INTRODUCTION

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In a variety of different cell types, cellular responses to hormones and/or growth factors are mediated by the activation of tyrosine kinases (1). Phosphorylation of tyrosine residues in specific proteins is due to the activity of not only tyrosine kinases, but also phosphotyrosine phosphatases. Specific phosphatase inhibitors, such as sodium vanadate (2), are useful tools in the study of hormone- and growth factor-induced tyrosine phosphorylation. Vanadium salts have been shown to have "insulin-like" effects both in intact animals and in cell cultures (3). H₂O₂, like vanadate, partially mimics the effects of insulin in different cell culture systems (4, 5). Furthermore, it has been reported that pervanadate, which is generated by vanadate peroxidation in the presence of H₂O₂, is a new powerful insulin-like agent that exceeds the sum of both vanadate and H₂O₂ effects (6). Intraperitoneal injection of pervanadate into mice leads to tyrosine phosphorylation of numerous proteins in the liver and kidney within minutes (7). Thus, pervanadate may be a useful tool to study the role of tyrosine-phosphorylated proteins involved in signaling pathways.

In rat pancreatic acinar cells, enzyme secretion is stimulated in response to secretagogues such as cholecystokinin or bombesin (8). The signal cascade involves activation of receptor-coupled G-proteins, leading to the stimulation of phospholipase C activity, which is followed by production of inositol 1,4,5-trisphosphate and diacylglycerol. Whereas inositol 1,4,5-trisphosphate releases Ca²⁺ from intracellular stores (9), diacylglycerol leads to activation of protein kinase C (PKC)¹ (10). Furthermore, tyrosine phosphorylation of a number of proteins could be observed following stimulation of the cells with cholecystokinin (11). It was suggested that tyrosine phosphorylation could amplify the secretory response rather than provide an obligate signal for enzyme secretion. On the other hand, tyrosine kinase inhibitors such as genistein and tyrphostin 25 were shown to inhibit agonist-induced amylase secretion and phospholipase C activation in rat pancreatic acinar cells (12, 13), indicating that tyrosine kinases are involved in receptor-mediated stimulation of amylase secretion.

In Swiss 3T3 cells, bombesin-induced tyrosine phosphorylation of two proteins, the cytosolic protein kinase p125^FAK and the cytoskeletal-associated protein paxillin, has been described as an early event in hormone-mediated cell growth (14, 15). The increase in tyrosine phosphorylation of both p125^FAK and paxillin is accompanied by profound changes in the organization of the actin cytoskeleton and in the assembly of the focal adhesion plaques, which represent the sites of cell attachment to the extracellular matrix (16, 17). Both p125^FAK and paxillin are localized in these distinct areas. They are assumed to be regulatory components of the complex of cytoskeletal proteins that link the actin cytoskeleton to the plasma membrane (reviewed in Ref. 18).

It was the purpose of this study to investigate if protein tyrosine phosphorylation is a step in the signal cascade of stimulus-secretion coupling in rat pancreatic acinar cells. We have used the tumor cell line AR4-2J, which has been described as a model system for long-term studies on pancreatic acinar cell function (19, 20).

Our results suggest that protein tyrosine phosphorylation downstream of protein kinase C activation is involved in stimulation of enzyme secretion from pancreatic AR4-2J cells. An increase in cytosolic free Ca²⁺ concentration accelerated amylase secretion, but had no effect on either PMA- or pervanadate-induced tyrosine phosphorylation. We therefore assume that Ca²⁺ is involved in protein secretion distal to protein tyrosine phosphorylation in such a way that priming of target proteins by tyrosine phosphorylation is a prerequisite for Ca²⁺-dependent activation of secretion.

	EXPERIMENTAL PROCEDURES

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Materials-- Genistein, tyrphostin B56, phorbol 12-myristate 13-acetate, Ro 31-8220, and thapsigargin were obtained from Calbiochem (Bad Soden, Germany) and were prepared in dimethyl sulfoxide as stock solutions. The anti-phosphotyrosine antibodies (clone PY20) were purchased from Santa Cruz (Heidelberg, Germany), anti-p125^FAK antibody (clone 2A7) from Biomol (Hamburg, Germany), and anti-paxillin antibody (clone 349) and protein G PLUS/protein A-agarose from Dianova (Hamburg). Peroxide of vanadate (pervanadate) was prepared by mixing vanadate (Sigma, Deisenhofen, Germany) with H₂O₂ (Merck, Darmstadt, Germany) for 15 min at room temperature, followed by addition of catalase (Sigma) to remove residual H₂O₂ as described by Fantus et al. (21). Fetal calf serum was obtained from PAA Laboratories (Cölbe, Germany), and penicillin/streptomycin from Life Technologies, Inc. (Eggenstein, Germany). Leupeptin was purchased from Serva (Heidelberg), and trypsin inhibitor (hen egg white) from Boehringer (Mannheim, Germany). Dulbecco's modified Eagle's medium and all other reagents (of analytical grade) were from Sigma.

