Protein tyrosine phosphorylation in pancreatic acini:
differential effects of VIP and CCK
Manfred P.
Lutz1,
Albrecht
Piiper2,
Herbert Y.
Gaisano3,
Danuta
Stryjek-Kaminska2,
Stefan
Zeuzem2, and
Guido
Adler1
1 Department of Internal
Medicine I, University of Ulm, 89070 Ulm;
2 II. Medical Department,
University of Frankfurt am Main, 60590 Frankfurt, Germany; and
3 Department of Medicine,
University of Toronto, Toronto, Ontario, Canada MSS 1A8
 |
ABSTRACT |
Cholecystokinin (CCK) and vasoactive
intestinal peptide (VIP) stimulate enzyme secretion from pancreatic
acini by binding to heptahelical receptors without intrinsic tyrosine
kinase activity. Signal transduction by the CCK receptor involves
activation of phospholipase C by
Gq proteins and activation of
tyrosine kinases, whereas occupation of VIP receptors stimulates
adenylyl cyclase through binding to
Gs proteins. Here, we use
electrophoretic separation of cellular proteins and antiphosphotyrosine
immunoblotting to demonstrate a VIP-stimulated rapid and dose-dependent
increase in tyrosine phosphorylation of proteins migrating at 130, 115, and 93 kDa in freshly isolated rat pancreatic acini. Phosphorylation of
these proteins was increased after direct stimulation of adenylyl cyclase or the adenosine 3',5'-cyclic monophosphate
(cAMP)-dependent protein kinase with forskolin or dibutyryl cAMP and
was inhibited by the tyrosine kinase inhibitors genistein or tyrphostin
23. Compared with VIP, CCK stimulated tyrosine phosphorylation of additional proteins migrating at 60, 66, and 72/78 kDa. Using two-dimensional electrophoretic separation or immunoprecipitation, the
72/78-kDa phosphoprotein was identified as paxillin. We
propose that paxillin might be involved in CCK- but not in VIP-induced exocytosis.
cholecystokinin; vasoactive intestinal peptide; paxillin; G
protein-coupled receptors; adenylyl cyclase
 |
INTRODUCTION |
CHOLECYSTOKININ (CCK) and vasoactive intestinal peptide
(VIP) are gastrointestinal peptide hormones that stimulate enzyme secretion from pancreatic acinar cells by binding to heptahelical non-tyrosine kinase receptors. Whereas the intracellular signaling system activated by the CCK receptor involves activation of
phospholipase C (PLC) through the
Gq/11 subtype of heterotrimeric G
proteins, VIP is known to increase adenylyl cyclase activity through G
proteins of the Gs subtype.
Subsequent activation of phospholipid-,
Ca2+-, and adenosine
3',5'-cyclic monophosphate (cAMP)-dependent protein kinases
and phosphorylation of proteins on serine and threonine residues by
these kinases mediates at least part of the cellular response (32). In
addition, CCK has recently been demonstrated to increase tyrosine
phosphorylation of several proteins and to increase protein tyrosine
kinase activity in pancreatic acini, apparently via protein kinase C-
as well as Ca2+-dependent pathways
(2, 8, 17, 19, 20). Whereas several reports demonstrate the
ability of other phospholipase C (PLC)-activating G protein-coupled
receptors to stimulate protein tyrosine phosphorylation (23),
receptor-mediated or direct activation of adenylyl cyclase or
cAMP-dependent protein kinase has not been related to increased tyrosine kinase activity. Similarly, protein tyrosine phosphorylation in response to stimulation of cells with VIP has not been
reported.
