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Cell Growth & Differentiation Vol. 13, 375-385, August 2002
© 2002 American Association for Cancer Research

c-Jun NH2-terminal Kinase Pathway in Growth-promoting Effect of the G Protein-coupled Receptor Cholecystokinin B Receptor

A Protein Kinase C/Src-dependent-Mechanism1

Stephanie Dehez, Christiane Bierkamp, Aline Kowalski-Chauvel, Laurence Daulhac, Chantal Escrieut, Christiane Susini, Lucien Pradayrol, Daniel Fourmy and Catherine Seva2

INSERM U.531, Groupe de Recherche de Biologie et Pathologie Digestives, Institut Louis Bugnard, Bat. L3, 31403 Toulouse, France


    Abstract
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
The proliferative effects of gastrin on normal and malignant gastrointestinal tissues have been shown to be mediated by a G protein-coupled receptor (GPCR), the cholecystokinin B receptor. The c-Jun NH2-terminal kinase (JNK) pathway has been implicated in the regulation of mitogenesis by growth factors or cytokines. However, the contribution of this signaling cascade to the proliferative effects of GPCR remains largely unknown. Here, we show that cholecystokinin B receptor occupancy by gastrin leads to the activation of the JNK pathway. The mechanism involves certain protein kinase C isoforms and Src family kinases other than p60Src. The complex p130Cas/CrkII, known to be involved in JNK activation, is also activated in response to gastrin by a protein kinase C- and Src-dependent mechanism. However, gastrin-induced CrkII and JNK pathways are independent. Using a dominant negative mutant of c-Jun, we blocked the ability of gastrin to induce DNA synthesis, demonstrating a major role of the JNK pathway in the growth-promoting effect of a GPCR agonist.


    Introduction
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
The peptide hormone gastrin is a potent stimulant of gastric acid secretion and a growth factor for normal and malignant gastrointestinal tissues. The effects of gastrin are mediated through a GPCR,3 the CCKBR (also known as CCK-2). The trophic action of gastrin has been observed on numerous cancer cell lines derived from colon, stomach, and pancreas (1, 2, 3, 4) . In addition, gastrin and its receptors are often up-regulated in human colon cancers and pancreatic adenocarcinomas (5, 6, 7) . More recent studies on transgenic mice overexpressing gastrin or its receptors have shown that this hormone is involved in gastric and pancreatic tumor formation (8) .

The MAPK pathways comprise the ERK pathway, the stress-activated protein kinase/JNK pathway, and the p38-MAPK pathway. The role of the ERK pathway in the regulation of cellular growth is well documented. This signaling cascade has been shown to be implicated in the proliferative effects induced by tyrosine kinases receptors, cytokines receptors, and GPCRs, including the CCKBR (9) .

The two other MAPK cascades, JNK and p38-MAPK, were initially identified as two pathways mediating cellular stress induced by UV radiation, proinflammatory cytokines, heat, and osmotic shocks. More recent studies have shown that JNK and p38-MAPK are also involved in the regulation of cell proliferation by growth factors, such as fibroblast growth factor 2, EGF, hepatocyte growth factor (10, 11, 12) , or cytokines such as TNF-{alpha}, prolactin, granulocyte colony-stimulating factor, IL-2, and IL-7 (12, 13, 14, 15) . Recently, we have demonstrated that the p38-MAPK pathway can also play a critical role in the growth-promoting effects of GPCR, such as the CCKBR (16) . The contribution of the JNK pathway to the proliferative effects mediated by GPCR remains largely unknown.

Here, we have investigated whether stimulation of the human CCKBR expressed in CHO-CCKB cells activates JNK, and we have analyzed the role of this signaling pathway in the growth-promoting effect of gastrin. We have also examined the molecular intermediates that may connect the CCKBR to the JNK pathway.

Because we have reported previously in CHO-CCKB cells that ERK activation by gastrin is mediated by a signaling cascade including the phosphorylation of Shc proteins by Src-like tyrosine kinases (17) , we therefore analyzed the possibility that kinases of this family could serve as intermediates between the CCKBR and the activation of the JNK pathway.

Several recent reports have shown the activation of the JNK pathway by two SH2/SH3 adaptor proteins, the proto-oncogene CrkII and a putative Src substrate, p130Cas (18, 19, 20, 21, 22) . The p130Cas/CrkII complex, interacting directly with JNK1 through the NH2-terminal SH3 domain of CrkII, could serve as a scaffolding structure involved in the specific activation of JNK1 (23) . The activation of such a complex by the CCKBR has never been reported. Here, we have examined whether the human CCKBR induces the tyrosine phosphorylation of p130Cas by Src family kinases, its association with the SH2 domain of CrkII, and whether this complex would be involved in gastrin-induced JNK1 activation.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Gastrin Stimulates the Phosphorylation and Activity of JNK.
The members of the MAPK family have been previously shown to be activated by phosphorylation on tyrosine and threonine residues. To determine whether gastrin regulates the JNK pathway, we therefore examined whether JNK could be phosphorylated in response to this peptide.

