J Biol Chem, Vol. 274, Issue 44, 31261-31271, October 29, 1999
Cholecystokinin Activates PYK2/CAK
by a Phospholipase
C-dependent Mechanism and Its Association with the
Mitogen-activated Protein Kinase Signaling Pathway in Pancreatic
Acinar Cells*
Jose A.
Tapia
,
Heather A.
Ferris§,
Robert T.
Jensen§¶, and
Luis J.
García
From the § Digestive Diseases Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892 and
Departamento de Fisiología, Universidad de
Extremadura, Cáceres 10071, Spain
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ABSTRACT |
PYK2/CAK
is a recently described cytoplasmic
tyrosine kinase related to p125 focal adhesion kinase
(p125FAK) that can be activated by a number of
stimuli including growth factors, lipids, and some G protein-coupled
receptors. Studies suggest PYK2/CAK may be important for coupling
various G protein-coupled receptors to the mitogen-activated protein
kinase (MAPK) cascade. The hormone neurotransmitter cholecystokinin
(CCK) is known to activate both phospholipase C-dependent
cascades and MAPK signaling pathways; however, the relationship between
these remain unclear. In rat pancreatic acini, CCK-8 (10 nM) rapidly stimulated tyrosine phosphorylation and
activation of PYK2/CAK by both activation of high affinity and low
affinity CCKA receptor states. Blockage of CCK-stimulated
increases in protein kinase C activity or CCK-stimulated increases in
[Ca2+]i, inhibited by 40-50% PYK2/CAK but
not p125FAK tyrosine phosphorylation. Simultaneous blockage
of both phospholipase C cascades inhibited PYK2/CAK tyrosine
phosphorylation completely and p125FAK tyrosine
phosphorylation by 50%. CCK-8 stimulated a rapid increase in
PYK2/CAK kinase activity assessed by both an in vitro
kinase assay and autophosphorylation. Total PYK2/CAK under basal
conditions was largely localized (77 ± 7%) in the membrane
fraction, whereas total p125FAK was largely localized
(86 ± 3%) in the cytosolic fraction. With CCK stimulation, both
p125FAK and PYK2/CAK translocated to the plasma
membrane. Moreover CCK stimulation causes a rapid formation of both
PYK2/CAK-Grb2 and PYK2/CAK-Crk complexes. These results
demonstrate that PYK2/CAK and p125FAK are regulated
differently by CCKA receptor stimulation and that PYK2/CAK is probably an important mediator of downstream signals by
CCK-8, especially in its ability to activate the MAPK signaling pathway, which possibly mediates CCK growth effects in normal and
neoplastic tissues.
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INTRODUCTION |
Cholecystokinin (CCK)1
functions as a neurotransmitter in the central nervous system and
gastrointestinal tract as well as a hormone in the gastrointestinal
tract and has numerous biologic effects (1). In the central nervous
system, CCK has such widespread effects as functioning as a potent
regulator of satiety and morphine-induced analgesia, modulating
dopamine release, stimulating panic attacks, and stimulating vagal
afferent transmission (1-5). In the gastrointestinal tract, CCK is a
physiological regulator of pancreatic secretion, gallbladder
contraction, gastric emptying, and colonic motility (1, 6-9). The
physiological and cellular basis of action of CCK have been extensively
studied in pancreatic acinar cells, where CCK stimulates activation of
c-fos, c-jun, and c-myc oncogene expression (10), enzyme secretion, enzyme synthesis (1, 6), growth (8),
and development (11). Extensive studies show that CCK causes activation
of phospholipase A2, phospholipase D, and phospholipase C
(PLC) (12). The activation of phospholipase C results in generation of
diacylglycerol and increased inositol 1,4,5-trisphosphate with
subsequent activation of protein kinase C (PKC), stimulation of
increases in intracellular calcium concentration, and activation of
mitogen-activated protein kinase (MAPK) signaling pathways (12-16).
Recent studies have demonstrated that CCKA receptor activation (17-19), similar to the activation in different tissues by
integrins (20, 21), bioactive lipids (22, 23), various growth factors
(19, 21, 24), and some G protein-coupled receptors (19, 25-28), causes
stimulation of tyrosine phosphorylation of a number of proteins. With
other stimulants, activation of this important intracellular pathway
has been shown to be particularly important in mediating cellular
growth and motility and adapting to cellular stresses (21, 25, 29).
Similar to a number of other G protein-coupled receptors (GPCRs), the
relationship between activation of the phospholipase C cascade, the
MAPK cascade, and tyrosine phosphorylation of various proteins
stimulated by CCKA receptor activation remains unclear. Recent studies (30-34) suggest that with activation of some GPCRs (30,
32-35), which cause increases in cellular calcium as well as some
integrins (31, 36, 37), growth factors (37-39), or phospholipids (32,
40), one protein, the cytoplasmic tyrosine kinase proline-rich kinase 2 (PYK2) (30) (also called cell adhesion kinase (CAK) (41),
related adhesion focal tyrosine kinase (RAFTK) (42), focal adhesion
kinase 2 (FAK2), and calcium-dependent tyrosine kinase
(CADTK) (40)) may be a particularly important tyrosine-phosphorylated
substrate responsible for coupling these intracellular cascades.
Studies with some GPCRs and various growth factors demonstrate that
activation of PYK2/CAK can activate the MAPK cascade and that
activation of PYK2/CAK in some cells, but not others, is completely
dependent on PLC-mediated increases in cytoplasmic calcium, activation
of PKC, or both (30, 33-35, 39, 43, 44). Numerous mechanisms for the
ability of GPCRs to activate the MAPK cascade by PYK2/CAK activation
have been described, including involvement of the Src kinase family
(32, 34, 45, 46), transactivation of the epidermal growth factor receptor (44, 47, 48), tyrosine phosphorylation of the adapter protein
Shc (33, 35, 49, 50), and participation of other cytoplasmic tyrosine
kinases such as Lyn and Syk (47, 51), as well as the ability of
resultant activation of subunits of heterotrimeric G proteins (34, 45,
46, 49, 50, 52) to cause subsequent activation of the MAPK cascade.
With different GPCRs, the coupling to PYK2/CAK and the mechanisms of
the possible coupling of these different intracellular cascades vary widely.
At present, it is unknown whether CCKA receptor activation
stimulates tyrosine phosphorylation of PYK2/CAK or whether its ability to activate one or both limbs of the PLC cascade is important for this tyrosine phosphorylation if it occurs. It is also unknown whether PYK2/CAK activation might be an important intermediate in
coupling the CCKA receptor activation to MAPK cascade
activation, which is important in mediating the potent effects of this
receptor's activation on normal and neoplastic tissues. In the present
study, the ability of CCKA receptor activation to stimulate
PYK2/CAK tyrosine phosphorylation and its relationship with these
other intracellular signaling cascades was explored in pancreatic
acini, which are one of the main physiological sites of action of CCK.
