©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cholecystokinin Stimulates the Down-regulation of CTP:Phosphocholine Cytidylyltransferase in Pancreatic Acinar Cells (*)

(Received for publication, September 13, 1994; and in revised form, November 9, 1994)

Guy E. Groblewski (1)(§) Yuli Wang (2) Steven A. Ernst (3) Claudia Kent (2) John A. Williams (1) (4)

From the  (1)Departments of Physiology, (2)Biological Chemistry, (3)Anatomy and Cell Biology, and (4)Internal Medicine, The University of Michigan, School of Medicine, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of rat pancreatic acinar cells with cholecystokinin (CCK) is known to result in a significant inhibition of CTP:phosphocholine cytidylyltransferase (CT), a rate-limiting enzyme in phosphatidylcholine biosynthesis. Immunoprecipitation of CT from P-labeled acinar cells revealed that CCK treatment also caused a marked reduction in CT phosphate levels. The effects of CCK were maximal over 60 min and dependent on concentration, exhibiting an EC of 800 pM. Other calcium mobilizing secretagogues such as carbamylcholine (100 µM) and bombesin (10 nM) also reduced CT phosphate levels to 20 and 39% of control, respectively. Treatment of cells with thapsigargin and/or 12-O-tetradecanoylphorbol-13-acetate established that a combination of increased intracellular Ca and protein kinase C activation was necessary to decrease phosphorylated CT content. Conversely, secretin (10 nM) or 8-(4-chlorophenylthio)-cAMP (100 µM) added alone had no effects. Use of the compound JMV-180 indicated CCK was acting through the low affinity state of the CCK(A) receptor to reduce CT phosphate levels. Further, the decrease in phosphorylated CT caused by CCK was blocked by the phosphatase inhibitors okadaic acid (3 µM) and calyculin A (100 nM). Finally, immunoblotting from whole cell lysates revealed CT was partially degraded in response to CCK, providing a novel mechanism by which the inhibition of CT enzyme activity occurs in response to the hormone. Moreover, this degradation was also blocked by a phosphatase inhibitor. These data suggest that the dephosphorylation of either CT itself or some other regulatory molecule(s) which mediates the CCK-induced protease activation may play a central role in reducing CT enzyme levels in acinar cells.


INTRODUCTION

Phosphatidylcholine metabolism in mammalian cells is thought to play a significant role in the production of the cellular messenger diacylglycerol(1) . Evidence for this in pancreas arises from studies utilizing acini that were prelabeled with [^3H]choline (2) or [^3H]myristic acid (3) demonstrating an activation of phospholipase C and D by secretagogues such as cholecystokinin (CCK), (^1)carbamylcholine, and bombesin. Similarly, it has also been shown that carbamylcholine treatment of acini which were prelabeled with [^14C]glycerol resulted in a significant decrease in the levels of [^14C]phosphatidylcholine(4) . Recently, a detailed investigation of the CDP-choline biosynthetic pathway in acinar cells revealed that these agonists actually inhibited phosphatidylcholine synthesis by reducing choline uptake into cells and by inhibiting activity of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) (CT)(5) . Because CT was inhibited by CCK treatment and this inhibition was blocked by calmodulin antagonists, it was proposed that phosphorylation of CT by a calcium-calmodulin-activated kinase led to its inhibition(5) .

CT activity is regulated by a number of factors in mammalian cells (reviewed in Kent(6) ). While the enzyme is present in both cytosolic and membrane-associated cell fractions, it is the membrane bound form that appears to be most highly active(6) . Phosphorylation is also believed to play an essential role in regulating CT. Initial evidence for this came from the finding that, in the presence of phosphatidylcholine vesicles, phosphorylation of CT in vitro by protein kinase A significantly inhibited CT activity(7) . Immunoprecipitation of CT from Hela cells later established that the enzyme was in fact phosphorylated in vivo(8) . More recently, it was demonstrated that the addition of exogenous phospholipase C to Chinese hamster ovary (CHO) cells produced a marked dephosphorylation of CT which coincided with its translocation from cytosolic to membrane-associated fractions and its subsequent activation(9) . Similar results were also reported in HeLa cells that had been stimulated with oleate(10) . The observation that the phosphatase inhibitor okadaic acid blocked both translocation and activation of CT in CHO cells suggested that these events were mediated by dephosphorylation of CT(9) . However, a recent in vitro study utilizing membranes from oleate- and phospholipase C-treated hepatocytes suggested that dephosphorylation of CT is not a prerequisite for its translocation but rather occurs after insertion into the membrane(11) . Thus, although the precise mechanism(s) by which CT is regulated in cells is uncertain, it seems clear that phosphorylation plays a substantial role in modulating the activity of this enzyme.

