AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 285: G726-G734, 2003. First published June 11, 2003; doi:10.1152/ajpgi.00111.2003
0193-1857/03 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/4/G726    most recent
00111.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schäfer, C.
Right arrow Articles by Groblewski, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schäfer, C.
Right arrow Articles by Groblewski, G. E.

HORMONES AND SIGNALING

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Claus Schäfer,1 Hanna Steffen,1 Karen J. Krzykowski,2 Burkhard Göke,1 and Guy E. Groblewski2

1Department of Internal Medicine II, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, 81377 Munich, Germany; and 2Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706

Submitted 11 March 2003 ; accepted in final form 3 June 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112–2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 µM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

exocrine pancreas; protein phosphatase; cyclic adenosine 5'-monophosphate; secretin; cholecystokinin; Ca2+-regulated heat-stable protein of 24 kDa

PANCREATIC ACINAR CELLS ARE responsible for the synthesis, storage, and regulated secretion of digestive enzymes that are essential for nutrient digestion and absorption. Both the synthesis and secretion of digestive enzymes are physiologically regulated by gastrointestinal hormones such as cholecystokinin (CCK) and secretin that activate a host of intracellular kinases and phosphatases (for review, see Refs. 31 and 32). Stimulation of the secretin-VIP-glucagon receptor family leads to the activation of adenylate cyclase via G proteins in acinar cell plasma membranes and results in an elevation of cAMP levels (9). Binding of cAMP to the regulatory subunit of protein kinase A (PKA) promotes the dissociation and activation of the catalytic subunit of the kinase. Alternatively, CCK, bombesin, and muscarinic cholinergic receptors in acini are also coupled to heterotrimeric G proteins that stimulate phosphoinositide metabolism through activation of phospholipase C (31, 32). This results in cleavage of phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-triphosphate and diacylglycerol, which in turn, respectively, liberates Ca2+ from intracellular stores and activates protein kinase C (PKC). Thus secretagogue stimulation of cAMP and phospholipase C signaling pathways leads to the acute activation of kinases and phosphatases that are pivotal to the regulation of acinar cell function (9, 31, 32).

Previous studies aimed at identifying acinar cell regulatory proteins that respond to secretagogue stimulation have utilized a proteomic approach of metabolically labeling cells with [32P]orthophosphate, followed by protein separation using one- and two-dimensional electrophoresis (3, 4, 8, 26, 36). This method has led to the identification of a number of key regulatory proteins that mediate acinar cell protein translation (2, 18), actin cytoskeletal dynamics (10, 21), and membrane trafficking events necessary for acinar secretion (13, 24, 25). Additionally, this strategy was used to isolate a novel physiological substrate to the Ca2+-calmodulin-regulated protein phosphatase calcineurin (also known as PP2B) termed Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) (11, 14). CRHSP-24 was purified from rodent pancreas on the basis of the finding that its acute dephosphorylation on serine residues was inhibited by the immunosupressant drugs cyclosporin A and FK-506, which are potent and selective inhibitors of calcineurin (14).

CRHSP-24 is a paralog of the brain-specific H3 histone mRNA-binding protein PIPPin (5, 20). PIPPin and CRHSP-24 contain a highly conserved cold-shock domain that is flanked on each side by RNA-binding motifs. PIPPin is thought to function as a translational regulatory protein, because it was shown to bind to the polyadenylated 5'-untranslated region of the H1° and H3.3 histone mRNAs and thereby inhibit translation of these messages in vitro (20). Interestingly, CRHSP-24 was recently reported to coimmunoprecipitate with the STYX/dead phosphatase enzyme from spermatid lysates (35). STYX is a phosphoserine/phosphothreonine/phosphotyrosine binding protein that is homologous to the dual-specificity phosphatase family but is inactivated catalytically by the endogenous substitution of an essential cysteine to glycine in the active site of the enzyme (33). As such, STYX is an inactive or dead phosphatase that complexes to phosphoserine/phosphothreonine and phosphotyrosine motifs in proteins and may act as an antagonist to protein tyrosine phosphatases (34). Genetic deletion of the STYX protein in rodents disrupts spermatid development (35).

