Differential translocation of protein kinase C isozymes by
phorbol esters, EGF, and ANG II in rat liver WB cells
Judith A.
Maloney,
Oxana
Tsygankova,
Agnieszka
Szot,
Lijun
Yang,
Quiyang
Li, and
John R.
Williamson
Department of Biochemistry and Biophysics, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
The protein kinase
C (PKC) family represents an important group of enzymes whose
activation is associated with their translocation from the cytosol to
different cellular membranes. In this study, the spatial distribution
of PKC-
, -
and -
in rat liver epithelial (WB) cells has been
examined by Western blot analysis after subcellular fractionation.
Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtained
from cells stimulated with phorbol 12-myristate 13-acetate (PMA),
angiotensin II (ANG II), or epidermal growth factor (EGF). PMA caused
most of the PKC-
, -
and -
initially present in the cytosol to
be transported to the membrane and nuclear fractions. In contrast, both
ANG II and EGF induced only a minor translocation of PKC-
to the
membrane fraction but caused a statistically significant
membrane-directed movement of PKC-
and -
. Translocation of
PKC-
and -
to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC-
and -
were present in the cytoskeleton of resting cells, but although PMA,
ANG II, and EGF caused some changes in their content, these were
variable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK).
However, although PKC depletion inhibited EGF-induced mitogenesis, the
maximum EGF-induced activation of the ERK pathway was only slightly
retarded. We hypothesize that PKC-
and -
are involved in
mitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation to
distinct intracellular sites.
mitogen-activated protein kinase; extracellular regulated protein
kinase; Raf-1; mitogenesis; angiotensin II; epidermal growth factor
 |
INTRODUCTION |
PROTEIN KINASE C (PKC) is composed of a family of
serine/threonine kinases that modulate the function of a variety of
signal transduction pathways leading to gene expression, cell
proliferation, and differentiation. PKC isoforms can be classified into
three subgroups. The conventional PKC isozymes, namely,
,
I,
II, and
, are activated by
Ca2+, phosphatidylserine (PS) and
diacylglycerol (DAG), or phorbol esters [phorbol 12-myristate
13-acetate (PMA)]. The novel PKC isozymes, consisting of PKC-
,
-
, -
, -
, and -µ, are activated by PS and DAG or PMA but are
insensitive to Ca2+. The atypical
isoforms,
and
/
, are not affected by
Ca2+, DAG, or PMA but are
dependent on PS for activation (for review, see Ref. 33).
The evolution of numerous isoforms of PKC and their differential
expression in various tissues implies that they may possess specific,
possibly unique functions. Furthermore, various agonists induce the
translocation of different PKC isoforms to distinct subcellular
locations. For example, by overexpressing the different isoforms of PKC
into NIH/3T3 cells, which contain insignificant levels of isoforms
other than PKC-
, Goodnight et al. (14) were able to show that each
isoform translocates to a unique subcellular location after stimulation
with phorbol esters. These results indicate that the different isoforms
may have distinct roles in signal transduction pathways. This
suggestion is supported by evidence from studies in which selected
isoforms were either overexpressed or underexpressed using antisense
technology. In these studies, PKC-
was demonstrated to be involved
in differentiation and decreased cell proliferation (27, 30, 40),
whereas PKC-
and -
have been implicated in increased cell
proliferation (24, 30). However, although different isoforms appear to
be involved in different aspects of cell growth and differentiation,
their specific roles in signal transduction pathways have not been
elucidated.
The translocation of PKC from the cytosol to membranes has been used as
an indication of its activation. Early studies concerning PKC
translocation were performed with crude membrane fractions that
consisted of both plasma membrane and nuclear components. However, with
the consideration of its role in proliferation and differentiation, an
involvement of PKC, either directly or indirectly, in nuclear events is
indicated (9). In addition, activation of PKC induces cytoskeletal
reorganization (6, 32), and several PKC binding proteins have been
shown to associate with the actin cytoskeleton (1, 25). PKC has also
been shown to be involved in the activation of extracellular regulated
protein kinase (ERK), a major signaling pathway leading to cellular
proliferation in many cell types. Therefore, it was of interest to
investigate the role of PKC in the activation of ERK and one of its
upstream activators, Raf-1, by angiotensin II (ANG II) and epidermal
growth factor (EGF) in a nontransformed rat liver epithelial WB cell line.
In this study, we have shown that although PMA induced the
translocation of PKC-
, -
, and -
to the membrane and nuclear fractions in WB cells, there was a significant translocation of only
the
- and
-isoforms to these sites after ANG II or EGF stimulation. Furthermore, phorbol ester-sensitive PKC isoforms are
shown to play a role when the ERK pathway is stimulated by ANG II, but
they have a limited effect on EGF-induced ERK activation. Therefore, it
appears that PKC-
and/or PKC-
affect ANG II-induced mitogenesis via an ERK-dependent pathway, whereas the effect of PKC on
EGFinduced mitogenesis is essentially ERK independent.
 |
MATERIALS AND METHODS |
Materials.
EGF was purchased from UBI (Lake Placid, NY). ANG II, PMA, and myelin
basic protein (MBP) were obtained from Sigma Chemical (St. Louis, MO).
Rabbit polyclonal antibodies to PKC-
, PKC-
, and Raf-1 were from
Santa Cruz Biotechnology (Santa Cruz, CA), whereas monoclonal
antibodies to PKC-
were obtained from Transduction Laboratories
(Lexington, KY). A plasmid encoding an inactive
glutathione-S-transferase-coupled ERK kinase
(GST-MEK-1) was provided by Michael J. Weber (University of Virginia, Charlottesville, VA).
[
-32P]ATP was
purchased from Amersham Life Sciences (Arlington Heights, IL).
Cell culture.
