From the
The initiation of saliva formation by parotid acinar cells,
which comprise the majority of cells in this salivary gland, is
initiated by the release of neurotransmitters (acetylcholine, substance
P) from parasympathetic nerves. In response to substance P and the
muscarinic agonist carbachol, two ligands that activate phospholipase
C-linked receptors, which stimulate fluid secretion, PKC
The protein kinase C (PKC)
Parotid acinar cells have been a
focus of study for the production of inositol 1,4,5-trisphosphate and
sn-1,2-diacylglycerol, the release of Ca
Several studies have
overexpressed various PKC isoforms to examine the functions of specific
isoforms. These and other studies have reported that PMA promoted the
tyrosine phosphorylation of PKC
The present study utilizes freshly
isolated dispersed cells from rat parotid gland. The rapid tyrosine
phosphorylation of PKC
Samples were subjected to
electrophoresis on a sodium dodecyl sulfate (SDS)-7% polyacrylamide
separating gel with a 3% stacking gel. Proteins were transferred to 0.2
µm pore-size nitrocellulose filters, and the filters were blocked
with TBS (20 mM Tris (pH 7.6), 137 mM NaCl), 2% (w/v)
BSA for 1 h. The filters were washed in TTBS (TBS, 0.2% (v/v) Tween 20)
three times. The nitrocellulose filters were exposed to anti-PKC
In
some cases the immobilized phosphorylated PKC
The results presented in this study demonstrate that the
activation of muscarinic and substance P receptors and phorbol ester
stimulated the tyrosine phosphorylation of PKC
The results presented here,
which were obtained using freshly isolated salivary epithelial cells,
demonstrate that this biochemical event is one of the signal
transduction events that normally occurs in response to acetylcholine
and substance P released from parasympathetic nerves at the parotid
gland. In this system, neurotransmitters released from parasympathetic
nerves initiate fluid secretion, while norepinephrine release from
sympathetic nerves promotes protein secretion (exocytosis) via the
activation of
Of interest to the studies presented in this
manuscript, staurosporine partially blocked the carbachol-promoted
release of amylase but did not block the carbachol-promoted release of
K
As in many
cellular systems, phorbol esters have been reported to have a wide
range of effects on various salivary cells, ranging in areas as diverse
as promoting the activation of early response genes (Yeh et
al., 1992), modulation of Ca
As in several other recent studies, the protein tyrosine
kinase responsible for the tyrosine phosphorylation of PKC
Our findings that carbachol,
substance P, and phorbol ester promote the rapid tyrosine
phosphorylation of PKC
The increase
in tyrosine phosphorylation of PKC
We thank Margaret Lubkin for excellent technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
was
phosphorylated on tyrosine residues. The maximal agonist-dependent
tyrosine phosphorylation occurred within seconds of the addition of
either agonist and then returned rapidly to a smaller increased level.
Phorbol ester also caused a rapid increase in tyrosine phosphorylation,
which reached a maximal level 5 min after the addition of phorbol
12-myristate 13-acetate. The increase in tyrosine phosphorylation of
PKC
was blocked by tyrosine kinase inhibitors genistein and
staurosporine. Ionophore-mediated elevation of
[Ca
]
or activation of
the
-adrenergic receptor, epidermal growth factor receptor, or
insulin receptor did not promote the tyrosine phosphorylation of
PKC
. These results indicate that tyrosine phosphorylation plays a
role in early signal transduction events promoted by the activation of
muscarinic and substance P receptors and suggests that the tyrosine
phosphorylation of PKC
has a role in the activation of fluid
secretion by neurotransmitters binding to phospholipase C-linked
receptors.
(
)
family of
proteins currently consists of 12 members that are
phospholipid-dependent serine/threonine-specific protein kinases (for
review, see Nishizuka(1992) and Hug and Sarre(1993)). The family is
subdivided into those members that are calcium-dependent conventional
PCKs (
,
I,
II,
), calcium-independent novel PKCs
(
,
,
,
), and atypical PKCs (
and
/
). Another subgroup may include PKCµ, a recent addition
to the PKC family. With the exception of the atypical PKC group, all
members are activated by phorbol esters, which bind to the site at
which the endogenous activator sn-1,2-diacylglycerol binds. In
addition to the activation of PKC by phospholipids and/or
Ca
, other phospholipids, notably phosphoinositides
produced by the activation of phosphatidylinositol 3-kinase, have been
found to activate members of the conventional PKC and novel PKC family
(Singh et al., 1993; Toker et al., 1994; Nakanishi
et al., 1993). Different cells have different profiles of PKC
isoforms, and recent studies suggest that isoforms may have distinct
and different functions in cells (Li et al. (1994b) and
Mischak et al. (1993a), and for a review, see Dekker and
Parker(1994)). Multiple members of the PKC family of enzymes exist in
parotid acinar cells, including PKC
(Terzian et al.,
1993).