To date, studies investigating the regulation of CT have utilized a number of approaches in cultured cells to modulate the enzyme, such as treatment with exogenous phospholipase C(9) , oleate(10) , cAMP(9, 12) phorbol ester(8, 13) , and choline deprivation(14) . The observation that CT activity is strongly inhibited in secretagogue-stimulated pancreatic acinar cells (5) provides a unique model to study this enzyme in acutely isolated cells responding to a physiological agonist. Therefore, in the present study, the effects of CCK and other secretory stimuli to regulate CT in pancreatic acinar cells was investigated.


EXPERIMENTAL PROCEDURES

Materials

Affinity-purified N antibody that was raised against a peptide corresponding to the amino-terminal 17 amino acids of rat liver cytidylyltransferase was previously characterized(10, 15) . Immobilized protein A-agarose beads were purchased from Pierce; [P]orthophosphate (9000 Ci/mmol) from DuPont-NEN; thapsigargin from LC Services (Boston MA); soybean trypsin inhibitor, benzamidine, paramethylsulfonylfluoride, 12-O-tetradecanoylphorbol-13-acetate (TPA), and 8-(4-chlorophenylthio)- (CPT-)-cAMP) from Sigma; and leupeptin and pepstatin from Boehringer Mannheim. CCK octapeptide was a gift from the Squibb Institute (Princeton, NJ); the CCK analogue JMV-180 was obtained from Research Plus (Bayonne, NJ).

Isolation of Rat Pancreatic Acini

Pancreatic acinar cells were isolated from adult, male, Sprague-Dawley rats (150-200 g) by collagenase digestion as described previously(16) . Approximately 1 ml or less of packed acini were suspended in 12 ml of HR buffer containing (in mM) 10 HEPES (pH 7.4), 137 NaCl, 4.7 KCl, 0.56 MgCl(2), 1 Na(2)HPO(4), 1.28 CaCl(2), 5.5 D-glucose, 2 L-glutamine, and an essential amino acid solution. The buffer was supplemented with 0.1 mg/ml soybean trypsin inhibitor, 0.1% bovine serum albumin and gassed with 100% O(2). For immunoprecipitation experiments involving P-labeling of cells, Na(2)HPO(4) was omitted from the buffer.

Immunoprecipitation

Acinar cells were incubated in HR buffer containing 0.3 mCi/ml [P]orthophosphate for 1.5 h at 37 °C prior to and throughout treatments. In experiments utilizing thapsigargin, ionomycin, TPA, okadaic acid, calyculin A, and JMV-180, these compounds were dissolved in dimethyl sulfoxide and added to the cells, producing a final vehicle concentration not exceeding 0.1%. In all cases, control cells received an equal concentration of vehicle. Following treatments, cells were quickly pelleted and suspended in 0.5 ml of ice-cold lysis buffer containing (in mM) 40 Na(4)P(2)O(7) (pH 7.4), 400 NaCl, 100 NaF, 10 EDTA, 1 benzamidine, 0.5% (v/v) Nonidet P-40, 1% 2-mercaptoethanol, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. All subsequent procedures were performed at 4 °C. The suspensions were sonicated, allowed to sit for 30 min, and then centrifuged at 150,000 times g for 30 min. The supernatant (whole cell lysate) was then precleared by mixing with 60 µl of protein A-linked agarose beads for 1 h. Samples were quickly centrifuged to remove the protein A beads, and protein concentrations were determined using a Bio-Rad assay. Antiserum to CT (N antibody) was then added (1:400) to 0.4 ml of each sample containing equal concentrations protein (approximately 0.8 mg total) and incubated overnight. Immune complexes were precipitated by incubation with 50 µl of protein A-linked agarose for 2 h. The beads were then washed five times with 1 ml of buffer containing (in mM) 50 Tris-HCl, 150 NaCl, 1 EDTA, 0.5% (v:v) Nonidet P-40, and then denatured by boiling in 15 µl of SDS sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% (w:v) SDS, 5% (v:v) 2-mercaptoethanol, and 10% glycerol. Samples were then separated by SDS-PAGE on 12.5% gels and visualized by autoradiography. Phosphorylation intensity of the CT band was quantified using a Bio-Rad model GS-250 imaging system.

