and generate DAG, thereby
activating PKC (4, 13, 14). As shown in Fig. 13, CCH elicited an increase in
basolateral endocytosis, an effect that persisted at least an hour
after addition, well after termination of the associated
Cl
secretory response.
However, the Ca2+-ATPase inhibitor
thapsigargin, which is not known to activate PKC directly, did not
increase endocytosis (data not shown), indicating that the effect of
CCH on endocytosis can be dissociated from its effects on
transepithelial secretion. As also shown in Fig. 13, CCH induced
PKC-
but not PKC-
translocation within 30 min, consistent with
its ability to induce basolateral endocytosis, and the effect on
endocytosis was inhibited by Gö-6850 but not Gö-6796. Taken
together, these data suggest that a physiological PKC stimulus such as
muscarinic stimulation enhances basolateral endocytosis.

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Fig. 13.
Carbachol (CCH) activates PKC- and elicits an increase in
basolateral endocytosis. A: monolayers
grown on 75-cm2 permeable supports
were treated with 100 µM CCH for 30 min, and "cytosolic" (C)
and "membrane" (M) fractions of homogenates were obtained as
described in EXPERIMENTAL PROCEDURES.
Fractions were subjected to SDS-PAGE and blotted with specific
antibodies against PKC- and PKC- to examine redistribution of the
2 PKC isoforms in response to CCH. PKC- , but not PKC- ,
translocated to membrane fraction within 30 min of CCH treatment.
Representative experiment shown was repeated 3 times.
B: Monolayers grown on
4.7-cm2 permeable supports were
treated with either 5 µM Gö-6850 or 5 µM Gö-6976 for 1 h followed by 100 µM CCH for 30 min in presence of inhibitors.
Basolateral uptake of FITC-dextran was subsequently measured and
compared with control. CCH increased basolateral endocytosis
(n = 15, * P < 0.001), which was
inhibitable by pretreatment with Gö-6850
(n = 3, P < 0.05) but not Gö-6976
(n = 3, P < 0.05).
|
|
 |
DISCUSSION |
This study demonstrates that phorbol esters such as PMA selectively
enhance basolateral but not apical endocytosis of a fluid-phase marker
in polarized human T84 intestinal epithelial monolayers. This action is
shared not only by bryostatin 1 and a cell-permeant DAG analog but also
by CCH (an analog of acetylcholine, which is an endogenous regulator of
intestinal transport function). The sensitivity of this response to a
panel of putative isoform-selective inhibitors of PKC as well as the
temporal correlation with PKC isoform translocation implicates a role
for the novel PKC-
isozyme. In response to PMA, PKC-
translocated
from the cytosolic to the membrane/particulate fraction within a time
frame consistent with the early enhancement of endocytosis. Information
regarding the role of specific PKC isoforms in plasma membrane dynamics
is scant, but the available evidence suggests that individual isoforms
may positively or negatively affect membrane traffic. For example, PKC-
was shown to associate with caveolae (31), implicating a role
for this isozyme in clathrin-independent endocytosis; however, activation of PKC-
by phorbol esters appears to inhibit rather than
enhance their internalization (41). Consistent with this notion, the
conventional PKC inhibitor Gö-6796 slightly enhanced rather than
inhibited PMA-elicited basolateral endocytosis in our experiments. The
specific function(s) of PKC-
are poorly understood, but available
data have suggested that it plays a role in, among other processes,
cell differentiation (34), oncogenesis (10), growth factor-stimulated
proliferation (5), transduction of mechanical shear stress (44),
synaptic function, and mucin exocytosis (23).
Surface expression of a given membrane protein represents a dynamic
balance between its rate of exocytic insertion and endocytic retrieval.
Activation of PKC has been shown to increase the internalization of a
number of membrane markers, including receptors for transferrin, asialoglycoprotein, lactate dehydrogenase, the chemokine CXCR4, and
various growth factors. Whether PKC enhances the endocytic retrieval of NKCC1 or other transport proteins from the basolateral membrane of secretory intestinal epithelial cells remains to be directly examined, although functional evidence to date supports such
an effect. A similar hypothesis was recently proposed in A6 renal
epithelia on the basis of the observation that PMA decreased basolateral binding of the
Na+-K+-ATPase
inhibitor ouabain in parallel with a decrease in basolateral membrane
surface area as well as an increase in fluid-phase endocytosis. NKCC1
contains several potential PKC phosphorylation sites, raising the
possibility that phosphorylation of NKCC1 at a specific PKC site could
alter its affinity for the recycling process. However, it has been
convincingly demonstrated that PMA-elicited endocytosis of the
transferrin receptor and
Na+-K+
ATPase does not involve their PKC-dependent phosphorylation, because
enhanced internalization persists despite the presence of PKC site
mutations. The possible role of the PKC-
isoform has not, to our
knowledge, been heretofore addressed.
Membrane trafficking is a complex process that involves vesicle
budding, specific transport, vesicle docking, and membrane fusion
events. A bewildering number of proteins have been identified in recent
years that may participate in or regulate these various steps.