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EXPERIMENTAL PROCEDURES |
Materials
Male Harlan Sprague Dawley rats (150-200 g) were obtained from
the Small Animals Section, Veterinary Resources Branch, National Institutes of Health (Bethesda, MD) or from the Animal Farm, Faculty of
Veterinary (Universidad de Extremadura, Cáceres, Spain); purified collagenase (type CLSPA) was from Worthington; COOH-terminal
octapeptide of cholecystokinin (CCK-8) was obtained from Peninsula
Laboratories (Belmont, CA); CCK-JMV was obtained from Research Plus
Inc. (Bayonne, NJ); phosphate-buffered saline, pH 7.4, was from
Biofluids (Rockville, MD); anti-proline-rich tyrosine kinase 2 (PYK2)
monoclonal antibody (mAb), anti-p125 focal adhesion kinase
(p125FAK) mAb, anti-phosphotyrosine mAb (PY20), anti-Crk
mAb, and anti-Grb2 mAb were from Transduction Laboratories (Lexington,
KY); recombinant protein A-agarose was from Upstate Biotechnology Inc.
(Lake Placid, NY); GF109203X, the calcium ionophore A23187, and
thapsigargin were from Calbiochem; soybean trypsin inhibitor,
Me2SO, Triton X-100, 12-O-tetradecanoylphorbol
13-acetate (TPA), ATP, poly(Gly-Tyr) (4:1), and deoxycholic acid were
from Sigma; phenylmethanesulfonyl fluoride was from Fluka (Ronkonkoma,
NY); basal medium Eagle amino acids and basal medium Eagle vitamin
solution were from Life Technologies, Inc.; bovine serum albumin
fraction V was from Miles Inc. (Kankakee, IL); aprotinin, leupeptin,
and HEPES were from Roche Molecular Biochemicals; rabbit anti-mouse IgG
and anti-mouse IgG-horseradish peroxidase conjugate were from Pierce;
SDS, 2-mercaptoethanol, protein assay dye reagent, Tris/Glycine/SDS
buffer (10× concentrated), and Tris/glycine buffer (10× concentrated)
were from Bio-Rad; BAPTA acetoxymethyl ester (BAPTA/AM) and fura-2
acetoxymethyl ester (fura-2/AM) were from Molecular Probes, Inc.
(Eugene, OR); [
-32]ATP (3000 Ci/mmol), Hyperfilm ECL,
and enhanced chemiluminescence detection reagents were from Amersham
Pharmacia Biotech; and nitrocellulose membrane was from Schleicher & Schuell.
Methods
Tissue Preparation--
Dispersed rat pancreatic acini were
prepared according to the modifications (53) of the procedure published
previously (54). Unless otherwise stated, the standard incubation
solution contained 25.5 mM HEPES (pH 7.4), 98 mM NaCl, 6 mM KCl, 2.5 mM
NaH2PO4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 11.5 mM glucose, 0.5 mM CaCl2, 1 mM MgCl2, 2 mM glutamine, 1% (w/v)
albumin, 1% (w/v) trypsin inhibitor, 1% (v/v) vitamin mixture, and
1% (w/v) amino acid mixture. The incubation solution was equilibrated with 100% O2, and all incubations were performed with
100% O2 as the gas phase.
Intracellular Ca2+ Measurements--
After
isolation, pancreatic acini were resuspended in NaHEPES medium
containing 130 mmol/liter NaCl, 5 mmol/liter KCl, 20 mmol/liter HEPES,
1.2 mmol/liter KH2PO4, 10 mmol/liter
D-glucose, 1 mmol/liter CaCl2, 0.1 mg/ml
trypsin inhibitor and loaded with 2 µM fura2/AM for
30-40 min at 25 °C. Changes in [Ca2+]i were
assessed using a spectrofluorophotometer RF-5001PC (Shimadzu Europe
GmbH, Duisburg, Denmark) as described previously (55). Fluorescence was
measured at 500 nm after excitation at 340 nm
(F340) and 380 nm (F380).
Values for [Ca2+]i were calculated, after
subtraction of background autofluorescence, as described previously
(55).
Immunoprecipitation--
Immunoprecipitation of
tyrosine-phosphorylated proteins was performed as described
previously (17, 56). Dispersed acini from one rat were preincubated
with standard incubation solution without or with different inhibitors
for 3 hours at 37 °C. Aliquots (1 ml) were then incubated at
37 °C with different agonists at the concentrations and times
indicated. Acinar lysates were obtained as described (17, 56). For
tyrosine phosphorylation determination, lysates (500 µg/ml) were
incubated with 4 µg of anti-phosphotyrosine monoclonal antibody
(PY20) or 3 µg of anti-PYK2/CAK mAb and 3 µg of rabbit
anti-mouse IgG and 25 µl of protein A-agarose overnight at 4 °C.
For co-immunoprecipitation studies, lysates (500 µg) were incubated
with 4 µg of anti-Crk mAb or 4 µg of anti-Grb2 mAb for 2 h at
4 °C. Then the immune complexes were incubated with 4 µg of rabbit
anti-mouse IgG and 25 µl of protein A-agarose for 1 h at
4 °C. The immunoprecipitates were washed three times with
phosphate-buffered saline and further analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting.
Subcellular Fractionation--
Acinar cell fractionation was
carried out according to the procedure published previously (57) with
minor modifications. Briefly, acinar cells were separated by
centrifugation, washed with phosphate-buffered saline with 0.2 mM Na3VO4 at 4 °C, centrifuged, and resuspended in 1 ml of lysis buffer without Triton and deoxycholate and homogenized with the use of a Polytron homogenizer (Brinkmann Instruments, Westburg, NY) for 20 s at power level 6 at 4 °C. Homogenates were centrifuged first at 500 × g for 10 min at
4 °C to remove nuclei and debris and then for 30 min at 100,000 × g at 4 °C to obtain membrane and cytosol fractions. Precipitates were washed with phosphate-buffered saline with 0.2 mM
Na3VO4 at 4 °C and resuspended in 0.5 ml of
lysis buffer and sonicated for 5 s at 4 °C. Lysates were
centrifuged at 15,000 × g for 15 min to remove
insoluble substances. Protein concentration was measured by the Bio-Rad
protein assay reagent.
Western Blotting--
Western blotting was performed as
described previously (17, 56). Immunoprecipitates or subcellular
fractions were subjected to 10% SDS-PAGE gels; proteins were
transferred to nitrocellulose membranes (0.45 and 0.2 µm for proteins
higher and lower than 60 kDa, respectively). Membranes were blocked and
incubated for 2 h at 25 °C with 0.5 µg/ml anti-PYK2/CAK
mAb, 1 µg/ml anti-phosphotyrosine mAb, 0.25 µg/ml
anti-p125FAK, 0.5 µg/ml anti-Crk mAb, or 0.5 µg/ml
anti-Grb2 mAb. Membranes were washed twice and incubated for 45 min at
25 °C with anti-mouse IgG-horseradish peroxidase conjugate. The
membranes were washed, and proteins were visualized using ECL reagents
and Hyperfilm ECL. Density of bands was measured using a scanning
densitometer (Molecular Dynamics, Inc., Sunnyvale, CA). When necessary,
membranes were reprobed as described previously (56).
