Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02215
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ABSTRACT |
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Protein kinase C (PKC) becomes tyrosine phosphorylated in rat parotid acinar cells exposed to
muscarinic and substance P receptor agonists, which initiate fluid
secretion in this salivary cell. Here we examine the signaling
components of PKC
tyrosine phosphorylation and effects of
phosphorylation on PKC
activity. Carbachol- and substance P-promoted
increases in PKC
tyrosine phosphorylation were blocked by inhibiting
phospholipase C (PLC) but not by blocking intracellular
Ca2+ concentration elevation, suggesting that
diacylglycerol, rather than D-myo-inositol
1,4,5-trisphosphate production, positively modulated this
phosphorylation. Stimuli-dependent increases in PKC
activity in
parotid and PC-12 cells were blocked in vivo by inhibitors of Src
tyrosine kinases. Dephosphorylation of tyrosine residues by PTP1B, a
protein tyrosine phosphatase, reduced the enhanced PKC
activity.
Lipid cofactors modified the tyrosine phosphorylation-dependent PKC
activation. Two PKC
regulatory sites (Thr-505 and Ser-662) were
constitutively phosphorylated in unstimulated parotid cells, and these
phosphorylations were not altered by stimuli that increased PKC
tyrosine phosphorylation. These results demonstrate that PKC
activity is positively modulated by tyrosine phosphorylation in parotid
and PC-12 cells and suggest that PLC-dependent effects of secretagogues
on salivary cells involve Src-related kinases.
serine/threonine phosphorylation; parotid acinar; protein kinase C
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INTRODUCTION |
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THE INVOLVEMENT OF
TYROSINE phosphorylation in heterotrimeric GTP-dependent protein
(G protein)-coupled receptor-mediated events has been documented in
many cellular systems, including freshly isolated cells and cultured
cell lines. These events may involve the activation of nonreceptor
tyrosine kinases such as Src and/or the transactivation of growth
factor receptors (1, 6-8, 30, 43). Previously, we
observed that carbachol and substance P promoted rapid increases in the
tyrosine phosphorylation of protein kinase C (PKC) in salivary
gland epithelial cells (rat parotid acinar cells) (46). In
these cells, the muscarinic and substance P receptors are linked to
phospholipase C (PLC)-
via a Gq-type G protein. The
activation of these G protein-coupled receptors (GPCRs) produces rapid
increases in diacylglycerol (DAG) and
D-myo-inositol 1,4,5-trisphosphate production,
which promote the activation of PKC and increases in intracellular
Ca2+ concentration ([Ca2+]i),
resulting in the initiation of fluid secretion (saliva formation) by
these cells. The rapid (within seconds) nature of tyrosine phosphorylation of PKC
after activation of these receptors suggests that PKC
may play a role in fluid secretion or other
neurotransmitter-promoted events in these cells. Phorbol esters,
which activate many PKCs, including PKC
, also produce many
regulatory effects on salivary cells, including the modulation of early
response genes (55), Ca2+-dependent
K+ channels (33), RNA synthesis
(54), cAMP response to
-adrenergic stimuli
(53), and exocytosis (56). The present study
had several goals, including the following: 1) to determine
the factors involved in tyrosine phosphorylation of PKC
in freshly
isolated salivary acinar cells; 2) to determine whether
secretagogues alter PKC
enzyme activity and whether such alterations
are dependent on tyrosine phosphorylation; and 3) to
determine whether Src-related tyrosine kinases contribute to PKC
enzyme activity and tyrosine phosphorylation in salivary and other cells.
PKC is a member of the PKC family of proteins, which is subdivided
into three classes: Ca2+-dependent classic PKCs (cPKCs),
consisting of
,
I,
II, and
; novel PKCs (nPKCs), consisting
of
,
,
, and
; and atypical PKCs (aPKCs), consisting of
and
/
. The cPKCs and nPKCs bind and are activated by DAG, for
which phorbol esters can substitute. PKC
becomes tyrosine
phosphorylated in cells exposed to various stimuli, including growth
factors (10, 26, 27) and other receptor ligands (44,
46, 47, 50), phorbol esters (10, 19, 46), and
H2O2 (21, 49), and in cells
transformed with rasHa or v-src (9, 58). A
number of groups have reported that PKC
can be tyrosine
phosphorylated in vitro by c-Abl, Src, Lyn, and other members of the
Src family of tyrosine kinases (2, 10, 14, 19, 47, 49, 50,
58). However, there are conflicting reports concerning the
contribution of tyrosine phosphorylation to the enzymatic activity of
PKC
(13). There was an increase in the tyrosine
phosphorylation of PKC
in cells in which the oncogenes v-src
(58) or rasHa (9) were
overexpressed, and this was associated with a decrease in PKC
enzymatic activity. In other cells, there was a positive association
between tyrosine phosphorylation and PKC
enzyme activation in
response to various stimuli (27, 28, 37), and other
studies failed to find any contribution of tyrosine phosphorylation in PKC
activity (26).