Immunoblotting

Treatment of cells, as well as the preparation of whole cell lysates, was carried out under conditions identical to those described for immunoprecipitation of CT. Following the 150,000 times g centrifugation, lysates were mixed in SDS sample buffer and boiled. An aliquot of each lysate was analyzed for protein content using a Bio-Rad assay. Approximately 90 µg of protein from each sample was separated by 10% SDS-PAGE using a mini protein II apparatus from Bio-Rad. Immunoblotting was carried out as detailed previously (10) using the N antibody (1:3000).

CT Assay

Acini were treated under conditions identical to those described for immunoprecipitation of CT. Following treatments, cells were quickly pelleted and suspended in 0.5 ml of digitonin release buffer containing (in mM) 10 Tris (pH 7.4), 250 sucrose, 33 NaF, 0.033 NaVO(4), 3.3 EDTA, 3.3 EGTA, 2 dithiothreitol, 1 phenylmethylsulfonyl fluoride, 0.8 mg/ml digitonin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. Following a 3-min incubation at 4 °C, samples were used for whole cell activity measurements or centrifuged at 10,000 times g for 5 min. The supernatant (soluble fraction) was removed, and the pellet (particulate fraction) was suspended in 0.5 ml of digitonin release buffer. Protein content was determined using a Bio-Rad assay. CT activity was assayed in the presence of 0.2 mM phosphatidylcholine and 0.2 mM oleate as described previously(17) .

Immunofluorescence

Acini that were incubated in HR buffer for 2.5 h were fixed in suspension with 2% paraformaldehyde in phosphate-buffered saline for 2 h at 4 °C. After rinsing with 5% sucrose in phosphate-buffered saline, acini were cryoprotected by infiltration with graded concentrations of sucrose in phosphate-buffered saline and frozen in isopentane cooled with liquid nitrogen following the procedures of Barthel and Raymond(18) . Small tissue blocks of intact pancreas were fixed and processed in a similar manner. Procedures for indirect immunofluorescence microscopy with 5-µm thick cryostat sections were as described previously(19) . Affinity-purified N antibody was used over a range of dilutions (1:100 to 1:1200). To determine specificity of immunofluorescence staining, competitions were carried out by preincubating 1:100 dilutions of antibody with 1 µg of the peptide used to generate the antibody.


RESULTS

CCK Decreases CT Phosphate Levels in Pancreatic Acini

Immunoprecipitation of whole cell lysates from P-labeled acini using anti-CT N antibody resulted in a prominent phosphoprotein of approximately 42 kDa corresponding to the molecular mass of CT (Fig. 1, arrow). A number of additional minor phosphoproteins were also present. Preabsorbtion of the antibody with the peptide it was raised against completely abolished the appearance of the 42-kDa band while having no effect on any of the other phosphorylations, establishing the specificity of this antibody toward CT. Identical results were also obtained by preabsorbing the antibody with purified CT protein (not shown). Performing the assay in the absence of antibody revealed that the additional phosphoproteins present resulted from nonspecific binding to the protein A-agarose beads. Illustrated in Fig. 1, these nonspecific bands were partially detectable even after preclearing the lysates three times with 60 µl of protein A beads prior to the addition of antibody.


Figure 1: Immunoprecipitation of CT in pancreatic acinar cells. Acinar cells, prelabeled with [P]orthophosphate, were treated as control or with 10 nM CCK for 1 h before preparation of whole cell lysates. Lysates were precleared with protein A-agarose beads three times and then incubated for 15 h with N antibody (lanes 1 and 2); N antibody that was preincubated for 1 h with 40 µg of CT peptide (lane 3); or without N antibody (lane 4). Immunecomplexes were precipitated using protein A-agarose beads, separated by SDS-PAGE, and autoradiographed.



Treatment of acini with 10 nM CCK for 1 h resulted in a complete loss of phosphorylated CT ( Fig. 1and Fig. 2). A time course for the effects of 10 nM CCK on CT phosphorylation indicated no changes were present after 15 min; however, a large decrease to 13% of control was seen at 30 min, with a complete loss of phosphate occurring by 60 min (Fig. 2A). The effects of CCK were also concentration dependent (Fig. 2B). Following 1-h treatments with CCK, a reduction in phosphorylated CT was detectable at concentrations as low as 10 pM, but became statistically significant at 100 pM (p < 0.05), reducing phosphorylation to 82% of control; the EC for CCK was approximately 800 pM.


Figure 2: Time course and concentration dependence of a CCK-induced decrease in CT phosphate levels. A, acini were prelabeled with [P]orthophosphate and then treated with or without 10 nM CCK for indicated times before immunoprecipitating CT from whole cell lysates. B, prelabeled acini were treated with the indicated concentrations of CCK for 1 hour before immunoprecipitating CT from whole cell lysates. The data in A and B are the mean and S.E. of three experiments. In B each experiment was performed in duplicate. A representative autoradiograph is shown above each graph.