CHO DG44 cells stably expressing the human CCKBR (CHO-CCKB cells) were treated with 10 nM gastrin for varying lengths of time. Cell lysates were analyzed by Western blot using an antibody against the phosphorylated form of JNK. We observed in Fig. 1ACitation a rapid increase of JNK phosphorylation by gastrin detectable at 1 min and maximal at 3 min (5.8 ± 0.9-fold).



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Fig. 1. Gastrin stimulates the phosphorylation and activity of JNK. CHO cells expressing the human CCKBR were stimulated for the times indicated with 10 nM gastrin. A, whole cell lysates were immunoblotted with an anti-phospho-Thr183 and -Tyr185 JNK antibody. B, after IP with an anti-JNK1 antibody, JNK activity using GST-c-Jun-(1–79) as an exogenous substrate was assayed as described in "Materials and Methods." As shown (bottom panels), comparable amounts of JNK1 proteins were detected in whole cell lysates or immunoprecipitates of all of the above-mentioned experiments. The autoradiograms were densitometrically analyzed, and the data from at least three separate autoradiograms were plotted as fold stimulation and presented as means ± SE.

 
We then analyzed the ability of gastrin to induce the kinase activity of JNK. After stimulation of the cells, in vitro JNK assays were performed on anti-JNK1 immunoprecipitates using GST-c-Jun-(1–79) as a substrate. Fig. 1BCitation shows that the kinetics of JNK activation in response to gastrin is biphasic. The maximal activation of the early phase (4.4 ± 0.8-fold) was observed at 5 min and decreased to basal level at 15 min. An early phase of JNK activation has also been observed on fibroblasts in response to stimuli that induce cell proliferation, including serum (24) . Gastrin also induced a second increase of JNK activation, maximal at 60 min, that remained elevated for at least 2 h.

As expected, nontransfected cells did not display gastrin-stimulated JNK activation (data not shown).

Role of the JNK Pathway in Gastrin-induced DNA Synthesis.
We have reported previously that gastrin stimulates the growth of CHO cells expressing the human CCKBR (16 , 17) . To analyze the role of the JNK pathway in gastrin-induced cell proliferation, we stably transfected a dominant negative mutant of c-Jun (TAM67) lacking the NH2-terminal transactivation domain or its empty vector pCMV in CHO-CCKB cells. As expected, Western blotting with a specific anti-c-Jun antibody, which recognizes the DNA-binding site of c-Jun, revealed overexpression of a Mr 29,000 protein corresponding to the mutant in three clones selected (TAM67 clones 5, 9, and 10; Fig. 2ACitation ). We verified by binding studies with 125I-radiolabeled CCK8 that the expression of the CCKBR was not affected by the transfection of the plasmids. Bmax values were 2.87 ± 0.2, 3.40 ± 0.37, 2.81 ± 0.36, 2.55 ± 0.2, and 3.32 ± 0.23 pmol/106 cells, respectively, for CHO-CCKB; TAM67 clones 5, 9, and 10; and CHO-CCKB transfected with the empty vector pCMV.



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Fig. 2. Role of the JNK pathway in gastrin-induced DNA synthesis. A, equal amounts of cellular proteins from CHO-CCKB cells, CHO-CCKB cells overexpressing TAM67 (clones 5, 9, and 10), and CHO-CCKB cells transfected with the empty vector pCMV (pCMV) were immunoblotted with an anti-c-Jun antibody directed against the DNA-binding region of the transcription factor. B, after a 30-min preincubation with or without 10 µM SP-600125 (SP), CHO cells expressing the human CCKBR were stimulated for 5 min with 10 nM gastrin. After IP with an anti-JNK1 antibody, JNK activity using GST-c-Jun-(1–79) as an exogenous substrate was assayed as described in "Materials and Methods." C, after a 30-min preincubation with or without 10 µM SP-600125 (SP), CHO cells expressing the human CCKBR were stimulated for 5 min with 10 nM gastrin. Whole cell lysates were immunoblotted with an anti-phospho-Thr202 and -Tyr204 ERK antibody. Comparable amounts of proteins were detected in immunoprecipitates or whole cell lysates of all of the above-mentioned experiments (data not shown). D, [3H]thymidine incorporation was measured, as described in "Materials and Methods," in CHO-CCKB cells, CHO-CCKB cells transfected with the empty vector pCMV (pCMV), CHO-CCKB cells overexpressing TAM67 (clones 5, 9, and 10), and CHO-CCKB cells pretreated with 10 µM SP-600125 (SP). The results are means ± SE of three experiences performed in triplicate.