WB cells are an epithelial cell that was originally isolated from the
liver of an adult Fischer rat (38). The cells were plated onto 100-mm
tissue culture plates and incubated in Richter's improved essential
medium containing L-glutamine
and insulin (Irvine Scientific, Santa Ana, CA) plus 10% fetal bovine
serum until confluent. Cells were incubated overnight in Richter's
medium without serum before the start of the experiment. Cells were
used between passages 20 and 40.
Subcellular fractionation.
Serum-starved WB cells were treated with agonist for 0-60 min as
indicated. Cells were washed twice with ice-cold PBS and scraped into
homogenization buffer containing 25 mM
Tris · HCl, pH 7.4, 2 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 mM
-mercaptoethanol, 10% glycerol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Cells were allowed to swell for 10 min and then
homogenized with 30 strokes of a Dounce homogenizer using a
tight-fitting pestle. This produced complete lysis of the cells as
determined by phase-contrast microscopy. Nuclei were pelleted by
centrifugation at 500 g for 5 min, and
the low-speed supernatant was centrifuged at 100,000 g for 30 min. The high-speed
supernatant constituted the cytosolic fraction. The pellet was washed
three times and extracted in ice-cold homogenization buffer containing 1% Triton X-100 for 30-60 min. The Triton-soluble component
(membrane fraction) was separated from the Triton-insoluble material
(cytoskeletal fraction) by centrifugation at 100,000 g for 15 min. The cytoskeletal fraction was washed three times with homogenization buffer, resuspended in the same buffer, and dispersed by sonication.
Nuclei (low-speed pellet) were resuspended in nuclear buffer containing
25 mM Tris · HCl, pH 7.4, 3 mM
MgCl2, 1 mM PMSF, 10 mM
-mercaptoethanol, and 0.05% Triton X-100 and homogenized with 10 strokes of a Dounce homogenizer to remove contaminating membrane components. They were centrifuged for 5 min at 500 g, resuspended in nuclear buffer
without Triton X-100, layered over 45% sucrose, and centrifuged at
1,900 g for 30 min. The purified
nuclei, which were visually free of cytoplasmic/cytoskeletal
attachments as assessed by phase-contrast microscopy, were resuspended
in homogenization buffer containing 1% Triton X-100 and incubated for
30-60 min. The small amount of insoluble material was removed by
centrifugation at 100,000 g for 15 min
at 4°C. Protein concentration was measured by the method of
Bradford (4) using BSA as a standard.
The purity of the subcellular fractions was assessed biochemically by
measuring lactate dehydrogenase (LDH) as a cytosolic marker and
oubain-sensitive
Na+-K+-ATPase
as a measure of plasma membrane contamination (13, 34). LDH activity
was 4.9 ± 0.3 U/mg protein in the cytosolic fraction, as compared
with 0.2 ± 0.03 and 0.5 ± 0.08 U/mg protein in the nuclear and
membrane fractions, respectively. The specific activity of
Na+-K+-ATPase
was 18 nmol · mg
1 · min
1
in the membrane fraction, whereas it was 0.7 nmol · mg
1 · min
1
in the nuclear fraction.
Western blot.
Ten to thirty micrograms of protein were applied to a 10%
SDS-polyacrylamide gel and electrophoresed. The amount of protein applied was routinely within the linear range for densitometric studies. The proteins were transferred to nitrocellulose membranes. Equal protein loading and the efficiency of protein transfer were assessed by staining the nitrocellulose membranes with Ponseau S. Membranes were blocked with 5% BSA in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 1 h and were then incubated with
isoform-specific anti-PKC antibodies for 1 h at room temperature, or
overnight at 4°C. Nitrocellulose membranes were washed three times
with PBST and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min. Protein bands were visualized by
enhanced chemiluminescence (ECL; Amersham). The results were analyzed
by densitometry, which was kept in the linear range of exposures, using
a Hewlett Packard scanner and SigmaGel software. In some experiments,
the nitrocellulose membrane was stripped by incubation for 1-2 h
in Immunopure Elution Buffer (Pierce, Rockford, IL), washed twice with
PBST, and then reblotted with a different antibody as described above.
Raf-1 assay.
After stimulation with ANG II or EGF, WB cells were lysed for 30 min on
ice with lysis buffer [10 mM Tris · HCl, pH
7.5, 100 mM NaCl, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 2 mM
Na3VO4,
1 mM 4-(2-aminoethyl)benzenesulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin]. Precleared cell
lysates were incubated with antibody against Raf-1 for 1 h on ice
followed by incubation with protein A-agarose with rotation for 1 h at 4°C. An irrelevant rabbit antibody was used as a negative control. The agarose beads were washed three times with the lysis buffer and
twice with kinase buffer (10 mM PIPES, pH 7.0, and 10 mM
MgCl2). The reactions were
carried out by addition of 5 µCi
[
-32P]ATP and 10 µg/ml GST-MEK to the kinase buffer at 30°C for 10 min, and
stopped by heating at 95°C for 5 min after the addition of Laemmli
sample buffer. The kinase assay samples were subjected to 10% SDS-PAGE
followed by gel drying and exposure to X-ray film at
86°C.
The results were analyzed by densitometry of the autoradiograms, which
was kept in the linear range of exposures, using a Hewlett Packard
scanner and SigmaGel software.
ERK assay.
Stimulation of WB cells with ANG II or EGF and immunoprecipitation of
ERK were carried out as described above using anti-ERK-2 antiserum.
Immunoprecipitates were washed once with lysis buffer, twice with
modified RIPA buffer (10 mM MOPS, pH 7.0, 150 mM NaCl, 0.1% SDS, 1%
Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, and 1 mM
Na3VO4)
and twice with kinase buffer. MBP (5 µg/ml) was used as substrate for
the ERK assay. The reaction was initiated by addition of 5 µCi
[
-32P]ATP and
carried out at 30°C for 15 min. The kinase assay samples were
subjected to 15% SDS-PAGE and analyzed as described for the Raf-1
assay.
DNA synthesis.
DNA synthesis was determined by
[3H]thymidine
incorporation into DNA. WB cells were serum starved for 48 h and
incubated with ANG II, EGF, or PMA for 24 h.