The present study demonstrates that CRHSP-24 dephosphorylation is differentially activated in cells by Ca2+-, PKC-, and cAMP-mediated pathways. Furthermore, it is shown that, although CRHSP-24 dephosphorylation in response to Ca2+-mobilizing stimuli is regulated by calcineurin, the effects of PKC and cAMP are mediated through a calyculin A-, okadaic acid (OA)-, and fostriecin-sensitive serine phosphatase, likely PP2A or PP4. Collectively, these data indicate that multiple kinase and phosphatase signaling pathways acutely regulate CRHSP-24 phosphorylation, suggesting that this molecule plays a pivotal role in acinar cell metabolism.


   EXPERIMENTAL METHODS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Secretin and CCK were purchased from Research Plus. The cell-permeable cAMP analog 5,6-dichloro-1-{beta}-D-ribo-furanosylbenzimidazole-3,5-monophosphorothioate Sp isomer (cBIMPS) was obtained from Biolog (Bremen, Germany). Streptolysin-O was from Wellcome Diagnostics, and calyculin A, OA, and cyclosporin A were from Calbiochem. Purified collagenase was from Worthington Biochemicals, and minimal essential amino acids were from GIBCO. The agents 12-o-tetradecanoylphorbol-13-acetate (TPA), 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), fostriecin, and all other chemicals were obtained from Sigma. Peroxidase-conjugated secondary antibodies were from Amersham. The CRHSP-24-specific polyclonal antiserum was previously characterized (14).

Preparation of pancreatic acini. The preparation of pancreatic acini has been described previously (3, 4). Briefly, pancreas from Sprague-Dawley rats was digested with purified collagenase, mechanically dispersed, and passed through a 150-µm mesh nylon cloth. Acini were purified by centrifugation in 4% BSA and then suspended in incubation buffer consisting of a HEPES-buffered Ringer solution (pH 7.40) containing 0.1% BSA. Where indicated, acini were preincubated for 30 min with phosphatase inhibitors before protein phosphorylation was measured or secretory studies were initiated.

32P-labeling of isolated acini. Isolated acini were suspended in HEPES-buffered Ringer solution without added phosphate and were labeled with radioactive 32PO4 (800 µCi/ml) for 2 h at 37°C with or without calyculin A present during the last 30 min. Aliquots of cells were stimulated with cBIMPS and TPA for 5 min before sonication in isoelectric focusing (IEF) solubilization buffer containing 9 M urea, 4% Nonidet P-40, 2% ampholytes, and 1% mercaptoethanol. Protein content was determined with a Bio-Rad protein assay kit. IEF was conducted on acinar cell protein (100 µg) by using a Millipore two-dimensional electrophoresis system. Gels were exposed to Kodak x-Omat AR films at –80°C for different times ranging from 6 to 36 h.

IEF and immunoblotting. Acinar cells were pretreated for 30 min with calyculin A or cyclosporin A before stimulation for 5 min with indicated agents. Samples were sonicated in IEF solubilization buffer. Equal amounts of protein (30 µg) were separated by IEF in urea-containing gels and then were transferred to nitrocellulose membranes. Membranes were immunoblotted with CRHSP-24-specific antiserum (1:1,000) and were identified by using peroxidase-conjugated secondary antibodies (1:5,000). Intensity of the CRHSP-24 phosphoisoforms was quantified by densitometry by using a PDI model DNA35 scanner interfaced with the Protein and DNA Imageware system (PDI, Huntington Station, NY). Each phosphoisoform is expressed as a percentage of the total CRHSP-24 phosphoisoforms present in each treatment condition.

{alpha}-Toxin-permeabilized cells. Acini were suspended in a permeabilization buffer containing (in mM) 20 1,4-piperazinediethanesulfonic acid (pH 6.6), 139 K+-glutamate, 4 EGTA, 1.78 MgCl2, and 2 MgATP, with 0.1 mg/ml soybean trypsin inhibitor, 1 mg/ml bovine serum albumin, and 200 U/ml {alpha}-toxin. The {alpha}-toxin was allowed to bind to the cells on ice for 10 min at 4°C. Acini were then aliquoted into Microfuge tubes containing the indicated amounts of OA or fostriecin. Cell suspensions were immersed in a 37°C water bath for 10 min before being stimulated with CPT-cAMP for an additional 5 min. Cells were quickly pelleted in a Microfuge and solubilized in IEF buffer.