 
Both basal and gastrin-induced [3H]thymidine incorporation were repressed in CHO-CCKB cells overexpressing TAM67 (clones 5, 9, and 10) compared with control cells (CHO-CCKB and CHO-CCKB transfected with the empty vector pCMV; Fig. 2DCitation ). Similar results were obtained when cells were pretreated with 10 µM of the specific JNK inhibitor SP-600125 (Fig. 2D)Citation . At this concentration, the activation of JNK1 by gastrin was totally blocked (Fig. 2B)Citation , whereas gastrin-induced ERK activity was not affected (Fig. 2C)Citation .

Several publications support a role for the JNK pathway in transducing the mitogenic signal of growth factors that bind tyrosine kinase receptors or cytokine receptors. Bost et al. (11) have previously reported that inhibition of the JNK pathway by JNK antisense oligonucleotide or a dominant negative c-Jun mutant abolished the EGF-stimulated proliferation of lung carcinoma cells. Proliferative activities of TNF-{alpha} or hepatocyte growth factor (12) in rat hepatocytes have also been shown to be blocked by dominant negative mutants of JNK or c-Jun. Similarly, a recent study has reported the involvement of JNK in prolactin-induced cell proliferation (13) . The contribution of the JNK pathway to the proliferative effects mediated by GPCR remains largely unknown. In this study, using a well-characterized dominant negative mutant of c-Jun, we completely blocked the ability of gastrin to induce DNA synthesis in CHO cells transfected with the CCKBR. These results demonstrate that the JNK pathway can play a central role in the growth-promoting effect of a GPCR agonist.

PKC and Src Family Tyrosine Kinases Are Required for Gastrin-induced JNK Activation.
Gastrin-dependent activation of the CCKBR has been shown to induce the rapid hydrolysis of phosphatidylinositol-biphosphates by phospholipase C to generate inositol triphosphates and diacyl-glycerol, which respectively mobilize intracellular calcium and stimulate PKC (25 , 26) . In addition, we have reported previously (27) that gastrin can activate the ERK pathway by a PKC-dependent mechanism. We therefore investigated whether PKC contributes to the activation of JNK.

In Fig. 3, A and BCitation , direct activation of phorbol ester-sensitive PKC isoforms by 100 nM PMA for 20 min induced a large increase in JNK activation. The magnitude of stimulation was not significantly different from that observed with gastrin. Pretreatment of the cells with a potent PKC inhibitor, GF109203X, prevented PMA- and gastrin-stimulated JNK phosphorylation (Fig. 3A)Citation and activation (Fig. 3B)Citation , indicating that PKC is involved in gastrin-mediated JNK activation. Similar results were observed with another PKC inhibitor, Gö6983, (Fig. 3C)Citation , confirming those obtained with GF109203X. In addition, we show in Fig. 3DCitation that both PKC inhibitors have no effect on JNK activation by anisomycin, which is known to enhance JNK activity through a PKC-independent mechanism.



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Fig. 3. PKC and Src family tyrosine kinases are required for JNK activation induced by gastrin. After a 30-min preincubation with (+) or without (-) 5 µM GF109203X (GF), 1 µM Gö6983, or 10 µM PP2, CHO cells expressing the human CCKBR were stimulated for 3 min (A, C, and E) or 5 min (B and F) with 10 nM gastrin, for 20 min with 100 nM PMA (A and B), or for 30 min with 10 µg/ml anisomycin (D). A, C, D, and E, whole cell lysates were immunoblotted with an anti-phospho-Thr183 and -Tyr185 JNK antibody. B and F, after IP with an anti-JNK1 antibody, JNK activity using GST-c-Jun-(1–79) as an exogenous substrate was assayed as described in "Materials and Methods." Comparable amounts of proteins were detected in whole cell lysates or immunoprecipitates of all of the above-mentioned experiments (data not shown). The autoradiograms were densitometrically analyzed, and the data from at least three separate autoradiograms were plotted as fold stimulation and presented as means ± SE.

 
We have reported previously (17 , 28) that gastrin can stimulate p60Src kinase activity in different cellular models, including CHO cells transfected with the CCKBR. In this cellular model, gastrin-induced p60Src kinase activity is totally abolished by the PKC inhibitor GF109203X (16) . In this study, we have analyzed whether Src family tyrosine kinases are involved in gastrin-induced activation of JNK. To determine the contribution of Src family kinases to the activation of JNK by gastrin, we measured the phosphorylation and activity of this MAPK in CCKBR-expressing CHO cells pretreated with a specific Src-like inhibitor, PP2. In the presence of 10 µM PP2, which totally abolished the gastrin-stimulated Src kinase activity (16) , the phosphorylation and kinase activity of JNK were completely inhibited (Fig. 3, E and F)Citation . This indicates that gastrin regulates the JNK pathway through a Src-like dependent mechanism.