[3H]thymidine (2.5 µCi/ml) was added 16 h before the end of the incubation. The cells
were quickly washed three times with ice-cold phosphate-buffered
saline, incubated for 10 min with 2 ml of 10% (wt/vol)
trichloracetic acid (TCA), and washed twice with 2 ml of 10% TCA and
three times with 2 ml of 95% ethanol. The acid-insoluble precipitate
was incubated for 60 min in 800 µl of 0.2 N NaOH, and the solution
was neutralized with HCl. The radioactivity was determined by liquid
scintillation counting.
Statistical analysis.
Data are expressed as means ± SE. Comparison of the effect of
various hormonal treatments was performed by Student's
t-test. Differences with a
P value of <0.05 were considered
statistically significant.
 |
RESULTS |
Distribution of PKC-
in WB cells.
In unstimulated cells, PKC-
appeared as a single band at 80 kDa and
was present mainly in the cytosolic fraction (Fig.
1A). Similar findings were observed in insulinoma
-cells (21) and vascular smooth muscle cells (15). With longer times of ECL development, however, PKC-
could be detected as a faint band in the
membrane fraction of control cells, suggesting that it is present in
very low abundance in the membrane. Stimulation with 1 µM PMA induced
a rapid translocation of PKC-
from the cytosol to the membrane and
nuclear fractions (Fig. 1). Translocation of PKC-
was evident at 30 s, maximal at 15 min, and subsequently declined over the next 45 min.
This decline in membrane- and nuclear-bound PKC-
from 15 to 60 min
is probably because of proteolytic degradation. Very little PKC-
was
detected in the cytoskeleton fraction in control cells, and this was
not increased by treatment with PMA (data not shown). After stimulation
with ANG II, cytosolic levels of PKC-
were 76 ± 4% of control
after 30 s but returned to control levels by 1 min (Fig.
2). This corresponded to a transient
fourfold increase of PKC-
in the membrane fraction compared with a
>10-fold increase 5 min after PMA treatment. Similar results were
obtained with EGF (data not shown). Neither ANG II nor EGF induced the translocation of PKC-
to the nuclear or cytoskeleton fractions.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 1.
Phorbol 12-myristate 13-acetate (PMA)-induced translocation of protein
kinase C (PKC)- to membrane and nuclear fraction of WB cells.
Serum-starved WB cells were stimulated with 1 µM PMA for 0-60
min. A: subcellular fractions were
subjected to SDS-PAGE and immunoblotted with anti-PKC- antibodies.
B: Western blot analysis of
subcellular fractions immunoblotted with anti-PKC- antibody. Western
blots were quantitated by densitometric scanning. Results are means ± SE from 3 independent experiments.
* P < 0.05.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Angiotensin II (ANG II)-induced translocation of PKC- to membrane
fraction of WB cells. Serum-starved WB cells were stimulated with 1 µM ANG II for 0-60 min. Cytosolic and membrane fractions were
subjected to SDS-PAGE and Western blotting with anti-PKC-
antibodies. Similar results were obtained in 2 separate experiments.
|
|
Distribution of PKC-
in WB cells.
In unstimulated cells, PKC-
was present in the cytosolic, membrane,
and cytoskeletal fractions but was not detectable in the nuclear
fraction (Fig.
3A).
These findings are in agreement with other studies in which PKC-
has
been shown to be constitutively associated with the membrane component
of unstimulated cells (21, 37). PMA induced the translocation of
PKC-
from the cytosol to the membrane, nuclear, and cytoskeleton
fractions, but with different kinetics. By 5 min, PKC-
was
undetectable in the cytosolic fraction and within 1 min appeared in the
membrane, nuclear, and cytoskeleton fractions. Thereafter, there was an
additional PMA-induced translocation of PKC-
to the membrane and
nuclear fractions, whereas that in the cytoskeleton became depleted
(Fig. 3B). From 30 to 60 min after
PMA addition, there was a loss of PKC-
from the membrane and nuclear
fractions, as observed for PKC-
(Fig. 1).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 3.
PMA-induced translocation of PKC- to membrane, nucleus, and
cytoskeletal (CSK) fractions of WB cells. Serum-starved WB cells were
stimulated with 1 µM PMA for 0-60 min. Subcellular fractions
were subjected to SDS-PAGE and immunoblotted with anti-PKC-
antibodies. A: Western blot analysis
of subcellular fractions immunoblotted with anti-PKC- antibodies.
B: Western blots were quantitated by
densitometric scanning. Results are means ± SE of 3 or 4 independent experiments. * P < 0.05.
|
|
In the cytosol, membrane, and cytoskeletal fractions, PKC-
appeared
as a doublet of ~76 and 78 kDa. In the nucleus, however, PKC-
appeared as a single band with an apparent molecular mass of 78 kDa. In
the membrane and cytoskeletal fractions, PMA induced a time-dependent
mobility shift of PKC-
. When WB cells were treated with PMA,
immunoprecipitated with antiphosphotyrosine antibodies, and
immunoblotted with anti-PKC-
antibodies, there was an increased tyrosine phosphorylation of PKC-
(data not shown). This finding is
consistent with previous observations demonstrating tyrosine phosphorylation of PKC-
after PMA stimulation (26, 37).
Both ANG II and EGF caused a rapid, statistically significant
(P < 0.05) translocation of PKC-
from the cytosol to the membrane fraction, where it remained elevated
for the 1-h duration of the experiment (Fig.
4B).
With ANG II stimulation, there was also a rapid, sustained
translocation of PKC-
to the cytoskeleton fraction, with a peak at 1 min (Fig. 4C), but with EGF
stimulation the changes of PKC-
in the cytoskeleton fraction were
not statistically significant (Fig.
