Báo cáo khoa học: Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini Formation of CrkII–protein complexes docx

Cholecystokinin rapidly stimulates CrkII function
in vivo
in rat
pancreatic acini
Formation of CrkII–protein complexes
Alberto G. Andreolotti
1
, Maria J. Bragado
2
, Jose A. Tapia
3
, Robert T. Jensen
3
and Luis J. Garcia-Marin
1
1
Departamento de Fisiologia, Universidad de Extremadura, Caceres, Spain;
2
Departamento de Bioquimica, Biologia Molecular y
Genetica, Universidad de Extremadura, Caceres, Spain;
3
Digestive Diseases Branch, National Institute of Diabetes and Digestive
and Kidney Diseases of the National Institutes of Health, Bethesda, MD, USA
Crk belongs to a family of adapter proteins whose structure
allows interaction with tyrosine-phosphorylated proteins
and is therefore an important modulator of downstream
signals, representing a convergence of the actions of
numerous stimuli. Recently, it was demonstrated that chole-
cystokinin (CCK) induced tyrosine phosphorylation of
proteins related to fiber stress formation in rat pancreatic
acini. Here, we investigated whether CCK receptor activa-

tion signals through CrkII and forms complexes with tyro-
sine-phosphorylated proteins in rat pancreatic acini. We
demonstrated that CCK promoted the transient formation
of CrkII–paxillin and CrkII–p130
Cas
complexes with maxi-
mal effect at 1 min. Additionally, CCK decreased the elec-
trophoretic mobility of CrkII. This decrease was time- and
concentration-dependent and inversely related with its
function. Carbachol and bombesin also decreased CrkII
electrophoretic mobility, whereas epidermal growth factor,
vasoactive intestinal peptide, secretin or pituitary adenylate
cyclase-activating polypeptide had no effect. CCK-induced
CrkII electrophoretic shift was dependent on the Src family
of tyrosine kinases and occurred in the intact animal, sug-
gesting a physiological role of CrkII mediating CCK actions
in the exocrine pancreas in vivo.
Keywords: Crk; protein complex; CCK; transduction path-
ways; pancreatic acini.
Cholecystokinin (CCK) is a peptide acting as a hormone/
neurotransmitter that controls several physiological effects
in the gastrointestinal tract [1,2] and in the central nervous
system (CNS) [3,4]. Additionally, CCK has potent growth
effects both in normal tissues, such as pancreas [3], and in
neoplasic tissues, such as stomach and pancreas adenocar-
cinomas [3,5]. It has been clearly established that its
physiological effects on the gallbladder, pancreas and
CNS are mediated in part by the CCK
A
receptor, a member

of the G-protein-coupled receptor (GPCR) superfamily
[1,2]. Intracellular pathways of CCK
A
receptor activation
have been investigated extensively in pancreatic acini [1,2].
In these cells, we reported recently that CCK stimulates
tyrosine phosphorylation of several proteins related with
fiber stress formation such as the focal adhesion kinase
p125
FAK
, paxillin [6], p130
Cas
[7] and PYK2 [8], dissecting
alternative transduction cascades involved in several cellular
functions, such as cystoskeleton reorganization [9].
A central role in signal transduction pathways down-
stream of different stimuli is played by the adapter protein,
Crk. A new oncogene identified from a chicken tumor that
activated a cellular adapter-type SH2-SH3-containing
G-protein led to the name of crk (chicken tumor virus
regulator of kinase) [10]. The product of the proto-oncogene
in human and mouse, c-Crk, is expressed as two distinct
proteins. c-CrkI contains one SH2 and one SH3 domain,
while c-CrkIIhas anadditional SH3 [11]. The SH2 domain of
Crk binds to phosphotyrosine-containing proteins such as
p130
Cas
, paxillin or Cbl [10,12]. The first SH3 domain
(N-terminal) of CrkII binds to guanine nucleotide exchange
factors such as C3G, which in turn activate transduction

cascades involving small GTP-binding proteins [10,12].
Recent investigations reported that CrkII complex formation
is induced by oncogenes such as Bcr-Abl [13], integrins [14],
growth factors [15,16] and ligands of G-protein-coupled
receptors (such as bombesin [17] or angiotensin II [18]) and
thus CrkII represents a convergence of the signal transduc-
tion cascades of different stimuli. Summarizing these data,
the Crk family seems to be involved in different signaling
systems including the formation of focal adhesion complexes
and actin cytoskeleton regulation, receptor tyrosine kinases
signaling pathways and pathogenesis of different leukemias
related with the Bcr/Abl tyrosine kinases [10,12].
A peculiar feature of CrkII is that, unlike some other
adapter proteins, CrkII itself is tyrosine phosphorylated by
tyrosine kinases such as the cytoplasmic c-Abl and the
epidermal growth factor (EGF) receptor [19–21]. It is
Correspondence to L. J. Garcia-Marin, Departamento de Fisiologia,
Facultad de Veterinaria, Universidad de Extremadura,
Avda. de la Universidad, s/n, 10071 Caceres, Spain.
Fax: + 34 927 257 110, Tel.: + 34 927 257 154,
E-mail:
Abbreviations: BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-
N,N,N¢,N¢-tetraacetic acid tetrakis (acetoxymethyl ester); CCK,
cholecystokinin; CNS, central nervous system; crk, chicken tumor
virus regulator of kinase; EGF, epidermal growth factor; GPCR,
G-protein-coupled receptor; PKC, protein kinase C; TPA,
12-O-tetradecanoylphorbol 13-acetate.
(Received 29 August 2003, accepted 6 October 2003)
Eur. J. Biochem. 270, 4706–4713 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03869.x
proposed that tyrosine phosphorylation of CrkII creates a