We then used a different approach to block Src family kinases. A negative regulatory protein of Src, CSK, which inactivates Src kinases by phosphorylation (29) , was stably transfected in CHO cells expressing the CCKBR. Overexpression of CSK in these cells completely blocked gastrin-induced p60Src kinase activity (17) . Here, we used a pool of CSK-transfected cells showing no alteration of the expression of the receptor (Bmax, 2.53 ± 0.2 pmol/106 cells) and demonstrated that both the phosphorylation of JNK (Fig. 4A)Citation and the activity of the enzyme (Fig. 4B)Citation in response to gastrin were abolished, confirming the involvement of Src family tyrosine kinases in the activation of the JNK pathway by gastrin. These results and the inhibition of cell proliferation obtained when the JNK pathway is blocked by a c-jun mutant (Fig. 2)Citation are in agreement with our previous observations showing that gastrin-induced cell growth is blocked in CHO-CCKB cells stably transfected with CSK.



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Fig. 4. Role of Src family tyrosine kinases in gastrin-induced JNK activation. CHO cells expressing CCKBR (CHO-CCKB) and CHO-CCKB cells stably transfected with the CSK or the p60Src kinase-inactive mutant (SRC- clones 1 and 2) were stimulated for the time indicated with 10 nM gastrin. A, C, and E, whole cell lysates were immunoblotted with an anti-phospho-Thr183 and -Tyr185 JNK antibody or an anti-p60Src antibody as indicated. B and F, after IP with an anti-JNK1 antibody, JNK activity was measured using GST-c-Jun-(1–79) as an exogenous substrate as described in "Materials and Methods." D, after IP with an anti-p60Src antibody, Src kinase activity was assayed using enolase as an exogenous substrate as described in "Materials and Methods." Comparable amounts of proteins were detected in whole cell lysates or immunoprecipitates of all of the above-mentioned experiments (data not shown).

 
To examine the role of p60Src specifically, we overexpressed, in CHO-CCKB cells, a kinase-inactive mutant of p60Src (Lys295->Met) that has previously been used to specifically block p60Src (30) . Several clones were obtained (two clones are shown Fig. 4CCitation ) with no alteration of the CCKBR expression (Bmax, 4.3 ± 0.2 and 2.81 ± 0.2 pmol/106 cells respectively). p60Src kinase activation by gastrin was measured in anti-p60Src immunoprecipitates using enolase as a substrate (Fig. 4D)Citation . In SRC- cells, gastrin-induced p60Src activity was inhibited. In contrast, the phosphorylation and activity of JNK stimulated by gastrin were not affected by overexpression of the p60Src mutant (Fig. 4, E and F)Citation .

Expression of the v-Src oncoprotein has been shown to strongly stimulate JNK in different cell types such as fibroblasts, HeLa cells, or breast cancer cells (18 , 31 , 32) . In addition, Src-like kinases have been found to be important mediators in the activation of the JNK pathway by certain tyrosine kinase receptors including Kit or nerve growth factor receptors (33 , 34) . Little is known about the contribution of Src family kinases to the signaling pathways linking GPCR to JNK activation. Recent studies have demonstrated that JNK activation by G protein {alpha} subunits (G{alpha}q11 and G{alpha}12) can involve Src family protein kinases (35 , 36) . Here, we show that a GPCR known to be coupled to G{alpha}q (37) activates the JNK pathway through a mechanism that involves a Src family kinase other than p60Src. Among the other ubiquitous Src-like kinases, Fyn is the only one that has been shown to be involved in the activation of the JNK pathway (38) . However, in the CHO-CCKB cells, we did not observe the activation of this tyrosine kinase in response to gastrin (data not shown). Additional studies will be required to identify the Src family kinase stimulated by gastrin that may be responsible for the activation of the JNK pathway.

Gastrin Stimulates the Phosphorylation of p130Cas and Its Association with CrkII.
We then analyzed the role of the p130Cas/CrkII complex that was shown to behave as a scaffolding structure involved in JNK1 activation (23) .

We first investigated whether gastrin was capable of inducing phosphorylation of the adaptor protein p130Cas. Cells were treated with gastrin for varying times. Lysates were immunoprecipitated with an anti-phosphotyrosine antibody and analyzed by Western blotting with anti-p130Cas antibodies. A transient increase in tyrosine phosphorylation of p130Cas was observed in response to gastrin (Fig. 5A)Citation with a maximal stimulation (3.67 ± 0.2-fold) between 1 and 3 min.