PKC has multiple tyrosine residues, and sites of tyrosine
phosphorylation have been localized to the regulatory domain at the
NH2-terminal side [Tyr-52 (50) and Tyr-187
(26)], the catalytic domain at the COOH-terminal side of
PKC
[Tyr-512 and Tyr-523 (21, 49)], and at a site
between the regulatory and catalytic domains [Tyr-311
(2)]. Recent studies demonstrated that PKC
is
phosphorylated on serine and threonine sites (23, 39, 40)
similar to those on cPKC family members (4, 20). In
serum-starved cells, serum promoted the phosphorylation of a
COOH-terminal hydrophobic site (Ser-662) on PKC
via a mammalian target of rapamycin (mTOR)-dependent pathway, and a site (Thr-505) in
the activation loop of the kinase domain was phosphorylated by
3-phosphoinositide-dependent protein kinase-1 (PDK1), which itself is activated by the lipid kinase phosphatidylinositol (PtdIns) 3-kinase. These sites and another one (Ser-643) were critical for the
functional activity of PKC
. To put PKC
tyrosine phosphorylation in the context of Thr-505 and Ser-662 phosphorylation, we used phosphospecific antibodies to examine the phosphorylation status of
PKC
on Thr-505 and Ser-662 in freshly isolated parotid cells exposed
to stimuli that increase the tyrosine phosphorylation of PKC
. We
also examined the active involvement of mTOR and PtdIns 3-kinase in
these phosphorylations in these cells.
On the basis of our previous findings, we examined various aspects
concerning the mechanism of tyrosine phosphorylation of PKC in
freshly isolated rat parotid acinar cells. Little information is
available concerning the role of tyrosine phosphorylation as a
signaling mechanism by exposure of these salivary gland cells to GPCR
ligands that promote fluid secretion. We investigated postreceptor
events that could contribute to the tyrosine phosphorylation of PKC
,
including PLC activation, [Ca2+]i elevation,
and the involvement of protein tyrosine kinases and phosphatases. A
major part of the present study was to determine whether tyrosine
phosphorylation produced any alterations on the enzymatic activity of
PKC
in parotid cells in response to secretory stimuli. To extend
correlative observations that indicated that increases in PKC
tyrosine phosphorylation produced increases in PKC
activity, the
reversibility of changes in enzymatic activity was examined by
dephosphorylating PKC
in vitro using PTP1B, a protein tyrosine
phosphatase. We also examined the in vitro and in vivo contributions of
Src and Src-related kinases to PKC
activation and tyrosine
phosphorylation. As a comparison for evaluating the contribution of
tyrosine phosphorylation to PKC
activity in parotid acinar
epithelial cells, we also examined this in PC-12 cells, a nonepithelial
cell type.
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MATERIALS AND METHODS |
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Chemicals.
All chemicals were reagent grade or better. Carbamylcholine
(carbachol), orthovanadate, and H2O2 were
purchased from Sigma (St. Louis, MO). Pervanadate was formed by
combining H2O2 and vanadate in a 1:1 molar
ratio and was made fresh on the day of the experiment. Substance P was
obtained from Peninsula Laboratories (Belmont, CA). PMA (phorbol
12-myristate 13-acetate) was obtained from Life Technologies (Grand
Island, NY). U-73122 was purchased from BioMol (Plymouth Meeting, PA).
4-Amino-5- (4-methylphenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine (PP1) was purchased from Calbiochem (La
Jolla, CA) and BioMol. Anti-phosphotyrosine antibody was a generous
gift of Dr. Thomas Roberts (Dana Farber, Boston, MA). Anti-PKC
polyclonal antibody (SC-213) and SRC2 antibody (SC-18; reactive toward
p60Src and other selected Src-related kinases, including
p62Yes and p59Fyn) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-PKC
monoclonal antibody
(MAb; P36520) was purchased from Transduction Laboratories (Lexington,
KY). Phospho-PKC
(Thr-505) and phospho-PKC (pan) antibodies, which
recognize the Thr-505 and Ser-662 sites, respectively, on PKC
were
obtained from Cell Signaling (New England Biolabs). Anti-Src MAb
(327) was a generous gift from Dr. Joan Brugge (Harvard
Medical School, Boston, MA). Protein A-Sepharose beads and protein
G-Sepharose beads were bought from Amersham Pharmacia.
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) was purchased from Molecular Probes
(Eugene, OR). Igepal was purchased from ICN Biomedicals (Costa Mesa,
CA). Recombinant PTP1B (14) was purchased from
Upstate Biotechnology (Lake Placid, NY) and was also a generous gift
from Dr. Benjamin Neel (Beth Israel Deaconess Medical Center, Boston, MA). DAG and phosphatidylserine (PS) were purchased from Avanti Polar
Lipids (Alabaster, AL). Dulbecco's modified Eagle's medium (DMEM) was
purchased from Bio Whittaker (Walkersville, MD).
Cell preparation and solutions.
Parotid acinar cells were prepared from male Sprague-Dawley rats
(200-250 g; Charles River Laboratories, Kingston, NY, or Taconic,
Germantown, NY) using previously established techniques (45). Briefly, rat parotid glands were removed and treated
with trypsin and collagenase to get a suspension of single cells and small groups of cells. Cells were suspended at 1-1.5 mg of
protein/ml in a medium composed of the following: 116.4 mM NaCl, 5.4 mM
KCl, 1 mM NaH2PO4, 25 mM sodium HEPES, 1.8 mM
CaCl2, 0.8 mM MgCl2, 5 mM sodium butyrate, and
5.6 mM glucose, pH 7.4. Cells were maintained on ice before use.