In Vitro Kinase Assay and Autophosphorylation of
PYK2/CAK--
After incubation with 10 nM CCK-8 for the
times indicated above, pancreatic acini were lysed with kinase lysis
buffer, which contained 50 mM Tris-HCl (pH 7.4), 1% Triton
X-100, 1 mM Na3VO4, 150 mM NaCl, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 1 mM phenylmethanesulfonyl
fluoride. PYK2/CAK was immunoprecipitated as described above. The
immunoprecipitates were washed two times with kinase lysis buffer and
two times with kinase buffer (50 mM Tris-HCl, pH 7.4, 5 mM MnCl2, 5 mM MgCl2)
and resuspended in 20 µl of kinase buffer supplemented with 20 µM of ATP including 10 µCi of
[
32P]ATP (3000 Ci/mmol) with (in vitro
assay) or without (autophosphorylation assay) 40 µg of poly(Glu-Tyr)
(4:1). After 10 min at room temperature, the reaction was stopped by
the addition of 10 µl of 4× SDS sample buffer and boiled for 5 min.
Samples were resolved using 4-20% SDS-PAGE gels. Gels were dried,
analyzed by autoradiography, and quantified by densitometry.
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RESULTS |
When pancreatic acini were incubated with 10 nM CCK-8
for 2.5 min (a concentration and time that causes a maximal stimulation of tyrosine phosphorylation of p125FAK and paxillin in
previous studies (17, 18)), immunoprecipitated with
anti-phosphotyrosine mAb (PY20), and analyzed by Western blotting with
the same antibody (Fig. 1, left
upper panel), an increase in the tyrosine phosphorylation of at
least two major components of molecular mass 115-140 kDa and 65-80
kDa was seen. Following stripping of the membrane from
anti-phosphotyrosine antibodies, immunoblotting with a specific
anti-PYK2 mAb that does not cross-react with p125FAK
revealed that, under basal conditions, very low levels of
tyrosine-phosphorylated PYK2/CAK existed; however, 10 nM
CCK-8 induced a dramatic increase in the tyrosine phosphorylation of
PYK2/CAK (Fig. 1, right upper panel). An identical result
was obtained when the acinar lysates were first immunoprecipitated with
anti-PYK2 mAb and then analyzed with anti-phosphotyrosine mAb (Fig. 1,
left bottom panel). Following stripping of this latter
membrane from anti-phosphotyrosine antibodies, Western blotting with
the specific anti-PYK2 mAb showed that the recovery of PYK2/CAK from
cell lysates was not altered by treatment with CCK-8 (Fig. 1,
right bottom panel). These results demonstrate that
PYK2/CAK is expressed in rat pancreatic acini and is phosphorylated upon pancreatic acini activation by CCK-8.
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Fig. 1.
CCK-8 stimulation of
PYK2/CAK tyrosine phosphorylation in rat
pancreatic acinar cells. Rat pancreatic acini were treated for 2.5 min with no additions or with 10 nM CCK-8 and then lysed.
Whole lysates were immunoprecipitated (IP) with
anti-phosphotyrosine monoclonal antibody ( PTyr mAb,
upper panel) or anti-PYK2/CAK monoclonal
antibody (PYK2 mAb, bottom panel).
Immunoprecipitates were analyzed by Western blotting (WB)
using either anti-phosphotyrosine mAb (upper and
bottom left panels) or PYK2 mAb
(upper and bottom right
panels). Positions of molecular mass markers are shown on
the left. The position of PYK2/CAK is indicated with the
molecular mass marked 115 kDa. The autoradiograms are representative of
three independent experiments.
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Tyrosine phosphorylation of PYK2/CAK was a rapid consequence of the
addition of 10 nM CCK-8 to pancreatic acini (Fig.
2, left panel). Tyrosine
phosphorylation after the addition of CCK-8 reached a maximum within 1 min with a 50 ± 10-fold increase and then decreased rapidly after
2.5 min (Fig. 2, left panel). However, even after a 20-min
incubation with CCK-8 PYK2/CAK tyrosine phosphorylation had still
not returned to control values, remaining 11.5 ± 2-fold over
control (Fig. 2, left panel). The effect of CCK-8 on
PYK2/CAK tyrosine phosphorylation was
concentration-dependent (Fig. 2, right panel).
CCK-8 caused a 18 ± 8% increase at 0.1 nM,
half-maximal effect at 0.3 nM, and maximal effect at 1 nM for PYK2/CAK tyrosine phosphorylation (Fig. 2,
right panel).
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Fig. 2.
Time course (left
panel) and concentration dependence (right
panel) of CCK-8 stimulation of
PYK2/CAK tyrosine phosphorylation in rat
pancreatic acinar cells. Rat pancreatic acinar cells were treated
with the indicated concentrations of CCK-8 for the indicated times and
then lysed. Whole cell lysates were immunoprecipitated with
anti-phosphotyrosine mAb (PY20). Immunoprecipitates were
analyzed by SDS-PAGE followed by transfer of proteins of molecular mass
>60 kDa to nitrocellulose membrane and anti-PYK2/CAK immunoblotting
as described under "Methods." Bands were visualized using ECL, and
quantitation of phosphorylation was performed by scanning densitometry.
Left panel, the upper part shows
results from a representative experiment with CCK-8 (10 nM)
at the indicated times. These results are representative of three
others in duplicate. The values shown in the bottom
part are mean ± S.E. of four independent experiments
and are expressed as -fold increase over the pretreatment level
(experimental/control). Right panel, rat pancreatic acinar
cells were incubated for 2.5 min with the indicated concentrations of
CCK-8. The upper panel shows PYK2/CAK tyrosine
phosphorylation results from a representative experiment with no
additions or with various concentrations of CCK-8. These results are
representative of three others in duplicate. The bottom
part shows the quantitation of PYK2/CAK tyrosine
phosphorylation. Values are the mean ± S.E. (n = 4) expressed as the percentage of maximal increase caused by 10 nM CCK-8 above control unstimulated values.
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PYK2/CAK tyrosine phosphorylation has been shown to increase in
parallel with PYK2/CAK kinase activity (40). Therefore, an
assessment of in vitro kinase assay was performed in
immunoprecipitates of pancreatic acini treated with 10 nM
CCK-8 at different times (Fig. 3,
bottom panel). CCK-8 causes a rapid increase in kinase activity, with maximal activation at 1-2.5 min (Fig. 3,
bottom), similar to its time course for CCK-stimulated
PYK2/CAK tyrosine phosphorylation (compare Fig. 3, bottom
panel, and Fig. 2, left panel). With activation,
PYK2/CAK also causes autophosphorylation in other cell systems
(58-60). CCK-8 (10 nM) caused a time-dependent increase in PYK2/CAK autophosphorylation (Fig. 3, top
panel).
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Fig. 3.