Tyrosine phosphorylation of proteins participates in the regulation of
various cellular functions, including cell proliferation, differentiation, and the formation of focal adhesions (23, 26). Increased tyrosine kinase activity is observed in response to stimulation of cells with growth factors, with several neuropeptides, or with a variety of other stimuli. Most growth factors, such as
platelet-derived growth factor or epidermal growth factor, stimulate
tyrosine phosphorylation through activation of receptors with intrinsic
tyrosine kinase activity (23), whereas neuropeptides such as
angiotensin II, vasopressin, bradykinin, or bombesin bind to
heptahelical receptors that lack intrinsic tyrosine kinase activity,
thus increasing tyrosine phosphorylation, probably by recruiting
intracellular non-receptor tyrosine kinases (1, 30). In fibroblasts
(23, 31, 35), vascular smooth muscle cells (27), and renal glomerular
mesangial cells (5), these agents cause prominent tyrosine
phosphorylation of proteins migrating between 110 and 130 kDa and
around 70 kDa, some of which have been identified as focal adhesion
kinases p125FAK and paxillin (9, 14, 23). After phosphorylation, both
proteins localize to the plasma membrane of cultured cells, where they
seem to participate in the regulated assembly of focal adhesion
complexes (23). In pancreatic acinar cells, protein tyrosine kinase
inhibitors such as genistein or tyrphostin 25 inhibit CCK-induced
tyrosine phosphorylation as well as amylase secretion, and incubation
of permeabilized cells with recombinant protein tyrosine phosphatase stimulates Ca2+-mediated secretion
(8, 17, 19, 20), indicating that tyrosine phosphorylation of one or
more proteins might be involved in the regulation of CCK-induced enzyme
secretion. However, the identity of the regulated phosphoproteins has
not been determined.
In this study we demonstrate VIP-, dibutyryl cAMP (DBcAMP)-, and
forskolin-induced protein tyrosine phosphorylation in rat pancreatic
acini. Whereas VIP and CCK both stimulated phosphorylation of proteins
migrating at 115 and 130 kDa, CCK induced tyrosine phosphorylation of
additional proteins migrating at 60, 66, and 72/78 kDa. One of these
proteins was identified as paxillin. Because protein tyrosine kinase
inhibitors reduce CCK- but not VIP-induced amylase release (3, 19), we
propose that protein tyrosine phosphorylation of paxillin may
participate in regulating the secretory response to CCK in pancreatic
acini.
 |
MATERIALS AND METHODS |
Materials and animals.
VIP, forskolin, DBcAMP, and the monoclonal antiphosphotyrosine antibody
(PT-66) were obtained from Sigma Chemical (St. Louis, MO).
CCK-8 (sulfated) was from Bachem (Bubendorf, Switzerland), peroxidase-conjugated affinity-purified rabbit anti-mouse
immunoglobulin G was purchased from Dianova (Hamburg, Germany), soybean
trypsin inhibitor and reagents for the amylase assay were from
Boehringer (Mannheim, Germany), and collagenase was from Worthington
Cell Systems (Hamburg, Germany). Tyrphostin 23 and genistein were from Calbiochem (La Jolla, CA), and essential and nonessential amino acids
were purchased from GIBCO (Gaithersburg, MD). Enhanced
chemiluminescence reagents and films were obtained from Amersham
(Braunschweig, Germany). The monoclonal antipaxillin antibody (clone
349) was from ICN (Costa Mesa, CA). All other reagents were analytical grade. Male Wistar rats (150-200 g) were bred at the Animal Care and Treatment Facility of the University of Ulm.
Preparation of isolated rat pancreatic acini.
The preparation of isolated rat pancreatic acini was performed
essentially as described (17). Acini were washed two times in
oxygenated
Krebs-Ringer-N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid (HEPES) buffer consisting of 104 mM NaCl, 5 mM KCl, 1 mM KH2PO4,
1.2 mM MgCl2, 2 mM
CaCl2, 0.2% (wt/vol) bovine serum
albumin, 0.01% (wt/vol) soybean trypsin inhibitor, 10 mM glucose, and
25 mM HEPES-NaOH, pH 7.4, and supplemented with minimal essential amino
acid solution and glutamine. Cell viability, as assessed by trypan blue
exclusion, exceeded 95%. All preincubation and incubation steps were
carried out at 37°C.