Aliquots (1.5 ml) of the cell suspension were equilibrated at 37°C
for at least 10 min before exposure to stimuli. In experiments designed
to examine the contribution of [Ca2+]i to the
tyrosine phosphorylation of PKC, cells were exposed to 25 µM
BAPTA-AM or vehicle (DMSO) for 30 min at 37°C before they were
exposed to stimuli. In some experiments, 5 mM EGTA was also added to
the BAPTA-loaded cells before the addition of stimuli.
Immunoprecipitations and Western blotting.
Parotid cells were exposed to various agents or vehicle (water or DMSO)
and were then collected by a brief spin in a microcentrifuge (Brinkmann
5414). The supernatant was removed, and cells were lysed in 1 ml of
ice-cold lysis buffer [137 mM NaCl, 20 mM Tris base, pH 7.5, 1 mM
EGTA, 1 mM EDTA, 10% (vol/vol) glycerol, and 1% vol/vol Nonidet P-40
(NP-40) or Igepal] containing the following phosphatase and protease
inhibitors: 1 mM vanadate, 1 mM ZnCl2, 4.5 mM sodium
pyrophosphate, 47.6 mM NaF, 9.26 mM -glycerophosphate, 0.5 mM
dithiothreitol (DTT), 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotinin, and 2 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. The lysates were vortexed and then sedimented at 16,000 g for 15 min at 4°C. In experiments conducted
using PC-12 cells, the cells were exposed to stimuli in DMEM containing 0.1% serum for the designated times, washed twice with ice-cold phosphate-buffered saline (PBS) solution (136.9 mM NaCl, 2.68 mM KCl,
1.47 mM KH2PO4, and 15.65 mM
NaH2PO4, pH 7.4), lysed in 1 ml of lysis
buffer, vortexed, and then sedimented at 16,000 g for 15 min
at 4°C. For parotid and PC-12 cell lysates, the cleared supernatants
were added to fresh tubes, and proteins were immunoprecipitated using
various antibodies in the following amounts: ~0.5 µg/ml MAb PKC
;
6 µg/ml anti-P-Tyr; 2 µg/ml MAb Src; and ~1 µg/ml polyclonal PKC
. Proteins were collected using protein A-Sepharose beads (4 mg/ml of lysate) except for the use of protein G-Sepharose (4 mg/ml) in
experiments in which Src and PKC
were immunoprecipitated individually or simultaneously with antibodies to both proteins. The
composition of the buffers and the washing protocols for the immunoprecipitations were performed as described previously, as were
the Western blotting conditions (46). For Western
blotting, the nitrocellulose filters were exposed to the following
dilutions of antibodies: anti-PKC
(polyclonal; SC-213), 0.2 µg/ml;
phospho-PKC
(Thr-505), 1:1,000 dilution; phospho-PKC (pan), 1:1,000
dilution; anti-P-Tyr, 1 µg/ml; anti-Src (MAb 327), 1 µg/ml; and
SRC2, 0.2 µg/ml.
PKC activity assay.
Cells were exposed to various agents and then lysed in 1 ml of ice-cold
lysis buffer. After the lysates were cleared by centrifugation for 15 min at 16,000 g, anti-PKC
MAb (~0.5 µg/ml) was added
to the lysate for ~3 h at 4°C, and protein A-Sepharose (4 mg/ml) was added for 1 h to collect PKC
. The immunoprecipitates were washed twice in PBS/NP-40 (1%) or PBS/Igepal (1%), once in 0.1 M
Tris (pH 7.5)/LiCl (0.5 M), and twice in 25 mM Tris (pH 7.5)/0.5 mM EGTA/5 mM MgCl2/0.5 mM DTT. All wash solutions were used
ice cold. The beads were resuspended in a final 100-µl reaction
buffer [5 mM MgCl2, 0.5 mM EGTA, 10 µM PKC
synthetic
substrate peptide (AKRKRKGSFFYGG), 1 mM DTT, and 25 mM
Tris · HCl (pH 7.5)]. As noted, in some experiments the assays
were conducted in the additional presence of 20 µg/ml PS or the
combination of 10 µM DAG plus 20 µg/ml PS in the assay buffer. The
assays were initiated with the addition of ATP {50 µM ATP and 10 µCi [
-32P]ATP (10 µCi/µl specific activity)}.
The samples were incubated for 30 min at 30°C with intermittent
mixing, and then duplicate 10-µl aliquots of each sample were spotted
onto p81 phosphocellulose paper. Background activity was measured using
lysates to which protein A-Sepharose was added without antibody. The
p81 papers were washed five times in 0.425% phosphoric acid, and the
amount of 32P was determined by liquid scintillation
counting. The duplicate values from each immunoprecipitate were
averaged and treated as one sample. Duplicate samples (2 separate
immunoprecipitates) were usually collected for each of the various
conditions in each experiment. The duplicate samples were averaged and
treated as the results from one cell preparation (n = 1). Statistics were performed on data from three or more separate
preparations/experiments. These assay conditions were similar to those
in a previous study in which the sequence of the substrate peptide,
which is based on the pseudosubstrate region of PKC
, was determined
to be optimal for PKC
(35). The basal and stimulated
PKC
activities were completely blocked in vitro by the presence of
various PKC inhibitors (1 µM RO31-8220 or 1 µM GF-109203X) in the
assay mixture (not shown). Typical basal values ranged between 5,000 and 15,000 counts per minute, depending on the cell preparation.