Amylase secretion. Acini were pretreated with calyculin A for 30 min and then resuspended in permeabilization buffer containing 0.5 IU/ml streptolysin-O, 5 mM EGTA, and 1 mM MgATP. The amount of CaCl2 and MgCl2 to be added to give final concentrations of 1 mM free Mg2+ and 10 µM free Ca2+ was calculated using a computer program as previously described (17). Permeabilized acini were incubated in the presence or absence of calyculin A for 20 min at 30°C before measuring amylase content in the medium using the Boehringer Mannheim Monotest. All experiments were conducted in duplicate. Amylase release was also assayed in duplicate and expressed as the percentage of total cellular amylase present at the start of the experiment. Statistical analysis of the data was performed with the SYSTAT computer program (Systat, Evanston, IL). Analysis of variance was followed by Duncan's multiple-range test.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Phosphatase inhibition alters TPA- and cAMP-mediated acinar cell protein phosphorylation. Metabolic labeling of isolated pancreatic acini with [32P]orthophosphate followed by phosphoprotein analysis using two-dimensional gel electrophoresis allows the distinction of up to 300 phosphoproteins, ~30 of which have been shown to be acutely regulated by secretagogues (36). A number of these regulated phosphoproteins have been identified in acini by protein purification and use of specific antibodies (10, 13, 14, 18, 36). A typical phosphoprotein pattern from 32P-labeled acini is shown in Fig. 1. Previous studies indicate that incubation of acini with high concentrations of OA, which inhibits type 1, 2A, and 2B serine/threonine phosphatases, dramatically alters the overall pattern of phosphoproteins on two-dimensional gels (28). In the present study, treatment of cells with a high concentration (100 nM) of the type 1 and 2A serine/threonine phosphatase inhibitor calyculin A by itself increased the overall phosphorylation intensity of acinar proteins but did not alter the phosphoprotein pattern seen under basal conditions.



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 1. Effects of phosphatase inhibition by calyculin A on basal protein phosphorylation in isolated acinar cells. Autoradiographs of total acinar cell phosphoproteins (100 µg) analyzed under control conditions (A) and following incubation with 100 nM calyculin A (B) are shown. Isolated acini were incubated in [32P]orthophosphate for 2 h, with calyculin A present during the last 30 min of incubation. Proteins were separated by 2-dimensional electrophoresis and detected by autoradiography. Regions marked with boxes indicate positions of the myristoylated alanine-rich C kinase substrate (MARCKS) protein (A, top) and Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24; A, bottom), which are analyzed in detail in Figs. 2 and 3. The positions of the {alpha}- and {beta}-phosphoisoforms of CRHSP-24 are indicated.

 



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 2. CRHSP-24 is dephosphorylated in response to cAMP and phorbol ester stimulation in acinar cells. Acinar cells were labeled with [32P]orthophosphate and preincubated with or without 100 nM calyculin A (Cal-A) before stimulation with 100 µM of the cAMP analog 5,6-dichloro-1-{beta}-D-ribo-furanosylbenzimidazole-3,5-monophosphorothioate (cBIMPS) or 1 µM 12-o-tetradecanoylphorbol-13-acetate (TPA) for 5 min. CRHSP-24 was detected by autoradiography following 2-dimensional electrophoresis. A: representative gels from each treatment condition. The position of the {alpha}-, {beta}-, and {gamma}-phosphoisoforms of CRHSP-24 are indicated in the basal state. Arrows indicate increase ({uparrow}), decrease ({downarrow}) or no change (->) in the {alpha}-phosphoisoform compared with basal levels. The area of the gel containing CRHSP-24 is shown in Fig 1. B: quantitative densitometric analysis of CRHSP-24 phosphorylation ({alpha}-isoform). Data are means ± SE of at least 3 independent experiments.