Examination of protein tyrosine phosphorylation.
To examine secretagogue-induced tyrosine phosphorylation, isolated
pancreatic acini were equilibrated for 10 min at 37°C. In some
experiments the acini were preincubated for an additional 10 min with
protein kinase inhibitor or appropriate vehicle. Secretagogue or
vehicle was then added for the indicated time periods, and the
incubation was terminated by suspending the acini in an excess volume
of ice-cold Krebs-Ringer-HEPES buffer. The acini were then pelleted by
centrifugation at 300 g (4°C). For
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
cellular protein was extracted by trituration in 400 µl lysis buffer
consisting of 20 mM tris(hydroxymethyl)aminomethane
(Tris) · HCl, pH 7.4, 30 mM
Na4P2O7,
95 mM NaCl, 1% (wt/vol) Triton X-100, 0.5 mM sodium orthovanadate,
0.1% (wt/vol) soybean trypsin inhibitor, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, 1 mM benzamidine, and 5 µM
ZnCl2. After removal of insoluble
material by centrifugation, 50 µg of soluble protein was separated
according to the method of Laemmli (13). For two-dimensional
separation, cells were extracted in sample buffer (12.25 M
urea, 0.125 mM dithiothreitol, 2.5% Triton X-100, 2.5%
Servalyte 7-9). In the first dimension, 40 µg of protein was
separated in polyacrylamide tube gels containing 2% Servalyte
3-10, 3% Servalyte 4-6.5, and 4.5% Servalyte 5-8, using a Mini-Protean II system (Bio-Rad, Hercules, CA) according to the
manufacturer's guidelines. SDS-PAGE was then performed as described
above. Gel-resolved proteins were electrophoretically transferred to
polyvinylidene difluoride membranes (Immobilon P; Millipore). Membranes
were incubated overnight in blocking buffer [50 mM
Tris · HCl, pH 7.8, 100 mM NaCl, 0.5% (wt/vol) Tween 20, 2% (wt/vol) bovine serum albumin], followed by two washes with Tris-buffered saline supplemented with 0.1% (wt/vol) Tween 20. The membranes were then incubated for 1 h with primary antibodies (1:3,000) in blocking buffer. After an additional three washes with
Tris-buffered saline supplemented with 0.1% (wt/vol) Tween 20, antigen-antibody complexes were visualized using secondary peroxidase-conjugated antibody and the enhanced chemiluminescence system by exposure to Kodak X-OMAT AR films for 1-2 min.
Quantitation was performed by densitometry using Phoretix 1D gel
analysis software (Phoretix, Newcastle upon Tyne, UK).
Data presentation.
All experiments were repeated at least three times with acini from
different preparations. Data shown are means ± SE. Statistical analysis was performed using Student's
t-test for paired values.
 |
RESULTS |
Tyrosine phosphorylation in response to VIP.
Phosphotyrosine-containing proteins were examined in freshly isolated
rat pancreatic acini under basal conditions and after stimulation with
VIP. Probing Western blots with antiphosphotyrosine antibodies revealed
that several protein bands were already phosphorylated under basal
conditions. Incubation of the acini with VIP resulted in an increase in
tyrosine phosphorylation of several protein bands, with the most
prominent phosphoproteins migrating at 130, 115, and 93 kDa on
SDS-polyacrylamide gels. These proteins are therefore referred to as
p130, p115, and p93 (Fig.
1A).
The phosphorylation signal of the VIP-responsive proteins was maximal
after 2-5 min of stimulation (Fig.
1B). Densitometric quantitation of
the bands revealed that the maximum intensity of tyrosine
phosphorylation of these phosphoproteins was 1.6- to 2-fold greater
than signal intensity under basal conditions and was observed at VIP
concentrations of 10 nM or higher (Fig.
1C). As shown in Fig.