Dephosphorylation of PKC using PTP1B.
In some experiments, immunoprecipitates were exposed to recombinant
PTP1B (0.25 µg, specific activity 67.8 nmol · min
1 · µg
1 using
p-nitrophenylphosphate). The immunoprecipitates were washed twice in PBS/Igepal (1%), once in 0.1 M Tris (pH 7.5)/LiCl (0.5 M),
and twice in 25 mM Tris (pH 7.5)/0.5 mM EGTA/5 mM MgCl2/5 mM DTT. Dephosphorylations or mock dephosphorylations (no added PTP1B)
were performed in 100 µl of this same buffer for 30 min at 30°C
with intermittent mixing. After dephosphorylation, the immunoprecipitates were washed twice in PBS/1% NP-40 or Igepal, once
in 0.1 M Tris (pH 7.5)/LiCl (0.5 M), and twice in 25 mM Tris (pH
7.5)/0.5 mM EGTA/5 mM MgCl2/0.5 mM DTT. PKC
activity
assays were performed as described above.
Joint immunoprecipitation of Src and PKC.
In experiments in which the effect of Src on PKC
activity was
measured, cells were lysed in RIPA buffer (0.1% SDS, 1% NP-40 or
Igepal, 0.5% deoxycholate, 158 mM NaCl, and 20 mM Tris, pH 8.0), and
PKC
was immunoprecipitated using anti-PKC
MAb alone or a
combination of anti-PKC
antibody and anti-Src (MAb 327) antibodies.
In some experiments, Src was immunoprecipitated in the absence of
PKC
using anti-Src antibody (MAb 327). The immunoprecipitates were
collected with protein G-Sepharose beads and were washed three times in
RIPA and twice in 20 mM HEPES (pH 7.4)/5 mM MgCl2/5 mM
MnCl2. Some immunoprecipitates were subjected to in vitro
phosphorylation reactions in the same buffer (± 100 µM ATP) for 45 min at 30°C with intermittent mixing. The immunoprecipitates were
washed twice in PBS/1% Igepal, once in 0.1 M Tris (pH 7.5)/LiCl (0.5 M), and twice in 25 mM Tris (pH 7.5)/0.5 mM EGTA/5 mM
MgCl2/0.5 mM DTT. PKC
activity assays were performed as
described above. Some immunoprecipitates used for PKC
activity
assays were subsequently used for Western blotting. For these samples,
2× sample buffer was added to the immunoprecipitates after the PKC
assays were complete, and the samples were subjected to SDS-PAGE.
Data.
The mean value ± SE of n number of different
experiments are as indicated. The percent inhibition of PKC activity
by various conditions was calculated as the difference between
stimulated and basal activities in the inhibitory condition (PP1 in
vivo or PTP1B in vitro) compared with the difference between stimulated and basal under control conditions. Statistics were performed using a
paired t-test. Western blots are representative of at least
three different experiments.
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RESULTS |
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Tyrosine phosphorylation of PKC is blocked by U-73122 in parotid
cells exposed to carbachol or substance P but not PMA or pervanadate.
In rat parotid acinar cells, the tyrosine phosphorylation of PKC
is
increased in cells exposed to carbachol and substance P, which are
agonists to PLC-linked GPCRs, as well as in cells exposed to PMA
(46). The GPCR-dependent changes in tyrosine phosphorylation occur very rapidly and are maximal within the first
minute of exposure to receptor ligands (46). To
investigate whether PLC activation was involved in promoting the
tyrosine phosphorylation of PKC
, parotid cells were exposed to the
PLC inhibitor (3, 12, 17, 57) U-73122 (10 µM), which
blocked the elevation of [Ca2+]i in parotid
acinar cells (not shown). The effects of carbachol and
substance P were blocked in U-73122-treated cells (Fig.
1A), suggesting that the
receptor-mediated tyrosine phosphorylation of PKC
was initiated by
the production of DAG or the elevation of
[Ca2+]i. The tyrosine phosphorylation of
PKC
was also increased by pervanadate, an inhibitor of PTP1B
(18), and other protein tyrosine phosphatases. Pervanadate
(1 min) increased the tyrosine phosphorylation to a much greater level
than carbachol (1 min) or PMA (5 min) (Fig. 1, B and
C).1 The effects
of PMA and pervanadate on the tyrosine phosphorylation of PKC
were
not blocked by U-73122 (Fig. 1, B and C),
indicating that the effects of PMA and pervanadate are independent of
PLC activity. The responses to pervanadate, which also increased the tyrosine phosphorylation of other proteins (not shown), suggest that
the inhibition of a tyrosine phosphatase is sufficient to promote the
tyrosine phosphorylation of PKC
in these cells. Unlike the effects
of pervanadate, equivalent exposures (100 µM, 1 min) of
H2O2 or vanadate were ineffective in producing
large increases in the tyrosine phosphorylation of PKC
or other
proteins (not shown). The effect of PMA on U-73122-treated cells
suggests that the production of DAG is also sufficient to promote an
increase in PKC
tyrosine phosphorylation. In addition, either the
constitutive activity of a tyrosine kinase or a kinase that is
activated by carbachol, substance P, PMA, and pervanadate is able to
promote the tyrosine phosphorylation of PKC
in these epithelial
cells. Additional experiments suggested that Src or a related protein kinase is the tyrosine kinase that phosphorylates PKC
in both parotid and PC-12 acinar cells.