Ca-mobilizing Agonists Decrease Phosphorylated CT Levels

To evaluate the role of other known pancreatic hormones and neurotransmitters in CT phosphorylation, the calcium mobilizing secretagogues carbamylcholine and bombesin as well as secretin, which acts predominantly through adenylate cyclase, were tested (Fig. 3). Similar to the effects seen for CCK, exposure of cells to 100 µM carbamylcholine or 10 nM bombesin for 1 h resulted in a marked reduction of phosphorylated CT to 20 and 39% of control, respectively. Conversely, treatment with 10 nM secretin had no effects on CT. Thus, the decrease in CT phosphate levels appeared to be mediated by agents whose receptors are coupled to the activation of phospholipase C and mobilization of free ionized intracellular Ca ([Ca](i)).


Figure 3: Multiple agonists decrease CT phosphate levels. Acini were prelabeled with [P]orthophosphate before treatment with 100 µM carbamylcholine (CCh), 10 nM bombesin (Bmb), 10 nM secretin (Sec), 10 nM cholecystokinin (CCK), and/or 10 µM JMV-180 (JMV) for 1 h. CT was then immunoprecipitated from whole cell lysates separated by SDS-PAGE and autoradiographed. The data are the means and S.E. of three experiments.



CCK Acts via a Low Affinity State of the CCK(A) Receptor to Decrease Phosphorylated CT Levels

Previous studies have established that rat pancreatic acinar cells express the A form of the CCK receptor (CCK(A)) which is known to possess at least two affinity states(20) . That the IC for the CCK-induced reduction in CT phosphorylation was approximately 800 pM suggested this effect was mediated through the low affinity state of the CCK(A) receptor. Discrimination between the low and high affinity states was achieved using the compound JMV-180 which acts as a high affinity agonist and low affinity antagonist to the CCK(A) receptor(20, 21) . Treatment of cells with 10 µM JMV-180 for 1 h to activate the high affinity receptor had no measurable effects on CT phosphorylation (Fig. 3). Conversely, use of the compound as a low affinity receptor antagonist by its simultaneous addition with CCK completely blocked the reduction in CT phosphate levels normally induced by the CCK. Together, these results suggest that CCK was acting through the low affinity state of the CCK(A) receptor to decrease CT phosphorylation.

Both Ca and Protein Kinase C Activation Are Required to Decrease CT Phosphate Levels

Previous studies have demonstrated that stimulation by CCK leads to intracellular increases in diacylglycerol, [Ca](i) and, with high concentrations of CCK, cAMP in rat pancreas(22) . To assess the role of these signaling pathways in CT phosphorylation; thapsigargin, which elevates [Ca](i) by inhibiting the Ca ATPase in the endoplasmic reticulum; TPA, a potent activator of protein kinase C; and CPT-cAMP, a nonhydrolyzable analogue of cAMP, were tested (Fig. 4). A 1-h incubation with any of these compounds alone produced no effects on CT phosphorylation. This was also true when 2 µM ionomycin was used to elevate [Ca](i) (data not shown). Similarly, CPT-cAMP in combination with either thapsigargin or TPA was also without effect on CT. However, treatment with thapsigargin and TPA together evoked a prominent reduction in CT phosphate content to 30% of control. Moreover, a combination of all three agents resulted in reducing CT phosphate levels to 6% of control, an effect similar to that seen using 10 nM CCK. Thus, at a minimum, an increase in [Ca](i) as well as activation of protein kinase C was required to reduce CT phosphate levels; however, a complete loss of phosphate also required activation of the cAMP pathway.


Figure 4: Multiple cellular messengers are required to reduce phosphorylated CT. Acini were prelabeled with [P]orthophosphate before treatment with 100 µM CPT-cAMP, 2 µM thapsigargin (TG), 1 µM TPA, or 10 nM CCK for 1 h. CT was then immunoprecipitated from whole cell lysates, separated by SDS-PAGE and autoradiographed. The data are the mean and S.E. of four or five experiments. Treatment with CPT-cAMP in combination with TG or TPA was performed twice with identical results and does not appear in graphical form.