binding site for its SH2 domain that inhibits the inter-
molecular interactions mediated by both SH2 and SH3
domains of CrkII [19,20]. Thus, changes in CrkII phos-
phorylation state would explain the regulation of CrkII–
protein complex formation. Stimuli that induce CrkII
tyrosine phosphorylation include growth factors other than
EGF, such as nerve growth factor and insulin-like growth
factor, sphingosine 1-phosphate [15,16,22,23], the engage-
ment of T-cell receptor or B-cell antigen receptor [24,25].
Although it has been demonstrated that activation of
GPCRs stimulates the formation of CrkII–protein com-
plexes [7,17,18], nothing is known about the effect of a
GPCR activation, such as CCK
A
, on CrkII signaling and
its subsequent effect on the formation of CrkII–protein
complexes in rat pancreatic acini. Thus, in the present work,
we investigated whether in vivo activation of the CCK
A
receptor regulates CrkII function to form protein complexes
in rat pancreatic acini, one of its main physiological cell
targets [1,2]. We also studied the electrophoretic mobility
shift of CrkII observed after CCK treatment and its possible
contribution to the regulation of CrkII function. Moreover,
we have investigated whether CCK intracellular actions in
the pancreas of the intact animal involved a CrkII signal.
Materials and methods
Materials
Male Wistar rats (150–200 g) were obtained from the
Animal Section (Veterinary Resources Branch, NIH,

Bethesda, MD, USA) or from the Veterinary Faculty
(UEX, Spain); purified collagenase (CLSPA) from Worth-
ington Biochemicals (Freehold, NJ, USA); COOH-ter-
minal octapeptide of cholecystokinin (CCK-8) from
Peninsula Laboratories (Belmont, CA, USA); EGF,
thapsigargin, A23187, tyrphostin B44, PP2, PP3 from
Calbiochem; 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-
tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-
AM), bombesin, pituitary adenylate cyclase-activating
polypeptide, secretin, vasoactive intestinal peptide, 12-O-
tetradecanoylphorbol 13-acetate (TPA) from Bachem AG
(Switzerland); anti-Crk mAb, anti-p130
Cas
mAb, anti-
paxillin mAb, anti-phosphotyrosine mAb (PY20) from
Transduction Laboratories (Lexington, KY, USA) and
vitamin/aminoacid mixture from Sigma.
In vivo
injection of CCK and preparation of pancreatic
homogenates
Experiments performed using animals were in line with the
Ethical Principles and Guidelines for Scientific Experiments
on Animals of the Swiss Academy of Medical Sciences. Male
Wistar rats weighing 150–200 g and fed a standard diet were
injected with either saline or 15 lgÆkg
)1
of CCK (intraperi-
toneal) between 09:00 and 11:00 h. Rats were killed after
10 min and the pancreas was removed and homogenized
(Polytron homogenizer) in seven volumes of lysis buffer:

50 m
M
Tris/HCl, pH 7.5, 150 m
M
NaCl, 1% Triton X-100,
1% deoxycholate, 1 m
M
EGTA, 0.4 m
M
EDTA,
2.5 lgÆmL
)1
aprotinin, 2.5 lgÆmL
)1
leupeptin, 1 m
M
phenylmethanesulfonyl fluoride, and 0.2 m
M
Na
3
VO
4
.The
homogenate was centrifuged at 10 000 g (10 min, 4 °C) and
the supernatant containing microsomes and soluble proteins
was used to analyze the CrkII phosphorylation state.
Rat pancreatic acini preparation
Dispersed rat pancreatic acini were isolated according to
modifications [6] of the procedure published previously [26].
Unless otherwise stated, the standard incubation solution

contained 25.5 m
M
Hepes, (pH 7.4), 98 m
M
NaCl, 6 m
M
KCl, 2.5 m
M
NaH
2
PO
4
,5m
M
sodium pyruvate, 5 m
M
sodium fumarate, 5 m
M
sodium glutamate, 11.5 m
M
glu-
cose, 0.5 m
M
CaCl
2
,1m
M
MgCl
2
,2m

M
glutamine, 1%
(w/v) albumin, 1% (w/v) trypsin inhibitor 1% (v/v) vitamin
mixture and 1% (w/v) amino acid mixture with 100% (v/v)
O
2
as the gas phase.
Immunoprecipitation
Pancreatic acini isolated from one rat were preincubated
with standard incubation solution for 3 h at 37 °C. Acini
were then incubated with agonists at concentrations and
times indicated, washed with phosphate buffered saline
(NaCl/P
i
) with 0.2 m
M
Na
3
VO
4
andsonicated(5sat4°C)
in lysis buffer. Lysates were centrifuged at 10 000 g for
15 min. Protein concentration in the supernatant was
standardized to 500 lgÆmL
)1
and 1 mL was incubated
overnight at 4 °C with anti-phosphotyrosine (PY20) mAb
(4 lg) or anti-Crk Ig (6 lg), bridging antibody (4 lg) and
25 lL of protein A-agarose. Immunoprecipitates were
washed three times with NaCl/P

i
andanalysedbySDS/
PAGE and Western blotting.
Western blotting
Proteins in total cellular lysates, immunoprecipitates or
pancreatic homogenates were resolved by SDS/PAGE and
transferred to nitrocellulose membranes. Western blotting
was performed as described previously [7,8] using the
following primary antibody concentrations: 1 lgÆmL
)1
anti-phosphotyrosine (PY20), 0.25 lgÆmL
)1
anti-Crk,
0.25 lgÆmL
)1
anti-p130
Cas
or 0.025 lgÆmL
)1
anti-paxillin.
Statistical analyses
All data provided are reported as mean ± SEM. Data were
analysed using Student’s t-test and only values with
P < 0.05 were accepted as statistically significant.
Results
CCK induced CrkII association with p130
Cas
and paxillin
to form protein complexes in rat pancreatic acini
To identify the presence of Crk on rat pancreatic acini,