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Elevation of [Ca2+]i does not contribute
to PKC tyrosine phosphorylation.
Although we previously found that exposure of salivary gland
epithelial cells to the Ca2+ ionophore ionomycin did not
promote the tyrosine phosphorylation of PKC
(46), there
was a possibility that elevations in
[Ca2+]i supported the stimulatory effects of
carbachol and substance P on PKC
tyrosine phosphorylation. To
examine this possibility, we loaded parotid acinar cells with the
Ca2+ chelator BAPTA, which buffers the carbachol-promoted
rise in [Ca2+]i in these cells
(45). BAPTA-loaded cells responded to carbachol in a
similar manner as those cells that were not loaded with BAPTA (Fig.
2). The carbachol-promoted increase in
PKC
tyrosine phosphorylation was also observed in BAPTA-loaded cells
that were exposed to EGTA to deplete extracellular Ca2+
(not shown). These results indicate that a rapid rise in
[Ca2+]i was not required for the stimulatory
effect of carbachol on PKC
tyrosine phosphorylation and demonstrate
that another aspect of muscarinic receptor activation was responsible
for the rapid increase in PKC
tyrosine phosphorylation. In
conjunction with the inhibitory effects of U-73122 on the
substance P- and carbachol-promoted PKC
tyrosine phosphorylations
(Fig. 1) and the positive effect of PMA on PKC
tyrosine
phosphorylation, these studies are consistent with DAG production
playing the main role in the receptor-mediated increases in the
tyrosine phosphorylation of PKC
.
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Tyrosine phosphorylation of PKC increases the activity of PKC
in parotid acinar cells and PC-12 cells.
We performed in vitro substrate phosphorylation assays using PKC
immunoprecipitated from cells exposed to various stimuli. The exposure
of parotid acinar cells to carbachol (10
4 M, 1 min)
increased the PKC
activity to 1.8 ± 0.1 (n = 4) times the basal level found in unstimulated cells. PMA (100 nM, 5 min) and pervanadate (100 µM, 1 min) each increased PKC
activity
by a much greater amount, to 4.1 ± 1.0 (n = 6)
and 4.6 ± 1.0 (n = 4), respectively, times the
basal level. These results suggested that the tyrosine phosphorylation
of PKC
promoted an increase in PKC
activity in salivary acinar cells.
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Stimuli-dependent increases in PKC activity did not occur in
presence of lipid cofactors.
The alterations in PKC
activity in parotid and PC-12 cells presented
above were measured in the absence of lipid cofactors and reflect an
increase in PKC
activity that is intrinsic to the immunoprecipitated
protein and not due to increases in protein levels. When PS was present
in the assay mixture (see MATERIALS AND METHODS), the
PKC
activity in immunoprecipitates from PMA (100 nM, 5 min)-stimulated PC-12 cells was significantly greater than the activity
in unstimulated cells, but the degree of stimulation was less than that
measured in assays conducted in the absence of added lipids (Fig.
5). Note, however, that the basal
activity in the presence of PS in vitro was approximately three times
that measured in the absence of PS (see Fig. 5 legend). When DAG and PS
were both present in the assay mixture, the basal PKC
activity in
anti-PKC
immunoprecipitates from unstimulated PC-12 was increased to
28.9 ± 4.2 (n = 3) times the activity measured in
the absence of lipid cofactors. However, in the presence of DAG plus
PS, the PKC
activity found in immunoprecipitates from stimulated
PC-12 cells was not enhanced compared with the activity in
immunoprecipitates from unstimulated cells. For example, under these
conditions (PS+DAG) the activity in PMA (100 nM, 5 min)-treated cells
was 0.9 (n = 2) times that in untreated cells (Fig. 5),
and the activity in PMA (100 nM, 5min) plus pervanadate (100 µM, 5 min)-treated cells was 1.0 ± 0.1 (n = 4) times
the activity in untreated cells. Thus the contribution of tyrosine
phosphorylation to PKC
activity was not manifested when DAG and PS
were both present in the assay, as was also reported by others
(21, 36).
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Src phosphorylates PKC and increases its enzymatic activity.