A Role for Serine/Threonine Protein Phosphatases in Decreasing CT Phosphate Levels

CT is known to be phosphorylated on multiple serine residues in cultured cells(6, 9, 23) . Thus, the role of serine/threonine phosphatases in mediating the phosphorylation of CT in acinar cells was investigated (Fig. 5). Pretreatment of cells for 20 minutes with the potent serine/threonine phosphatase inhibitors okadaic acid (3 µM) or calyculin A (100 nM) prior to the addition of CCK completely inhibited the reduction in phosphorylated CT. Both of these compounds are potent inhibitors of type 1 and 2A protein phosphatases(24) . Inhibition of the CCK effect on CT by either compound required high concentrations. Indeed, the effects of okadaic acid were only evident when used at concentrations greater than 300 nM (not shown). This is in agreement with concentrations of okadaic acid previously shown to affect amylase secretion and pancreatic phosphoproteins(16) . Further, inhibition by calyculin A also occurred over a very narrow range; no effects were seen below 30 nM with full inhibition occurring at 100 nM (not shown). In addition to blocking the decrease in phosphorylated CT, the inhibitors also caused the appearance of a slightly slower migrating form of CT both in control and CCK-stimulated cells (Fig. 5). Formation of this doublet suggested an enhanced level of phosphorylation of CT with phosphatase inhibition, however a significant increase in total phosphorylation intensity was not detected. At micromolar concentrations, okadaic acid is also known to inhibit protein phosphatase 2B(25) . To investigate a possible role for this phosphatase, cells were treated with the immunosuppressant cyclosporin A, a potent and specific inhibitor of 2B phosphatase in acinar cells(26) . At a concentration known to fully inhibit phosphatase 2B in acinar cells (1 µM), cyclosporin A had no effects on CT phosphorylation either in control or CCK stimulated cells.


Figure 5: Inhibition of CCK-induced decreases in CT phosphate levels by phosphatase inhibitors. Acini were prelabeled with [P]orthophosphate for 1.5 h before the addition of 100 nM calyculin A (cal-A), 3 µM okadaic acid (OA), or 1 µM cyclosporin A (CsA). Following a 20-min preincubation with phosphatase inhibitors, cells were treated with or without CCK for 1 h in the continued presence of phosphatase inhibitor. CT was then immunoprecipitated from whole cell lysates, separated by SDS-PAGE, and autoradiographed. Results shown are representative of three experiments.



Immunofluorescence Localization of CT

The subcellular distribution of CT was determined with cryostat sections of isolated pancreatic acini (Fig. 6). Immunofluorescence staining was confined predominantly to acinar nuclei at high dilutions (1:300-1:1200) of antibody. Although nuclei for the most part were uniformly stained, immunofluorescence in some nuclei was restricted primarily to the region of the nuclear envelope (arrow). These results are consistent with biochemical and immunocytochemical evidence localizing CT to the nuclei of CHO, HepG2, NIH-3T3, and L-cells(27) . No significant changes in CT distribution were detected in cells that had been treated with 10 nM CCK for 1 h (data not shown). At high concentrations of antibody (1:100), a prominent staining in the basal cytoplasm was also present (data not shown). Similar patterns of fluorescence staining for these dilutions of antibody used on acini were seen with cryostat sections of intact, formaldehyde-fixed pancreas (data not shown). While both the nuclear and cytoplasmic staining were abolished by preincubation with the peptide antigen used to generate the antibody, the specificity of the cytoplasmic staining remains uncertain due to the high concentrations of antibody necessary to detect this signal.


Figure 6: Immunofluorescence localization of CT in pancreatic acini. Acinar nuclei were stained at a 1:1200 dilution of affinity purified anti CT N antibody. In some nuclei, staining was confined to the periphery, likely corresponding to the nuclear envelope (arrow).



Western Analysis of CT

Whole cell lysates prepared from acini treated identically to those used for immunoprecipitations were separated by SDS-PAGE and immunoblotted using N antibody (Fig. 7). Consistent with previous studies(10, 15) , a band with a molecular mass of approximately 42 kDa was seen in control cells. Intensity of the 42-kDa protein was found to decrease upon treatment with increasing concentrations of CCK. In addition, a second immunoreactive band of approximately 38 kDa was detected with CCK treatment (Fig. 7A). The presence of this smaller protein also appeared to increase with increasing concentrations of CCK, suggesting that CT was in fact undergoing partial degradation. Because this antibody recognizes the amino terminus of CT, the results suggest that proteolysis was occurring at the carboxyl end of the protein. Further, the absence of this 38-kDa band in the immunoprecipitations also suggest this fragment was not phosphorylated. Pretreatment of cells with 100 nM calyculin A was found to prevent the degradative effects of CCK and abolished the appearance of the 38-kDa band. When the same samples were subjected to electrophoresis for a longer period of time to achieve a greater separation, the 42-kDa protein was found to exist as a doublet (Fig. 7B, arrows). Slower migrating forms of CT have previously been shown to be the result of a differential phosphorylation of the protein (9) . Consistent with a dephosphorylation of CT, the enzyme was found to shift to the faster migrating form upon treatment with increasing amounts of CCK. Moreover, pretreatment of cells with calyculin A before CCK stimulation shifted the protein to the slower migrating form, suggesting an increased phosphate content of the molecule. Under these conditions the 38-kDa band ran off the gel. Samples were also analyzed from soluble and particulate fractions of acini prepared using a digitonin release buffer as detailed in Methods indicating that CCK treatment caused no apparent translocation of the enzyme (data not shown).