Cell Culture-- AR4-2J cells were obtained from American Type Culture Collection (ATCC CRL1492; Rockville, MD). The cells were seeded at 750,000 cells/35-mm Petri dish and were routinely grown for 72 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. Differentiation of the cells was induced by addition of 100 nM dexamethasone. After 72 h, the cells were washed with the appropriate buffer and used for the assays.

Amylase Release-- For measurement of amylase release, AR4-2J cells were washed three times with KRH buffer (130 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 1.2 mM KH₂PO₄, 1 mM CaCl₂, 20 mM Hepes, 10 mM glucose, and 0.1 mg/ml trypsin inhibitor, pH 7.4) and preincubated for 10 min with or without kinase inhibitors at 37 °C. The cells were stimulated by addition of agonists with or without pervanadate, and at the indicated times, aliquots of the supernatants were removed for the determination of amylase released by the cells. For measurement of the total amount of amylase, the cells were broken and scraped off in a buffer containing 0.1% Triton X-100, 5 mM Hepes, pH 7.0, 280 mM mannitol, 10 mM KCl, 1 mM MgCl₂, 1 mM benzamidine, 1 µM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, and 20 µg/ml trypsin inhibitor. Amylase activity was determined using the Phadebas amylase test (Pharmacia, Freiburg, Germany) and is expressed as a percent of the total cellular content of amylase.

Tyrosine Phosphorylation and Immunoprecipitation-- For determination of tyrosine phosphorylation, AR4-2J cells were washed three times with KRH buffer and preincubated with or without tyrosine kinase inhibitors for 10 min at 37 °C. Bombesin, PMA, or pervanadate was added for the indicated times, followed by washing the cells with ice-cold phosphate-buffered saline containing 0.5 mM sodium orthovanadate. Cells were then scraped off and lysed in lysis buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 0.5 mM sodium orthovanadate, 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 0.5% Triton X-100). After sonication of the cell lysates, 3× concentrated electrophoresis sample buffer (22) was added, and the samples were boiled for 5 min to denature proteins. For immunoprecipitation, 500 µl of lysate containing 500 µg of total protein was incubated with 1.5-3 µg of primary antibody for 60 min on ice. After this time, 30 µl of a 30% suspension of protein G PLUS/protein A-agarose in phosphate-buffered saline was added, and the samples were incubated for an additional 60 min with agitation at 4 °C. The agarose beads were centrifuged and washed three times with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Triton X-100. The beads were then resuspended in electrophoresis sample buffer and boiled for 5 min to release precipitated proteins from the beads.

Electrophoresis and Immunoblotting-- Proteins were electrophoretically separated on SDS-polyacrylamide gels (22). Electrotransfer to nitrocellulose membranes was done in a buffer containing 48 mM Tris, pH 8.6, 39 mM glycine, 0.0375% SDS, and 20% methanol for 60 min at 0.8 mA/cm². After staining with 0.2% Ponceau S to check the efficiency of the transfer, free binding sites of the membranes were blocked with 1% bovine serum albumin in Tris-buffered saline (10 mM Tris-HCl, pH 8.0, and 150 mM NaCl) for 60 min, followed by a 60-min incubation with anti-phosphotyrosine or anti-paxillin antibodies diluted in Tris-buffered saline plus 0.2% Tween 20. Bound antibodies were visualized with secondary antibodies conjugated to horseradish peroxidase using an enhanced chemiluminescence detection kit (Amersham Corp., Braunschweig, Germany) and Reflection^TM autoradiography film (NEN Life Science Products, Köln, Germany). The density of bands on the film was measured using a scanning densitometer (Fröbel Labortechnik, Lindau, Germany).

Protein Determination-- Protein was assayed as described by Bradford (23) using bovine serum albumin as a standard.