proteins from whole acinar lysates immunoprecipitated with
anti-Crk antibody were separated using SDS/PAGE and
analysed by Western blotting. In acinar lysates (Fig. 1A)
anti-Crk specific Ig revealed the presence of two bands at the
suitable molecular mass of CrkII (40/42 kDa) [23]. In
unstimulated acini, the majority of CrkII was present in the
lower band which shows higher electrophoretic mobility
Ó FEBS 2003 Regulation of Crk by cholecystokinin (Eur. J. Biochem. 270) 4707
(Fig. 1A, lane 1). In acini stimulated with 10 n
M
CCK-8 the
majority of CrkII is shifted to the upper band, showing lower
electrophoretic mobility (lanes 4, 5). These results were
confirmed by immunoprecipitation of acinar proteins with
anti-CrkII Ig followed by Western blotting with the same
antibody (Fig. 1B). Two bands with different electrophoretic
mobility were also visualized in these conditions. The same
membranes containing anti-CrkII immunoprecipitated pro-
teins were subsequently probed with anti-paxillin Ig (Fig. 1C)
to analyze the regulation by CCK of the interaction between
the adapter protein, CrkII and paxillin. Data shown in
Fig. 1C demonstrated that 10 n
M
CCK-8 treatment caused a
maximal effect on CrkII–paxillin complex formation at
1 min which decreased rapidly with almost no effect after
5 min of CCK-8 addition (Fig. 1C). We have confirmed that
CCK regulates the protein complex formation of CrkII with
other proteins such as p130
Cas

in rat pancreatic acini.
Coimmunoprecipitation studies perfomed in the same sam-
ples as above are shown in Fig. 1D and show that treatment
with CCK-8 stimulated the formation of the CrkII–p130
Cas
complex with a maximum effect at 1 min that decreased
rapidly (Fig. 1D), confirming previous results [7].
CCK-8 induced an electrophoretic mobility shift of CrkII
in a time- and concentration-dependent manner
We next investigated the differences in the electrophoretic
mobility of CrkII observed in Fig. 1. CCK-induced CrkII
electrophoretic shift was time-dependent (Fig. 2A) with an
increase in the upper band detected within 1 min after
addition of CCK-8 and a maximum reached within 5 min
Fig. 1. Identification of CrkII in rat pancreatic acini (A, B) and CCK-
dependent induction of CrkII function to form CrkII-protein complexes
in vivo (C, D). Rat pancreatic acini were incubated with 10 n
M
CCK-8
for the indicated times and then lysed. Lysates were immunoprecipi-
tated with anti-Crk mAb. Resulting immunocomplexes (B) or 10 lg
protein acinar lysates (A) were analysed by SDS/PAGE and Western
blotting with anti-Crk mAb or anti-paxillin mAb (C) or anti-p130
Cas
mAb (D). CrkII positions are indicated on the left. Results shown are
representative of three independent experiments.
Fig. 2. Time-course and concentration-dependence of CCK-8 stimula-
tion of CrkII electrophoretic mobility shift. Rat pancreatic acini were
treated with CCK-8 at concentrations and times indicated and then
lysed. Cell lysates were analysed by Western blotting using anti-Crk

mAb. Quantification of bands was performed by scanning densito-
metry and is represented in the graphs. Results shown are represen-
tative of four independent experiments, each one performed in
duplicate. (A) The upper panel shows a representative experiment with
CCK-8 at the indicated times. Values shown in the graph are means ±
SEM, expressed as the percentage of CrkII upper band with respect to
total CrkII (upper and lower band). (B) The upper panel shows a
representative experiment where acini were incubated for 2.5 min with
indicated CCK-8 concentrations. Values are means ± SEM expressed
as the percentage of maximal increase caused by 10 n
M
CCK-8 above
control unstimulated values.
4708 A. G. Andreolotti et al. (Eur. J. Biochem. 270) Ó FEBS 2003
(65% of total CrkII was shifted to the upper band). The
degree of electrophoretic mobility shift decreased at 40 min
but remained elevated (50% of total CrkII was shifted). The
lower CrkII electrophoretic mobility state induced by CCK-
8 was dose-dependent (Fig. 2B) with a half-maximal effect
at 0.1 n
M
. A weak increase was detected after 5 min
incubation with 0.01 n
M
CCK-8 and the maximum effect
was observed at 100 n
M
CCK-8.
CCK induces the electrophoretic mobility shift of CrkII
in the intact animal

To study whether CCK has a similar effect in the
electrophoretic mobility of CrkII in vivo,weinjectedrats
with 15 lgÆkg
)1
of CCK. The injection of CCK reduced the
electrophoretic mobility of CrkII in pancreatic homogenates
(41.8 ± 1.5% of total CrkII was shifted to the upper band)
as shown in Fig. 3. This electrophoretic shift effect observed
in the intact rat is comparable to the effect obtained in rat
isolated pancreatic acini after 10 n
M
CCK treatment for
20 min.
Effect of different agonists on the electrophoretic
mobility of CrkII in pancreatic acini
Different agonists belonging to the GPCR family [1] were
tested to study whether the shift in CrkII electrophoretic
mobility also occurred with other stimuli. Bombesin and
carbachol induced a marked decreased in CrkII electro-
phoretic mobility shift (Fig. 4, lanes 3, 4), comparable to the
effect obtained with CCK-8 (lane 1). The CCK-8 analog,
CCK-JMV-180 (1 l
M
), an agonist at the high-affinity state
of the CCK receptor and antagonist at the low-affinity state
[1], had no effect on CrkII electrophoretic mobility (lane 5).
We also evaluated whether CrkII electrophoretic shift was
induced by growth factors. EGF had no effect on CrkII
electrophoretic mobility (Fig. 4, lane 6). We further con-
firmed the lack of effect of EGF by incubating pancreatic