Time course of the ability of CCK to
stimulate autophosphorylation of PYK2/CAK
(top panel) or increase its kinase
activity (bottom panel). Top
panel, pancreatic acini were incubated for the indicated time with
10 nM CCK-8, lysed, PYK2/CAK-immunoprecipitated,
resuspended in kinase buffer with 10 µCi of
[-32P]ATP and 20 µM ATP, and incubated
for 10 min at room temperature. Samples were resolved using 4-20%
SDS-PAGE gel and analyzed by autoradiography. This result is
representative of three others. Bottom panel, methods were
the same as in the top panel except that 40 µg
of poly(Glu-Tyr) (4:1) was added to the kinase assay. At the
top is shown a typical autoradiograph, and at the
bottom is shown the mean ± S.E. results from four
experiments. Kinase activity is expressed as the -fold increase as a
ratio of the experimental value at the indicated time over the control
value without CCK-8 added.
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CCK-JMV is reported to be an agonist at the CCKA high
affinity receptor state and an antagonist at the low affinity
CCKA receptor state in rat pancreatic acini (61-63).
CCK-JMV (1 µM) caused a 12 ± 2-fold increase in
PYK2/CAK tyrosine phosphorylation, which was 25 ± 4% of the
maximal stimulation caused by CCK-8 (1 nM) (Fig.
4, upper panel, compare
lanes 2 and 3). Because
CCK-JMV-stimulated PYK2/CAK tyrosine phosphorylation did not attain
the full response that was obtained by CCK-8, we tested both analogues
in combination. Acini were stimulated with 1 µM CCK-JMV
alone (Fig. 4, upper panel, lane
3, and bottom panel) and with 1 nM CCK-8 alone (Fig. 4, upper panel,
lane 2, and bottom panel)
or in combination with different concentrations of CCK-JMV (0.03, 0.1, 0.3, and 1 µM) (Fig. 4, upper panel,
lanes 4-7, and bottom
panel). Inhibition of CCK-8-stimulated PYK2/CAK tyrosine
phosphorylation was noted with 0.3 µM CCK-JMV (Fig. 4,
lane 6, upper panel). With
1 µM CCK-JMV, CCK-8-stimulated tyrosine phosphorylation
decreased to the same level as that of 1 µM CCK-JMV alone
(Fig. 4, upper panel, compare lanes
3 and 7, and bottom
panel).
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Fig. 4.
Effect of CCK-8 and CCK-JMV alone or in
combination on PYK2/CAK tyrosine
phosphorylation. Acini were stimulated with 1 nM
CCK-8, with 1 µM CCK-JMV or with 1 nM CCK-8
with various concentrations of CCK-JMV for 2.5 min and then lysed.
PYK2/CAK tyrosine phosphorylation was determined by
immunoprecipitation and Western blotting as described in the Fig. 2
legend. The upper panel shows results from a
representative experiment. These results are representative of three
others in duplicate. The bottom panel shows the
results of PYK2/CAK tyrosine phosphorylation, and the results are
expressed as the percentage of the maximal increase caused by 1 nM CCK-8. Values are mean ± S.E. for four experiments
in duplicate. An asterisk indicates significant differences
as compared with 1 nM CCK-8 (p < 0.05 with
Student's t test for unpaired samples).
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In addition to increasing the production of inositol phosphate, which
causes mobilization of cellular calcium, activation of phospholipase C
by CCK-8 promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to production of diacylglycerol, which in
turn activates PKC (12). Results from a number of recent studies in
other cell systems (30, 32-34, 58, 64) suggest that increases in
cytosolic calcium and activation of PKC may be important for the
agonist to stimulate PYK2/CAK tyrosine phosphorylation and kinase
activity (40, 55). We next attempted to determine whether CCK-8's
increases in intracellular calcium or activation of PKC or both were
needed for its ability to cause PYK2/CAK tyrosine phosphorylation in
pancreatic acini. To determine whether direct activation of PKC
increased the tyrosine phosphorylation of PYK2/CAK, pancreatic acini
were treated with the phorbol ester, TPA. TPA (1 µM)
stimulated a rapid increase in PYK2/CAK tyrosine phosphorylation,
which reached a maximum at 10 min with a 31 ± 6-fold increase and
was maintained for at least 20 min (Fig.
5). To determine whether increased
cytosolic calcium either alone or in combination with activation of PKC
could alter PYK2/CAK tyrosine phosphorylation, we compared the
ability of the calcium ionophore A23187 and thapsigargin, an agent that
specifically inhibits the endoplasmic reticulum
Ca2+-ATPase, depletes Ca2+ from intracellular
compartments, and increases calcium influx (65, 66), to cause
PYK2/CAK tyrosine phosphorylation in pancreatic acini when present
alone or with TPA (Fig. 6). Calcium
ionophore A23187 (1 µM) and thapsigargin (10 µM) under conditions previously shown to increase
[Ca2+]i (66, 67) caused a 14 ± 5-fold
increase and a 25 ± 9-fold increase in PYK2/CAK tyrosine
phosphorylation, which were 28 ± 9 and 56 ± 7% of that
caused by a maximally effective 1 nM concentration of
CCK-8, respectively (Fig. 6, upper panel, lanes 2-4, and bottom
panel). The simultaneous stimulation with both TPA (1 µM) and A23187 (1 µM) or TPA (1 µM) and thapsigargin (10 µM) increased
PYK2/CAK tyrosine phosphorylation to 130 ± 26 and 173 ± 36% of the stimulation caused by CCK-8 (1 nM) alone, respectively (Fig. 6, upper panel,
lanes 5 and 6, and bottom
panel). The increase with both agents together was greater
than the sum of the values obtained with each alone (Fig. 6) and was
greater than the stimulation caused by a maximally effective
concentration of CCK-8 (i.e. 1 nM) (Fig. 6,
upper panel, compare lanes
5-7, and bottom panel).
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Fig. 5.
Time course of the ability of the phorbol
ester TPA to stimulate PYK2/CAK tyrosine
phosphorylation in rat pancreatic acinar cells. Rat pancreatic
acinar cells were treated with 1 µM of TPA at the
indicated times and then lysed. PYK2/CAK tyrosine phosphorylation
was determined by immunoprecipitation and Western blotting as described
in the Fig. 2 legend. The upper panel shows that
PYK2/CAK tyrosine phosphorylation results from a representative
experiment with TPA (1 µM) at the indicated times. These
results are representative of three others in duplicate. The
bottom panel show the quantitation of PYK2/CAK
tyrosine phosphorylation. Values are the mean ± S.E.
(n = 4) expressed as the percentage of maximal increase
caused by treatment for 20 min with 1 µM TPA above
control unstimulated values, which was a 31 ± 6-fold
increase.
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Fig. 6.
Effect of the calcium ionophore A23187 or
thapsigargin, alone or in combination with the phorbol ester TPA, on
stimulation of PYK2/CAK tyrosine
phosphorylation in rat pancreatic acinar cells. Pancreatic acinar
cells were treated with the indicated agents for 5 min, and PYK2/CAK
tyrosine phosphorylation was determined as described in the legend to
Fig. 2. Results shown in the upper panel are from
a typical experiment representative of three others in duplicate.
Results in the bottom panel are the mean ± S.E. of four experiments expressed as the percentage of the maximal
increase of PYK2/CAK tyrosine phosphorylation caused by 1 nM CCK-8 above control (i.e. 50 ± 10-fold
increase).