4F). As seen from the PKC-
immunoblot in Fig. 5A,
translocation of PKC-
to the nuclear fraction occurred more quickly
under the influence of ANG II than with EGF, but the amount translocated was much less than that induced by PMA. This kinetic difference was confirmed using an immunofluorescence approach with
primary antibodies to PKC-
and secondary antibodies labeled with
Texas red. Again, translocation induced by EGF was slower than that
with ANG II (data not shown).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
ANG II- and epidermal growth factor (EGF)-induced translocation of
PKC- to membrane and cytoskeletal fractions of WB cells.
Serum-starved WB cells were stimulated with either 1 µM ANG II
(A-C)
or 200 ng/ml EGF
(D-F)
for 0-60 min. Subcellular fractions were subjected to SDS-PAGE and
immunoblotted with anti-PKC- antibodies. Western blots were
quantitated by densitometric scanning and expressed either as percent
of control (cytosol, A and D) or percent of
maximal response [membrane (B and E) and
cytoskeleton (C and F) fractions]. Results are
means ± SE of 3-5 independent experiments.
* P < 0.05.
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 5.
ANG II- and EGF-induced translocation of PKC- to nuclear fraction of
WB cells. Serum-starved WB cells were stimulated with either 1 µM ANG
II or 200 ng/ml EGF for 0-60 min. Nuclear fractions were subjected
to SDS-PAGE and immunoblotted with anti-PKC- antibodies. Similar
results were obtained in 3 separate experiments.
|
|
Distribution of PKC-
in WB cells.
As previously observed in mouse neuroblastoma × rat glioma
(NG 108-15) cells (3) and insulinoma
-cells (21), PKC-
ran as a doublet in immunoblots of the cytosol and cytoskeletal fractions (Fig. 6). However, in the nuclear and
membrane fractions, PKC-
appeared as a single band. Additionally,
there was another 130-kDa band recognized by the PKC-
antibody that
did not change after PMA or hormonal stimulation (data not shown). This
band has been previously detected in insulinoma
-cells
(21), but its identity remains unknown.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
PMA-induced translocation of PKC- to membrane, nucleus, and
cytoskeletal fractions of WB cells. Serum-starved WB cells were
stimulated with 1 µM PMA for 0-60 min. Subcellular fractions
were subjected to SDS-PAGE and immunoblotted with anti-PKC-
antibodies. A: Western blot analysis
of subcellular fractions immunoblotted with anti-PKC- antibodies.
B: Western blots were quantitated by
densitometric scanning. Results are means ± SE of 3 or 4 independent experiments. * P < 0.05.
|
|
As with PKC-
and -
, PMA caused a complete loss of PKC-
from
the cytosol within 1 min (Fig. 6B,
top left). Translocation of PKC-
to both the membrane and nuclear fractions was observed after 30 s,
increased to a peak at 15 min, and remained elevated for up to 60 min
(Fig. 6B, top
right and bottom
left). PKC-
was present in the cytoskeleton of
resting cells, fell by 90% after 1 min, and subsequently increased
gradually to 60% of control values after 1 h (Fig.
6B, bottom
right). Interestingly, the most marked difference
between the translocations of PKC-
and PKC-
, as affected by PMA,
was to the cytoskeleton fraction where the initial increase observed
with PKC-
was not apparent with PKC-
.
After stimulation of the cells with ANG II, PKC-
decreased rapidly
in the cytosol, and like PKC-
subsequently returned partially to
basal levels (cf. Figs. 4A and
7A),
whereas the decrease of PKC-
observed with EGF was slower and
completely reversible (cf. Figs. 4D
and 7D). Removal of PKC-
from the
cytosol induced by both EGF and ANG II was associated with
translocation of PKC-
to the membrane fraction, where it increased
twofold (Fig. 7, B and
E). In contrast, the hormone-induced
translocation of PKC-
to the cytoskeletal fraction was small and did
not reach statistical significance. As observed for PKC-
, ANG II and
EGF both caused a small transient increase of PKC-
in the nucleus,
with the EGF-induced translocation being slightly more delayed than
that induced by ANG II (data not shown). The major effect of hormonal
stimulation in WB cells, therefore, was to elicit a rapid, substantial
translocation of both PKC-
and PKC-
from the cytosol to the
membrane fraction.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
ANG II- and EGF-induced translocation of PKC- to membrane and
cytoskeletal fractions of WB cells. Serum-starved WB cells were
stimulated with either 1 µM ANG II
(A-C)
or 200 ng/ml EGF
(D-F)
for 0-60 min. Subcellular fractions were subjected to SDS-PAGE and
immunoblotted with anti-PKC- antibodies. Western blots were
quantitated by densitometric scanning, and results are expressed either
as percent of control (cytosol, A and D) or
percent of maximal response [membrane (B and E)
and cytoskeleton (C and F) fractions]. Results
are means ± SE of 4 or 5 independent experiments.
* P < 0.05.
|
|
Effect of PKC downregulation on ANG II- and EGF-induced stimulation
of DNA synthesis and activation of Raf-1 and ERK2 in WB cells.
As shown in Figs. 1, 3, and 6, PMA acutely administered induced a rapid
translocation of PKC-
, -
, and -
to the nucleus, indicating
that PKC may exert a direct regulation of nuclear events. Alternatively, PKC may be translocated and activated at the plasma membrane with subsequent phosphorylation of substrates that convey signals to the nucleus through the mitogen-activated protein (MAP) kinase cascade (5). This latter possibility may be the primary one by
which signals are transmitted from activated receptors, since the
effects of EGF and ANG II on translocation of PKC isoforms directly to
the nucleus were small and transient (Fig. 5).
To investigate the effects of PKC activation on cell function in WB
cells, conventional and novel isoforms of PKC (which include PKC-
,
-
and -
) were downregulated by prolonged treatment of the cells
with PMA. Figure 8 shows the results of an
experiment that illustrates this phenomenon. The disappearance of
PKC-
, -
and -
isoforms, as determined by immunoblotting, was
followed over a 25-h period. Immunoreactive PKC-
was no longer
observed at the first assayed time point after 5 h, whereas PKC-
and
-
exhibited slower kinetics of downregulation.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 8.