	RESULTS

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Amylase Secretion Induced by Pervanadate and Bombesin-- In the presence of pervanadate, amylase secretion from AR4-2J cells increased with time. However, the kinetics of amylase secretion induced by different concentrations of pervanadate were different compared with those for bombesin-induced amylase secretion (Fig. 1A). Whereas bombesin stimulated amylase secretion mostly during the initial 10 min, pervanadate-stimulated amylase release above basal levels was detectable after 15 min and increased continuously within 60 min of observation. After 60 min of incubation, maximal amylase release was obtained with 100 µM pervanadate, which was comparable to that obtained by a maximally effective bombesin concentration. In Fig. 1B, the dose-response relationship of pervanadate-stimulated amylase release in the absence and presence of bombesin is shown. After 30 min of incubation, secretion was stimulated by the maximally effective concentration of 100 µM pervanadate from 7.0 ± 0.7% of total amylase in control cells to 13.3 ± 1.2%. Half-maximal stimulation occurred at 20 µM pervanadate. At a maximally effective concentration of bombesin, amylase secretion over 30 min (19.4 ± 1.8%) was not significantly increased by pervanadate (23.1 ± 2.3%). After 60 min of incubation, amylase secretion was stimulated from 17.4 ± 0.8 to 34.3 ± 1.0% of total amylase by 100 µM pervanadate and was in the same range as the amylase release induced by a maximally effective bombesin concentration (31.6 ± 0.2%). Bombesin-stimulated amylase release was not further increased by any of the tested pervanadate concentrations. Taken together, these results indicate that bombesin-stimulated amylase secretion is probably mediated by a tyrosine kinase. As shown in Table I, the single components that are necessary to prepare pervanadate did not increase basal or bombesin-stimulated amylase secretion. H₂O₂ alone had an inhibitory effect on both unstimulated and bombesin-stimulated amylase secretion. This effect is probably due to free oxygen radicals because no inhibition of amylase secretion was observed when H₂O₂ was preincubated with catalase (Table I).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Time and dose dependence of pervanadate-stimulated amylase secretion from AR4-2J cells. A, time course of unstimulated and pervanadate- and bombesin-stimulated amylase release. AR4-2J cells were incubated in KRH buffer for up to 60 min in the absence and presence of pervanadate (pv) at the indicated concentrations or with 10 nM bombesin (bn). At the indicated time points, aliquots were removed from the medium, and amylase secreted into the medium was quantified. Amylase release is expressed as a percent of total amylase content of the cells present at the beginning of the incubation (n = 4). B, effect of various concentrations of pervanadate on basal and bombesin-stimulated amylase secretion. AR4-2J cells were incubated in KRH buffer for 30 or 60 min at 37 °C in the absence or presence of pervanadate at the indicated concentrations without (control (c)) and with 10 nM bombesin (bn). Amylase secreted into the medium after 30 and 60 min of incubation was quantified and is expressed as a percent of total amylase content of the cells present at the beginning of the incubation (n = 4).