acini with 10 n
M
EGF for different times (data not shown).
EGF did not modify CrkII mobility at any time point
studied. We also analysed whether occupation of receptors
coupled to an enhancement of intracellular cAMP led
to CrkII electrophoretic shift. Neither pituitary adenylate
cyclase-activating polypeptide, secretin nor vasoactive intes-
tinal peptide affected CrkII electrophoretic shift in pancre-
atic acini (Fig. 4, lanes 7–9). All these agonists have been
demonstrated to have biological effects on pancreatic acinar
cells at concentrations similar to the one used by us in this
work [1,8,27].
Effect of intracellular calcium on CrkII electrophoretic
shift induced by CCK in pancreatic acini
CCK receptor occupation causes activation of phospho-
lipase C (PLC), resulting in the generation of inositol
phosphates and diacylglycerol releasing intracellular cal-
cium and activation of protein kinase C (PKC), respectively
[1,2]. To investigate the involvement of calcium in CCK-
induced CrkII electrophoretic mobility shift, we preincu-
bated pancreatic acini with thapsigargin and BAPTA-AM
in a calcium-free medium (with 5 m
M
EGTA) before
addition of CCK-8. Incubation with each compound alone
did not affect the electrophoretic mobility of CrkII (Fig. 5,
lanes 2, 3 and 4). The calcium-free medium, which decreases
calcium influx in response to CCK-8 in pancreatic acini
[2,8], significantly decreased the CCK-8-induced CrkII

mobility shift by 41.5 ± 2.9% (Fig. 5, lane 6 compared
with 5). Preincubation with thapsigargin, which totally
abolished the CCK-8-stimulated increase of intracellular
Fig. 3. Exogenous CCK enhances CrkII electrophoretic mobility shift in
the intact animal. Rats were injected with 15 lgÆkg
)1
of CCK-8 and
10 min later pancreas was removed and homogenized. Western blot-
ting analysis of total CrkII after SDS/PAGE was performed and the
electrophoretic mobility shift of CrkII assessed. The top panel shows a
representative Western blot and the bottom panel shows means ±
SEM of four independent experiments [expressed as percentage of
CrkII upper band with respect to total CrkII (upper and lower band)].
Duplicate samples were analysed for each rat.
Fig. 4. Effect of different stimuli on CrkII electrophoretic mobility shift in rat pancreatic acini. Acini were treated 5 min with the indicated
concentrations of agonists and lysed. CrkII was identified by Western blotting as described. Results are representative of three experiments
performed in duplicate.
Ó FEBS 2003 Regulation of Crk by cholecystokinin (Eur. J. Biochem. 270) 4709
calcium in pancreatic acini [8], significantly reduced the
CCK-induced CrkII electrophoretic mobility shift by
35.5 ± 4.0% (Fig. 5, lane 7 compared with 5). Depletion
of intracellular calcium by preincubation with BAPTA-AM
markedly decreased the CCK-induced mobility shift (by
84 ± 11.6%; Fig. 5, lane 8 compared with 5). Direct
activation of PKC by 1 l
M
TPA or simultaneous treatment
with TPA and calcium ionophores did not modify CrkII
electrophoretic mobility (data not shown).
CrkII electrophoretic mobility shift induced by CCK

involves tyrosine phosphorylation
It is well established that CCK stimulates the tyrosine
phosphorylation of several intracellular proteins in rat
pancreatic acini [2,6–8]. It is also known that CrkII can be
phosphorylated on tyrosine [19–25]. To investigate whether
CrkII electrophoretic mobility shift induced by CCK was
related with its tyrosine phosphorylation, we pretreated
pancreatic acini with B44, a general inhibitor of protein
tyrosine kinases. Previously, we showed that pretreatment
of these cells with 300 l
M
B44 for 2 h caused almost a
complete inhibition of CCK-stimulated tyrosine phosphory-
lation [7,8]. Pretreatment of acini with this tyrosine kinase
inhibitor prevented the CrkII electrophoretic mobility shift
induced by CCK-8 (Fig. 6A, lane 4 compared with 2) where
the majority of CrkII protein from whole acinar cell lysates
remained in the lower band in presence of B44. To check
further whether CrkII was tyrosine phosphorylated in
response to CCK, proteins from the same acinar lysates
were immunoprecipitated with an anti-phosphotyrosine Ig
and analysed by Western blotting using a specific anti-Crk
Ig (Fig. 6B). Two bands were visible in untreated acini (lane
1). Treatment with CCK-8 caused an increase in the
intensity of the upper, slower migrating band (lane 2),
showing that it contains the more tyrosine phosphorylated
band of Crk. Pretreatment with tyrosine kinase inhibitor
alone increased the intensity of the lower migrating band
(lane 3) and markedly reduced the CCK-8-induced elec-
trophoretic mobility shift to the upper, slower migrating

band resulting in an increase of the lower migrating band
(lane 4). Similar results were observed when antibodies were
used in the reverse order, (immunoprecipitation with an
anti-Crk Ig followed by Western blotting using an anti-
phosphotyrosine Ig (Fig. 6C).
Involvement of Src family of tyrosine kinases
on the CCK-induced CrkII electrophoretic mobility shift
We next examined the effect of a specific Src family tyrosine
kinase inhibitor, PP2 and its inactive analog, PP3 [28,29] on
CrkII electrophoretic mobility shift. Pretreatment of pan-
creatic acini with 20 l
M
PP2 (1 h) did not modify unstim-
ulated CrkII electrophoretic migration (Fig. 7, lane 4),
but significantly reduced the CCK-8-induced CrkII
electrophoretic mobility shift by 33.5 ± 4.5 and
Fig. 5. Calcium dependence of CCK-8 stimu-
lation of CrkII electrophoretic mobility shift in
pancreatic acini. Acini were pretreated 30 min
at 37 °C in a calcium-free medium (with
EGTA 5 m
M
) either in absence or presence of
thapsigargin (10 l
M
)orBAPTA/AM(50 l
M
).
Acini were further incubated for 5 min with
no addition or with CCK-8 (10 n