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Fig. 5. PKC and Src family tyrosine kinases are required for the p130Cas/CrkII complex formation induced by gastrin. A and B, CHO cells expressing the human CCKBR were stimulated for the times indicated with 10 nM gastrin. C-F, after a 30-min preincubation with (+) or without (-) 5 µM GF109203X (GF) or 10 µM PP2, CHO cells expressing the human CCKBR were stimulated for 1 min with 10 nM gastrin or for 20 min with 100 nM PMA. Proteins from cell lysates were prepared and immunoprecipitated (IP) with anti-phosphotyrosine (P-Tyr; A, C, and E) or anti-CrkII (B, D, and F) antibodies and then subjected to p130Cas immunoblotting. Comparable amounts of proteins were detected in immunoprecipitates or whole cell lysates of all of the above-mentioned experiments (B, D, and F and data not shown). The autoradiograms were densitometrically analyzed, and the data from at least three separate autoradiograms were plotted as fold stimulation and presented as means ± SE.

 
We also determined the effect of gastrin on the association between p130Cas and CrkII. IP of p130Cas from cell extracts followed by Western blot analysis for CrkII (Fig. 5B)Citation revealed a time-dependent association of p130Cas with CrkII in response to gastrin. A maximal association was observed at 1 min (3.5 ± 0.7-fold).

PKC and the Tyrosine Kinase p60Src Are Required for p130Cas/CrkII Complex Formation Induced by Gastrin.
To assess the role of phorbol ester-sensitive PKC isoforms in the formation of the p130Cas/CrkII complex, we treated the cells with 100 nM PMA for 20 min. Stimulations of both the phosphorylation of p130Cas and its association with CrkII were comparable with those obtained with gastrin and were abolished by the PKC inhibitor GF109203X. Similar to what we observed for the stimulation of JNK by gastrin, the p130Cas/CrkII complex formation and phosphorylation induced by this peptide were also inhibited by GF109203X (Fig. 5, C and D)Citation .

To determine the role of Src family tyrosine kinases in p130Cas/CrkII complex formation by gastrin, we used three different approaches: (a) pretreatment of cells with the specific Src-like inhibitor PP2 (Fig. 5, E and F)Citation ; (b) overexpression of the negative regulator of Src, CSK (Fig. 6, A and B)Citation ; and (c) overexpression of a kinase-inactive mutant of p60Src (Fig. 6, C and D)Citation . In all these experiments, gastrin failed to stimulate the tyrosine phosphorylation of p130Cas and the association between p130Cas and CrkII, indicating the crucial role played by a Src family kinase, namely, p60Src, in the activation of the p130Cas/CrkII complex by gastrin.



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Fig. 6. Role of p60Src in the p130Cas/CrkII complex formation induced by gastrin. CHO cells expressing the CCKBR (CHO-CCKB) and CHO-CCKB cells stably transfected with the CSK or the p60Src kinase-inactive mutant (SRC- clone 2) were stimulated for the time indicated with 10 nM gastrin. Proteins from cell lysates were prepared and immunoprecipitated (IP) with anti-phosphotyrosine (P-Tyr; A and C) or anti-CrkII (B and D) antibodies and then subjected to p130Cas immunoblotting. Comparable amounts of proteins were detected in whole cell lysates or immunoprecipitates of all of the above-mentioned experiments (data not shown).

 
Gastrin Stimulates JNK Activation through a CrkII-independent Mechanism.
To determine whether CrkII was involved in gastrin-induced JNK activation, we stably transfected in CHO-CCKB cells a SH3 dominant negative mutant of CrkII unable to bind its effectors (18) . Here, we used a pool of cells overexpressing this mutant (Fig. 7A)Citation , and we demonstrated that neither the phosphorylation of JNK (Fig. 7B)Citation nor the activity of the enzyme (Fig. 7C)Citation in response to gastrin was attenuated, indicating that gastrin activates the JNK pathway through a mechanism independent of CrkII.



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Fig. 7. Gastrin stimulates JNK activation through a CrkII-independent mechanism. CHO cells expressing the CCKBR stably transfected with dominant negative mutant of CrkII (CRK-) or the empty vector (CHO-CCKB) were stimulated for the times indicated with 10 nM gastrin. A and B, whole cell lysates were immunoblotted with an anti-CrkII (A) or anti-phospho-Thr183 and -Tyr185 JNK antibody (B). C, after IP with an anti-JNK1 antibody, JNK activity using GST-c-Jun-(1–79) as an exogenous substrate was assayed as described in "Materials and Methods." Comparable amounts of proteins were detected in whole cell lysates or immunoprecipitates of all of the above-mentioned experiments (data not shown).