(
)
from intracellular stores, and the subsequent entry of
Ca
across the plasma membrane via capacitative entry
mechanisms (Komabayashi et al.(1992) and Takamura and
Putney(1989) and for a review, see Baum et al. (1993)). These
events lead to the activation of Ca
-sensitive
channels and the initiation of saliva formation, events that are
controlled by the release of neurotransmitters (acetylcholine,
substance P) from parasympathetic nerves. While examining the effects
of growth factors and other receptor ligands on the tyrosine
phosphorylation pattern of parotid acinar cells, we found that the
muscarinic agonist carbachol and substance P promoted the tyrosine
phosphorylation of an
80-kDa protein and that phorbol ester
promoted the tyrosine phosphorylation of the same band, which was
identified as the
isoform of PKC.
. PKC
(and PKC
)
expression was increased in c-Ha-ras-transformed fibroblasts
(Borner et al., 1992b), and PKC
was constitutively
tyrosine phosphorylated in c-Ha-ras-transformed keratinocytes
(Denning et al., 1993). In a myeloid progenitor cell line (32D
cells) and in NIH 3T3 cells that overexpressed PKC
, phorbol ester
stimulated the tyrosine phosphorylation of PKC
(Li et
al., 1994a). In 32D cells in which PKC
and the
platelet-derived growth factor receptor were overexpressed,
platelet-derived growth factor stimulated the tyrosine phosphorylation
of PKC
and myeloid differentiation (Li et al., 1994b). In
an earlier study using 32D cells, differentiation was promoted in cells
in which PKC
and -
were overexpressed, but not in cells in
which PKC
II,
,
, or
was overexpressed (Mischak
et al., 1993a). In NIH 3T3 cells, overexpression of PKC
produced changes in cell morphology and slowed cell growth, while very
different changes (increase in growth rate and in cell density at
confluence) were obtained by overexpression of PKC
in these cells
(Mischak et al., 1993b).
in the acinar cells suggests that this
biochemical event contributes to the normal physiological responses
promoted by the release of neurotransmitters from parasympathetic
nerves that promote fluid secretion and saliva formation in this
exocrine gland.
Chemicals
All chemicals were reagent grade or
better. PMA and 4-PMA were obtained from Life Technologies, Inc.
(Grand Island, NY). Carbamyl choline (carbachol) and substance P were
purchased from Sigma. Anti-phosphotyrosine was a generous gift of Dr.
Tom Roberts (Dana Farber, Boston, MA).
[
P]PO
was purchased from DuPont NEN.
Anti-protein kinase C
antibody was purchased from Santa Cruz
Biotechnology. Male Sprague-Dawley rats (Charles River Laboratories,
Kingston, NY, or Taconic, Germantown, NY), 200-250 g, were used
for all experiments.
Cell Preparation and Solutions
Freshly dispersed
parotid acinar cells were prepared as described previously (Soltoff
et al., 1989). The cells were suspended at 2 mg/ml in a
medium (Solution A) of the following composition: 116.4 mM
NaCl, 5.4 mM KCl, 1 mM NaH
PO
,
25 mM HEPES, 1 mM CaCl
, 0.8 mM
MgCl
, 5 mM butyrate, 5.6 mM glucose, pH
7.4. Cells were kept on ice prior to use. Samples (1.5 ml) of the cell
suspension were stirred and equilibrated at 37 °C for 10-15
min prior to use.
Immunoprecipitations and Western Blotting
After
the cells were exposed to the agonists or inhibitors, the cells were
pelleted by a brief spin in a microcentrifuge (Brinkmann 5414). The
supernatant was removed and replaced by 1 ml of ice-cold lysis buffer
(137 mM NaCl, 20 mM Tris, 0.2 mM vanadate, 1
mM EGTA, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v)
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml
aprotinin, 4 µg/ml leupeptin, 4 µg/ml pepstatin, 2 mM
dithiothreitol, pH 7.5). The lysates were vortexed and centrifuged at
16,000 g at 4 °C for 15 min. The cleared
supernatants were transferred to fresh 1.5-ml microcentrifuge tubes. A
portion (5-10% of the volume) of the lysate was removed and
combined with an equal volume of 2
sample buffer (62.5
mM Tris, pH 6.8, 10% (v/v) glycerol, 6.25% (v/v) SDS, 0.72
N
-mercaptoethanol, bromphenol blue for color). The
remainder was incubated with either anti-P-Tyr (6.6 µg/ml) or
anti-PKC
(1-2 µg/ml) antibodies and protein A-Sepharose
(4 mg/ml lysate), and the samples were rocked at 4 °C for 3 h or
overnight. At the end of the incubation, the immunoprecipitates were
collected by centrifugation, washed 3 times in ice-cold
phosphate-buffered saline (PBS) (137 mM NaCl, 15.7 mM
NaH
PO
, 1.47 mM
KH
PO
, 2.68 mM KCl, 1% Nonidet P-40, pH
7.4), two times in 0.1 M Tris (pH 7.5), 0.5 M LiCl,
and two times in TNE (10 mM Tris, 100 mM NaCl, 1
mM EDTA, pH 7.5). All wash solutions contained 0.2 mM
vanadate. The majority of the TNE was removed, the remaining volume was
diluted with an equal volume of 2
sample buffer, and the
samples were boiled for 5-10 min. The immunoprecipitated proteins
and the lysate fractions were subjected to electrophoresis or stored at
- 80 °C prior to electrophoresis.