M
). CrkII
electrophoretic mobility shift was assayed by
Western blotting as described. Results shown
in the upper panel are representative of four
experiments, each one performed in duplicate.
Results at the bottom panel are means of
CrkII upper band ± SEM expressed as a
percentage of CrkII maximal electrophoretic
mobility shift (obtained with CCK treatment
in a medium with normal calcium concentra-
tion). **P < 0.01.
Fig. 6. Phosphotyrosine dependence of the CCK-8 induction of CrkII
electrophoretic mobility shift in pancreatic acini. Rat pancreatic acini
preincubated for 2 h with the tyrosine kinase inhibitor, B44 (300 l
M
),
were treated for 5 min with no addition (lanes 1, 3) or with 10 n
M
CCK-8 (lanes 2, 4) and then lysed. Proteins from whole cell lysates
were immunoprecipitated with anti-phosphotyrosine (B) or anti-Crk
(C) Igs. Cell lysates (A) or immunoprecipitates (IP; B and C) were
analysed by Western blotting (WB) using anti-Crk mAb (A and B) or
anti-phosphotyrosine mAb (C). CrkII positions are indicated on the
left. Results shown are representative of three independent experi-
ments.
4710 A. G. Andreolotti et al. (Eur. J. Biochem. 270) Ó FEBS 2003
47.0 ± 6.5% at 5 and 40 min of CCK-8 treatment (Fig. 7,
lanes 5 and 6), respectively. The inactive analog of the
inhibitor of the Src family tyrosine kinase, PP3 (20 l

M
),
showed no effect on basal nor CCK-stimulated CrkII
electrophoretic mobility shift (Fig. 7, lanes 7–9).
Discussion
In this study, we have demonstrated that CCK rapidly
promotes the formation of CrkII–protein complexes,
CrkII–paxillin and CrkII–p130
Cas
, in rat pancreatic acini.
Recently, it has been demonstrated that CCK activates
different intracellular pathways in rat pancreatic acinar cells
[2]. We have demonstrated previously in these cells that
CCK is a potent activator of the tyrosine phosphorylation
of different proteins such as p130
Cas
[7] and paxillin [6],
creating potential binding sites for the SH2 domain of
CrkII, necessary for the formation of protein complexes.
Our results demonstrated that CrkII is present in rat
pancreatic acini as two bands with different electrophoretic
mobility. Moreover, we have shown an inverse correlation
between the electrophoretic mobility and the formation of
CrkII protein complexes. Concerning the regulation of CrkII
complex formation, it has been proposed that the SH2
domain of CrkII intramolecularly binds to the CrkII
phosphorylated Tyr221 residue and that this association
inhibits the intermolecular interactions mediated by both the
SH2 and SH3 Crk domains [10,19,30]. The electrophoretic
mobility shift of CrkII appears to be due to the phosphory-

lation state of the protein; thus, the upper band corresponds
to the more phosphorylated CrkII and the lower band
corresponds to the less phosphorylated state of the CrkII
protein [23]. By using two different approaches, our results
demonstrated that CCK induced both the apparition of a
slower electrophoretic migrating band of CrkII and an
increase in its tyrosine phosphorylation content. Under our
conditions we can suggest that, at least partially, the CCK-
induced electrophoretic mobility shift is correlated with an
increase in the phosphorylated tyrosine content of CrkII. In
this regard, it is well documented that CrkII itself, unlike
other adapter proteins, is tyrosine phosphorylated in
response to growth factors EGF, nerve growth factor,
insulin-like growth factor and also by sphingosin 1-phos-
phate [15,16,22,23], engagement of T-cell receptor or B-cell
antigen receptor [24,25]. Tyrosine kinases, such as the
cytoplasmic c-Abl and the EGF receptor, phosphorylate
CrkII in tyrosine [19,21]. However we cannot rule out other
possibilities that would explain the CrkII electrophoretic
mobility shift induced by CCK in our study.
According to this model of inhibition of CrkII complex
formation, regulated by the intramolecular binding of SH2 to
phosphorylated Tyr221, there is an established sequence of
mechanisms regulating CrkII complex formation where (a)
different proteins have to be phosphorylated on tyrosine
residues to allow the complex formation with CrkII through
the SH2 domain of this adapter protein; (b) CrkII is
phosphorylated on Tyr221 to allow (c) the intramolecular
SH2 domain bind to it, which results in (d) inhibition of the
intermolecular interactions of CrkII mediated by the SH2

domain and (e) subsequent blockade of the downstream
CrkII-mediated pathways [30]. Based on our results, we
propose that a similar sequential model of the regulation of
CrkII function by CCK may occur in rat pancreatic acini: (a)
CCK stimulates the rapid tyrosine phosphorylation of
proteins such as p130
Cas
and paxillin [6,7]; (b) immediately
afterwards, CCK promotes the rapid formation of CrkII–
p130
Cas
and CrkII–paxillin complexes, probably complexing
to the SH2 domain of CrkII (rate is maximal at 1 min and
declines after 5 mins; (c) the CrkII electrophoretic mobility
shift appears visible within 1 min and is maximal at 5 min
Fig. 7. Effect of PP2, a specific inhibitor of Src
family tyrosine kinases, and its inactive analog,
PP3 on the CCK-8 induction of CrkII elec-
trophoretic mobility shift in rat pancreatic acini.
Pancreatic acini were pretreated 2 h at 37 °C
in either absence or presence of 20 l
M
PP2
(lanes 4–6) or 20 l
M
PP3 (lanes 7–9). Acini
were further incubated 5 or 40 min with 10 n
M
CCK-8 and then lysed. Results shown are
representative of three experiments in dupli-