 
To verify that the dominant negative mutant of CrkII was functional, we tested its ability to reverse a biological effect dependent on this adaptor protein. There is now good evidence that Crk is an important regulator of cell morphology, leading to changes involved in cell migration (39) . We therefore analyzed the effects of the CrkII mutant on morphological modifications induced by gastrin. Strong changes of cellular morphology were observed after 24 h of treatment with gastrin (Fig. 8, B and D)Citation compared with control cells (Fig. 8, A and C)Citation . Gastrin induced cell elongation and projection of long extensions (arrows in Fig. 8, B and CCitation ). Nonstimulated cells, on the other hand, were two to three times shorter (arrows in Fig. 8, A and CCitation ). In Fig. 8, E and FCitation , CHO-CCKB cells were transiently transfected with a plasmid encoding the dominant negative mutant of CrkII in combination with an EYFP coding plasmid and stimulated with gastrin. Proper expression of the mutant protein was verified by performing anti-CrkII immunofluorescence staining and analyzing CrkII-positive cells (arrows in Fig. 8ECitation ) for EYFP fluorescence (arrows in Fig. 8FCitation ). We observed that the CrkII mutant reversed the morphological changes mediated by gastrin (arrows in Figs. 8, E and FCitation ). In Fig. 8GCitation , cells transiently transfected with increasing concentrations of CrkII mutant together with the EYFP vector were scored for their ability to generate long extensions as described in "Materials and Methods." In response to gastrin, 80% of the cells showed an elongated phenotype. A dose-dependent decrease in the percentage of elongated cells (50% to 33%) was observed when increasing concentrations of CrkII mutant plasmid were transfected (0.5–1.5 µg).



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Fig. 8. Transient transfection with Crk mutant reverses morphological changes induced by gastrin. A and B, CHO cells expressing CCKBR (CHO-CCKB) were grown on culture dishes, serum-starved for 18 h (2% FCS), and then incubated for 24 h in the absence (A) or in presence (B) of 10 nM gastrin. Cells were photographed on an inverted phase-contrast microscope. Bar (A and B), 50 µm. C-F, cells were seeded on coverslips and transiently transfected with the EYFP coding plasmid alone (C and D) or together with 1 µg of the dominant negative mutant of CrkII (E and F). After serum starvation, cells were incubated for 24 h in the absence (C) or presence (D-F) of 10 nM gastrin. Cells were fixed and analyzed for EYFP fluorescence (C and D). Proper expression of the mutant protein was verified by performing anti-CrkII immunofluorescence staining and analyzing CrkII-positive cells for EYFP fluorescence (E and F). Bar (C and D), 60 µm; bar (E and F), 120 µm. G, cells were transiently transfected with either 0 (control), 0.5, 1.0, or 1.5 µg of dominant negative mutant of CrkII-encoding plasmid (Crk-) together with 0.5 µg of EYFP-encoding plasmid and stimulated with 10 nM gastrin for 24 h. Cells were scored for the ability to generate long extensions as described in "Materials and Methods." Arrows point toward elongated cells in B and D and toward short cells in A, C, E, and F.

 
Other mechanisms have been described in the literature for JNK activation. In particular, the adaptor molecules Shc and Grb2 have been shown to be involved in the activation of the JNK pathway by the EGF receptor (40 , 41) . In CHO cells expressing the CCKBR, we have previously demonstrated the role of the Shc/Grb2 complex upstream of the gastrin-mediated ERK activation using a stably expressed dominant inhibitory mutant of Shc (17) . Therefore, we have examined the ability of this mutant to block gastrin-induced JNK activation. We did not observe an inhibition of the JNK activity stimulated by gastrin (data not shown), indicating that Shc does not participate in the regulation of the JNK pathway by this peptide. It has been proposed that multidomain scaffold proteins that interact with the different components of the JNK pathway (MAPK/ERK kinase kinase, MAPK kinase, and JNK), such as the JNK-interacting proteins (42, 43, 44) , ß-arrestin, or axin, regulate JNK activation in response to different stimuli. Whether one of these scaffold factors plays a role in gastrin-induced JNK activation remains to be determined.

Gastrin Stimulates JNK Activation through a p85/p110-PI 3K-independent Mechanism.
Several groups have reported that p85/p110-PI3K can play an essential role in the activation of the JNK pathway (33 , 45, 46, 47, 48, 49) . Because we have previously shown in CHO cells expressing the CCKBR that gastrin stimulates p85/p110-PI3K (17) , we therefore investigated the role of this kinase in gastrin-induced JNK activation using two different PI3K inhibitors, wortmannin and LY294002. Pretreatment of the cells with increasing concentrations of inhibitor did not attenuate the phosphorylation of JNK stimulated by gastrin (Fig. 9, A and B)Citation . Both inhibitors (used at 10 nM and 10 µM, respectively) totally abolished gastrin-induced PI3K activation (data not shown).



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Fig. 9. Gastrin stimulates JNK activation through a PI3K-independent mechanism. After a 30-min preincubation with or without the indicated concentrations of wortmannin (A) or LY 294002 (B), CHO cells expressing the human CCKBR were stimulated for 3 min with 10 nM gastrin. Whole cell lysates were immunoblotted with an anti-phospho-Thr183 and -Tyr185 JNK antibody. Comparable amounts of proteins were detected in whole cell lysates of all of the above-mentioned experiments (data not shown).