Protein Tyrosine Phosphorylation Induced by Pervanadate and Bombesin-- To further assess the role of tyrosine phosphorylation in stimulus-secretion coupling, we compared both pervanadate- and bombesin-induced protein tyrosine phosphorylation in AR4-2J cells by immunoblot analysis using a monoclonal antibody to phosphotyrosine. Fig. 2A shows that treatment of cells with 100 µM pervanadate leads time-dependently to a strong tyrosine phosphorylation of multiple proteins. The earliest phosphorylation of proteins migrating with apparent molecular masses of 145, 120-140, 82, and 70 kDa was observed already after 3 min of incubation of the cells with pervanadate and increased during the observed period of 25 min. During this time, more and more proteins became tyrosine-phosphorylated in a dose-dependent manner (Fig. 2B). At pervanadate concentrations of 2 and 5 µM, a protein band of 145 kDa and one of 82 kDa appeared, respectively. After 25 min of incubation of cells with 50 µM pervanadate, protein bands were detected that migrated with apparent molecular masses of 180, 145, 120-140, 98, 82, 70, 62, 58, 55, 51, 42, and 37 kDa (Fig. 2B). Bombesin-induced protein tyrosine phosphorylation, which was maximally stimulated after 15 min of incubation with 10 nM bombesin, showed a different pattern (Fig. 2B, bn). The most prominent bands observed migrated with apparent molecular masses of 120-140 and ~70 kDa. These bands probably correspond to the focal adhesion proteins p125^FAK and paxillin, respectively, known to be tyrosine-phosphorylated after bombesin treatment in Swiss 3T3 cells (14, 15). Immunoprecipitation and immunoblotting showed that previous treatment of AR4-2J cells with pervanadate increased tyrosine phosphorylation of p125^FAK and paxillin ~5- and 39-fold, respectively, compared with basal phosphorylation in control cells (Fig. 3). Whereas tyrosine phosphorylation of p125^FAK induced by bombesin was also ~3-fold of the control, tyrosine phosphorylation of paxillin was increased only by ~12-fold above basal phosphorylation. In the anti-paxillin immunocomplexes of bombesin- and pervanadate-stimulated cells, anti-phosphotyrosine immunoreactivity migrated as broad diffuse bands with an apparent molecular mass of 66-75 kDa. After pervanadate stimulation, the anti-paxillin antibody detected several protein bands between 63 and 73 kDa (see Fig. 5B, lower panel), indicating that paxillin is present in several higher phosphorylated forms, whereas after bombesin stimulation, the anti-paxillin antibody detected a shift from 63 kDa to the phosphorylated form at 64-70 kDa (see Fig. 5B, lower panel). This indicates that tyrosine phosphorylation of paxillin by the physiological stimulus bombesin is lower compared with the nonspecific stimulus pervanadate. Furthermore, the finding that the anti-paxillin antibody detected bands with a lower molecular mass compared with the anti-phosphotyrosine antibody (see Fig. 5B, compare lower and upper panels) suggests that these bands represent minor phosphorylated or unphosphorylated forms of paxillin.

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Effect of pervanadate on protein tyrosine phosphorylation in AR4-2J cells. AR4-2J cells were incubated in KRH buffer with 100 µM pervanadate for the indicated times (A) or at different pervanadate concentrations for 25 min or with 10 nM bombesin (bn) for 15 min at 37 °C (B). The different incubation times for pervanadate and bombesin correspond to the time at which each agent had its maximal effect on tyrosine phosphorylation. Cell lysates were subjected to SDS-PAGE, followed by electrotransfer and immunoblotting with anti-phosphotyrosine antibodies as described under "Experimental Procedures." The experiment shown is representative of two separate experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Effect of bombesin and pervanadate on tyrosine phosphorylation of p125FAK and paxillin. AR4-2J cells were incubated in the absence (control (c)) or presence of 10 nM bombesin (bn) or 100 µM pervanadate (pv) for 15 min at 37 °C. After lysis of the cells, immunoprecipitation of p125FAK and paxillin was performed. Immunoprecipitates were subjected to SDS-PAGE, and immunoblotting using anti-phosphotyrosine antibodies was carried out as described under "Experimental Procedures." The experiment shown is representative of three separate experiments.