2, preincubation with the protein kinase
inhibitors genistein (0.1 mM) or tyrphostin 23 (0.1 mM) decreased the
VIP-induced change in protein tyrosine phosphorylation of p130
completely and reduced the signal intensity of p115 to less than basal
levels. In comparison to other studies (3, 17), the current experiments
do not show prominent phosphoproteins migrating below 60 kDa, which may
be explained by molecular weight-dependent changes in the blotting
efficiency. In the present setting, conditions of electrophoretic
transfer of proteins from the gel to the membrane were adjusted to
allow maximum transfer of high molecular weight proteins, whereas some
of the low molecular weight proteins migrated through the blotting
membrane.

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Fig. 1.
Vasoactive intestinal peptide (VIP)-stimulated protein tyrosine
phosphorylation in rat pancreatic acini. Representative immunoblot
analysis of isolated rat pancreatic acini after stimulation with
10 8 M VIP for various time
periods (A) and densitometric
quantitation (B). Acini were
incubated with indicated concentrations of VIP for 2 min
(C). Values are means ± SE of at
least 3 independent experiments.
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Fig. 2.
Inhibition of VIP-stimulated protein tyrosine phosphorylation by
tyrosine kinase inhibitors. Cells were preincubated with genistein or
tyrphostin 23 for 10 min before stimulation with 10 nM VIP.
Representative immunoblot of 3 independent experiments.
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Tyrosine phosphorylation in response to forskolin or DBcAMP.
The VIP-induced signaling cascade involves activation of adenylyl
cyclase via Gs proteins, which
leads to an increase in the intracellular cAMP level (32) and to
stimulation of the cAMP-dependent protein kinase. To examine whether
this signaling pathway mediates VIP-induced protein tyrosine
phosphorylation, pancreatic acini were stimulated with forskolin or
DBcAMP, which activate adenylyl cyclase or the cAMP-dependent protein
kinase directly, thus bypassing receptor activation of G proteins. As
shown in Fig. 3, forskolin (0.1 mM) or
DBcAMP (2 mM) increased tyrosine phosphorylation of the same protein
bands as VIP, i.e., p130, p115, and to a lesser extent p93. Moreover,
the magnitude and the time course of the effect of forkolin or DBcAMP
on tyrosine phosphorylation of these phosphoproteins were similar to
those observed in response to VIP.

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Fig. 3.
Forskolin (left)- and dibutyryl cAMP
(right)-stimulated protein tyrosine
phosphorylation in rat pancreatic acini. Immunoblot analysis of
isolated rat pancreatic acini after stimulation with forskolin (0.1 mM
for 2 min) or with dibutyryl cAMP (2 mM for 3 min). Shown are
representative immunoblots of at least 3 independent experiments.
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Comparison of VIP- and CCK-induced protein tyrosine phosphorylation
patterns.
Both VIP and CCK are able to induce tyrosine phosphorylation of several
cellular proteins in a dose- and time-dependent manner. However, only
CCK-stimulated acinar cell secretion is sensitive to inhibition of
tyrosine kinases (3, 19). We have therefore examined whether this
differential sensitivity toward tyrosine kinase inhibition is
accompanied by a differential pattern of protein tyrosine
phosphorylation. Acini were incubated with maximal stimulatory
concentrations of VIP (10 nM) or CCK (10 nM) with respect to tyrosine
phosphorylation to enable direct comparison between VIP and CCK-induced
protein tyrosine phosphorylation. As shown in Fig.
4, the VIP-sensitive phosphoproteins p130
and p115 were phosphorylated on tyrosine residues in response to both VIP and CCK. Most important, CCK caused a strong increase in tyrosine phosphorylation of a broad band migrating at 72/78 kDa and to a lesser
extent of two proteins migrating at 66 and 60 kDa, whereas VIP had no
effect on the phosphorylation state of these proteins.

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Fig. 4.