 
Numerous recent reports have shown that PI3K can be involved in the activation of JNK by growth factors such as EGF (45) and platelet-derived growth factor (46) , cytokines such as TNF-{alpha} (47) and IL-1ß (48) , ligands binding to GPCRs such as cannabinoids (49) , or the ß{gamma} subunits of the heterotrimeric G proteins (50) . In contrast to what was observed in these studies, PI3K is not required for regulation of the JNK pathway by gastrin in CHO cells transfected with the CCKBR.

In summary, our study demonstrates an essential role for the JNK pathway in transducing the proliferative activity of a GPCR ligand. We report the activation of the JNK pathway by the CCKBR through a mechanism that involves PKC and a Src family kinase other than p60Src. Our data also show that gastrin activates the p130Cas/CrkII complex by a Src family kinase, namely, p60Src, in a PKC-dependent manner. However, we found that the CrkII and JNK pathways activated by gastrin are independent.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Cell Culture.
CHO DG44 cells, which do not express CCKA receptor or CCKBR (data not shown), were stably transfected with an expression plasmid encoding the human CCKBR (Bmax, 2.87 ± 0.2 pmol/106 cells). Cells were grown in {alpha}-MEM supplemented with 10% FCS at 37°C in a 95% air/5% CO2 atmosphere.

Expression Vector and Transfection.
The plasmid pSV-CSK encoding for CSK, the plasmid pSGT-SrcK- encoding for p60Src inactive kinase mutant (K295M), or the plasmid pCMV-67 encoding for a c-Jun protein (TAM67) lacking the transactivation domain (residues 2–122) was cotransfected with pTK-Hyg selection vector in CHO cells expressing the human CCKBR (CHO-CCKB cells), using FuGENE 6 reagent. Transfected cell lines obtained after 3 weeks of hygromycin selection (400 µg/ml) were characterized and compared with control cells. The plasmids pSV-CSK and pSGT-SrcK- were obtained from Dr. S. Roche (Montpellier, France). The plasmid pCMV-TAM67 was a gift from Dr. M. Birrer (Rockville, MD). The plasmid pMex-neo-CrkIIW169L encoding for SH3 mutant of CrkII was a gift from Dr. M. Matsuda (Tokyo, Japan). The cDNA encoding CrkII mutant was amplified by PCR and recloned into pcDNA3.1(zeo)-. The plasmid pcDNA3.1(zeo)-CrkIIW169L was stably transfected into CHO cells stably expressing the human CCKBR, using FuGENE 6 reagent. Transfected cells obtained after 2 weeks of zeocin selection (750 µg/ml) were characterized and compared with control cells.

Receptor Binding Studies.
Binding experiments were performed as described previously (51) . Briefly, cells seeded into 24-well plates were incubated with 60 pM 125I-Bolton hunter-(Thr,Nle-CCK1–9s) with or without an excess of unlabeled gastrin. Nonspecific binding, which was determined in the presence of 1 µM gastrin, was <10% that of total binding.

IPs and Western Blotting Analysis.
Cells were serum-starved for 18 h before the addition of peptide. After stimulation, the cells were washed with buffer A [50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, and 2 mM orthovanadate] and homogenized in 500 µl of lysis buffer (buffer A containing 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 10 µg/ml aprotinin) for 15 min at 4°C. The soluble fractions containing identical levels of proteins were immunoprecipitated with the indicated antibodies as described previously (17) . Proteins from immunoprecipitates or whole cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blotted as described previously (17) with the indicated antibodies. Proteins were visualized by autoradiography using 125I-protein A or horseradish peroxidase chemiluminescent reagent (for phospho-JNK and phospho-ERK detection according to the manufacturer’s instructions).

c-Src Kinase Assays.
Immunoprecipitates were washed twice with lysis buffer and three times with Tris-buffered saline [25 mM Tris (pH 7.5), 150 mM NaCl, and 0.2 mM vanadate]. Kinase assays were carried out at 30°C for 10 min in 40 µl of kinase buffer containing 20 mM HEPES (pH 7.5), 10 mM MnCl2, 1 mM DTT, 3.75 µM ATP, 5 µCi of [{gamma}-32P]ATP, and 5 µM acid-denatured enolase as an exogenous substrate. Samples were analyzed by SDS-PAGE. After electrophoresis, the gels were treated with 1 N KOH at 50°C for 1 h and autoradiographed.

Jun Kinase Assays.
Immunoprecipitates were washed twice with lysis buffer containing 1% Triton X-100 and three times with buffer B (50 mM HEPES, 150 mM NaCl, 0.1% Triton, and 10% glycerol) and then incubated for 30 min in buffer B containing 10 mM magnesium acetate, 2 µg of GST-c-Jun-(1–79), 5 µM ATP, and 0.3 µCi of [{gamma}-32P]ATP. The reaction was stopped by the addition of Laemmli sample buffer and boiling. Samples were analyzed by SDS-PAGE, and the gels were autoradiographed.