Effect of the Tyrosine Kinase Inhibitors Genistein and Tyrphostin B56 on Pervanadate- and Bombesin-stimulated Amylase Secretion and Protein Tyrosine Phosphorylation-- The effects of pervanadate on amylase secretion and tyrosine phosphorylation indicate that a tyrosine kinase is involved in these processes. We therefore tested the effects of tyrosine kinase inhibitors on pervanadate- and bombesin-stimulated amylase secretion and on protein tyrosine phosphorylation. As shown in Fig. 4, 100 µM genistein reduced both pervanadate- and bombesin-stimulated amylase secretion to basal levels, whereas 100 µM tyrphostin B56 nearly completely inhibited basal and pervanadate- and bombesin-stimulated amylase secretion. Similarly, both genistein and tyrphostin B56 also reduced pervanadate-induced protein tyrosine phosphorylation by ~60 and 90%, respectively (Fig. 5A), whereas bombesin-induced protein tyrosine phosphorylation was reduced to basal phosphorylation by genistein and was completely blocked by tyrphostin B56. As shown in Fig. 5B (upper panel), pervanadate-stimulated tyrosine phosphorylation of paxillin was ~39-fold of the control and was reduced by genistein and tyrphostin B56 by ~70 and 90%, respectively, whereas bombesin-induced tyrosine phosphorylation was ~14-fold of the control and was reduced to basal phosphorylation by genistein and was completely blocked by tyrphostin B56. The apparently lower content of paxillin, as detected with the anti-paxillin antibody, in immunoprecipitates of bombesin- and pervanadate-stimulated cells compared with that of control cells is likely due to the shift of paxillin (Fig. 5B, lower panel). Densitometric scanning showed that the total amount of paxillin in the immunoprecipitates of stimulated cells was 82.5 and 135.7% after bombesin and pervanadate stimulation, respectively, compared with that of control cells. This indicates that the differences in the phosphotyrosine content of paxillin are due to different levels of phosphorylation and not to various amounts of protein (Fig. 5B, lower panel). In the samples from genistein-treated cells, the amount of precipitated paxillin was about half of the control and was probably due to degradation of paxillin. Fig. 5C shows that pervanadate-induced tyrosine phosphorylation of p125^FAK was 4-fold of the control and was reduced to basal levels by genistein and by ~90% by tyrphostin B56. Bombesin-stimulated tyrosine phosphorylation was 5-fold of the control and was also reduced to basal levels by genistein and was completely inhibited by tyrphostin B56. Since the anti-p125^FAK antibody is not useful for immunoblot analysis, it was not possible to control the amounts of protein precipitated with this antibody. However, equal amounts of immunoglobulins in each lane detected by immunoblot analysis suggested that equal amounts of p125^FAK had been precipitated.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of genistein and tyrphostin B56 on bombesin- and pervanadate-stimulated amylase secretion. AR4-2J cells were preincubated in KRH buffer in the presence or absence of 100 µM genistein or 100 µM tyrphostin B56 for 10 min at 37 °C. Cells were then stimulated by addition of 10 nM bombesin or 100 µM pervanadate, and unstimulated or stimulated amylase secretion over 30 min was measured as described in the legend to Fig. 1 (n = 3).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Effect of genistein and tyrphostin B56 on bombesin- or pervanadate-induced tyrosine phosphorylation of p125FAK and paxillin. AR4-2J cells were preincubated in KRH buffer in the absence (- -) or presence of 100 µM genistein (gen) or 100 µM tyrphostin B56 (tyr) for 10 min at 37 °C. Then, cells were left unstimulated (addition of a corresponding volume of vehicle; control (c)) or were stimulated by addition of 10 nM bombesin (bn) or 100 µM pervanadate (pv) for 15 min at 37 °C. After lysis of the cells, immunoprecipitation of p125FAK and paxillin was performed. Total cell lysates (A) and immunoprecipitates of paxillin (B) and of p125FAK (C) were subjected to SDS-PAGE, and immunoblotting was performed using anti-phosphotyrosine and anti-paxillin antibodies as described under "Experimental Procedures." The experiment shown is representative of two (immunoprecipitation) and four (total cell lysates) separate experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effects of genistein on bombesin-induced amylase release and tyrosine phosphorylation of p125FAK and paxillin. For measurement of amylase release, AR4-2J cells were preincubated with the indicated concentrations of genistein for 10 min at 37 °C. Cells were then stimulated by addition of 10 nM bombesin, and after 15 min, amylase release was determined as described in the legend to Fig. 1. Results are given as a percent of maximally stimulated amylase release induced by 10 nM bombesin (n = 2-5) (circles). The effects of various genistein concentrations on bombesin-induced tyrosine phosphorylation of p125FAK (squares) and paxillin (triangles) were tested as described in the legend to Fig. 5 and under "Experimental Procedures." The level of tyrosine phosphorylation was quantified by densitometric scanning and is expressed as a percent of maximal phosphorylation induced by 10 nM bombesin (n = 2-4).

Effect of Pervanadate on Phospholipase C Activity-- Previous reports on rat pancreatic acinar cells suggest that tyrosine kinases are involved in the activation of phospholipase C induced by different secretagogues such as cholecystokinin, bombesin, or carbachol (12, 13). To test if pervanadate-stimulated amylase secretion is induced by pervanadate-mediated activation of phospholipase C, AR4-2J cells were treated with 100 µM or 1 mM pervanadate for 10 s to 50 min and with 10 nM bombesin for 15 s for comparison. Phospholipase C activity was determined by measurement of inositol 1,4,5-trisphosphate production. At no time could any significant change in inositol 1,4,5-trisphosphate production be observed in response to pervanadate compared with the control, whereas bombesin increased inositol 1,4,5-trisphosphate levels to ~5-fold of the control (data not shown).