antibody (0.2 µg/ml) or anti-P-Tyr antibody (1 µg/ml) in
TTBS/1% BSA for
16 h at 4 °C. The filters were washed three
times in TTBS, and exposed to anti-rabbit (for anti-PKC
) or
anti-mouse (for anti-P-Tyr) horseradish peroxidase
(Boehringer-Mannheim) at a 1:10,000 dilution in TTBS/1% BSA for 1 h.
All washes and exposure to the secondary antibody were performed at
room temperature. Filters were washed three times with TTBS and twice
with TBS, and were visualized on x-ray film (Kodak) using a
chemiluminescence system (Amersham or DuPont NEN). In some experiments,
the filters were stripped by exposing them to 62.5 mM Tris (pH
6.8),
-mercaptoethanol (0.1 M), 2% (w/v) SDS at 70 °C
for 40 min. The stripped filters were washed several times in TTBS,
once in TBS, blocked with TBS, 2% BSA for one hour, and reprobed with
antibody for 16 h. Blots were then treated as described above.
In Vivo Labeling and Phosphoamino Acid
Analysis
In vivo phosphorylation studies were performed
by labeling the cells with [P]PO
.
Cells were suspended in solution A, except NaH
PO4 was
omitted. A suspension of cells was incubated at 37 °C and exposed
to [
P]PO
(2-3 mCi/ml cells)
for
1-2 h, after which they were resuspended in an identical
pre-warmed medium in the absence of
[
P]PO
. Cells were exposed to PMA,
carbachol, or vehicle (Me
SO). The cells from each
experimental condition were collected and pelleted, the supernatant was
removed and the tube was rinsed with phosphate-buffered saline without
dislodging the pellet. The cell pellet was lysed in ice-cold lysis
buffer, to which anti-PKC
(2-3 µg/ml) antibody was
added. The antibody was collected using protein A-Sepharose and washed
as described above. The proteins were transferred to 0.45 µm pore
size Immobilon-P (Millipore) using a Bio-Rad Trans-Blot system and a
transfer buffer of the following composition: 25 mM Tris base,
192 mM glycine in 15% (v/v) methanol, pH 8.2-8.3.
proteins were cut
out and subjected to acid hydrolysis for two-dimensional phosphoamino
acid analysis as described previously (Boyle et al., 1991).
Cell Fractionation (Translocation)
Experiments
Fractionation experiments were conducted with
modifications of the protocol outlined in Olivier and Parker(1994).
Cells were suspended and stirred at 2 mg of protein/ml and
equilibrated at 37 °C for 10 min prior to use. Upon treatment,
cells were quickly pelleted, the physiological solution was replaced
with Buffer A (25 mM Tris-HCl, pH 7.5, 250 mM
sucrose, 2.5 mM magnesium acetate, 10 mM sodium
fluoride, 2 mM dithiothreitol, 10 mM benzamidine),
and this was removed and replaced with 2 ml of homogenization buffer
(Buffer A plus 5 mM EGTA, 5 mM EDTA, 4 µg/ml of
aprotinin, leupeptin, and pepstatin; 1 mM phenylmethylsufonyl
fluoride). The cells were resuspended, and the suspension was
homogenized for 40 strokes in a tight Dounce homogenizer followed by a
5-s sonication (Branson Ultrasonics, Danbury, CT; setting 7) repeated 3
times. The homogenate was centrifuged for 30 min at 70,000 rpm at 4
°C in a Beckman Optima TLX ultracentrifuge. A portion (
5%) of
the supernatant (cytosolic fraction) was collected as cytosolic lysate,
and an equal volume of 2
sample buffer was added, and the
samples were heated at 100 °C for 8 min. The detergent-soluble
proteins were solubilized by adding Buffer B (25 mM Tris-HCl,
pH 7.5, 10 mM sodium fluoride, 5 mM EDTA, 1% Nonidet
P-40 detergent, 4 µg/ml of aprotinin, leupeptin, and pepstatin, 1
mM phenylmethylsufonyl fluoride, 5 mM dithiothreitol,
10 mM benzamidine) to the pellet; the solution was vortexed,
transferred to a Dounce homogenizer, and broken apart with 20 strokes.