Figure 7: Down-regulation of CT in acinar cells following CCK treatment. Acinar cells, maintained under conditions identical to those used in immunoprecipitation experiments, were treated as control (lane 1) or with 0.1, 1, or 10 nM CCK for 1 h (lanes 2, 3, and 4, respectively). Cells were also preincubated with 100 nM calyculin A for 20 min prior to and throughout the addition of 10 nM CCK for 1 h (lane 5). A, whole cell lysates separated by 10% SDS-PAGE and immunoblotted using anti CT N-antibody which reacted with two proteins of approximately 42 and 38 kDa (arrows). B, the same samples were electrophoresed for a longer time, revealing that the 42-kDa protein migrates as two forms (arrows).



CT Activity Is Inhibited by CCK

Matozaki et al.(5) have previously demonstrated that a 1-h treatment with 1 nM CCK partially inhibits both cytosolic and particulate CT activity in acinar cells. Consistent with these findings, whole cell CT activity was found to decrease to 63% of control following a 1-h treatment with 10 nM CCK (Table 1). Interestingly, treatment of cells with 100 nM calyculin A for 1 h also partially but significantly inhibited CT activity to 81% of control. Exposure of cells to both calyculin A and CCK partially prevented the inhibition to a level of activity consistent with the phosphatase inhibitor alone; although this treatment was not statistically significant from the activity measured for CCK alone. Also in agreement with previous studies(5) , CT activity was inhibited by CCK in both soluble and particulate fractions prepared by digitonin permeabilization (data not shown).




DISCUSSION

In the present study, CT was found to be a regulated phosphoenzyme in isolated pancreatic acinar cells. Immunoprecipitation of CT from P-labeled acini demonstrated the molecule to be highly phosphorylated in control cells and to undergo a marked decrease in phosphate levels in response to pancreatic secretagogues. Furthermore, the results presented here also support a mechanism in which stimulation by CCK triggers CT down-regulation within the cell leading to a reduction of its enzymatic activity.

The CCK-induced reduction in CT phosphate levels followed a time course consistent with the inhibition of phosphatidylcholine synthesis previously reported by Matozaki et al.(5) . Relatively high concentrations of CCK were required to elicit these effects; the IC for inhibition of phosphatidylcholine synthesis was 100 pM(5) , while the EC for the attenuation of phosphorylated CT was approximately 800 pM. Both of these CCK concentrations are consistent with an activation of the low affinity state of the CCK(A) receptor(20) . Also in agreement with the inhibitory effects of CCK on phosphatidylcholine synthesis (5) , a decrease in phosphorylated CT was only apparent upon stimulation with Ca-mobilizing secretagogues and could not be stimulated by the cAMP signaling hormone secretin nor through protein kinase activation using TPA or CPT-cAMP. However, whereas inhibition of phosphatidylcholine synthesis could be stimulated using the calcium ionophore A23187(5) , the present study demonstrated that a reduction in phosphorylated CT was accomplished only by a combination of increased [Ca](i) and activation of protein kinase C. Moreover, a complete loss of CT phosphate also required addition of CPT-cAMP to cells. Neither thapsigargin nor the Ca ionophore ionomycin altered CT phosphate levels. The reason for this difference at present is unknown. One possibility is that Ca may have effects on CT separate from promoting a decrease in phosphorylated CT. However, it was reported that physiological concentrations of Ca did not significantly alter CT activity in vitro(5) . It is also possible that Ca may influence other aspects of the CDP-choline pathway such as choline uptake, a process that was also partially inhibited by CCK(5) .