Previously, we found that a kinase inhibitor, staurosporine, and
genistein, a nonselective tyrosine kinase inhibitor, blocked the
tyrosine phosphorylation of PKC
(5). Because several
investigators have suggested a role for Src, Src-related kinases, and
other tyrosine kinases in the tyrosine phosphorylation of PKC
, we
conducted several experiments to determine whether Src-related proteins are involved in the activation of PKC
by receptor-mediated and nonreceptor-mediated agents in parotid cells and PC-12 cells. In one
series of in vivo experiments, parotid acinar cells were treated with
PP1, an inhibitor of Src and Src family members (16), and
then the cells were exposed to agents that promoted increases in PKC
tyrosine phosphorylation and activity. Treatment of parotid cells with
PP1 blocked carbachol- and pervanadate-promoted increases in tyrosine
phosphorylation (Fig. 6A). PP1
also blocked the effects of PMA on PKC
tyrosine phosphorylation (not
shown). PP1 treatment of parotid cells also blocked the increases in
PKC
activity promoted by carbachol, PMA, and pervanadate by 78.2%
(n = 2), 83.1 ± 6.6% (n = 3),
and 67.3 ± 21.3% (n = 3), respectively (Fig.
6B). PP1 treatment did not significantly reduce the basal
activity in parotid cells (Fig. 6B), consistent with a role
for Src in the increase of PKC
activity but not in basal activity.
PP1 blocked the tyrosine phosphorylation of PKC
in PMA (± pervanadate)-treated PC-12 cells (Fig. 6C), and it also
blocked the PMA-promoted increase in PKC
activity by 74.8%
(n = 2). These results suggest that the in vivo activity of Src or an Src-like protein is responsible for the increases
in PKC
tyrosine phosphorylation and the resulting increases in
PKC
activity in both parotid acinar cells and PC-12 cells. These
results also suggest that a Src-related kinase is involved in tyrosine
phosphorylation that is promoted by diverse agents: PLC-linked receptor
agonists, phorbol ester, and a phosphatase inhibitor.
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Tyrosine phosphorylation of PKC does not alter constitutive
serine and threonine phosphorylations.
PKC
is also phosphorylated on particular serine and threonine
residues that can affect PKC
activity. To put PKC
tyrosine phosphorylation in the context of Thr-505 and Ser-662 phosphorylation, we examined the phosphorylation of PKC
on Thr-505 and Ser-662 in rat
parotid acinar cells exposed to carbachol and PMA. Because PtdIns
3-kinase and mTOR activities are upstream of the phosphorylation of
these sites (39, 40), we treated cells with the PtdIns 3-kinase inhibitor LY-294002 and the mTOR inhibitor rapamycin. In
parotid cells, Thr-505 and Ser-662 sites were constitutively phosphorylated under basal conditions. These phosphorylations were not reduced by exposure of cells to LY-294002 (100 µM) or rapamycin (20 nM; Fig. 8) for times up to
30 min. The PtdIns 3-kinase inhibitor wortmannin (100 nM) gave similar
results to LY-294002 (not shown). No alterations in the constitutive
Thr-505 or Ser-662 phosphorylations were observed in cells exposed to
100 µM carbachol for 1-10 min (not shown) or PMA for 5 min (Fig.
8) or 10 min (not shown) in control cells or in cells exposed to PtdIns
3-kinase and mTOR inhibitors. The increases in PKC
tyrosine
phosphorylation promoted by PMA (Fig. 8) and carbachol (not shown) were
not affected by the presence of these inhibitors. In stimuli-treated
cells, the more slowly migrating top band of PKC
was phosphorylated on Thr-505 and Ser-662 in addition to tyrosine residues, but the bottom
band was only phosphorylated on Thr-505 and Ser-662. These results
indicate that tyrosine phosphorylation of PKC
in freshly isolated
parotid cells occurs in the absence of any change in Thr-505 and
Ser-662 phosphorylation.
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DISCUSSION |
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A major reason for undertaking these studies was to examine the
contribution of tyrosine phosphorylation to PKC enzyme activity in
rat parotid acinar cells, which respond to secretory stimuli with an
increase in PKC
tyrosine phosphorylation. We also examined PC-12
cells as a second cell model. From our results, we conclude the
following: 1) the activation of PLC and the production of DAG account for muscarinic and substance P-mediated increases in PKC
tyrosine phosphorylation in parotid acinar cells; 2)
increases in the tyrosine phosphorylation of PKC
by
receptor-mediated and nonreceptor-mediated agents have a positive
effect on PKC
enzyme activity in both parotid and PC-12 cells;
3) the activation of PKC
is reversible upon the
dephosphorylation of the tyrosine residues; 4) Src or
Src-related proteins phosphorylate PKC
on tyrosine in vivo and in
vitro, and this increases its activity; and 5) the
stimuli-dependent tyrosine phosphorylation of PKC
in freshly
isolated salivary cells occurs subsequent to its phosphorylation on
Thr-505 and Ser-662.
PKC was activated by receptor-mediated stimuli (carbachol), by PMA,
which bypasses PLC (Fig. 1), and by pervanadate. Pervanadate does not
appear to act by stimulating PLC (Fig. 1); presumably, it promotes the
tyrosine phosphorylation of PKC
by inhibiting tyrosine phosphatase
activity. Increases in carbachol-mediated PKC
tyrosine
phosphorylation in parotid cells were blocked by inhibiting PLC (Fig.