Detergent-soluble proteins were collected by spinning the sample at
16,000
g for 15 min at 4 °C. A portion (
5%)
of the supernatant was collected as membrane lysate, sample buffer was
added, and the samples were heated. In some experiments, the remainder
of the supernatants of the cytosolic fraction and membrane fraction
were used for immunoprecipitation studies using anti-PKC
antibody
(2 µg/ml) or anti-P-Tyr antibody (6.6 µg/ml). After addition of
protein A-Sepharose as above, the immunoprecipitates were rocked at 4
°C for 3 h or overnight and collected and washed as described
above.
Data
All experiments were conducted at least twice
with similar results to those shown in the figures.
Carbachol, Substance P, and PMA Stimulate the Tyrosine
Phosphorylation of PKC
Carbachol and substance P, two
receptor ligands that stimulate the production of diacylglycerol in
parotid cells, and PMA, which binds to the diacylglycerol binding site
on PKC, all stimulated the tyrosine phosphorylation of PKC.
Differences among the stimuli were highlighted in a comparison of their
time courses (Fig. 1, A and B). In cells
exposed to carbachol or substance P for varying periods of time up to
15 min, the increase in tyrosine phosphorylation of PKC
was
greatest for cells exposed to these stimuli for
15 s, and then it
diminished to a lower level. Within 15 min, the level of tyrosine
phosphorylation was close to that found in untreated cells. Previous
studies on inositol trisphosphate production and
[Ca
]
elevation in parotid cells demonstrated that the stimulatory
effects of substance P rapidly diminished due to homologous receptor
desensitization, although the effects of carbachol were maintained for
up to 10 min or longer (McMillian etal., 1987;
Sugiya etal., 1987). However, the tyrosine
phosphorylation of PKC
elicited by both carbachol and substance P
displayed a very early increase and then a decline. The decrease in
tyrosine-phosphorylated PKC
(Fig. 1, A and
B) without a decrease in the mass of the PKC
protein
(Fig. 1C) indicates that a protein-tyrosine phosphatase
was activated. This suggests that the activation of tyrosine
phosphatase activity is also involved in the stimulation of these cells
by carbachol and substance P.Effects of PMA were observed at
times as early as
15 s (Fig. 1, A and B).
However, in contrast to the effects of the two receptor-mediated
ligands, the PMA-promoted phosphorylation of PKC
continued to
increase up to 5 min of exposure. For all three stimuli, similar
findings were observed when proteins were immunoprecipitated using
anti-PKC
antibody and immunoblotted with anti-P-Tyr antibody
(Fig. 1A) or when proteins were immunoprecipitated with
anti-P-Tyr antibody and immunoblotted with anti-PKC
antibody
(Fig. 1B). The results suggest that PKC
is involved
in early signal transduction events subsequent to the activation of the
muscarinic and substance P receptors in the parotid acinar cell.
Figure 1:
Time dependence of the increase in
PKC tyrosine phosphorylation promoted by the exposure of parotid
acinar cells to carbachol, substance P, and PMA. Cells were
equilibrated at 37 °C and exposed to carbachol (1
10
M), substance P (1
10
M), or PMA (200 nM) for 0.2, 1,
5, or 15 min. Unstimulated cells (0) were exposed to vehicle
(Me
SO or water). Cells were lysed, and proteins were
immunoprecipitated using anti-PKC
antibody (A and
C) or anti-P-Tyr antibody (B). Proteins were
separated by SDS-PAGE, transferred to nitrocellulose filters, and
probed with anti-P-Tyr antibody (A) or anti-PKC
antibody
(B and C) as indicated. Tyrosine-phosphorylated
PKC
was immunoprecipitated by anti-P-Tyr antibody or by
anti-PKC
antibody. Proteins were visualized on x-ray film using
enhanced chemiluminescence. Molecular mass markers (in kDa) are
indicated on the left. The arrow on the right designates the location of the tyrosine phosphorylated form of
PKC
.