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CCKA receptor activation results in the release of
Ca2+ from intracellular stores followed by the influx of
Ca2+ from the medium (12, 66). Since calcium ionophore and
thapsigargin, under conditions that increase calcium influx, induced
the tyrosine phosphorylation of PYK2/CAK in pancreatic acini, we
examined the role of extracellular calcium in CCK-8-stimulated
PYK2/CAK tyrosine phosphorylation. The responses of pancreatic acini
to CCK-8 resuspended in either calcium-free media, with EGTA (5 mM), or calcium-containing media were compared (Fig.
7). Calcium influx was decreased when the
pancreatic acini were stimulated by CCK-8 in the absence of calcium in
the medium with the result that the [Ca2+]i
increase by CCK-8 returned to base line sooner (Fig. 7, left
panels). However, inhibition of the calcium influx had no
significant effect on the increase in PYK2/CAK tyrosine
phosphorylation caused by CCK-8 in pancreatic acini (Fig. 7,
right upper panel, lanes 3 and
4, and right bottom panel).
Next, we examined the role of intracellular calcium changes in
CCK-8-stimulated PYK2/CAK tyrosine phosphorylation by using two
different approaches. Pretreatment of pancreatic acini for 30 min with
thapsigargin (1 µM) in a calcium-free medium (with 5 mM EGTA) inhibited completely the
[Ca2+]i increase stimulated by CCK-8 (1 nM) (Fig. 8, left middle panel). Moreover, pretreatment of
pancreatic acini for 30 min with BAPTA/AM (50 µM), an
intracellular calcium chelator, in a calcium-free medium (with 5 mM EGTA) prevented the CCK-8-induced increase in
[Ca2+]i due to mobilization of cellular calcium
stores (Fig. 8, left bottom panel).
Pretreatment of pancreatic acini with thapsigargin or with BAPTA/AM in
a calcium-free medium decreased CCK-8-stimulated PYK2/CAK tyrosine
phosphorylation by 69 ± 3 and 64 ± 3%, respectively (Fig.
8, right top and bottom
panels). However, neither pretreatment with thapsigargin nor
pretreatment with BAPTA/AM had an effect on the increase in
p125FAK tyrosine phosphorylation caused by CCK-8 under
identical conditions, demonstrating that neither BAPTA/AM nor
thapsigargin pretreatment was having a nonspecific inhibitory effect
(Fig. 8, right upper panel).
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Fig. 7.
Extracellular calcium dependence of CCK-8
stimulation of PYK2/CAK tyrosine
phosphorylation in rat pancreatic acinar cells. Pancreatic acini
were pretreated for 10 min at 37 °C in a calcium-containing medium
or in a calcium-free medium (with EGTA 5 mM) and then
incubated for a further 2.5 min with no additions or with CCK-8 (1 nM). PYK2/CAK tyrosine phosphorylation was determined as
described in the Fig. 2 legend. The left panels
show the effect of 1 nM CCK-8 on
[Ca2+]i in pancreatic acini in a
calcium-containing medium (upper panel) or in a
calcium-free medium (bottom panel) for a single
experiment representative of three others. Results shown in the
upper right panel are from a typical
experiment representative of three others in duplicate. Results in the
bottom right panel are the mean ± S.E. of four experiments in duplicate expressed as the percentage of
the maximal increase of PYK2/CAK tyrosine phosphorylation caused by
1 nM CCK-8 above control in a medium with a normal calcium
concentration.
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Fig. 8.
Intracellular calcium dependence of CCK-8
stimulation of PYK2/CAK and
p125FAK tyrosine phosphorylation in rat pancreatic acinar
cells. Pancreatic acinar cells were pretreated for 30 min at
37 °C in a calcium-free medium (with EGTA 5 mM) either
in the absence or presence of thapsigargin (1 µM) or
BAPTA/AM (50 µM). Acini were then incubated for a further
2.5 min with no additions or with CCK-8 (1 nM). PYK2/CAK
and p125FAK tyrosine phosphorylation were determined as
described in the Fig. 2 legend and under "Methods." The
left panels show the effect of 1 nM
CCK-8 on [Ca2+]i in pancreatic acini in a
calcium-containing medium (upper panel), in a
calcium free-medium with thapsigargin (1 µM)
(medium panel) or in a calcium-free medium with
BAPTA/AM (50 µM) (bottom panel) for
a single experiment representative of three others. Results shown in
the upper right panel are from a
typical experiment representative of three others in duplicate. Results
in the bottom right panel are the
mean ± S.E. of four experiments expressed as the percentage of
the maximal increase of PYK2/CAK tyrosine phosphorylation caused by
1 nM CCK-8 above the control unstimulated values, in a
medium with a normal calcium concentration. (p < 0.01 with Student's t test for unpaired samples).
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To determine whether PKC activation might be involved in mediating
CCK-8-stimulated increases in PYK2/CAK tyrosine phosphorylation, we
examined the effect of a PKC inhibitor, GF109203X (68). Previously, we
have shown that pretreatment of pancreatic acinar cells with 5 µM of GF109203X for 2 h caused complete inhibition
of p125FAK and paxillin tyrosine phosphorylation induced by
activation of PKC with TPA (17). Pretreatment of pancreatic acinar
cells with GF109203X (5 µM) for 2 h attenuated the
increase in PYK2/CAK tyrosine phosphorylation in response to CCK-8
(1 nM) by 50 ± 7% (Fig.
9, upper panel,
compare lanes 4 and 5, and
bottom left panel) but had no effect
on CCK-8-stimulated p125FAK tyrosine phosphorylation in
parallel experiments (Fig. 9, upper panel,
compare lanes 4 and 5) as we have
shown previously (17).
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Fig. 9.
Effect of GF109203X either alone or in
combination with thapsigargin on CCK-8 stimulation of
PYK2/CAK and p125FAK tyrosine
phosphorylation in rat pancreatic acini. Pancreatic acini were
pretreated with 1 µM thapsigargin for 30 min in a
calcium-free-medium (with EGTA 5 mM) or with GF109203X (5 µM) for 2 h either alone or in combination. Acini
were then incubated for a further 2.5 min with no additions (control)
or with CCK-8 (1 nM). PYK2/CAK or p125FAK
tyrosine phosphorylation was determined as described in the Fig. 2
legend and under "Methods." The upper panel
shows a single experiment representative of three others in duplicate.
In the lower panel are shown the mean ± S.E. from four experiments, and the data are expressed as the
percentage of the maximal increase in phosphorylation caused by 1 nM CCK-8 above the control unstimulated values in a medium
with normal calcium concentration. (p < 0.01 with
Student's t test for unpaired samples).
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Since increases in intracellular calcium by the calcium ionophore
A23187 and activation of PKC by TPA have synergistic effects on
PYK2/CAK tyrosine phosphorylation (Fig. 6), and inhibition of both
limbs of the PLC cascade separately attenuate the increase in
PYK2/CAK tyrosine phosphorylation in response to CCK-8 by almost
50%, we examined the effect of a combination of inhibition of
CCK-8-induced increases in intracellular calcium and PKC activation (Fig. 9). Pretreatment of pancreatic acini with thapsigargin to inhibit
mobilization of intracellular calcium and with GF109203X to block PKC,
in a calcium-free medium, completely inhibited CCK-8 stimulation of
PYK2/CAK tyrosine phosphorylation (Fig. 9, top panel, compare lanes 4 and
6, and left bottom panel).