Downregulation of PKC- , - , and - by prolonged PMA treatment of
WB cells. Serum-starved WB cells were treated with 1 µM PMA for
0-25 h. Whole cell lysates were subjected to SDS-PAGE and
immunoblotted with anti-PKC- , - , and - antibodies. Film was
overexposed in regard to control lane to adequately visualize PKC
isoforms at later time points.
|
|
In further experiments, these PKC isoforms were downregulated by
pretreatment of the WB cells with PMA for 24 h to assess the potential
role of hormone-stimulated PKC on cell proliferation and activation of
the MAP kinase pathway. The cells were subsequently stimulated with
either 1 µM ANG II or 200 ng/ml EGF for various times, and the
activities of Raf-1 and ERK were measured. In untreated cells, both
hormones stimulated a three- to sixfold increase in the activities of
Raf-1 and ERK, and these kinases remained activated for the 90 min of
the experiment (Figs. 9 and
10). In PMA-pretreated cells, there was
little effect of PKC depletion on the ANG II-induced activations of
both Raf-1 and ERK within the first 5 min, but after 60 and 90 min,
both kinases were significantly inhibited (Fig. 9). These data suggest
that the ANG II-induced stimulation of the ERK cascade is at least
partially PKC dependent, possibly at the level of Raf-1. However, after
15 min of ANG II stimulation, ERK activity was significantly decreased
in PKC-depleted cells, although there was no effect on Raf-1 activity.
Additional data will be needed to verify this point, which suggests
that ANG II may also activate ERK in a Raf-1-independent manner.
Similarly, ERK activity was significantly decreased by EGF in
PKC-depleted cells after 5 min, whereas Raf-1 activity was not
significantly affected. The major point of interest is that there was
no effect of PKC depletion on the EGF-induced sustained activation of
Raf-1 or ERK (Fig. 10). These results indicate that the EGF-induced
activation of the ERK pathway is minimally affected by those PKC
isoforms that can be downregulated by PMA. PKC-
was shown by
immunoblotting experiments to be present in both the soluble and
particulate fractions prepared from WB cells, but their relative
amounts were not affected by acute or prolonged addition of PMA (data
not shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 9.
ANG II-stimulated activation of Raf-1
(A) and extracellular regulated
protein kinase (ERK) (B) in control
and PKC-depleted cells. WB cells were pretreated with DMSO alone (open
bars) or with 1 µM PMA (hatched bars) for 18 h, and then stimulated
with 1 µM ANG II for various time intervals up to 90 min. Immune
complex kinase assays for Raf-1 and ERK activities were performed as
described in MATERIALS AND METHODS.
Protein kinase activity in cells treated with DMSO alone was taken as
100%. Results are means ± SE of 4 independent experiments.
* P < 0.05.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 10.
EGF-stimulated activation of Raf-1
(A) and ERK
(B) in control and PKC-depleted
cells. WB cells were pretreated with DMSO alone (open bars) or with 1 µM PMA (hatched bars) for 18 h and then stimulated with 200 ng/ml EGF
for various time intervals. Immune complex kinase assays for Raf-1 and
ERK activities were performed as described in
MATERIALS AND METHODS. Protein kinase
activity in cells treated with DMSO alone was taken as 100%. Results
are means ± SE of 3 independent experiments.
* P < 0.05.
|
|
Stimulation of the ERK pathway in many cells is known to be correlated
with increased mitogenesis. Consequently, it was of interest to
determine the effects of PKC downregulation on ANG II- and
EGF-stimulated proliferation in WB cells. Figure
11 shows that addition of 10 nM PMA, 1 µM ANG II, and 100 ng/ml EGF to WB cells produced increasing
percentage effects on
[3H]thymidine
incorporation into DNA. Pretreatment of the cells for 24 h with 500 nM
PMA completely inhibited the effects of ANG II and PMA and greatly
diminished the effect of EGF on DNA synthesis. These results indicate
that activation of PKC-
, -
, or -
is an important component of
the mitogenic pathway in WB cells.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 11.
Effects of PKC depletion on EGF- and ANG II-induced
[3H]thymidine
incorporation into DNA in WB cells. Serum-starved WB cells were
pretreated for 24 h with 500 nM PMA before stimulation with 10 nM PMA,
1 µM ANG II, or 100 ng/ml EGF and
[3H]thymidine (2.5 µCi/ml) for an additional 16 h. Control experiments (open bars) were
pretreated with DMSO alone, whereas PMA pretreatment is shown by
hatched bars. Results are means ± SE of 6 independent
experiments.
|
|
 |
DISCUSSION |
The role of different PKC isoforms in signal transduction pathways
remains unclear despite extensive studies. It has been suggested,
however, that each isoform may perform distinct functions via its
translocation to discrete regions within the cell. The present study
was initiated to investigate this possibility by examining the
translocation of PKC-
, -
, and -
to different subcellular
fractions after stimulation of WB cells with PMA, EGF, or ANG II. PMA
induced the translocation of all three isoforms from the cytosol to the
membrane and nuclear fractions. PKC-
and -
were present in the
cytoskeleton fraction of resting cells, but the major effect of PMA was
to cause a depletion of these PKC isoforms in the cytoskeleton. PKC-
and -
were also the major isoforms translocated from the cytosol to
the membrane fraction after ANG II and EGF stimulation. However, unlike
the changes induced by PMA, ANG II and EGF caused very little
translocation of PKC-
and -
to the nucleus. On the other hand,
hormonal stimulation of WB cells was essentially ineffective in causing
a translocation of PKC-
to membrane or nuclear fractions, suggesting
that it may play a relatively minor role in the signal transduction
pathway induced by these two agonists in WB cells. This finding
contrasts with the fact that PKC-
is ubiquitously expressed in
diverse cells types. However, its translocation induced by various
hormones and growth factors is variable (15, 17), although PKC-
translocation to the nucleus has been described (15). Interestingly,
arachidonic acid has been shown to induce the translocation of PKC-
to the particulate fraction in WB cells (16). This indicates that
PKC-
may respond to signaling through the phospholipase
A2 pathway in WB cells.