1) but were not blocked by preventing the elevation of
[Ca2+]i (Fig. 2), suggesting that DAG
production initiated the activation of PKC
in parotid acinar cells
in a Ca2+-independent manner. Previously, we reported that
the tyrosine kinase inhibitor genistein blocked increases in the
tyrosine phosphorylation of PKC
(5). The data presented
in this paper suggests that Src or a Src-related kinase is the tyrosine
kinase that phosphorylates PKC
in these cells. In addition, these
studies suggest that both protein tyrosine kinases and phosphatases
affect the tyrosine phosphorylation and activity of PKC
.
A model for PKC tyrosine phosphorylation is as follows: the
production of DAG in response to PLC-linked receptor activation, or,
bypassing this step, the exposure of cells to PMA promotes the
recruitment of PKC
to the plasma membrane, where PKC
is a
substrate for Src or other Src-like proteins and is phosphorylated on
tyrosine residues that contribute to increases in PKC
activity. Previously, we demonstrated that PMA promoted the translocation of
PKC
from the cytosol to a membrane compartment of salivary gland
acinar cells, and the tyrosine phosphorylated form of PKC
was
exclusively found in the membrane compartment (46). Other investigators have made similar observations concerning the
localization of tyrosine-phosphorylated PKC
(13, 19, 24,
58), although an alternative finding of PMA-promoted
tyrosine-phosphorylated PKC
in the cytosol has also been reported
(42).
Presumably, membrane-localized PKC is a substrate for Src or a
Src-related protein that is constitutively active and able to
phosphorylate PKC
in cells exposed to GPCRs, PMA, and pervanadate. Other proteins known to be Src substrates are constitutively
phosphorylated on tyrosine residues in unstimulated parotid cells
(unpublished observations). Src can also be directly activated by
-subunits of G proteins (31), but this cannot account
for the stimulation promoted by PMA or pervanadate. Under the
conditions of our experiments, we did not find a physical association
between Src or Src-related proteins and PKC
. If this association
occurred, it did not survive our immunoprecipitation protocol or was
too subtle to detect by immunoblot techniques. In other cellular
systems, PKC
was coimmunoprecipitated with Src and members of the
Src family (42, 47, 58). PKC
and c-Abl associate in
response to oxidative stress (H2O2), and each
of these two proteins affects the activity of each other (49).
Studies of PKC using serum-starved cells indicate that serum can
promote increases in the phosphorylation of Thr-505 and Ser-662. Under
these conditions, these sites were crucial for PKC
activity, and the
phosphorylation of these sites were dependent on pathways involving
PtdIns 3-kinase and mTOR (39, 40). In our experiments
using freshly isolated parotid cells, these sites were constitutively
phosphorylated and were not changed by exposure to PtdIns 3-kinase and
mTOR inhibitors (Fig. 8). Presumably, cellular serine/threonine
phosphatases were ineffective in dephosphorylating Thr-505 and Ser-662
during the relatively brief (30-min) exposure of cells to these
inhibitors. This may not be surprising, because cultured cells must be
serum starved (23) and maintained in suspension (38,
40) to reduce the phosphorylation of these sites before
stimulation by serum. Phosphorylations of the basal serine and
threonine phosphorylation sites were also not affected by secretory
stimuli (carbachol) and other PKC-activating agents (PMA). These PKC
sites had already been phosphorylated in the salivary cells, and
tyrosine phosphorylation occurred subsequent to threonine and serine
phosphorylation. The basal phosphorylation status of the cells did not
allow us examine alterations in tyrosine phosphorylation under
conditions in which the enzyme was not phosphorylated on Thr-505 and
Ser-662, and, therefore, we cannot conclude that tyrosine
phosphorylation and its effects on PKC
activity absolutely require
the prior phosphorylation of these threonine and serine sites. It also
should be noted that a contrasting argument has been made that Glu-500,
an acidic residue in the activation loop, rather than phosphorylated
Thr-505, is required for the catalytic activity of PKC
(48).
There have been contrasting studies concerning the contribution of
tyrosine phosphorylation to the activity of PKC. Because there are
multiple tyrosine residues on PKC
, phosphorylation of the different
residues could have quite different effects on the absolute activity of
PKC
and/or the biological activity of PKC
. Evidence of the latter
was recently suggested (22). Thus our findings may be
restricted to activation of PKC
by PLC-linked receptor activation,
phorbol ester, and pervanadate treatment of these cells. In CHO-K1
cells, PKC
activity was increased by activation of a G
protein-coupled ATP-binding receptor by PMA and by
H2O2 (37). The degree of maximal
activation by these stimuli was H2O2
PMA > ATP, and the relative amount of tyrosine phosphorylation
also followed this relative order, although no significant degree of
phosphorylation was detected for ATP. These results are similar to the
relative order of stimuli acting on salivary gland cells:
pervanadate
PMA > carbachol.
The exposure of parotid cells to 100 µM pervanadate produced a much
larger increase in PKC tyrosine phosphorylation than did exposure of
the cells to 100 nM PMA (Figs. 1C and 4A), yet both agents produced about the same degree of increase in PKC
activity (Fig. 4B). This suggests that multiple tyrosine
residues on PKC
can be phosphorylated but that not all of the
phosphorylated tyrosine residues contribute to increases in PKC
activity. This may be true for increases in tyrosine phosphorylation
promoted by PMA and GPCR ligands as well as by pervanadate. On the
basis of mutational studies and an analysis of tryptic digests of
PKC
, Blake et al. (2) concluded that Src phosphorylated
Tyr-311 but that additional tyrosine sites were subsequently
phosphorylated. Thus the relative degree of stimuli-dependent increases
in PKC
activity is not necessarily quantitative with the relative
increases in tyrosine phosphorylation, especially when different
stimuli are compared.