In
lysates of parotid acinar cells, PKC appears as a doublet protein
at
80 kDa (Fig. 2). Although the doublet appears in both
stimulated and unstimulated cells in some experiments, in most
experiments the mass of the upper band appears to increase with
stimulation. This is most readily detected in cells treated with PMA,
but also it is seen in carbachol- or substance P-stimulated cells
(Fig. 1C). Anti-P-Tyr antibody preferentially
immunoprecipitates and immunoblots the upper form of the enzyme,
indicating that this is the tyrosine-phosphorylated form. The
anti-PKC
antibody immunoprecipitates and immunoblots both forms of
enzyme. This is observed easily when proteins immunoprecipitated using
anti-PKC
antibody are immunoblotted for both PKC
and tyrosine
phosphorylation, and are compared side by side with lysates
(Fig. 2). This also demonstrates that the tyrosine-phosphorylated
enzyme accounts for only a portion of the upper band of PKC
(Fig. 2). The identification of the upper band as the
tyrosine-phosphorylated form is consistent with the stimuli producing a
mobility shift to a slower migrating form of the isoenzyme due to an
increase in phosphorylation, which consists of both serine and tyrosine
phosphorylation (see below).
Figure 2:
Identification of tyrosine phosphorylated
PKC in anti-PKC
immunoprecipitates of carbachol- and
PMA-treated parotid acinar cells. Unstimulated cells (-) or cells
stimulated with PMA (200 nM) or carbachol (1
10
M) for 5 min were lysed. Proteins were
immunoprecipitated with anti-PKC
, separated by SDS-PAGE, and
transferred to nitrocellulose, and the blot was probed with
anti-PKC
(leftpanel). The nitrocellulose blot
was stripped and reprobed with anti-P-Tyr antibody (middlepanel). Lysates (rightpanel) are from
a separate experiment using cells treated as described. PKC
is
visualized as a doublet in cell lysates and in anti-PKC
immunoprecipitates. The upper band of the doublet was selectively
immunoprecipitated by anti-P-Tyr antibody (Fig. 1B). Proteins
were visualized on x-ray film using enhanced chemiluminescence.
Molecular mass markers (in kDa) are indicated on the left. The
arrow on the right designates the location of the
tyrosine phosphorylated form of PKC
.
PKC was immunoprecipitated from
PMA-treated rat parotid acinar cells in a concentration-dependent
manner using anti-P-Tyr antibody (Fig. 3). For cells treated with
PMA for 15 min, 20 nM PMA produced a measurable increase in
tyrosine phosphorylation. This was increased by 200 nM PMA,
and concentrations of 1 µM (Fig. 3) or 2
µM (not shown) did not produce an increase over that
promoted by 200 nM. The results obtained using inactive
phorbol ester (4
-PMA) were similar to those found in cells treated
with vehicle (Me
SO) alone (not shown).
Figure 3:
Concentration dependence of the
stimulatory effect of PMA on the tyrosine phosphorylation of PKC.
Cells were treated with 20-1000 nM PMA for 15 min or
treated with 0.2% Me
SO (0), and proteins were
immunoprecipitated with anti-P-Tyr and immunoblotted using
anti-PKC
antibody. Tyrosine phosphorylation is modest but
observable at 20 nM PMA and is maximal at 200 nM PMA.
Proteins were visualized on x-ray film using enhanced
chemiluminescence. Molecular mass markers (in kDa) are indicated on the
left. The arrow on the right designates the location of the tyrosine-phosphorylated form of
PKC
.
Carbachol and PMA Stimulate Phosphorylation of PKC
Carbachol and PMA both produced substantial increases in
the phosphorylation of PKC
in Vivo
in cells that were prelabeled with
[
P]PO
and then exposed to these
stimuli. A 2-min exposure to PMA increased the phosphorylation to 6
times that found in unstimulated cells, and carbachol increased it to 4
times the basal level (Fig. 4). Similar increases were observed
in cells treated for 5 or 15 min (not shown).
Figure 4:
Carbachol and PMA stimulate the in
vivo phosphorylation of PKC. Parotid acinar cells were
labeled with [
P]PO
, washed to remove
the extracellular isotope, and resuspended (see ``Experimental
Procedures''). Cells were treated with 0.2% Me
SO
(-), PMA (200 nM), or carbachol (1
10
M) for 2 min, after which they were
lysed. PKC
was immunoprecipitated using anti-PKC
antibody,
subjected to SDS-PAGE, transferred to Immobilon, and exposed to x-ray
film. The arrow on the right designates the location
of PKC
. Similar results were obtained using cells treated with
stimuli for 5 or 15 min.
In some experiments
the PKC band at
80 kDa was cut out and subjected to
two-dimensional phosphoamino acid analysis. Although the in vivo [
P]PO
labeling was sufficient
to demonstrate an increase in serine phosphorylation (not shown) in
PMA-treated cells, consistent with the activation of PKC
activity
by these stimuli, the in vivo labeling was not sufficient to
observe the increase in tyrosine phosphorylation. Thus, tyrosine
phosphorylation makes up only a small fraction of the enhanced in
vivo phosphorylation in the anti-PKC
immunoprecipitates shown
in Fig. 4. A similar conclusion concerning the relatively small
fraction of tyrosine phosphorylated PKC
was made from results
obtained from anti-P-Tyr immunoblots of anti-PKC
immunoprecipitates (Fig. 2).