 


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 3. Effect of type 1 and 2A phosphatase inhibition on TPA-stimulated MARCKS phosphorylation. Acinar cells were labeled with [32P]orthophosphate and preincubated with or without calyculin A before a 5-min stimulation with 100 µM of the cAMP analog cBIMPS or 1 µM TPA. MARCKS phosphorylation was analyzed by autoradiography following 2-dimensional electrophoresis. A: representative gels from each treatment condition. The area of the gel containing the MARCKS protein is shown in Fig 1. B: quantitative densitometric analysis of MARCKS phosphorylation. Data are means ± SE of at least 3 independent experiments.

 
Isolated acini were treated with the phorbol ester TPA or the cell-permeable cAMP analog cBIMPS, which was previously shown to stimulate amylase secretion and activate PKA in acini (22). Each compound induced distinct changes in acinar cell protein phosphorylation, the most notable of which were detected in proteins seen at 24 and 80 kDa. The 24-kDa protein, known as CRHSP-24, was previously purified from rat pancreas on the basis of its secretagogue-regulated dephosphorylation by the Ca2+-dependent phosphatase calcineurin (14), whereas the TPA-sensitive acidic phosphoprotein at 80 kDa was previously identified as the myristoylated alanine-rich C kinase substrate (MARCKS) protein utilizing anti-MARCKS specific antibodies (36). Because the secretagogue-regulated phosphorylation of CRHSP-24 and MARCKS was previously characterized in acini by two-dimensional electrophoresis, these proteins served as excellent markers to evaluate the effects of phosphatase inhibition by calyculin A on cAMP- and TPA-mediated signaling in acini.

Stimulation of cells with either cBIMPS or TPA induced a marked dephosphorylation of CRHSP-24, resulting in ~60% decrease in total radiolabeled phosphate incorporation into the protein (Fig. 2). Incubation of cells with 100 nM calyculin A was found to increase the basal phosphorylation of CRHSP-24 by ~20%. Moreover, dephosphorylation of CRHSP-24 in response to both cBIMPS and TPA was inhibited by calyculin A pretreatment. These findings were unexpected, because CRHSP-24 was previously shown to be regulated by PP2B in acinar cells based on the findings that it is markedly dephosphorylated in response to Ca2+-mobilizing secretagogues and furthermore that its dephosphorylation is inhibited by the immunosupressants cyclosporin A and FK-506 (11, 14). Calyculin A has been shown to inhibit protein phosphatases PP1 and PP2A with roughly equal potency (IC50 values 1–2 nM) but has little or no effect on PP2B or PP2C (16, 23). Consequently, these data suggested that CRHSP-24 is acted on by a serine phosphatase other than PP2B in response to cAMP and TPA stimulation.

Because MARCKS is a known substrate for PKC in cells (1), its phosphorylation was used to evaluate the specificity of cBIMPS- and TPA-stimulated kinase activation in acini. Incubation of cells with TPA induced approximately threefold increase in phosphate incorporation into MARCKS within 5 min, whereas treatment with cBIMPS had no effect on the protein (Fig. 3). Pretreatment of acini with 30 nM calyculin A altered neither basal nor TPA-induced phosphorylation of MARCKS. In contrast, pretreatment with 100 nM calyculin A significantly augmented the basal levels of MARCKS phosphorylation and modestly enhanced TPA-stimulated phosphorylation of the protein. The selective effects of TPA on MARCKS phosphorylation verified that cBIMPS and TPA were acting through distinct cellular pathways to regulate protein phosphorylation in acini.