The tyrosine phosphorylation of PKC produced at most an increase in
activity to levels four to five times the basal level (Fig.
6B). However, the tyrosine-phosphorylated form of PKC
, which migrated more slowly through an SDS-PAGE gel (Figs.
3A, 4, A and C, and 6A),
constituted at most one-half of the total PKC
protein in cells
exposed to PMA and pervanadate in both cell types. Thus the
tyrosine-dependent increases in PKC
activity would be at least twice
the values as those reported here if all of the PKC
in the samples
were composed of the tyrosine-phosphorylated form. In parotid cells
exposed to carbachol, the tyrosine-phosphorylated form of PKC
made
up a much lower fraction of the total PKC
(46), and
this would appear to account for the relatively smaller increase in
PKC
activity promoted by carbachol compared with PMA or pervanadate. Stimuli-dependent increases in activity due to tyrosine phosphorylation were not observed in the presence of DAG and PS, which increased the
assayed activity to a much greater level than that produced by tyrosine
phosphorylation, but stimuli-dependent increases in activity were
observed (albeit to a lesser relative extent) in the presence of PS
alone (Fig. 3). Presumably, tyrosine phosphorylation and lipid
production both affect PKC
activity in vivo, and the membrane lipid
composition in vivo may not be that which is idealized under in vitro
conditions. In vivo, PKC
may localize at a site that has a lipid
composition much different from that which permits the maximal
activation of the enzyme in vitro. PKC
also may have a biological
role in a nonmembrane compartment or in a membrane compartment distinct
from the plasma membrane. In fact, PKC
was found to sequentially
localize to nuclear and Golgi membranes after it translocated to the
plasma membrane in response to PMA and other phorbol ester derivatives
(51). In some cells, phorbol ester initiated the
translocation of PKC
to the mitochondria where it was involved in
promoting apoptosis (32). The translocation of
PKC
to a new location will increase PKC
activity at that location. In addition to affecting PKC
activity, tyrosine
phosphorylation can alter the substrate specificity of PKC
(15) as well as the lipid dependence (19).
Thus changes in location and changes in various enzymatic parameters
may affect the biological activity of PKC
.
PKC is an important signaling protein that produces various
effects on cells (for review, see Ref. 13), including
alterations of cell growth (29, 52). In PC-12 cells,
PKC
modulated the upregulation of L-type Ca2+ channels
in ethanol-treated cells (11) and also appeared to play a
role in nerve growth factor-mediated neurite outgrowth (36). PKC
was involved in receptor-mediated activation
of the Na+-K+-Cl
cotransport
system in human tracheal epithelial cells (25) and
affected the permeability of tight junctions in LLC-PK1
epithelial cells (34). We did not directly address the
physiological role of PKC
in rat parotid acinar epithelial cells,
but the activation of parotid muscarinic receptors, which increases
PKC
activity, stimulates both fluid secretion as well as a
Ca2+-sensitive component of exocytosis of protein secretory
granules (56). PKC
may play a role in these events.
PKC
plays a critical role in etoposide-induced apoptosis in
a cultured rat parotid acinar cell line (41). However,
this was mediated by the caspase-dependent cleavage of PKC
to its
constitutively activated 40-kDa form, which does not play a role in the
increases in PKC
activity presented in our studies.
In conclusion, these results demonstrate that Src-related protein
kinases and tyrosine phosphorylation participate in cellular events
initiated by secretagogues acting on freshly isolated salivary acinar
cells. DAG production, Src tyrosine kinases, and tyrosine phosphatases
play integrated roles in the regulation of PKC activity. In both
salivary gland epithelial cells and PC-12 cells, two cell types that
contain neither overexpressed PKC
nor overexpressed Src family
members, the tyrosine phosphorylation of PKC
produced a positive
effect on PKC
activity. This property of PKC
is important in our
understanding of the biological function of PKC
in these and other cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Yue Zheng for excellent technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Institute of Dental Research Grant DE-10877 (to S. P. Soltoff).
Address for reprint requests and other correspondence: S. P. Soltoff, Division of Signal Transduction, Dept. of Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Rm. 1025, 330 Brookline Ave., Boston, MA 02215 (E-mail: ssoltoff{at}caregroup.harvard.edu).
1
The exposure of parotid cells to 100 µM
pervanadate for 1 min produced a larger increase in PKC tyrosine
phosphorylation than the effects of the exposure of these cells to PMA
(100 nM) or carbachol (10
4 M) for any time between 0 and
15 min.
2
The relatively lower effectiveness of PTP1B
treatment in reducing the enhanced PKC activity in PC-12 cells
compared with salivary cells was due to the lower effectiveness of one
particular lot of PTP1B that was used to dephosphorylate PKC
immunoprecipitated from PC-12 cells.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 September 2000; accepted in final form 17 January 2001.
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