In comparison, pretreatment with thapsigargin and GF109203X caused
almost a 50% decrease in CCK-8-stimulated p125FAK tyrosine
phosphorylation in parallel experiments (Fig. 9, upper panel, compare lanes 4 and
6, and right bottom panel)
as shown previously (17).
Recent studies show that the integrity of the actin cytoskeleton is
important for tyrosine phosphorylation of some cellular proteins such
as p125FAK and paxillin (17, 39, 69). To determine whether
the integrity of the cytoskeleton network is needed for CCK-8-increased
PYK2/CAK tyrosine phosphorylation, we pretreated pancreatic acinar
cells for 2 h with cytochalasin D (3 µM), a
selective disrupter of the actin filament network (70), or colchicine,
a selective inhibitor of microtubule synthesis (71), and then incubated
with CCK-8 (1 nM) for another 2.5 min (Fig.
10). Treatment with cytochalasin D
completely inhibited CCK-8-stimulated PYK2/CAK tyrosine
phosphorylation (Fig. 10, lane 5). In contrast,
pretreatment with colchicine (0.3 µM) had no effect in
PYK2/CAK tyrosine phosphorylation stimulated by CCK-8 (1 nM) (Fig. 10, lane 6).
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Fig. 10.
Effect of cytochalasin D or colchicine on
CCK-8 stimulation of PYK2/CAK tyrosine
phosphorylation in rat pancreatic acini. Pancreatic acinar cells
were pretreated for 2 h at 37 °C either in the absence or
presence of 3 µM cytochalasin D or 0.3 µM
colchicine. Acini were then incubated for a further 2.5 min with no
additions or with 1 nM CCK-8 and then lysed. PYK2/CAK
tyrosine phosphorylation was determined as described in the legend to
Fig. 2. Shown are results from a typical experiment representative of
three others in duplicate.
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Cell activation by growth factors, oncogenes, and
neurotransmitter/hormones can result in a redistribution of
tyrosine-phosphorylated proteins (56, 72-75). To examine this
possibility for PYK2/CAK, we determined the distribution of
PYK2/CAK in membrane and cytosolic fractions under basal conditions
and under CCK-8 stimulation. In unstimulated pancreatic acini, total
PYK2/CAK and total p125FAK were found in both the
cytosolic and the membrane fractions (Fig. 11, upper left
panel, lanes 1 and 3, and
upper right panel, lanes 1 and 3). Densitometric analysis of immunoblots
indicated that in unstimulated pancreatic acini total PYK2/CAK was
largely localized (77 ± 7% of total) in the membrane fraction
(Fig. 11, top left panel, compare
lanes 1 and 3, and bottom
panel); however, total p125FAK was largely
localized (86 ± 3% of total) in the cytosolic fraction (Fig. 11,
upper right panel, compare
lanes 1 and 3, and bottom panel). Upon the addition of 1 nM CCK-8 for 2.5 min, there was a 56 ± 9% decrease in the amount of total
PYK2/CAK in cytosolic fractions (Fig. 11, left
upper panel, compare lanes
1 and 2, and bottom panel).
However, the increase in the total PYK2/CAK in the corresponding
membrane fractions did not reach significance after CCK-8 stimulation
(Fig. 11, upper left panel, compare
lanes 3 and 4, and bottom
panel). In comparison, the addition of 1 nM CCK-8 for 2.5 min caused an 86 ± 32% increase in the amount of total p125FAK in membrane fractions (Fig. 11,
top right panel, compare
lanes 3 and 4, and bottom
panel). However, no detectable increase in the amount of
total p125FAK in the corresponding cytosolic fractions was
found after CCK-8 stimulation (Fig. 11, upper
right panel, compare lanes
1 and 2, and bottom
panel).
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Fig. 11.
CCK-8 stimulation of translocation of total
PYK2/CAK and total p125FAK from
cytosol and membrane fractions in rat pancreatic acini. Rat
pancreatic acini were incubated for 2.5 min with or without 1 nM CCK-8 and then lysed. Cytosol fractions and membrane
fractions were isolated as describe under "Methods." Lysates of the
subcellular fractions (15 µg/well) were analyzed by Western blotting
with anti-PYK2 mAb (upper left panel)
or anti-p125FAK mAb (upper right
panel) as described under "Methods" without
immunoprecipitation prior to Western blotting. The upper
panels show results from a typical experiment representative
of three others. The bottom panel shows the
relative concentration of total PYK2/CAK and total
p125FAK expressed as the percentage of the maximal amount
of each one measure in unstimulated pancreatic acini; i.e.
100% is the amount of total PYK2/CAK in membrane fractions of
unstimulated acini or the amount of total p125FAK in the
cytosolic fraction of unstimulated pancreatic acini. Values are
mean ± S.E. (n = 4).
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Previous studies in rat pancreatic acini (15, 16, 76) demonstrate
activation of the MAPK signaling pathway after CCKA receptor activation by agonists. Moreover, in other cell systems recent
studies show that PYK2/CAK activation could be responsible for
activation of the Ras/MAPK signaling pathway by G protein-coupled receptors (30, 32, 34). To explore whether CCKA receptor activation could promote activation of the Ras/MAPK signaling pathway
by PYK2/CAK, we examined the interaction between the adapter protein
Grb2 with PYK2/CAK in pancreatic acini stimulated by CCK-8. We
assessed the formation of a complex between Grb2 with PYK2/CAK by
determining the results of co-immunoprecipitation of PYK2/CAK with
Grb2 after immunoprecipitation with Grb2 (Fig. 12, right
panels). With the addition of 1 nM CCK-8, there
was a rapid stimulation of the formation of a PYK2/CAK-Grb2 complex with a maximal effect seen at 2.5 min (Fig. 12, right
panels). This change was not due to a difference in protein
loading, because Western blotting with an anti-Grb2 mAb showed equal
loading (Fig. 12, right upper panel).
In other tissues, it has been shown that an alternative pathway for
activation of the MAPK signaling pathway could be the tyrosine
phosphorylation of the adapter protein c-Crk (77-79). To assess
whether CCKA receptor activation could promote the
association of PYK2/CAK with the adapter molecule c-Crk, we assessed
the formation of this complex by determining the results of
co-immunoprecipitation of PYK2/CAK with c-Crk after
immunoprecipitation with c-Crk mAb (Fig. 12, left
panel). CCK-8 stimulation of pancreatic acini caused the
rapid formation of a PYK2/CAK-Crk complex with a maximal effect seen
at 2.5 min (Fig. 12, left panel). The observed change in the amount of PYK2/CAK in c-Crk immunoprecipitates was not
due to differential recovery of c-Crk from CCK-8-treated cells because
when c-Crk mAb was used for Western blotting after immunoprecipitation
with c-Crk mAb, similar amounts of c-Crk were seen in all samples (Fig.