Comparison of effects of VIP and CCK on protein tyrosine
phosphorylation. Representative immunoblot analysis of isolated rat
pancreatic acini after stimulation with 10 nM VIP for 3 min
(left) or after stimulation with 10 nM CCK for 2 min (right).
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Identification of p72/78.
To clearly distinguish individual tyrosine-phosphorylated proteins,
total cellular extracts were separated by two-dimensional gel
electrophoresis and were again analyzed by antiphosphotyrosine immunoblotting. As shown in Fig.
5A,
several proteins were phosphorylated on tyrosine residues under basal
conditions, with subsets increasing in signal intensity after
stimulation with 10 nM CCK (Fig.
5B). As observed after
one-dimensional separation in SDS-polyacrylamide gels, the most
prominent protein migrated as a broad band between 70 and 80 kDa in the
pH range of ~6-7. Again, stimulation of cells with VIP did not
induce signal intensity of this phosphoprotein (Fig.
5C). Two-dimensional separation and
antiphosphotyrosine Western blotting further revealed tyrosine
phosphorylation of several additional proteins that were not recognized
after one-dimensional SDS-PAGE with the same antibody, whereas the
signals of higher molecular weight proteins became less intense. This
effect is probably due to better isoelectric focusing of lower
molecular weight proteins, whereas higher molecular weight proteins
might not be focused and run off or not enter the gel. Proteins that were stimulated by CCK as well as by VIP migrated at 50 kDa at an
estimated pH of 5.5 and 6 and at 40 kDa at an estimated pH of 7. All
signals could be suppressed by competition with soluble phosphotyrosine. Because activation of several
Gq protein-coupled heptahelical
receptors in cultured cell systems is known to cause phosphorylation of
paxillin (23), membranes were incubated with paxillin-specific
antibodies, and the signal was compared with the migration pattern of
tyrosine-phosphorylated proteins. As shown in Fig.
5D, the 72/78-kDa band
did indeed comigrate with paxillin. Similar results were obtained after
the one- and two-dimensional blots were stripped and reprobed with
antipaxillin antibody. Furthermore, the CCK-induced increase in
tyrosine phosphorylation of paxillin could be confirmed by
immunoprecipitation of paxillin from CCK-stimulated and control
extracts (data not shown).

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Fig. 5.
Two-dimensional analysis of tyrosine-phosphorylated proteins. Rat
pancreatic acini were stimulated with VIP (10 nM) or CCK (10 nM). Total
cellular extracts of control cells (A) and of cells after
stimulation with CCK (B and D) or VIP
(C) were separated by two-dimensional gel electrophoresis
and analyzed by antiphosphotyrosine immunoblotting
(A-C) or by staining with
antipaxillin antibodies (D).
Immunoblots shown are representative of at least 3 independent
experiments.
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 |
DISCUSSION |
CCK stimulates secretion of digestive enzymes as well as cell growth of
pancreatic acinar cells through binding to the CCK-A receptor (21). On
activation, this heptahelical transmembrane receptor then couples to
Gq proteins, which in turn
activate the inositol phospholipid-specific PLC-
(33). Furthermore,
CCK has been shown to increase protein tyrosine phosphorylation of cellular proteins as well as protein tyrosine kinase activity, and a
role for tyrosine phosphorylation events in mediating the physiological
cellular response to CCK has been suggested (8, 17, 19, 20). In
contrast, VIP stimulates pancreatic acinar cell enzyme secretion by
occupying receptors that couple to
Gs proteins and thus activate
adenylyl cyclase and the cAMP-dependent protein kinase (33).
In this study, we demonstrate that VIP stimulates protein tyrosine
phosphorylation in freshly isolated pancreatic acinar cells. Direct
activation of adenylyl cyclase with forskolin or DBcAMP elicited a
similar phosphorylation response, indicating that activation of
adenylyl cyclase in pancreatic acini and the subsequent stimulation of
cAMP-dependent protein kinase is sufficient to mediate at least part of
the VIP-induced protein tyrosine phosphorylation and that this can
occur independently from direct activation of tyrosine kinases by G
proteins. Even though tyrosine phosphorylation is a well-known
signaling mechanism of various Gq
protein-coupled receptors (15, 23, 30), stimulation of tyrosine
phosphorylation by heptahelical receptors that couple to
Gs proteins has not been reported.