Understanding the specific role of PKC in plasma membrane remodeling
and endocytosis is further complicated by the presence of multiple
pathways rather than a single pathway for endocytosis. At present, at
least five independent forms of endocytosis are recognized: a
clathrin-dependent pathway, macropinocytosis, a caveolar pathway, a
clathrin- and caveolin-independent pathway, and phagocytosis. The
molecular mechanisms involved in these pathways are distinct (37) and
appear to be independently regulated. Uptake of fluid-phase markers can
be differentially affected by stimuli that regulate clathrin-dependent
and -independent endocytosis. Interestingly, the five major endocytic
pathways all share the common feature of involvement of the actin
cytoskeleton, although agents that affect microfilament architecture
have been shown to exert opposite effects on these processes in many
instances. For example, in proximal tubule-derived opossum kidney
cells, phorbol esters and cytochalasin D enhanced fluid-phase uptake of
FITC-dextran but inhibited adsorptive endocytosis of albumin (18), and
in Vero kidney cells, the endocytic uptake of the membrane probe ricin
and the fluid-phase marker lucifer yellow could be selectively
modulated by cytochalasin, phorbol esters, and epidermal growth factor
without alteration of the clathrin-dependent uptake of transferrin
receptor (38).
The plasma membrane is intimately associated with a dense actin
filament cortex beneath the cytoplasmic leaflet of the lipid bilayer,
and it is perhaps not surprising that cortical actin interacts with
various pathways of endocytosis and exocytosis (18, 25, 40, 46).
Disruption of cortical actin appears to inhibit receptor-dependent
endocytic events, perhaps by altering the clustering of ligands within
coated pits or by affecting the molecular mechanisms of detachment of
newly formed membrane invaginations. However, cortical F-actin appears
also to act as a physical barrier to membrane fusion and budding
events, and disassembly of this layer by chemical manipulation of actin
polymerization or by agonist-regulated pathways appears to locally
destabilize the plasma membrane and thereby favor a generalized
increase in both endocytic and exocytic processes. Phorbol esters exert
profound effects on cell shape and membrane spreading, effects that
have been linked to their ability to reorganize actin filaments,
microtubules, and their associated proteins (2). The present
experiments suggest a link between the ability of PKC to remodel
F-actin and the ability to enhance basolateral fluid-phase endocytosis.
PKC-
has been proposed to affect membrane traffic in other cells by
an actin-dependent mechanism. For example, activated PKC-
has been
shown to bind actin within nerve endings and has been proposed to
thereby participate in the exocytic neurotransmitter release (35).
Cytochalasin D, like PMA, enhanced basolateral uptake of FITC-dextran;
moreover, the effects of PMA on endocytosis were blocked by two
chemically dissimilar stabilizers of F-actin, phalloidin and jasplakinolide.
The present experiments further suggest a role for MARCKS or a
MARCKS-like protein in PKC-regulated basolateral endocytosis. Although
MARCKS is one of the major cellular targets of PKC, its precise
physiological function remains to be clearly established. MARCKS
appears to associate with plasma membranes either directly via its
myristoylated tail or indirectly via an effector protein (1). Hartwig
et al. (21) showed that nonphosphorylated MARCKS acts as an actin
cross-linking protein. Phosphorylation of MARCKS by PKC at four serine
residues in its effector domain inhibits its actin cross-linking
activity and results in translocation of MARCKS from the membrane
(presumably cytoskeletally associated) environment to the cytosol. A
number of PKC isoforms, including PKC-
, have been shown to be MARCKS
kinases (45). Our data are consistent with a model in which PMA, acting
via PKC-
, induces MARCKS phosphorylation and releases it from the
basolateral membrane; as a result, MARCKS-mediated actin cross-linking
is disrupted, actin filament disassembly is promoted, and membrane
invagination and endocytosis are favored. In support of this concept,
we demonstrated that 1) PMA induces
MARCKS translocation in T84 cells from membrane to cytosolic fraction
with an appropriate time course that parallels endocytosis;
2) pharmacological blockade of
PKC-
inhibits MARCKS movement and PMA-elicited endocytosis; and
3) a myristoylated tet-Ser PSD
peptide blocks MARCKS movement and endocytosis in response to PMA, but
a tet-Ala PSD peptide blocks neither. Such data should be interpreted
with caution, because a number of other potential PKC targets such as
adducin and MARCKS-related protein (MRP, also called
MacMARCKS or F52) also contain a MARCKS-like PSD
(12) and thus could be functionally inhibited by these novel myristoylated PSD peptides. Agents that affect actin
assembly were shown in glioma cells to induce MARCKS translocation and to enhance PKC-induced MARCKS translocation (15), effects that we
confirm for intestinal epithelial cells in the present study.
In summary, the present study indicates that phorbol esters, acting via
the novel Ca2+-independent
-isoform of PKC, selectively enhance basolateral but not apical
membrane endocytosis in T84 cells by an F-actin-dependent mechanism. A
novel myristoylated PSD peptide inhibits both MARCKS translocation and
PMA-stimulated basolateral endocytosis, suggesting that MARCKS (or a
protein containing a MARCKS-like PSD) may represent a link between
PKC-stimulated actin rearrangement and endocytosis. This phenomenon may
account for the profound alteration of the transport characteristics of
the basolateral membrane of various polarized epithelia in response to
agents that affect PKC.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-48010 and DK-51630 and the
George H. A. Clowes, Jr., M.D., F.A.C.S. Memorial Career Development Award from the American College of Surgeons (J. B. Matthews).
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. B. Matthews,
Dept. of Surgery, Beth Israel Deaconess Medical Center, East Campus,
330 Brookline Ave., Boston MA 02215 (E-mail:
jmatthew{at}caregroup.harvard.edu).
Received 14 January 1999; accepted in final form 20 July 1999.
 |
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