Tyrosine Phosphorylation of PKC
Other stimuli were examined for
their ability to stimulate the tyrosine phosphorylation of PKC Is Not Promoted by
Protein Kinase A-linked Receptors, EGF and Insulin Receptor Tyrosine
Kinases, and Calcium Ionophore
(Fig. 5). Isoproterenol, a
-adrenergic agonist that
activates protein kinase A in these cells, was examined at
concentrations as high as 10
M and did not
promote the tyrosine phosphorylation of PKC
in any significantly
detectible manner. Ionomycin, a calcium ionophore, was without effect
at 10
or 10
M, which are
concentrations that cause large increases in
[Ca
]
. EGF (100 ng/ml)
produced an increase in tyrosine phosphorylation of multiple proteins
in parotid acinar cells, including the epidermal growth factor receptor
(not shown), but it did not promote the tyrosine phosphorylation of
PKC
. Insulin (100 nM) also was without effect on the
tyrosine phosphorylation of PKC
. These results suggest that the
carbachol-, substance P-, and phorbol ester-promoted tyrosine
phosphorylation of PKC
was not mediated by an increase in
[Ca
]
or an activation
of protein kinase A and that the activation of two receptor
protein-tyrosine kinases (the EGF and insulin receptors) also was
insufficient to promote the tyrosine phosphorylation of PKC
in
parotid acinar cells. EGF promotes increases in inositol
1,4,5-trisphosphate and diacylglycerol in some cells (Hughes et
al., 1991), and its lack of effect on PKC
tyrosine
phosphorylation in parotid cells may reflect a relatively low density
of EGF receptors and the sensitivity of detection of this event.
Figure 5:
Comparison of the effects of various
stimulatory agents on PKC tyrosine phosphorylation in parotid
acinar cells. Cells were treated with vehicle (-) or exposed to
the following concentrations of stimuli: PMA (200 nM),
carbachol (1
10
M), substance P (1
10
M), ionomycin (1
10
M), isoproterenol (10
M), EGF (100 ng/ml), and insulin (100 µM).
Cells were exposed for 5 min (PMA, carbachol, isoproterenol, EGF,
insulin), 1 min (ionomycin), or both times (substance P). At the end of
the exposure period, proteins were immunoprecipitated from lysed cells
using anti-P-Tyr antibody, subjected to SDS-PAGE, transferred to
nitrocellulose, and immunoblotted using anti-PKC
antibody.
Proteins were visualized on x-ray film by using enhanced
chemiluminescence. Only cells exposed to PMA, carbachol, and substance
P demonstrated a measurable increase in tyrosine phosphorylation of
PKC
. EGF (100 ng/ml), insulin (100 nM), and isoproterenol
(10
M) also did not produce a significant
effect on PKC
tyrosine phosphorylation at shorter times of agonist
exposure (0.2 and 1 min). The arrow on the right designates the location of the tyrosine phosphorylated form of
PKC
.
Genistein and Staurosporine Block PKC
The effects of two tyrosine kinase inhibitors,
genistein and staurosporine, were examined on the tyrosine
phosphorylation of PKC Tyrosine
Phosphorylation
. Cells were pretreated with the inhibitors
for 15 min prior to exposure to carbachol or PMA. The tyrosine
phosphorylation promoted by PMA was partially reduced at 0.1
µM staurosporine and was not observable at 1
µM or 5 µM staurosporine (not shown). The
effects of PMA or carbachol were partially reduced by 1 and 10
µM genistein, and 100 µM produced an almost
complete reduction in the PKC
tyrosine phosphorylation (not
shown). These experiments indicate that the effects of carbachol and
PMA are mediated via staurosporine- and genistein-sensitive tyrosine
kinase activity. Although staurosporine also inhibits PKC activity
directly (Tamaoki, 1991), presumably its inhibitory effect in these
studies is due to its effects in blocking a tyrosine kinase for which
PKC
is a substrate (see ``Discussion'').
Translocation of PKC
The effects of PMA and carbachol treatment of the
cells on the translocation of PKC to the Membrane by PMA and
Carbachol
to the plasma membrane were
investigated by fractionating the cells into cytosolic and membrane
components and immunoprecipitating PKC
from these individual
fractions. Immunoprecipitated proteins and lysates were subjected to
SDS-PAGE and immunoblotting using anti-P-Tyr or anti-PKC
antibodies. Tyrosine-phosphorylated PKC
was present only in the
membrane fraction of PMA- or carbachol-treated cells (Fig. 6,
leftpanel). Under basal conditions, a substantial
portion of PKC
was constitutively localized at the membrane in
addition to a cytosolic component (Fig. 6, rightpanel). Other investigators also reported that a
significant fraction of PKC
was associated constitutively with the
membrane fraction in the absence of treatment with phorbol ester
(Borner et al., 1992a; Mischak et al., 1993a; Olivier
and Parker, 1994). Nearly the entire cytosolic portion of PKC
disappeared in cells treated for 5 min with phorbol ester
(Fig. 6, rightpanel). In cells treated with
carbachol, a portion of the cytosolic PKC
was diminished, but this
occurred to a lesser extent than that produced by PMA treatment.