Role of Protein Kinase C in Pervanadate- and Bombesin-stimulated Amylase Secretion and Protein Tyrosine Phosphorylation-- It is well established that secretagogue-stimulated amylase secretion from rat pancreatic acinar cells is mediated by PKC (24, 25). To test if pervanadate-induced amylase release is also mediated by PKC activity and if a tyrosine kinase is involved in the PKC-activated pathway, the specific PKC inhibitor Ro 31-8220 (26, 27) was tested on amylase secretion and tyrosine phosphorylation. As shown in Fig. 7A, the time course of PMA-stimulated amylase secretion was nearly identical to that induced by pervanadate. Incubation of the cells with both PMA and pervanadate showed no higher stimulatory effect than with each component alone. Preincubation of AR4-2J cells with Ro 31-8220 inhibited PMA- and bombesin-stimulated amylase secretion by ~80 and 50%, respectively, whereas secretion induced by pervanadate alone or in combination with PMA was not significantly inhibited (Fig. 7B). These data suggest that PKC and the responsible tyrosine kinase are acting in the same signaling pathway and that PKC functions upstream of this tyrosine kinase.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Effect of the PKC inhibitor Ro 31-8220 on bombesin-, pervanadate-, or PMA-stimulated amylase secretion. A, time course of unstimulated and stimulated amylase release. AR4-2J cells were left unstimulated (addition of a corresponding volume of vehicle; control (c)) or were stimulated by addition of 10 nM bombesin (bn), 200 nM PMA, or 100 µM pervanadate (pv) or by a combination of both PMA and pervanadate. Amylase secretion was measured over 60 min as described in the legend to Fig. 1 (n = 3-5). B, stimulated amylase release over 60 min in the absence or presence of Ro 31-8220. AR4-2J cells were preincubated in KRH buffer in the absence or presence of 10 µM Ro 31-8220 for 10 min at 37 °C. Then, cells were left unstimulated or were stimulated as described for A (n = 3-5). The significance of the differences was calculated using the t test for paired values. n.s., not significant.

View larger version (38K): [in this window] [in a new window]   Fig. 8.   Effect of the PKC inhibitor Ro 31-8220 on bombesin-, PMA- or pervanadate-induced tyrosine phosphorylation in AR4-2J cells. AR4-2J cells were preincubated in KRH buffer in the absence or presence of 10 µM Ro 31-8220 for 10 min at 37 °C. Then, cells were left unstimulated (addition of a corresponding volume of vehicle; control (c)) or were stimulated by addition of 10 nM bombesin (bn), 200 nM PMA, or 100 µM pervanadate (pv) for 15 min at 37 °C. Total cell lysates were subjected to SDS-PAGE, and immunoblotting was performed using anti-phosphotyrosine antibodies as described under "Experimental Procedures." To compare the tyrosine phosphorylation patterns induced by bombesin, PMA, and pervanadate, respectively, the immunoblots with pervanadate-treated samples were exposed to x-ray film for 90 s, whereas immunoblots with bombesin- or PMA-treated samples were exposed for 5 min. The experiment shown is representative of three separate experiments.

Effect of Thapsigargin on PMA- and Pervanadate-stimulated Amylase Secretion and Protein Tyrosine Phosphorylation-- The slower time course of amylase release induced by pervanadate or PMA compared with bombesin indicates that Ca²⁺ release and Ca²⁺ entry, by which the initial rapid phase of bombesin-induced amylase secretion is mediated (29), were not affected by tyrosine kinase activity. Therefore, we tested if an increase in cytosolic free Ca²⁺ concentration could enhance amylase secretion within the first minutes in the presence of pervanadate or PMA using the Ca²⁺-ATPase inhibitor thapsigargin (30) or the calcium ionophore A23187. As shown in Fig. 9A, thapsigargin increased PMA-stimulated amylase release to levels comparable to those induced by bombesin, whereas thapsigargin alone increased amylase release only slightly within the first 5-10 min and thereafter showed no higher secretion rate than the unstimulated control cells. Amylase release induced by the combination of pervanadate and thapsigargin was increased within the first 10 min to the same extent as by thapsigargin alone (Fig. 9B). Thereafter, the rate of stimulation was similar to that with pervanadate alone, indicating that both pervanadate and thapsigargin have additive effects on amylase secretion (Fig. 9B). Similarly, the increase in cytosolic free Ca²⁺ concentration by the ionophore A23187 increased amylase secretion within the first 10 min. It was then additive with pervanadate and stimulated amylase secretion to levels comparable to those induced by bombesin within 30 min (Fig. 9B). An increase in cytosolic free Ca²⁺ concentration by thapsigargin or the calcium ionophore A23187 had no effect on basal or PMA- or pervanadate-induced protein tyrosine phosphorylation (data not shown), indicating that an increase in cytosolic free Ca²⁺ concentration, which leads to the initial increase in amylase secretion, affects the secretory mechanisms downstream of tyrosine phosphorylation. We therefore expected that the PKC inhibitor Ro 31-8220 would not inhibit PMA- plus thapsigargin-induced amylase secretion to the same extent as that induced by PMA alone. As shown in Table II, PMA- plus thapsigargin-stimulated amylase secretion after 60 min of incubation was inhibited by Ro 31-8220 by ~50% and was comparable to the inhibition of bombesin-stimulated amylase release by Ro 31-8220 (Fig. 7B and Table II ).