Therefore, our finding may be interpreted in at least two ways. One
explanation is that the freshly isolated acinar cells used in this
study represent a more sensitive experimental system than cultured or
receptor-transfected cells, which are commonly employed to examine
signal transduction mechanisms. In addition, acinar cells might employ
a signaling system that couples activation of adenylyl cyclase to the
stimulation of protein tyrosine kinases. Another possibility would be
cross-activation of Gq proteins or
phospholipases by the VIP receptor or other downstream signaling molecules similar to dual coupling of the luteinizing hormone receptor
(6). However, VIP is not known to stimulate second messenger pathways
other than the Gs protein-adenylyl
cyclase cascade, and we were able to stimulate phosphorylation by
direct activation of adenylyl cyclase or the cAMP-dependent protein
kinase (33). Hence VIP-stimulated tyrosine phosphorylation seems to represent a new signal transduction mechanism of the
Gs protein-coupled VIP receptor,
which is mediated by activation of the cAMP-dependent protein kinase
through adenylyl cyclase.
CCK and VIP both are able to stimulate pancreatic enzyme secretion. To
establish a role for tyrosine phosphorylation events in regulating
pancreatic acinar cell secretion, we and others have examined amylase
secretion from pancreatic acini in the presence of tyrosine kinase
inhibitors. In these experiments, genistein, tyrphostin 25, and
herbimycin were able to decrease CCK- or
Ca2+-induced amylase secretion
(17) but had no effect on VIP-stimulated secretion (3, 19). Similar
results were observed with tyrphostin 23 or with the peptide
pp60v-src-(137
157) in permeabilized acini, which was used as a more
specific approach for tyrosine kinase inhibition (A. Piiper and M. P. Lutz, unpublished observations). On the basis of these data it was
suggested that protein tyrosine kinases are involved in the
CCK-stimulated signal transduction cascade leading to amylase release
in response to activation of the
PLC-Ca2+ pathway.
To date, the tyrosine-phosphorylated proteins and protein tyrosine
kinases involved in the regulation of CCK-induced amylase secretion
from pancreatic acini have not been identified, even though several of
the phosphorylated proteins have been described (2, 4, 9, 28). As
shown, CCK and VIP both stimulate pancreatic acinar cell tyrosine
phosphorylation, yet only the CCK-induced tyrosine phosphorylation
events seem to be involved in enzyme secretion. Therefore,
identification of a subset of proteins that are tyrosine phosphorylated
in response to CCK but not in response to VIP might help to identify
proteins involved in the regulation of CCK-induced acinar cell
secretion. Comparison of VIP- and CCK-induced protein tyrosine
phosphorylation patterns revealed that several tyrosine-phosphorylated
proteins, namely p130, p115, and p93, are stimulated by both
secretagogues. Thus tyrosine phosphorylation of these substrates can be
achieved either by activation of the PLC or the adenylyl cyclase
pathways, and the observation that protein tyrosine kinase inhibitors
did not affect VIP-induced amylase secretion suggests that p130, p115, or p93 are unlikely to be involved in regulating this cellular function. In contrast, only CCK caused an increase in tyrosine phosphorylation of p66 and p72/78. Whereas phosphorylation of these
proteins was not observed in response to stimulation of acini with VIP,
forskolin, or DBcAMP, increased tyrosine phosphorylation of p72/78 has
been reported on incubation with a
Ca2+ ionophore or a COOH-terminal
phenylethylester analog of CCK (CCK-OPE), which elicits little or no
PLC activation but a clear intracellular Ca2+ response, or by direct
activation of protein kinase C with
12-O-tetradecanoylphorbol 13-acetate
(17), indicating that tyrosine phosphorylation of p72/78 is mediated by
Ca2+-protein kinase C pathway and
not by activation of adenylyl cyclase. In addition, tyrosine kinase
inhibitors were able to inhibit the secretory response to bombesin,
carbachol, CCK-OPE, and to Ca2+
ionophores (3, 17, 19), and we therefore propose that p72/78 might play
a role in the Ca2+-mediated
secretory response to CCK.