12, top left panel).
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Fig. 12.
Ability of CCK-8 to stimulate association of
PYK2/CAK with endogenous c-Crk
(left panel) or Grb2 (right
panel). Rat pancreatic acini were incubated with
1 nM CCK-8 for the indicated times and then lysed. The
lysates were immunoprecipitated (IP) with anti-Crk mAb
(Crk) (left panel) (4 µg) or
anti-Grb2 mAb (Grb2) (4 µg) for 2 h at 4 °C, and
the immunoprecipitate was fractionated using 10% (left
panel) or 14% (right panel)
polyacrylamide gels. Western blotting (WB) was performed
with anti-PYK2 mAb (both panels) or with anti-Crk
mAb (left panel). In the right
panel, Grb2 was detected using an anti-Grb2 mAb by ECL. The
upper panels show results from a typical
experiment representative of three others. The bottom
panels are the mean ± S.E. of four experiments with
results expressed as the ratio of the PYK2/CAK coupled to Crk
(left panel) or Grb2 (right
panel) of the experimental to that seen in the
control.
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DISCUSSION |
A number of recent studies demonstrate that in addition to
integrins (31, 36), bioactive lipids (32, 40), cellular stress
(radiation, ultraviolet light, osmotic changes) (80), and growth
factors (37-39), a number of G protein-coupled receptors (30, 32, 35,
39, 60, 81) also cause tyrosine phosphorylation and activation of the
cytoplasmic tyrosine kinase PYK2/CAK. A number of results in our
study support the conclusions that activation of both the high affinity
and the low affinity states of the CCKA receptor in rat
pancreatic acini can cause tyrosine phosphorylation of PYK2/CAK and
suggest that PYK2/CAK may have a significant role in
agonist-activated CCKA receptor-mediated intracellular signaling pathways. First, whether an anti-phosphotyrosine mAb is used
for immunoprecipitation followed by protein identification using an
anti-PYK2/CAK mAb or the same mAbs are used in reverse order, CCK-8
caused a marked increased in the tyrosine phosphorylation of a single
PYK2/CAK immunoreactive band. Second, PYK2/CAK tyrosine phosphorylation and activation were a rapid consequence of the addition
of CCK-8 to pancreatic acini, causing a maximal increase in both by
1-2.5 min. Third, the dose-response curve for PYK2/CAK tyrosine
phosphorylation occurred over the concentration range in which CCK-8
stimulates changes in cellular calcium (13, 17); generation of inositol
phosphates (17, 82); enzyme secretion (82); MAPK signaling pathway
activation (15); p125FAK, p130Cas, and paxillin
tyrosine phosphorylation (17, 56); and [3H]thymidine
incorporation (83). Fourth, stimulation of pancreatic acini with
CCK-JMV, a CCK analogue, caused a clear increase in the PYK2/CAK
tyrosine phosphorylation. Moreover, CCK-JMV antagonized the ability of
CCK to stimulate PYK2/CAK tyrosine phosphorylation to a greater
extent than that seen with CCK-JMV alone. Because CCK-JMV functions as
an agonist for the CCKA receptor high affinity state and an
antagonist at the low affinity CCKA receptor state in rat
pancreatic acini (18, 19, 61, 62), these results demonstrate that 20%
of the increase in tyrosine phosphorylation of PYK2/CAK by CCK is
due to activation of the high affinity state and 80% is due to
activation of the low affinity CCKA receptor state. Last,
this conclusion is further supported by the observation that CCK-8
stimulation of PYK2/CAK tyrosine phosphorylation occurred over the
concentration range for CCK-8 occupation of both CCKA receptor states, the high affinity state and the low affinity state
(17, 82).
Activation of the pancreatic CCKA receptor is known to
stimulate phospholipase C activity, resulting in the generation of inositol phosphates and diacylglycerol, which in turn mobilize cellular
calcium and activate protein kinase C (PKC), respectively (12, 13).
Recent studies demonstrate that the mechanism of changes in cellular
calcium in stimulating PYK2/CAK tyrosine phosphorylation in various
cells with different stimuli varies. With T cell receptor activation in
Jurkat cells (51), UTP activation of P2Y2 receptors in
PC-12 cells (35) or angiotensin II or platelet-derived growth factor in
rat vascular smooth muscle cells (39), EGTA partially inhibited
PYK2/CAK tyrosine phosphorylation, suggesting that calcium influx
was at least partially involved in mediating the tyrosine
phosphorylation. In contrast, stimulation of PYK2/CAK tyrosine
phosphorylation with bradykinin or lysophosphatidic acid in PC-12 cells
(30, 32, 51), stem cell factor in CMK human megakaryocytic cells (38),
and angiotensin II in GN4 rat liver cells (81) or rat vascular smooth
muscle cells (39) is either not altered by EGTA or partially or
completely inhibited by BAPTA/AM, suggesting that mobilization of
intracellular calcium is playing an important role. Similarly, studies
in other cell systems suggest the role PKC activation in modulating
PYK2/CAK tyrosine phosphorylation by receptors coupled to PLC may
differ in different cells. PKC inhibitors or PKC down-regulation by
preincubation with TPA had either minimal or no effect on stimulation
of PYK2/CAK tyrosine phosphorylation by bradykinin in PC-12 cells
(30) or angiotensin II in cardiac fibroblasts (43), suggesting that PKC
activation was not involved. In contrast, PYK2/CAK tyrosine
phosphorylation was either partially or completely inhibited by PKC
inhibitors or PKC down-regulation for the effect of thrombin in
platelets (64), stem cell factor in megakaryocytes (38),
P2Y2 receptor activation in PC-12 cells (35), or
platelet-derived growth factor in rat aortic smooth muscle cells (39),
suggesting PKC activation was essential for some or all of the
stimulation seen. In rat pancreatic acini, our results support the
conclusion that mobilization of intracellular calcium stores by CCK-8,
but not calcium influx, is responsible for almost 50% of the maximal
stimulation of PYK2/CAK tyrosine phosphorylation and that
CCKA receptor ability to stimulate PKC activation is
accounting for the other 50% of the PYK2/CAK tyrosine
phosphorylation. Therefore, our results demonstrate that in rat
pancreatic acini the ability of CCKA receptor activation to
stimulate PYK2/CAK tyrosine phosphorylation is completely dependent
on activation of both limbs of the PLC cascade.