View larger version (22K): [in this window] [in a new window]   Fig. 9.   Effect of thapsigargin or the calcium ionophore A23187 on PMA- or pervanadate-stimulated amylase secretion. AR4-2J cells in KRH buffer were left unstimulated (addition of a corresponding volume of vehicle; control (c)) or were stimulated by addition of 10 nM bombesin (bn), 200 nM PMA, 100 µM pervanadate (pv), 5 µM thapsigargin (tg), or 5 µM A23187 alone or in combinations as indicated. Amylase secretion over 30 min was measured as described in the legend to Fig. 1. For clarity, the effect of thapsigargin on PMA-stimulated amylase secretion is shown in A, and the effect of thapsigargin or A23187 on pervanadate-induced amylase release is shown in B. Data shown in A and B are from the same experiment, which is representative of three different experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Effect of genistein on PMA- and thapsigargin-stimulated amylase secretion. AR4-2J cells were preincubated in KRH buffer in the absence or presence of 100 µM genistein for 10 min at 37 °C. Then, cells were left unstimulated (addition of a corresponding volume of vehicle; control (c)) or were stimulated by addition of 10 nM bombesin (bn), 200 nM PMA, or 5 µM thapsigargin (tg) or by a combination of both PMA and thapsigargin. Amylase secretion over 60 min was measured as described in the legend to Fig. 1 (n = 3).

	DISCUSSION

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We have examined the role of protein tyrosine phosphorylation in amylase secretion from differentiated AR4-2J rat pancreatic acinar cells. Previous evidence had suggested that both an increase in cytosolic free Ca²⁺ concentration and activation of protein kinase C are critical steps in stimulus-secretion coupling (25, 29, 31). Whereas an initial rapid phase of hormone-induced enzyme secretion is mediated by production of inositol 1,4,5-trisphosphate followed by Ca²⁺ release from intracellular stores and Ca²⁺ influx into the cell, a second slower sustained phase is associated with production of diacylglycerol and activation of protein kinase C (25, 29). Both the Ca²⁺- and the protein kinase C-induced pathways act synergistically. Artificial activation of these pathways by elevation of cytosolic Ca²⁺ concentration in the presence of stimulators of protein kinase C, such as phorbol esters, could mimic the effect of secretagogues on enzyme secretion (29, 31, 32). Although different proteins have been shown to be phosphorylated in response to secretagogues of enzyme secretion, key targets in the Ca²⁺- and diacylglycerol-dependent cascade of stimulatory events have not yet been identified.