Whether the decrease in phosphorylated CT stimulated by CCK is a direct result of CT degradation or rather reflects a dephosphorylation of the protein is not clear. Although the portion of total enzyme that was phosphorylated in control cells is not known, these data do clearly demonstrate that maximal CCK treatment caused a complete loss of CT phosphate while decreasing CT protein levels by approximately 50%. It is possible that CT was present in both a phosphorylated and nonphosphorylated state in control cells. The total loss of CT phosphate may suggest that the phosphorylated form of the molecule is most susceptible to proteolysis following CCK treatment. Supporting this was the appearance of the 38-kDa CT fragment by immunoblotting, indicating that proteolysis was occurring at the carboxyl terminus of the molecule. A previous study has established that phosphorylation of CT in vivo is confined exclusively to serine residues near its carboxyl terminus(23) ; thus, cleavage of that end of the protein may potentially explain the loss of phosphate seen with CCK treatment. This is also supported by the absence of any P-labeled 38-kDa fragment with immunoprecipitation, suggesting that it was not phosphorylated. Inhibition of the CCK response by the phosphatase inhibitors may then be explained by their effects on CCK signaling rather than a direct inhibition of CT dephosphorylation. These compounds have previously been demonstrated to inhibit CCK-induced changes in protein phosphorylation as well as secretion at a point beyond the generation of second messengers in acinar cells(16, 28, 29) .

Alternatively, these results are also consistent with a mechanism in which the CCK-induced dephosphorylation of CT triggers its down-regulation in the cell. Here again the complete loss of CT phosphate in comparison to the partial degradation of the protein with CCK treatment may support this mechanism. Further, immunoblotting of CT revealed that not only did CCK treatment dose-dependently decrease CT protein levels, but also shifted the intact molecule to a faster migrating form on SDS-PAGE, suggesting the protein was being dephosphorylated prior to its degradation. Moreover, phosphatase inhibition not only blocked CT degradation, but also shifted the molecule to a slower and presumably more phosphorylated form. Finally, while pleiotropic effects of the phosphatase inhibitors cannot be ruled out, these compounds did fully inhibit the effects of CCK on CT phosphorylation as well as its down-regulation in acinar cells and therefore, may implicate a direct role for a serine/threonine phosphatase in mediating this response.

Studies performed in cultured cells (9, 10) and in vitro(7) have indicated that phosphorylation of CT may inhibit its activity. This mechanism may account for the present finding that, in the absence of CCK, addition of the phosphatase inhibitor calyculin A to acinar cells significantly attenuated CT activity. Enhanced phosphorylation of CT in response to calyculin A was suggested by the appearance of a slower migrating form of the protein following immunoprecipitation. Further, while the rate of phosphate turnover on CT was not measured, the cells were in the presence of phosphatase inhibitor for a total of 80 min before preparation of lysates, making the conditions favorable for an enhanced phosphorylation of the enzyme. Thus, although a significant increase in phosphorylation intensity was not detected, it is possible that the high basal levels of P could mask any subtle changes in phosphate content caused by the inhibitor. Alteratively, because CT is phosphorylated at multiple sites(6, 23) , treatment with calyculin A may have promoted a differential phosphorylation of CT in each band. A differential phosphorylation of CT appearing in two bands following immunoprecipitation of CT in cytosolic extracts of CHO cells has previously been demonstrated by phosphopeptide mapping(9) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-41122 and American Cancer Society Grant BE126. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Institutes of Health National Research Service Award fellowship DK-08819. To whom correspondence should be addressed: Dept. of Physiology, University of Michigan, School of Medicine, 7737 Med. Sci. II, Ann Arbor, MI. Tel.: 313-764-9456; Fax: 313-936-8813.

(^1)
The abbreviations used are: CCK, cholecystokinin; CT, CTP:phosphocholine cytidylyltransferase; CHO, Chinese hamster ovary cells; [Ca], free ionized intracellular calcium; TPA, 12-O-tetradecanoylphorbol-13-acetate; CPT-, 8-(4-chlorophenylthio)-; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Noel Badyna for her technical assistance with immunofluorescence localization.