[3H]Thymidine Incorporation.
DNA synthesis was estimated by measurement of [3H]thymidine incorporation into the trichloroacetic acid-precipitable material. [3H]Thymidine (0.6 µCi/ml) was added during the last 3 h of the 24-h gastrin treatment. The cells were washed with serum-free medium to remove unincorporated [3H]thymidine. DNA was precipitated with 10% trichloroacetic acid at 4°C for 15 min. Precipitates were washed twice with 95% ethanol, dissolved in 0.5 ml of 0.1 N NaOH, and analyzed in liquid scintillation counter.

Transient Transfections and Immunofluorescence.
Cells (3 x 104) were seeded on 6-well plates containing cover slides. After 24 h, the medium was exchanged for medium containing 2% FCS (starvation). After 18 h, 10 nM gastrin was added to the medium, and the cells were further incubated for additional 24 h. The cells were analyzed, and their morphological phenotype was photographed on an inverted phase-contrast microscope (Nikon). Cells were transiently transfected with either 0 (control), 0.5, 1.0, or 1.5 µg of pcDNA3.1(zeo)-CrkIIW169L-encoding plasmid together with 0.5 µg of EYFP-encoding plasmid using FuGENE 6 reagent in medium containing 2% FCS, 24 h after seeding. Cells were fixed in 2% paraformaldehyde, washed several times in PBS, permeabilized in 0.2% Triton X-100, blocked in 2% BSA, incubated with anti-CrkII primary antibody (1 µg/ml), washed several times in PBS, and incubated with secondary antibody coupled to CY-3. Slides were mounted in Mowiol and analyzed on a Nikon E400 microscope with a Sony DXC 950 camera and Visiolab 2000 software. Images were further assembled using Adobe Photoshop software. Transfected cells were scored for the ability to generate long extensions. A cell was scored as having an elongated phenotype if it had two extensions longer than the cell body and if the cell size was two to three times longer than that of nontreated cells. At least 130 cells coexpressing different concentrations of dominant negative mutant of CrkII together with EYFP were counted for each experiment. Data are the means ± SE of three independent experiments.

Materials.
125I-Na (2000 Ci/mmol) and [3H]thymidine (18 Ci/mmol) were obtained from Amersham Pharmacia Biotech, and [{gamma}-32P]ATP (7000 Ci/mmol) was obtained from Isotopchim. Anti-JNK1, anti-CrkII (directed against the COOH terminus), and GST-c-Jun were from Santa Cruz Biotechnology. Anti-c-Jun and anti-p60Src antibodies were from Oncogene Science. Anti-phosphotyrosine and anti-p130Cas antibodies were from Transduction Laboratories. Anti-phospho-Thr183 and -Tyr185 JNK and anti-phospho-Thr202 and -Tyr204 ERK antibodies were from New England Biolabs, and secondary antibodies coupled to CY-3 were from Jackson Immunoresearch Laboratories. PP2, wortmannin, LY294002, GF109203X, Gö6983, and Mowiol were obtained from Calbiochem. PMA, anisomycin, and paraformaldehyde were obtained from Sigma. SP-600125 was from Biomol, and gastrin was from Bachem. EYFP-encoding plasmid was from Clonetech, and pcDNA3.1(zeo)- was from Invitrogen.


    Acknowledgments
 
We thank Dr. S. Roche (Montpellier, France) for the plasmids pSV-CSK and pSGT-Src-, Dr M. Birrer (Rockville, MD) for the plasmid pCMV-TAM67, and Dr. M. Matsuda (Tokyo, Japan) for the plasmid pMex-neo-CrkIIW169L.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by funds from INSERM, Association pour la Recherche contre le Cancer Grants 9540 and 5481, Conseil Régional Midi Pyrénées Grant 97001932, and European Economic Community Grant QLG3-CT-1999-00908. Back

2 To whom requests for reprints should be addressed, at INSERM U.531, Groupe de Recherche de Biologie et Pathologie Digestives, CHU Rangueil, 1, avenue J. Poulhes, Institut Louis Bugnard, Bat. L3, 31403 Toulouse, France. Phone: 33-561322408; Fax: 33-561322403; E-mail: sevac{at}toulouse.inserm.fr Back

3 The abbreviations used are: GPCR, G protein-coupled receptor; CCK, cholecystokinin; CCKBR, CCKB receptor; CHO, Chinese hamster ovary; CSK, COOH-terminal Src kinase; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; EYFP, enhanced yellow fluorescent protein; GST, glutathione S-transferase; IL, interleukin; IP, immunoprecipitation; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; SH, Src homology; TNF-{alpha}, tumor necrosis factor {alpha}. Back

Received for publication 12/26/01. Revision received 4/25/02. Accepted for publication 6/24/02.


    References
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 

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