A number of reports have described the translocation of PKC-
, -
,
and -
to the Triton X-100-insoluble cytoskeletal fraction (19, 20,
35). In the present study, some differences were observed in the PMA-
or hormone-stimulated movement of PKC-
and -
to this fraction.
Phorbol ester and ANG II induced a rapid translocation of PKC-
to
the cytoskeleton, which was transient with PMA but sustained with ANG
II. On the other hand, there was no apparent effect of EGF in causing a
translocation of PKC-
to the cytoskeleton fraction. Interestingly,
there was a decrease in the amount of PKC-
bound to the cytoskeleton
after both PMA and ANG II treatment, but again EGF failed to give a
statistically significant movement of PKC-
to the cytoskeleton.
Taken together, these results suggest a differential activation
and/or subcellular targeting of PKC-
and -
after
stimulation with PMA, ANG II, and EGF.
In recent years, several PKC binding proteins have been identified that
associate with the membrane and the Triton X-100-insoluble cytoskeletal
fraction. These include receptors for activated C kinase (RACKS) (31),
myristoylated alanine-rich C-kinase substrate (MARCKS) (1), and the
adducins (10). Although there has been no definitive evidence that a
particular PKC isoform preferentially associates with a specific
binding protein in vivo, a peptide based on the RACK binding site of
PKC-
was shown to inhibit the translocation of PKC-
and -
after phorbol ester addition to cardiac myocytes or glucose stimulation
of pancreatic
-cells (36, 42). Further work showed that a peptide
based on the RACK binding site for PKC-
behaved similarly (18, 42).
These studies lend credence to the idea that the differential movement of PKC isoforms to the cytoskeleton and other subcellular structures may be at least partially because of a differential targeting to
distinct binding proteins within the cell.
In this study, the phorbol ester-sensitive PKC isoforms were shown to
play a major role in the EGF- and ANG II-induced
[3H]thymidine
incorporation in WB cells. Hence, the selective translocation of
PKC-
and -
by these agents supports the view that they may be
involved in the mitogenic pathway initiated by both G protein-coupled and tyrosine kinase receptors. To explore this possibility in greater
detail, we investigated the involvement of PKC in ERK activation.
The relative importance of PKC in activation of the ERK pathway has
been shown to depend on the agonist and the cell type (11, 17, 43).
Moreover, there has been conflicting data with regard to the role of
PKC in ANG II and EGF stimulation of the ERK pathway (2, 12, 23, 28,
29). Here, we demonstrate that PKC plays a role in the ANG II-induced
activation of ERK and Raf-1, especially in the later phase of their
activation. This is an interesting finding in light of the fact that a
sustained activation of ERK is believed to be necessary for mitogenesis (29). Consistent with this hypothesis, PKC depletion inhibited [3H]thymidine
incorporation into DNA induced by ANG II. Although the mechanism by
which Raf-1 is activated remains unresolved, it is believed to occur
via the translocation of Raf-1 to the plasma membrane, where it is
subsequently phosphorylated by kinases such as PKC (8). Recently,
PKC-
and -
have been shown to phosphorylate and activate Raf-1
(7, 22). Because we observed a sustained translocation of PKC-
to
the membrane fraction after ANG II stimulation, this isoform is a
possible candidate for Raf-1 activation in WB cells. Similarly, in
cardiac myocytes and rat aortic smooth muscle cells, it was suggested
that PKC-
is involved in endothelin- and ANG II-induced activation
of ERK, respectively (17, 28). However, PKC-
has also been
implicated in activation of the ERK pathway (39, 43). A prolonged
activation of PKC-
and/or -
may, therefore, be involved
in the mitogenic action of ANG II via promoting a sustained activation
of ERK in at least a partially Raf-1-dependent manner.
In contrast to the effects of ANG II, EGF-induced activation of ERK was
minimally affected in PKC-depleted cells. It should be noted, however,
that EGF produced a sustained activation of ERK, in accordance with the
well-established mitogenic effect of growth factors. Although PKC
depletion had no effect on this sustained phase of ERK activation, it
did decrease the EGF-induced [3H]thymidine
incorporation into DNA. This implicates an additional PKC-dependent
pathway that is required for DNA synthesis after EGF stimulation and is
consistent with the notion that ERK activation is required but alone is
insufficient for mitogenesis (41). Alternativelly, PKC isoforms that
are insensitive to PMA-induced downregulation may be involved in ERK
activation in WB cells. Therefore, although the phorbol ester-sensitive
PKC isoforms may have a minimal role in the ERK pathway induced by EGF,
they clearly play a major role in mitogenesis in these cells.
In conclusion, we observed the translocation of PKC-
and -
mostly
to the membrane fraction after ANG II and EGF stimulation. This
indicates that both isoforms are important for signaling events
initiated at the plasma membrane by each hormone. PKC depletion appears
to inhibit ANG II-induced mitogenesis via an ERK-dependent pathway,
whereas it inhibits EGF-induced mitogenesis in an ERK-independent pathway. The mechanism by which PKC-
or -
is involved in hormone stimulation of the ERK pathway and cellular proliferation remains to be
determined. Interestingly, translocation of PKC-
but not PKC-
to
the cytoskeleton was enhanced by ANG II, whereas EGF caused no
significant translocation of either PKC isoform to the cytoskeleton.
These data suggest PKC isozyme and hormone-directed specificity of
functions in liver epithelial WB cells.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Dr. Michael J. Weber for the generous
gift of the plasmid encoding the inactive GST-MEK.
 |
FOOTNOTES |
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases (NIDDK) Grants DK-15120 and DK-48494 (to
J. R. Williamson), an American Diabetes Association Career Development
Award (to L. J. Yang), and NIDDK Postdoctoral Fellowship DK-09404 (to
J. A. Maloney).
Address for reprint requests: J. R. Williamson, Dept. of Biochemistry
and Biophysics, Univ. of Pennsylvania, 601 Goddard Labs, 37th and
Hamilton Walk, Philadelphia, PA 19104.
