In unstimulated acini, the majority of CrkII was present in the lower band which shows higher electrophoretic mobility... In acini stimulated with 10 nMCCK-8 themajority of CrkII is shif
Trang 1Cholecystokinin rapidly stimulates CrkII function in vivo in rat
pancreatic acini
Formation of CrkII–protein complexes
Alberto G Andreolotti1, Maria J Bragado2, Jose A Tapia3, Robert T Jensen3and Luis J Garcia-Marin1
1 Departamento de Fisiologia, Universidad de Extremadura, Caceres, Spain; 2 Departamento de Bioquimica, Biologia Molecular y Genetica, Universidad de Extremadura, Caceres, Spain;3Digestive 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 CCKreceptor
activa-tion signals through CrkII and forms complexes with
tyro-sine-phosphorylated proteins in rat pancreatic acini We
demonstrated that CCKpromoted the transient formation
of CrkII–paxillin and CrkII–p130Cascomplexes with
maxi-mal effect at 1 min Additionally, CCKdecreased 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 CCKactions
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, CCKhas 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 CCKAreceptor, a member
of the G-protein-coupled receptor (GPCR) superfamily
[1,2] Intracellular pathways of CCKA receptor activation
have been investigated extensively in pancreatic acini [1,2]
In these cells, we reported recently that CCKstimulates
tyrosine phosphorylation of several proteins related with
fiber stress formation such as the focal adhesion kinase
p125FAK, paxillin [6], p130Cas[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-CrkII has an additional SH3 [11] The SH2 domain of Crk binds to phosphotyrosine-containing proteins such as p130Cas, 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: ljgarcia@unex.es
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)
Trang 2binding 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 CCKA, 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 CCKA
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 CCKtreatment and its possible
contribution to the regulation of CrkII function Moreover,
we have investigated whether CCKintracellular 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-p130CasmAb,
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 Animalsof 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)1of
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 mMTris/HCl, pH 7.5, 150 mMNaCl, 1% Triton X-100,
1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA,
2.5 lgÆmL)1 aprotinin, 2.5 lgÆmL)1 leupeptin, 1 mM
phenylmethanesulfonyl fluoride, and 0.2 mMNaVO The
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 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 glu-cose, 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 with 100% (v/v)
O2as the gas phase
Immunoprecipitation Pancreatic acini isolated from one rat were preincubated with standard incubation solution for 3 h at 37C Acini were then incubated with agonists at concentrations and times indicated, washed with phosphate buffered saline (NaCl/Pi) with 0.2 mMNa3VO4and sonicated (5 s at 4C)
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 4C 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/Pi and analysed by SDS/ 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)1anti-p130Casor 0.025 lgÆmL)1anti-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 p130Casand 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
Trang 3(Fig 1A, lane 1) In acini stimulated with 10 nMCCK-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 CCKof the interaction between
the adapter protein, CrkII and paxillin Data shown in
Fig 1C demonstrated that 10 nMCCK-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
CCKregulates the protein complex formation of CrkII with
other proteins such as p130Cas 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–p130Cas
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-p130Cas
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.
Trang 4(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 nM A weak increase was detected after 5 min
incubation with 0.01 nMCCK-8 and the maximum effect
was observed at 100 nMCCK-8
CCK induces the electrophoretic mobility shift of CrkII
in the intact animal
To study whether CCKhas a similar effect in the
electrophoretic mobility of CrkII in vivo, we injected rats
with 15 lgÆkg)1of 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
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 lM), an agonist at the high-affinity state
of the CCKreceptor 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 nMEGF 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
CCKreceptor 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 mM 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)1of 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.
Trang 5calcium 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 lMTPA 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 CCKstimulates 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 CCKwas
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 lM 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 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 lMPP2 (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 ) or BAPTA/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 CCKtreatment
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.
Trang 647.0 ± 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 lM),
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 CCKrapidly
promotes the formation of CrkII–protein complexes,
CrkII–paxillin and CrkII–p130Cas, in rat pancreatic acini
Recently, it has been demonstrated that CCKactivates
different intracellular pathways in rat pancreatic acinar cells
[2] We have demonstrated previously in these cells that
CCKis a potent activator of the tyrosine phosphorylation
of different proteins such as p130Cas [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 CCKinduced 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 CCKin 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 CCKmay occur in rat pancreatic acini: (a) CCKstimulates the rapid tyrosine phosphorylation of proteins such as p130Casand paxillin [6,7]; (b) immediately afterwards, CCKpromotes the rapid formation of CrkII– p130Casand 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.
Trang 7incubation 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
p130Casor 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–p130Cas 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
CCKand was dependent on the CCKconcentration At
present, little is known about the intracellular pathways
coupling receptor activation to CrkII, especially in the case
of the GPCRs such as the CCKA receptor It is well
established that the CCKconcentration range that regulates
CrkII also causes activation of CCKAreceptor [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
p125FAK, PYK2, paxillin and p130Castyrosine
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 CCKto
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 CCKstimulation Thus, our
results support the conclusion that CCKinduces 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 CCKA [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 p130Cas, p125FAKand 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 nM EGF induced a maximal increase in EFG-receptor, p125FAKand paxillin tyrosine phosphorylation [34]
The physiological importance of CrkII electrophoretic mobility shifts induced by CCKbecame more relevant when
we demonstrated that it occurred in the intact animal Exogenous CCKmarkedly 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 CCKphysiological 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 CCKA receptor
in vivoin rat pancreatic acini promotes the function of CrkII, resulting in complex formation (CrkII–paxillin and CrkII– p130Cas) Formation of both CrkII complexes in vivo is dependent on the incubation time with CCKand follows
an opposite kinetic than the electrophoretic mobility shift observed after CCKtreatment 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 CCKactions 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.
Trang 8Liddle, 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-ges in the actin cytoskeleton in rat pancreatic acini 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 CRKcDNA 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) Cellular interactions
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.
& 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) B cell antigen receptor 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.