Similar finding were observed in immunoblots of the lysates (not
shown).
Figure 6:
Carbachol and PMA stimulate the
translocation of cytosolic PKC and its tyrosine phosphorylation at
the membrane. Parotid acinar cells were treated with vehicle (-),
PMA (200 nM), or carbachol (1
10
M) for 5 min, lysed, and fractionated into a cytosolic
and membrane component. A lysate portion of each component was
retained, and the remainder was subjected to immunoprecipitation using
anti-PKC
antibody. Proteins were subjected to SDS-PAGE,
transferred to nitrocellulose, and immunoblotted using anti-P-Tyr
antibody (leftpanel). Tyrosine-phosphorylated
PKC
was detected only in the membrane fraction of treated cells.
The nitrocellulose blot was stripped and reprobed with anti-PKC
antibody (rightpanel). The cytosolic component of
PKC
was substantially diminished in cells treated with PMA and
carbachol. Proteins were visualized on x-ray film by using enhanced
chemiluminescence. The arrow on the right designates
the location of the tyrosine phosphorylated form of
PKC
.
In other experiments (not shown) using anti-P-Tyr as the
immunoprecipitating antibody, PKC was immunoprecipitated from the
membrane of PMA-treated cells but not from the cytoplasmic fraction,
which was similar to recent results obtained using PKC
-transfected
myeloid cells (Li et al., 1994b). The parotid cell
fractionation experiments indicate that carbachol and phorbol ester
promote the translocation of PKC
to the plasma membrane, and that
tyrosine phosphorylation occurs subsequent to translocation. Consistent
with this, neither translocation from the cytosolic fraction nor
tyrosine phosphorylation of the membrane fraction of PKC
was
observed in cells exposed to isoproterenol (10
M), EGF (100 ng/ml), and insulin (100 nM) (not
shown), suggesting that the two events are linked.
in rat parotid
acinar cells. The tyrosine phosphorylation of PKC
reached a
maximum within seconds of the addition of the agonists carbachol and
substance P, was blocked by genistein and staurosporine, and was not
promoted by agonists to other (
-adrenergic, EGF, insulin)
receptors present on parotid cells. Tyrosine-phosphorylated PKC
was immunoprecipitated using anti-P-Tyr antibody or anti-PKC
antibody and occurred subsequent to the translocation of cytosolic
PKC
to the plasma membrane. The rapid decrease in tyrosine
phosphorylation after the initial increase suggests the activation of
both a protein-tyrosine kinase and tyrosine phosphatase after the
addition of carbachol and substance P.
-adrenergic receptors. Carbachol and substance P
mobilize intracellular Ca
by binding to receptors
that are linked to phospholipase C via GTP-dependent proteins. This
promotes the phospholipase C-mediated hydrolysis of
phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate
and sn-1,2-diacylglycerol, which elevate the intracellular
free Ca
concentration
([Ca
]
) and activate
PKC, respectively (see Nishizuka, 1992). The elevation of
[Ca
]
via the
activation of muscarinic and substance P receptors activates
Ca
-sensitive ion channels that participate in the
initiation of saliva formation by the secretion of fluid and
electrolytes into the acinar lumen (Baum et al., 1993; Turner,
1993). In contrast, the activation of
-adrenergic receptors and
the resulting stimulation of cAMP production promote the secretion of
amylase but not much water secretion in the salivary system (Quissell,
1993). The lack of effect of isoproterenol, a
-adrenergic agonist,
on the tyrosine phosphorylation of PKC
suggests that this isoform
is not involved in the main pathway of activation of exocytosis in
these cells. In parotid acinar cells, amylase secretion also is
increased by phorbol ester or carbachol, but this stimulation is only
small fraction (<50%) of that produced by isoproterenol or cAMP
(Takuma and Ichida, 1986; McMillian et al., 1988; Shimomura
et al., 1988). Thus, the activation of parotid muscarinic
receptors produces a modest effect on amylase release, and this appears
to be due to the activation of protein kinase C, although
[Ca
]
may play a
modulating role in exocytosis in salivary glands (Tojyo et
al., 1992, 1993).
from rat parotid acini (Tojyo et al.,
1993). In a study of recombinantly produced PKC isoforms, it was
reported that the concentration of staurosporine that inhibits PKC
enzyme activity (EC
=
500 nM) was
about two log orders greater than that required to inhibit PKC
(McGlynn et al., 1992). If the inhibition of PKC
in
parotid cells has a similar sensitivity, then any potential inhibitory
effects of staurosporine on physiological processes that may involve
PKC
could be due to a direct blockade of PKC
enzyme activity
and/or to a blockade of its tyrosine phosphorylation.