cate. Results in the lower panel are means ±
SEM of four experiments expressed as a per-
centage of CrkII (upper band) with respect to
total CrkII (upper and lower band).
**P < 0.01.
Ó FEBS 2003 Regulation of Crk by cholecystokinin (Eur. J. Biochem. 270) 4711
incubation with CCK. If we suppose that this mobility shift is
at least partially due to the tyrosine phosphorylation of
CrkII, as suggested from this study, then at 5 min, the SH2
domain of CrkII could be intramolecularly bound to
phosphorylated tyrosine resulting in a blockade of any CrkII
protein complex mediated via the SH2 domain. In agreement
with this model, we have found that CrkII association with
p130
Cas
or paxillin is almost completely disrupted at 5 min,
which is in concordance with this temporal sequence model of
CrkII functional mechanism. Thus, changes in CrkII phos-
phorylation state, visualized by the subsequent electro-
phoretic mobility shift, would explain the CCK-mediated
regulation of CrkII–protein complex formation by a mech-
anism of open-closed configuration similar to that described
for Src family members [12].
Concerning the function of the CrkII protein complexes,
in rat-1 fibroblasts over-expressing human insulin receptor
(HIRc cells), a potential role of CrkII–p130
Cas
complex
formation (via the SH2 domain of CrkII) has been
suggested in the regulation of mammalian actin cytoskele-

ton [30]. Several studies have recently suggested that CrkII
may regulate cytoskeleton organization through activation
of the Rho/Rac family of small GTPases [30].
Electrophoretic mobility shift of CrkII was a rapid
consequence of the stimulation of rat pancreatic acini with
CCK and was dependent on the CCK concentration. At
present, little is known about the intracellular pathways
coupling receptor activation to CrkII, especially in the case
of the GPCRs such as the CCK
A
receptor. It is well
established that the CCK concentration range that regulates
CrkII also causes activation of CCK
A
receptor [1] activating
PLC, resulting in the subsequent PKC activation, inositol
phosphate generation and intracellular calcium release [1,2],
and also activates several transduction pathways such as
p125
FAK
, PYK2, paxillin and p130
Cas
tyrosine phosphory-
lation [6–8].
PKC activation is probably not part of an intracellular
pathway that mediates CCK-stimulated CrkII electropho-
retic mobility in pancreatic acini, as demonstrated by the
lack of effect of TPA or the PKC inhibitor, GF109203X
(data not shown). Moreover, simultaneous PKC and
intracellular calcium stimulation did not affect CrkII

electrophoretic mobility. In the present study, we have
demonstrated that intracellular calcium increase by itself did
not cause a change on CrkII electrophoretic migration but
the presence of intracellular calcium did play a permissive
role because its presence was necessary for CCK to
stimulate CrkII mobility shift.
In the present study we have found that PP2, a specific
inhibitor of Src family kinases [28,29], produced a significant
inhibition of CCK-stimulated CrkII electrophoretic mobil-
ity shift. This effect was specific as pretreatment with PP3,
an inactive analog of PP2 [28,29], had no effect at all on
CrkII migration induced by CCK stimulation. Thus, our
results support the conclusion that CCK induces CrkII
electrophoretic mobility shift by an intracellular pathway
that is at least partially mediated by the Src family of
tyrosine kinases in rat pancreatic acini. Src family tyrosine
kinase inhibitors have been demonstrated to abolish CrkII
phosphorylation induced by sphingosine 1-phosphate [31].
This observation reinforces our idea that the electrophoretic
mobility shift of CrkII is related directly with its tyrosine
phosphorylation. Various GPCRs, including CCK
A
[32],
activate Src family kinases [33]. In addition, v-Src and v-Crk
transformed cells display elevated tyrosine phosphorylation
on proteins related with CrkII and focal adhesions, inclu-
ding p130
Cas
,p125
FAK

and paxillin [10,33].
The CrkII electrophoretic mobility shift was also
observed after stimulation with agonists of pancreatic
receptors (belonging to the GPCR family) other than
CCK (such as bombesin and carbachol). Interestingly, EGF
or agonists of receptors coupled to an increase in cAMP did
not change the electrophoretic mobility of CrkII. The lack
of effect of EGF is not due to an inefficient dose as we have
shown previously in pancreatic acini that 10 n
M
EGF
induced a maximal increase in EFG-receptor, p125
FAK
and
paxillin tyrosine phosphorylation [34].
The physiological importance of CrkII electrophoretic
mobility shifts induced by CCK became more relevant when
we demonstrated that it occurred in the intact animal.
Exogenous CCK markedly altered CrkII electrophoretic
mobility in the intact animal at a dose that has been
demonstrated to regulate the initiation phase of protein
synthesis in rat pancreas in vivo [35]. At present, our results
support the idea that CrkII is probably an important
intracellular mediator of CCK physiological actions in vivo,
although its role mediating the physiological effects of CCK
in the intact pancreas deserves future research.
In summary, results in this study support the conclusion
that activation of the G-protein coupled CCK
A
receptor