Previous studies have shown that both PYK2/CAK and
p125FAK in the same tissues (64, 84) may become tyrosine
phosphorylated in response to similar stimuli. However, some recent
studies (37-39) suggest that with activation of some receptors
PYK2/CAK and p125FAK tyrosine phosphorylation can be
regulated differentially. For example, p125FAK tyrosine
phosphorylation is either not stimulated or minimally stimulated,
whereas PYK2/CAK tyrosine phosphorylation is markedly stimulated by
stem cell factor in megakaryocytes (38) and by platelet-derived growth
factor and angiotensin II in rat smooth muscle cells (37, 39). In
contrast, p125FAK, but not PYK2 tyrosine phosphorylation,
is stimulated by platelet aggregation (64), by activation of glutamate
receptors in rat hippocampal slices (84), and by adhesion to
fibronectin in rat aortic smooth muscle cells (37). Previous studies
have reported that PYK2/CAK has a more restricted tissue
distribution than p125FAK (41, 42, 85), suggesting that
both kinases are likely to be cell type-specific functional equivalents
(36, 42, 86). Furthermore, in recent studies in some cells expressing
both PYK2/CAK and p125FAK it was proposed that
PYK2/CAK and p125FAK, albeit highly homologous in
primary structure, appear to have different functions (37, 84, 86, 87).
Because pancreatic acinar cells express both endogenous PYK2/CAK and
p125FAK and tyrosine phosphorylation of both is stimulated
by CCK, they provide an ideal system to compare regulation of
agonist-activated G protein-coupled receptor stimulation of PYK2/CAK
and p125FAK tyrosine phosphorylation in the same cell. As
shown in the present study in parallel experiments and in a previous
study (17), a number of results suggest that in rat pancreatic acini,
tyrosine phosphorylation of PYK2/CAK and p125FAK after
CCKA receptor activation is differentially regulated, and this could account for a different function of these kinases in rat
pancreatic acini. First, CCK-stimulated PYK2/CAK tyrosine phosphorylation was more rapid and of greater magnitude than previously reported (17) for CCK stimulation of p125FAK tyrosine
phosphorylation. Second, stimulation of PYK2/CAK tyrosine phosphorylation by CCK was reduced by 50% after the inhibition of
increases in [Ca2+]i or PKC inactivation, but
CCK-stimulated p125FAK tyrosine phosphorylation was not
affected. Third, inhibition of both increases in
[Ca2+]i and PKC activation completely inhibited
CCK stimulation of PYK2/CAK tyrosine phosphorylation but caused only
a 50% decrease in CCK-stimulated p125FAK tyrosine
phosphorylation. These results show p125FAK tyrosine
phosphorylation with CCKA receptor activation is controlled by both PLC-dependent and -independent mechanisms, whereas
PYK2/CAK tyrosine phosphorylation is completely controlled by
PLC-dependent mechanisms. Similar to our results, a recent
study in rat hippocampal slices (84) demonstrated that PYK2/CAK and
p125FAK tyrosine phosphorylation were regulated differently
by changes in PKC or [Ca2+]i. However, in
contrast to our results, in rat hippocampal slices (84) ionomycin
stimulated p125FAK but not PYK2/CAK tyrosine
phosphorylation, whereas PKC activation by TPA caused the inverse.
These results suggest that the regulation of these two structurally
similar kinases by PLC-activated cascades appears to vary markedly in
different cells.
Numerous recent studies demonstrate that the integrity of the actin
cytoskeleton is important for various neuropeptides as well as
bioactive lipids such as lysophosphatidic acid and
sphingosylphosphocholine to stimulate tyrosine phosphorylation of the
PYK2/CAK structurally related kinase, p125FAK (69,
88-90). In rat pancreatic acini CCK-stimulated PYK2/CAK, tyrosine
phosphorylation was completely inhibited by disruption of the actin
cytoskeleton with cytochalasin D, but not disruption of the microtubule
network. These results are consistent with other reports indicating
that PYK2/CAK tyrosine phosphorylation after activation of G
protein-coupled receptors by thrombin or angiotensin II (39, 64, 81),
activation of growth factor receptors by platelet-derived growth factor
or stem cell factor (38, 39), or after integrin stimulation (36, 91)
was dependent on the integrity of the actin cytoskeleton.
A number of studies have investigated the distribution of PYK2/CAK
under basal conditions using immunocytochemical methods (31, 41, 86).
In chicken embryo cells (86) PYK2/CAK was diffusely distributed
throughout the cell with a small fraction in focal adhesions that are
attached to the plasma membrane. In another study (31), in the CMK
megakaryocyte cell line both PYK2/CAK and p125FAK were
found in focal adhesions, whereas in COS-7 cells expressing PYK2/CAK
it was found only in cell to cell contacts but not in focal adhesions
where p125FAK was localized (41). In the present study,
under basal conditions the majority (i.e. 77%) of
PYK2/CAK was localized in the membrane fraction. This was the
opposite pattern to p125FAK localization in which the
majority (i.e. 86%) was localized in the cytosol. Thus, our
results demonstrate a different localization of PYK2/CAK and
p125FAK in rat pancreatic acini under basal conditions,
which provides a foundation for a compartmentalization of these
kinases, and this may contribute to their differential regulation after
CCKA receptor activation in these cells. Moreover, the
compartmentalization of different kinases has been proposed to be
helped by formation of a cytoskeletal complex, which provides a
foundation for the interactions of kinases with different regulators
and substrates (36, 64). This proposal is consistent with our results
that show a clear relationship between the actin cytoskeleton and the tyrosine phosphorylation of both kinases, PYK2/CAK and
p125FAK.
Tyrosine-phosphorylated proteins such as p125FAK,
p130Cas, and p60src have been shown to alter their
cellular localization after cell activation (56, 72-75). Our results
show that in rat pancreatic acini after CCKA receptor
activation, both p125FAK and PYK2/CAK undergo a change
in cell distribution. There was a decrease in the amount of total
PYK2/CAK in cytosolic fractions after CCKA receptor
activation, whereas with total p125FAK, there was an
increase in the total amount in the membrane fraction. Although a
reciprocal increase in the amount of total PYK2/CAK detectable in
the corresponding membrane fractions or a decrease in the amount of
total p125FAK in cytosolic fractions was not detected,
these changes in the smaller fraction probably represent a
redistribution. This is the first report showing that PYK2/CAK can
alter its cellular localization after cell activation. What role the
redistribution plays in the cellular function of PYK2/CAK is at
present unclear.
Recent studies with lysophosphatidic acid (32), growth factors such as
colony-stimulating growth factor (92), and some G protein-coupled
receptors such as angiotensin II (43, 44), UTP (33, 35), bradykinin
(30, 32), and - and -adrenergic agents (34) demonstrate that
activation of PYK2/CAK tyrosine kinase is an intracellular regulator
of the ability of these stimuli to activate the MAPK signaling pathway
(30, 32-35, 51, 92). The MAPK signaling pathway represents an
important point of convergence of cell signaling, especially in the
regulation of cell division and growth (21, 93, 94). This cascade is
regulated by protein phosphorylation, with the MAPKs themselves being
serine/threonine kinases and including p38MAPK, the Jun
N-terminal kinase/stress-activated protein kinase, and the
extracellular signal-regulated kinases 1 and 2 (21, 93, 94). Previous
studies in rat pancreatic acini (15, 16, 76) demonstrate that
activation of the CCKA receptor causes Ras/MAPK signaling
pathway activation via a mechanism involving protein kinase C, calcium
mobilization, and tyrosine kinases. However, the full mechanism by
which CCK activates Ras/MAPK signaling pathway in rat pancre
TOP
ABSTRACT
INTRODUCTION