-dependent
K
channels (Moran and Turner, 1993), stimulation of
RNA synthesis (Woon et al., 1993), and the phosphorylation of
ribosomal protein S6 (Padel and Soling, 1985). Phorbol ester enhanced
the
-adrenergic receptor-mediated cAMP accumulation in mouse
parotid acini (Watson et al., 1993), suggesting that in these
cells, as in many others, there is cross-talk between two different
signal transduction pathways (e.g. phospholipase C-linked and
adenylyl cyclase-linked receptors). Phorbol ester also promoted the
differentiation of a neoplastic intercalated salivary duct cell line
into one that resembled acinar cells (Hayashi et al., 1987).
In this regard, it is interesting that a number of studies have
implicated a role for PKC in cell differentiation. As mentioned in the
introduction, the tyrosine phosphorylation of PKC
appeared to play
a role in the platelet-derived growth factor-initiated differentiation
of myeloid progenitor cells (Li et al., 1994b). In contrast,
staurosporine (acting as a protein kinase inhibitor) reduced the
tyrosine phosphorylation of PKC
and promoted the differentiation
of v-ras-transformed murine keratinocytes that exhibit a
constitutively tyrosine-phosphorylated PKC
(Denning et
al., 1993), which suggested that PKC
tyrosine phosphorylation
may block differentiation of these cells. Thus, a role for PKC
in
cellular differentiation may be dependent on the cell type and status,
and a specific role in parotid acinar cell differentiation is as yet
unrealized.
remains
to be determined. PKC
was phosphorylated in vitro by
various receptor and cytosolic tyrosine kinases, including Fyn, insulin
receptor, and the
-platelet-derived growth factor receptor (Li
et al., 1994a) and Src (Gschwendt et al., 1994). Two
receptor tyrosine kinases on parotid acinar cells, the EGF and insulin
receptors, did not promote a detectible tyrosine phosphorylation of
PKC
in vivo in parotid acinar cells (Fig. 5).
In vitro phosphorylation by src occurred only in the
presence of PMA, suggesting that a conformational change in PKC
may be required prior to its phosphorylation on tyrosine (Gschwendt
et al., 1994). This finding is consistent with observations
that tyrosine phosphorylation of PKC
occurred subsequent to its
recruitment to the membrane in phorbol agonist-treated cells (Li et
al., 1994b). Tyrosine phosphorylation of PKC
has been
reported to increase (Li et al., 1994a) or decrease (Denning
et al., 1993) its activity.
in parotid acinar cells suggest that the
physiological response to this biochemical event is a process that is
promoted by these agents due to their common stimulation of PKC
activity via sn-1,2-diacylglycerol/phorbol ester binding. As
indicated from the summary of secretory events outlined above,
potential physiological effects could be related to the major role that
these receptors play in fluid secretion, the minor role that they play
in protein exocytosis, or to another aspect of cellular physiology or
biochemistry. Numerous proteins involved in electrolyte movement have
been reported to be substrates for PKC, including the Na,K-ATPase
(Beguin et al., 1994) and various ion channels (Dunne, 1994;
West et al., 1991). In addition, carbachol produced
PKC-mediated changes in cell signaling and transport events in other
chloride-secreting epithelia, including promoting the serine
phosphorylation of the Na-K-Cl cotransporter in avian salt gland
(Torchia et al., 1994) and alterations in receptor-stimulated
oscillations in [Ca
]
in lacrimal cells (Bird et al., 1993).
that is promoted by carbachol
and substance P suggests that the signal transduction pathway promoted
by these Ca
-mobilizing agonists also involves an
increase in tyrosine phosphorylation, a biochemical event that is
usually associated with the activation of growth factor receptors.
Although the specific effects of PKC
tyrosine phosphorylation in
parotid acinar cells remains to be determined, the findings outlined in
the present report suggest that the tyrosine phosphorylation of a
specific isoform of PKC among several that are expressed in this tissue
activates an effector downstream of muscarinic and substance P
receptors. The fact that the largest effect on tyrosine phosphorylation
occurs in the first seconds after the ligands bind to their receptors
suggests that PKC
participates in the earliest part of the
responses mediated by muscarinic and substance P receptors. Thus,
PKC
tyrosine phosphorylation may be involved in mediating the
stimulation of fluid secretion in parotid acinar cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.