Received 1 July 1997; accepted in final form 23 January 1998.
 |
REFERENCES |
1.
Aderem, A.
The MARCKS brothers: a family of protein kinase C substrates.
Cell
71:
713-716,
1992[Medline].
2.
Arai, H.,
and
J. A. Escobedo.
Angiotensin II type 1 receptor signals through Raf-1 by a protein kinase C-dependent, Ras-independent mechanism.
Mol. Pharmacol.
50:
522-528,
1996[Abstract].
3.
Beckmann, R.,
C. Lindschau,
H. Haller,
F. Hucho,
and
K. Buchner.
Differential nuclear localization of protein kinase C isoforms in neuroblastoma × glioma hybrid cells.
Eur. J. Biochem.
222:
335-343,
1994[Abstract].
4.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
5.
Buchner, K.
Protein kinase C in the transduction of signals toward and within the cell nucleus.
Eur. J. Biochem.
228:
211-221,
1995[Medline].
6.
Bussolino, F.,
F. Silvagno,
G. Garbarino,
C. Costamagna,
F. Sanavio,
M. Arese,
R. Soldi,
M. Aglietta,
G. Pescarmona,
G. Camussi,
and
A. Bosia.
Human endothelial cells are targets for platelet-activating factor (PAF). Activation of
and
protein kinase C isozymes in endothelial cells stimulated by PAF.
J. Biol. Chem.
269:
2877-2886,
1994[Abstract/Free Full Text].
7.
Cai, H.,
U. Smola,
V. Wixler,
I. Eisenmann-Tappe,
M. T. Diaz-Meco,
J. Moscat,
U. Rapp,
and
G. M. Cooper.
Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.
Mol. Cell. Biol.
17:
732-741,
1997[Abstract].
8.
Carroll, M. P.,
and
W. S. May.
Protein kinase C-mediated serine phosphorylation directly activates Raf-1 in murine hematopoietic cells.
J. Biol. Chem.
269:
1249-1256,
1994[Abstract/Free Full Text].
9.
Chao, T.-S. O.,
M. Abe,
M. B. Hershenson,
I. Gomes,
and
M. R. Rosner.
Src tyrosine kinase mediates stimulation of raf-1 and mitogen-activated protein kinase by the tumor promoter thapsigargin.
Cancer Res.
57:
3168-3173,
1997[Abstract].
10.
Dong, L.,
C. Chapline,
B. Mousseau,
L. Fowler,
K. Ramsay,
J. L. Stevens,
and
S. Jaken.
35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.
J. Biol. Chem.
270:
25534-25540,
1995[Abstract/Free Full Text].
11.
Duan, R. D.,
and
J. A. Williams.
Cholecystokinin rapidly activates mitogen-activated protein kinase in rat pancreatic acini.
Am. J. Physiol.
267 (Gastrointest. Liver Physiol. 30):
G401-G408,
1994[Abstract/Free Full Text].
12.
Eguchi, S.,
T. Matsumoto,
E. D. Motley,
H. Utsunomiya,
and
T. Inagami.
Identification of an essential signaling cascade for mitogen-activated protein kinase activation by angiotensin II in cultured rat vascular smooth muscle cells.
J. Biol. Chem.
271:
14169-14175,
1996[Abstract/Free Full Text].
13.
Forbush, B., III.
Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin.
Anal. Biochem.
128:
159-163,
1983[Medline].
14.
Goodnight, J.,
H. Mischak,
W. Kolch,
and
J. F. Mushinski.
Immunocytochemical localization of eight protein kinase C isozymes overexpressed in NIH 3T3 fibroblasts.
J. Biol. Chem.
270:
9991-10001,
1995[Abstract/Free Full Text].
15.
Haller, H.,
P. Quass,
C. Lindschau,
F. C. Luft,
and
A. Distler.
Platelet-derived growth factor and angiotensin II induce different spatial distributions of protein kinase C-
and -
in vascular smooth muscle cells.
Hypertension
23:
848-852,
1994[Abstract].
16.
Hii, C. S. T.,
A. Ferrante,
Y. S. Edwards,
Z. H. Huang,
P. J. Hartfield,
D. A. Rathjen,
A. Poulos,
and
A. W. Murray.
Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.
J. Biol. Chem.
270:
4201-4204,
1995[Abstract/Free Full Text].
17.
Jiang, T.,
E. Pak,
H. Zhang,
R. P. Kline,
and
S. F. Steinberg.
Endothelin-dependent actions in cultured AT-1 cardiac myocytes: the role of the
isoform of protein kinase C.
Circ. Res.
78:
724-736,
1996[Abstract/Free Full Text].
18.
Johnson, J. A.,
M. O. Gray,
C.-H. Chen,
and
D. Mochly-Rosen.
A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function.
J. Biol. Chem.
271:
24962-24966,
1996[Abstract/Free Full Text].
19.
Kiley, S. C.,
and
S. Jaken.
Activation of
-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells.
Mol. Endocrinol.
4:
59-68,
1990[Abstract].
20.
Kiley, S. C.,
P. J. Parker,
D. Fabbro,
and
S. Jaken.
Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.
Mol. Endocrinol.
6:
120-131,
1992[Abstract].
21.
Knutson, K. L.,
and
M. Hoenig.
Identification and subcellular characterization of protein kinase-C isoforms in insulinoma
-cells and whole islets.
Endocrinology
135:
881-886,
1994[Abstract].
22.
Kolch, W.,
G. Heidecker,
G. Kochs,
R. Hummel,
H. Vahidi,
H. Mischak,
G. Finkenzeller,
D. Marme,
and
U. R. Rapp.
Protein kinase C
activates RAF-1 by direct phosphorylation.
Nature
364:
249-252,
1993[Medline].