REFERENCES

  1. Exton, J. (1990) J. Biol. Chem. 265, 1-4 [Abstract/Free Full Text]
  2. Matozaki, T., and Williams, J. A. (1989) J. Biol. Chem. 264, 14729-14734 [Abstract/Free Full Text]
  3. Rivard, N., Rydzewska, G., Lods, J.-S., Martinez, J., and Morisset, J. (1994) Am. J. Physiol. 266, G62-G70
  4. Rubin, R. P., Hundly, T. R., and Adolf, M. A. (1992) Biochim. Biophys. Acta 1133, 127-132 [Medline] [Order article via Infotrieve]
  5. Matozaki, T, Sakamoto, C., Nishisaki, H., Suzuki, T., Wada, K., Matsuda, K., Nakano, O., Konda, Y., Nagao, M., and Kasuga, M. (1991) J. Biol. Chem. 266, 22246-22253 [Abstract/Free Full Text]
  6. Kent, C. (1990) Prog. Lipid Res. 29, 87-105 [Medline] [Order article via Infotrieve]
  7. Sanghera, J. S., and Vance, D. E. (1989) J. Biol. Chem. 264, 1215-1223 [Abstract/Free Full Text]
  8. Watkins, J. D., and Kent, C. (1990) J. Biol. Chem. 265, 2190-2197 [Abstract/Free Full Text]
  9. Watkins, J. D., and Kent, C. (1991) J. Biol. Chem. 266, 21113-21117 [Abstract/Free Full Text]
  10. Wang, Y., MacDonald, J. I. S., and Kent, C. (1993) J. Biol. Chem. 268, 5512-5518 [Abstract/Free Full Text]
  11. Houweling, M., Jamil, H., Hatch, G. M., and Vance, D. E. (1994) J. Biol. Chem. 269, 7544-7551 [Abstract/Free Full Text]
  12. Watkins, J., Wang, Y., and Kent, C. (1992) Arch. Biochem. Biophys. 292, 360-367 [CrossRef][Medline] [Order article via Infotrieve]
  13. Utal, A. K., Jamil, H., and Vance, D. E. (1991) J. Biol. Chem. 266, 24084-24091 [Abstract/Free Full Text]
  14. Weinhold, P. A., Charles, L., and Feldman, D. A., (1994) Biochim. Biophys. Acta 1210, 335-347 [Medline] [Order article via Infotrieve]
  15. Watkins, J. D., and Kent, C. (1992) J. Biol. Chem. 267, 5686-5692 [Abstract/Free Full Text]
  16. Wagner, A. C. C., Wishart, M. J., Yule, D. I., and Williams, J. A. (1992) Am. J. Physiol. 262, C1172-C1180
  17. Weinhold, P. A., and Feldman, D. A. (1992) Methods Enzymol. 209, 248-258 [Medline] [Order article via Infotrieve]
  18. Barthel, L. K., and Raymond, P. A. (1990) J. Histochem. Cytochem. 38, 1383-1388 [Abstract]
  19. Ernst, S. A., Crawford, K. M., Post, M. A., and Cohn, J. A (1995) Am. J. Physiol. 267, C990-C1001
  20. Williams, J. A., and Blevins, G. T. (1993) Physiol. Rev. 73, 701-721 [Free Full Text]
  21. Matozaki, T., Goke, B., Tsunoda, Y., Rodriguez, M., Martinez, J., and Williams, J. A. (1990) J. Biol. Chem. 265, 6247-6254 [Abstract/Free Full Text]
  22. Yule, D. I., and Williams, J. A. (1994) in Physiology of the Gastrointestinal Tract (Johnson, L. R., ed) 3rd Ed., pp. 1447-1472, Raven Press, New York
  23. MacDonald, J. I. S., and Kent, C. (1994) J. Biol. Chem. 269, 10529-10537 [Abstract/Free Full Text]
  24. Ishihara, H., Martin, B. L., Brautigan, D. L., Karaki, H., Ozaki, H., Kato. Y., Fusetani, N., Watabe, S., Hashimoto, K., Uemura, D., and Hartshorne, D. J. (1989) Biochem. Biophys. Res. Commun. 159, 871-877 [Medline] [Order article via Infotrieve]
  25. Cohen P. (1989) Annu. Rev. Biochem. 58, 453-508 [CrossRef][Medline] [Order article via Infotrieve]
  26. Groblewski, G. E., Wagner, A. C. C., and Williams, J. A. (1994) J. Biol. Chem. 269, 15111-15117 [Abstract/Free Full Text]
  27. Wang, Y., Sweitzer, T. D., Weinhold, P. A., and Kent, C. (1993) J. Biol. Chem. 268, 5899-5904 [Abstract/Free Full Text]
  28. Wagner, A. C. C., Schafer, C., and Williams, J. A. (1992) Biochem. Biophys. Res. Commun. 189, 1606-1612 [Medline] [Order article via Infotrieve]
  29. Meyer-Alber, A., Fetz, I., Waschulewski, I. H., Hocker, M., Folsch, U. R., and Schmidt, W. E. (1994) Biochem. Biophys. Res. Commun. 201, 1470-1476 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.