in vivo in rat pancreatic acini promotes the function of CrkII,
resulting in complex formation (CrkII–paxillin and CrkII–
p130
Cas
). Formation of both CrkII complexes in vivo is
dependent on the incubation time with CCK and follows
an opposite kinetic than the electrophoretic mobility shift
observed after CCK treatment. CrkII complex formation is
maximal when the majority of CrkII is present in the lower
band (at 1 min) and is disrupted when the majority of CrkII
is present in the upper slower migrating band (at 5 min).
Intracellular calcium probably plays a permissive role in the
CCK-induced electrophoretic mobility shift of CrkII, which
is also partially mediated by the Src family of tyrosine
kinases. The molecular nature of this mobility shift remains
unclear but the fact that it occurred in the intact animal
reinforces the idea of a relevant physiological role of CrkII in
mediating some CCK actions in the exocrine pancreas in vivo.
Acknowledgements
We thank Mercedes Go
´
mez for her technical assistance. J.A. Tapia was
supported by a Postdoctoral Grant from Direccion General de
Universidades (MECD), Spain. This work was supported by DGICYT
grant PB97-0370, and Junta de Extremadura, Consejeria de Sanidad
grants 02/10 and 03/63.
References
1. Jensen, R.T. (1994) Receptors on pancreatic acinar cells. In
Physiology of the Gastrointestinal Tract (Johnson, L.R., Jacobsen,
E.D., Christensen, J., Alpers, D.H. & Walsh, J.H., eds), pp. 1377–

1446. Raven Press, New York.
2. Williams, J.A. (2001) Intracellular signaling mechanisms activated
by cholecystokinin-regulating synthesis and secretion of digestive
enzymes in pancreatic acinar cells. Annu. Rev. Physiol. 63, 77–97.
4712 A. G. Andreolotti et al. (Eur. J. Biochem. 270) Ó FEBS 2003
3. Reeve, J.R. Jr, Eysselein, V.E., Ho, F.J., Chew, P., Vigna, S.R.,
Liddle, R.A. & Evans, C. (1994) Natural and synthetic CCK-58.
Novel reagents for studying cholecystokinin physiology. Ann. NY
Acad. Sci. 713, 11–21.
4. Moran, T.H. & Schwartz, G.J. (1994) Neurobiology of cholecys-
tokinin. Crit. Rev. Neurobiol. 9, 1–28.
5. Smith, J.P., Kramer, S.T. & Cheung, J.Y. (1991) Effects of cho-
lecystokinin on cytosolic calcium in human pancreatic cancer cells.
Regul. Peptides 36, 299–310.
6. Garcia, L.J. & Jensen, R.T. (1998) Tyrosine phosphorylation of
p125
FAK
and paxillin by cholecystokinin and other neuropeptides.
In Advances in Molecular and Cellular Endocrinology (LeRoith,
D., ed.), pp. 117–151. JAI Press, Stamford.
7. Ferris, H.A., Tapia, J.A., Garcı
´
a, L.J. & Jensen, R.T. (1999)
CCKA receptor activation stimulates p130Cas tyrosine phos-
phorylation, translocation and association with Crk in rat pan-
creatic acinar cells. Biochemistry 38, 1497–1508.
8. Tapia, J.A., Ferris, H.A., Jensen, R.T. & Garcia, L.J. (1999)
Cholecystokinin activates PYK2/CAKbeta by a phospholipase C-
dependent mechanism and its association with the mitogen-acti-
vated protein kinase signaling pathway in pancreatic acinar cells.

J. Biol. Chem. 274, 1261–1271.
9. Schafer, C., Ross, S.E., Bragado, M.J., Groblewski, G.E., Ernst,
S.A. & Williams, J.A. (1998) A role for the p38 mitogen-activated
protein kinase/Hsp 27 pathway in cholecystokinin-induced chan-
gesintheactincytoskeletoninratpancreaticacini.J. Biol. Chem.
273, 24173–24180.
10. Feller, S.M. (2001) Crk family adaptors-signalling complex for-
mation and biological roles. Oncogene 20, 6348–6371.
11. Matsuda, M., Tanaka, S., Nagata, S., Kojima, A., Kurata, T. &
Shibuya, M. (1992) Two species of human CRK cDNA encode
proteins with distinct biological activities. Mol. Cell Biol. 12, 3482–
3489.
12. Buday, L. (1999) Membrane-targeting of signalling molecules by
SH2/SH3 domain-containing adaptor proteins. Biochim. Biophys.
Acta 1422, 187–204.
13. ten Hoeve, J., Kaartinen, V., Fioretos, T., Haataja, L., Voncken,
J.W.,Heisterkamp,N.&Groffen,J.(1994)Cellularinteractions
of CRKL, and SH2-SH3 adaptor protein. Cancer Res. 54, 2563–
2567.
14. Nojima, Y., Morino, N., Mimura, T., Hamasaki, K., Furuya, H.,
Sakai,R.,Sato,T.,Tachibana,K.,Morimoto,C.&Yazaki,Y.
(1995) Integrin-mediated cell adhesion promotes tyrosine phos-
phorylation of p130Cas, a Src homology 3-containing molecule
having multiple Src homology 2-binding motifs. J. Biol. Chem.
270, 15398–15402.
15. Ribon, V. & Saltiel, A.R. (1996) Nerve growth factor stimulates
the tyrosine phosphorylation of endogenous Crk-II and augments
its association with p130Cas in PC-12 cells. J. Biol. Chem. 271,
7375–7380.
16. Sorokin, A., Reed, E., Nnkemere, N., Dulin, N.O. & Schlessinger,