23.
Le Panse, R.,
V. Mitev,
L.-M. Houdebine,
and
B. Coulomb.
Protein kinase C-independent activation of mitogen-activated protein kinase by epidermal growth factor in skin fibroblasts.
Eur. J. Pharmacol.
307:
339-345,
1996[Medline].
24.
Leszczynski, D.,
S. Joenvaara,
and
M. L. Foegh.
Protein kinase C-
regulates proliferation but not apoptosis in rat coronary vascular smooth muscle cells.
Life Sci.
58:
599-606,
1996[Medline].
25.
Li, J.,
and
A. Aderem.
MacMARCKS, a novel member of the MARCKS family of protein kinase C substrates.
Cell
70:
791-801,
1992[Medline].
26.
Li, W.,
H. Mischak,
J. Yu,
L. Wang,
J. F. Mushinski,
M. A. Heidaran,
and
J. H. Pierce.
Tyrosine phosphorylation of protein kinase C-
in response to its activation.
J. Biol. Chem.
269:
2349-2352,
1994[Abstract/Free Full Text].
27.
Li, W.,
J.-C. Yu,
P. Michieli,
J. F. Beeler,
N. Ellmore,
M. A. Heidaran,
and
J. H. Pierce.
Stimulation of the platelet-derived growth factor
receptor signaling pathway activates protein kinase C-
.
Mol. Cell. Biol.
14:
6727-6735,
1994[Abstract].
28.
Malarkey, K.,
A. McLees,
A. Paul,
G. W. Gould,
and
R. Plevin.
The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells.
Cell. Signal.
8:
123-129,
1996[Medline].
29.
Mii, S.,
R. A. Khalil,
K. G. Morgan,
J. A. Ware,
and
K. C. Kent.
Mitogen-activated protein kinase and proliferation of human vascular smooth muscle cells.
Am. J. Physiol.
270 (Heart Circ. Physiol. 39):
H142-H150,
1996[Abstract/Free Full Text].
30.
Mischak, H.,
J. Goodnight,
W. Kolch,
G. Martiny-Baron,
C. Schaechtle,
M. G. Kazanietz,
P. M. Blumberg,
J. H. Pierce,
and
J. F. Mushinski.
Overexpression of protein kinase C-
and -
in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity.
J. Biol. Chem.
268:
6090-6096,
1993[Abstract/Free Full Text].
31.
Mochly-Rosen, D.,
H. Khaner,
J. Lopez,
and
B. L. Smith.
Intracellular receptors for activated protein kinase C.
J. Biol. Chem.
266:
14866-14868,
1991[Abstract/Free Full Text].
32.
Murphy, T. L.,
T. Sakamoto,
D. R. Hinton,
C. Spee,
U. Gundimeda,
D. Soriano,
R. Gopalakrishna,
and
S. J. Ryan.
Migration of retinal pigment epithelium cells in vitro is regulated by protein kinase C.
Exp. Eye Res.
60:
683-695,
1995[Medline].
33.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J.
9:
484-496,
1995[Abstract/Free Full Text].
34.
Pesce, A.,
R. H. McKay,
F. Stolzenbach,
R. D. Cahn,
and
N. O. Kaplan.
The comparative enzymology of lactic dehydrogenases. I. Properties of the crystalline beef and chicken enzymes.
J. Biol. Chem.
239:
1753-1762,
1964[Free Full Text].
35.
Prekeris, R.,
M. W. Mayhew,
J. B. Cooper,
and
D. M. Terrian.
Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function.
J. Cell Biol.
132:
77-90,
1996[Abstract].
36.
Ron, D.,
J. Luo,
and
D. Mochly-Rosen.
C2 region-derived peptides inhibit translocation and function of
protein kinase C in vivo.
J. Biol. Chem.
270:
24180-24187,
1995[Abstract/Free Full Text].
37.
Soltoff, S. P.,
and
A. Toker.
Carbachol, substance P, and phorbol ester promote the tyrosine phosphorylation of protein kinase C
in salivary gland epithelial cells.
J. Biol. Chem.
270:
13490-13495,
1995[Abstract/Free Full Text].
38.
Tsao, M. S.,
J. D. Smith,
K. G. Nelson,
and
J. W. Grisham.
A diploid epithelial cell line from normal adult rat liver with phenotypic properties of "oval" cells.
Exp. Cell Res.
154:
38-52,
1984[Medline].
39.
Ueda, Y.,
S. I. Hirai,
S. I. Osada,
A. Suzuki,
K. Mizuno,
and
S. Ohno.
Protein kinase C
activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf.
J. Biol. Chem.
271:
23512-23519,
1996[Abstract/Free Full Text].
40.
Wang, Q. J.,
P. Acs,
J. Goodnight,
T. Giese,
P. M. Blumberg,
H. Mischak,
and
J. F. Mushinski.
The catalytic domain of protein kinase C-
in reciprocal
and
chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes.
J. Biol. Chem.
272:
76-82,
1997[Abstract/Free Full Text].
41.
Wilkie, N.,
C. Morton,
L. L. Ng,
and
M. R. Boarder.
Stimulated mitogen-activated protein kinase is necessary but not sufficient for the mitogenic response to angiotensin II: a role for phospholipase D.
J. Biol. Chem.
271:
32447-32453,
1996[Abstract/Free Full Text].
42.
Yedovitzky, M.,
D. Mochly-Rosen,
J. A. Johnson,
M. O. Gray,
D. Ron,
E. Abramovitch,
E. Cerasi,
and
R. Nesher.
Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic
-cells.
J. Biol. Chem.
272:
1417-1420,
1997[Abstract/Free Full Text].
43.
Young, S. W.,
M. Dickens,
and
J. M. Tavare.
Activation of mitogen-activated protein kinase by protein kinase C isotypes
,
I and
, but not
.
FEBS Lett.
384:
181-184,
1996[Medline].
AJP Cell Physiol 274(4):C974-C982
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society