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: 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) 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. 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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 . Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini Formation of CrkII protein complexes Alberto G 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