A Protein-tyrosine Kinase Acts Downstream of Protein Kinase C in Bombesin-stimulated Amylase Secretion-- This study gives strong evidence that distal to the activation of protein kinase C, tyrosine kinase activation and tyrosine phosphorylation of one or more proteins play a crucial role in bombesin-induced amylase secretion. Our results show that two proteins, p125^FAK and paxillin, are mainly tyrosine-phosphorylated by stimulation of AR4-2J cells with bombesin (Fig. 3). The striking correlation of the dose-response curves for genistein-induced inhibition of both bombesin-stimulated amylase secretion and tyrosine phosphorylation of p125^FAK and paxillin indicates that tyrosine phosphorylation of these proteins might be involved in bombesin-induced amylase release (Fig. 6). Pervanadate, a powerful protein-tyrosine phosphatase inhibitor, increases tyrosine phosphorylation of multiple proteins, including p125^FAK and paxillin, and stimulates amylase release to the same extent as bombesin (Fig. 1). However, the time courses of amylase release induced by maximally effective concentrations of bombesin and pervanadate are quite different. Whereas amylase secretion rapidly increases within the first minutes after addition of bombesin, the second phase continues at a lower rate for the duration of agonist stimulation (Fig. 1A). In contrast, stimulation by pervanadate does not lead to a significant amylase release earlier, but 10-15 min after addition, and the level of bombesin-stimulated amylase release is reached after 60 min. This time course resembles that of phorbol ester-stimulated amylase secretion (Fig. 7A). The effects of both pervanadate and PMA are not additive. Since bombesin-stimulated sustained amylase secretion and pervanadate-stimulated amylase release are also not additive (Fig. 1B), it can be concluded that a common mechanism is involved in stimulation of amylase secretion by these substances. Furthermore, Ro 31-8220, a specific inhibitor of protein kinase C, inhibits bombesin- and PMA-induced amylase secretion (Fig. 7B) as well as protein tyrosine phosphorylation (Fig. 8), but has no effect on both pervanadate-induced amylase secretion and protein tyrosine phosphorylation. In addition, secretion induced by PMA or pervanadate is inhibited by the tyrosine kinase inhibitor genistein (Figs. 4 and 10). These observations are compatible with the interpretation that stimulation of protein kinase C precedes tyrosine kinase activation. This is in contrast to previous studies on freshly isolated rat pancreatic acinar cells that showed that genistein had no effect on the secretory response to PMA (11, 13). A possible explanation for this difference could be the use of freshly isolated pancreatic acinar cells in those studies compared with our cells kept in tissue culture.

Role of Ca²⁺ in Protein Tyrosine Phosphorylation and Amylase Secretion-- The slower time course of amylase release induced by pervanadate indicates that Ca²⁺ release and Ca²⁺ entry, by which the initial rapid phase of agonist-induced amylase secretion is mediated (29), are not affected by tyrosine kinase activity. This agrees with our observation that pervanadate affects neither unstimulated nor bombesin-stimulated inositol 1,4,5-trisphosphate production. An increase in cytosolic free Ca²⁺ concentration by addition of thapsigargin or A23187 rapidly increases amylase secretion within the first minutes after addition. This effect of elevated cytosolic free Ca²⁺ concentration is additive to those of PMA and pervanadate (Fig. 9) and indicates that both protein tyrosine phosphorylation and elevation of cytosolic free Ca²⁺ concentration act together in the stimulation of amylase secretion. Inhibition of PKC by Ro 31-8220 inhibits PMA-induced amylase secretion in the absence of elevated cytosolic Ca²⁺ concentration to a higher degree than in its presence (see Table II). This indicates that the initial rapid phase of agonist-stimulated amylase secretion is probably due to an effect of Ca²⁺ downstream of tyrosine kinase activity. In agreement with this, we did not observe any increase in protein tyrosine phosphorylation after elevation of intracellular Ca²⁺ concentration by thapsigargin or A23187 (data not shown). Moreover, PMA-induced protein tyrosine phosphorylation was neither increased nor accelerated within the initial 10 min by an increase in intracellular Ca²⁺ concentration. These data are consistent with the interpretation that tyrosine phosphorylation of target proteins in the secretory machinery is an essential requirement for exocytosis to occur. Both tyrosine kinase and Ca²⁺ could act in sequence on a common step in exocytosis in such a way that priming of target proteins by tyrosine phosphorylation facilitates Ca²⁺ activation of secretion. If tyrosine kinase is blocked by genistein, Ca²⁺-dependent secretion is also abolished (see Fig. 10).

Role of p125^FAK and Paxillin in Cellular Functions-- p125^FAK and paxillin had been originally described to be components of the focal adhesions anchoring cultured cells to extracellular matrix proteins (18). There is evidence from a number of cell types that p125^FAK and paxillin are regulatory components of cytoskeletal proteins that link the actin cytoskeleton to the plasma membrane. Paxillin binds to vinculin and is tyrosine-phosphorylated in adherent cells, presumably by one of the tyrosine kinases with which it associates (p125^FAK, Csk, Src, or Lck) (18, 33, 34). Recent in vitro findings demonstrate that p125^FAK phosphorylates paxillin at Tyr-118 (35). Furthermore, the time course of tyrosine phosphorylation for p125^FAK in Swiss 3T3 and AR4-2J cells preceding that of paxillin (36)² suggests that p125^FAK might be the tyrosine kinase itself that phosphorylates paxillin.