J. (1998) Crk protein binds to PDGF receptor and insulin receptor
substrate-1 with different modulating effects on PDGF- and
insulin-dependent signaling pathways. Oncogene 16, 2425–2434.
17. Casamassima, A. & Rozengurt, E. (1997) Tyrosine phosphoryla-
tion of p130 (cas) by bombesin, lysophosphatidic acid, phorbol
esters, and platelet-derived growth factor. Signaling pathways and
formation of a p130 (cas)-Crk complex. J. Biol. Chem. 272, 9363–
9370.
18. Takahashi, T., Kawahara, Y., Taniguchi, T. & Yokoyama, M.
(1998) Tyrosine phosphorylation and association of p130Cas and
c-Crk II by ANG II in vascular smooth muscle cells. Am. J.
Physiol. 274, H1059–H1065.
19. Feller, S.M., Knudsen, B. & Hanafusa, H. (1994) c-Abl kinase
regulates the protein binding activity of c-Crk. EMBO J. 13, 2341–
2351.
20. Rosen, M.K., Yamazaki, T., Gish, G.D., Kay, C.M., Pawson, T.
& Kay, L.E. (1995) Direct demonstration of an intramolecular
SH2–phosphotyrosine interaction in the Crk protein. Nature 374,
477–479.
21. Hashimoto, Y., Katayama, H., Kiyokawa, E., Ota, S., Kurata, T.,
Gotoh, N., Otsuka, N., Shibata, M. & Matsuda, M. (1998) Phos-
phorylation of CrkII adaptor protein at tyrosine 221 by epidermal
growth factor receptor. J. Biol. Chem. 273, 17186–17191.
22. Blakesley, V.A., Beitner-Johnson, D., Van Brocklyn, J.R., Rani,
S., Shen-Orr, Z., Stannard, B.S., Spiegel, S. & LeRoith, D. (1997)
Sphingosine 1-phosphate stimulates tyrosine phosphorylation of
Crk. J. Biol. Chem. 272, 16211–16215.
23. Beitner-Johnson, D. & LeRoith, D. (1995) Insulin-like growth
factor-I stimulates tyrosine phosphorylation of endogenous c-Crk.
J. Biol. Chem. 270, 5187–5190.

24. Reedquist, K.A., Fukazawa, T., Panchamoorthy, G., Langdon,
W.Y., Shoelson, S.E., Druker, B.J. & Band, H. (1996) Stimulation
through the T cell receptor induces Cbl association with Crk
proteins and the guanine nucleotide exchange protein C3G.
J. Biol. Chem. 271, 8435–8442.
25. Ingham, R.J., Krebs, D.L., Barbazuk, S.M., Turck, C.W., Hirai,
H.,Matsuda,M.&Gold,M.R.(1996)Bcellantigenreceptor
signaling induces the formation of complexes containing the Crk
adapter proteins. J. Biol. Chem. 271, 32306–32314.
26. Peikin, S.R., Rottman, A.J., Batzri, S. & Gardner, J.D. (1978)
Kinetics of amylase release by dispersed acini prepared from
guinea pig pancreas. Am. J. Physiol. 235, 743–749.
27. Garcia,L.J.,Pradhan,T.K.,Weber,H.C.,Moody,T.W.&Jen-
sen, R.T. (1997) The gastrin-releasing peptide receptor is differ-
entially coupled to adenylate cyclase and phospholipase C in
different tissues. Biochim. Biophys. Acta 1356, 343–354.
28. Hanke,J.H.,Gardner,J.P.,Dow,R.L.,Changelian,P.S.,Bris-
sette, W.H., Weringer, E.J., Pollok, B.A. & Connelly, P.A. (1996)
Discovery of a novel, potent, and Src family-selective tyrosine
kinase inhibitor. Study of Lck- and FynT-dependent T cell acti-
vation. J. Biol. Chem. 271, 695–701.
29. Touyz, R.M., Wu, X.H., He, G., Park, J.B., Chen, X., Vacher, J.,
Rajapurohitam, V. & Schiffrin, E.L. (2001) Role of c-Src in the
regulation of vascular contraction and Ca2+ signaling by
angiotensin II in human vascular smooth muscle cells. J. Hyper-
tens. 19, 441–449.
30. Nakashima, N., Rose, D.W., Xiao, S., Egawa, K., Martin, S.S.,
Haruta, T., Saltiel, A.R. & Olefsky, J.M. (1999) The functional
role of CrkII in actin cytoskeleton organization and mitogenesis.
J. Biol. Chem. 274, 3001.

31. Endo, A., Nagashima, K.I., Kurose, H., Mochizuki, S., Mat-
suda, M. & Mochizuki, N. (2002) Sphingosine-1-phosphate
induces membrane ruffling and increases motility of human
umbilical vein endothelial cells via vascular endothelial growth
factor receptor and CrkII. J. Biol. Chem. 277, 23747–23754.
32. Tsunoda, Y., Yoshida, H., Africa, L., Steil, G.J. & Owyang, C.
(1996) Src kinase pathways in extracellular Ca (2+)-dependent
pancreatic enzyme secretion. Biochem. Bioph. Res. Co. 227,
876–884.
33. Brown, M.T. & Cooper, J.A. (1996) Regulation, substrates and
functions of src. Biochim. Biophys. Acta 1287, 121–149.
34. Tapia, J.A., Camello, C., Jensen, R.T. & Garcı
´
a, L.J. (1999) EGF
stimulates tyrosine phosphorylation of focal adhesion kinase
(p125FAk) and paxillin in rat pancreatic acini by a phospholipase
C-independent process that depends on PI3-kinase, the small
GTP-binding protein, p21rho, and the integrity of the actin
cytoskeleton. Biochim. Biophys. Acta 1448, 486–499.
35. Bragado, M.J., Tashiro, M. & Williams, J.A. (2000) Regulation of
the initiation of pancreatic digestive enzyme protein synthesis by
cholecystokinin in rat pancreas in vivo. Gastroenterology 119,
1731–1739.
Ó FEBS 2003 Regulation of Crk by cholecystokinin (Eur. J. Biochem. 270) 4713