To identify p72/78 we did compare protein tyrosine phosphorylation
events in CCK-stimulated acinar cells with patterns reported after
stimulation of other heptahelical
Gq protein-coupled receptor systems. Interestingly, the pattern observed in acinar cells in response to CCK was strikingly similar to that reported in cultured cells in response to stimulation with vasopressin, bradykinin, angiotensin II, bombesin, or endothelins (15, 27, 35). In most of these
systems, paxillin was identified as one of the most prominent
phosphoproteins migrating at or around 70 kDa (23), and paxillin
phosphorylation in acinar cells has been reported in response to CCK
(9). Using two-dimensional electrophoretic protein separation as well
as immunoprecipitation of total cellular extracts with antipaxillin
antibody, we were able to confirm that p72/78 comigrates with paxillin.
Thus paxillin is phosphorylated on tyrosine residues in response to
stimulation of pancreatic acinar cells with CCK but not in response to
stimulation with VIP. Paxillin is a 68-kDa cytoskeletal protein
concentrated in focal adhesions, i.e., multimeric protein complexes
that occur at sites where cultured cells adhere to the extracellular
matrix (25). To date, the functional role of paxillin has not been firmly established. Phosphorylation of paxillin correlates with the
formation of focal adhesions in a human colon cancer cell line (24) and
in cultured rat aortic smooth muscle cells (29) and with the formation
of actin stress fibers as well as the assembly of focal contacts in
Swiss 3T3 fibroblasts (12), where it colocalizes with the focal
adhesion kinase and integrin receptors (18). Activation of these
proteins, e.g., by hormonal stimulation or adhesion to the
extracellular matrix, leads to changes in cell adhesion properties,
cell motility, and cell shape (22, 23). Paxillin has been implicated in
cell signaling because it directly binds to the paxillin binding site
on the focal adhesion kinase and associates with pp60src or the
COOH-terminal Src kinase (7). Even though paxillin is thought to be
involved in the formation of focal adhesions, these complexes are
typically observed in cultured cells only (10), and the presence of
focal adhesion complexes has not been demonstrated in pancreatic acinar
cells. However, acinar cells do need an intact actin filament system for regulated enzyme secretion (11), and it is likely that actin filaments in pancreatic acinar cells are attached to the plasma membrane by anchoring systems that resemble the focal adhesion complexes in adhering cultured cells. Therefore, we propose that phosphorylated paxillin binds to and may even participate in the regulation of protein complexes that mediate the turnover and composition of the actin filament system in acinar cells. Because the
actin filament system is thought to be essential for regulated fusion
of zymogen granules with the plasma membrane (11), paxillin might
participate in the regulation of CCK-induced enzyme secretion at this
final step. The role of VIP-induced protein tyrosine phosphorylation as
well as the tyrosine kinases activated by VIP remain to be identified.
 |
ACKNOWLEDGEMENTS |
We acknowledge the excellent technical help of Claudia
Längle, Tanja Wissling, Sandra Theimer, and Susanne Scherr. We
thank Thomas Gress and André Menke for helpful discussions.
 |
FOOTNOTES |
This work was supported by grants from the Deutsche
Forschungsgemeinschaft to M. P. Lutz (Lu 441/2-1) and to S. Zeuzem
(Ze 634/2-2).
Address for reprint requests: M. P. Lutz, Dept. of Internal Medicine I,
Univ. of Ulm, 89070 Ulm, Germany.
Received 10 January 1997; accepted in final form 7 August 1997.
 |
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