(Received for publication, September 13, 1994; and in revised form, November 9, 1994)
From the
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
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.
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
[H]choline (2) or
[
H]myristic acid (3) demonstrating an
activation of phospholipase C and D by secretagogues such as
cholecystokinin (CCK), (
)carbamylcholine, and bombesin.
Similarly, it has also been shown that carbamylcholine treatment of
acini which were prelabeled with [
C]glycerol
resulted in a significant decrease in the levels of
[
C]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.
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.
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.
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.
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.
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).
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).
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
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
]
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) .