Differential effects of PP2B and PP1/PP2A inhibition on CRHSP-24 phosphoregulation. It was previously demonstrated that, under basal conditions, CRHSP-24 is maximally phosphorylated on a minimum of four serine residues and is rapidly dephosphorylated on at least three of these sites in response to secretagogue stimulation of acinar cells (11, 14). These acute changes in CRHSP-24 phosphorylation are easily monitored by alterations in the IEF pattern of the molecule. Thus to fully evaluate the effects of TPA and cAMP analog treatment on CRHSP-24 phosphorylation, immunoblotting with CRHSP-24-specific antiserum was conducted in combination with IEF of acinar lysates (Fig. 4). In the basal state, CRHSP-24 is present mainly as two acidic phosphoisoforms in acini (labeled {alpha} and {beta} in Fig. 4A). Stimulation of cells with the Ca2+-mobilizing secretagogue CCK rapidly dephosphorylates CRHSP-24, and this is reflected by a pronounced alkaline shift in the molecule to two additional phosphoisoforms (labeled {gamma} and {delta} in Fig. 4A). Thus the CCK-induced dephosphorylation of CRHSP-24 is illustrated by reciprocal changes in the intensities of the CRHSP-24 phosphoisoforms seen by immunoblotting. Intensities of the acidic {alpha}- and {beta}-isoforms decrease, giving rise to increased intensities of the {gamma}- and {delta}-isoforms of the protein.



View larger version (50K):
[in this window]
[in a new window]
  		Fig. 4. Cholecystokinin (CCK)-stimulated CRHSP-24 dephosphorylation is partially regulated by PP2B. Isolated acinar cells were treated as control (A) or were preincubated with cyclosporin A (1 µM; B) or calyculin A (30 nM; C) for 30 min before stimulation with 100 µM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), 1 µM TPA, or 1 nM CCK for 5 min. Cell proteins (30 µg) were separated by isoelectric focusing (IEF) and were immunoblotted with CRHSP-24-specific antiserum (1:1,000). Right: representative gels for each experimental condition, indicating the position of the CRHSP-24 phosphoisoforms. Corresponding 32P-labeled isoforms are also indicated in control panels in Fig 1 and 2. Note the alkaline shift in CRHSP-24 phosphoisoforms following CCK stimulation. Left: CRHSP-24 phosphoisoforms ({alpha}, {beta}, {gamma}, and {delta}) were quantified by densitometry and expressed as %total CRHSP-24 present in each sample. Note the expanded y-axis in C reflecting the large increase in basal phosphorylation of CRHSP-24 by calyculin A treatment. Data are means ± SE of 3–5 independent experiments, each performed in duplicate. Statistical analysis was calculated by ANOVA, and significant differences from basal values were designated as: aP < 0.05, bP < 0.01, and cP < 0.001.

 

In agreement with 32P-labeling experiments (see Fig. 2), CRHSP-24 was significantly dephosphorylated following treatment of acini with the cell-permeable cAMP analog CPT-cAMP, resulting in a 25% decrease in the intensity of the {alpha}-isoform and a 60% increase in the {gamma}-isoform of the protein. Like cBIMPS, CPT-cAMP was previously shown to regulate protein phosphorylation in 32P-labeled acinar cells (12). Unlike CCK treatment, CPT-cAMP did not shift the protein to the most alkaline {gamma}-isoform. Treatment of cells with TPA also promoted CRHSP-24 dephosphorylation, as evidenced by a significant increase in the intensity of the {gamma}-isoform.

As previously reported (11, 14), complete inhibition of PP2B activity in acini by 1 µM cyclosporin A strongly inhibited CRHSP-24 dephosphorylation in response to CCK (Fig. 4B). However, close analysis of the blots revealed that CRHSP-24 remained significantly dephosphorylated by CCK in the presence of cyclosporin A compared with control cells; the intensity of the {gamma}-isoform remained elevated by approximately two-fold compared with basal levels. Similarly, CRHSP-24 dephosphorylation in response to CPT-cAMP or TPA treatment was not altered by calcineurin inhibition using cyclosporin A. Dephosphorylation of CRHSP-24 following a combined treatment with TPA and CPT-cAMP was not additive (data not shown), suggesting that these agents were acting by a similar mechanism.

Incubation of acini with 30 nM calyculin A induced a marked acidic shift of CRHSP-24 to the {alpha}-isoform under basal conditions and completely abolished the effects of CPT-cAMP and TPA to dephosphorylate the protein (Fig. 4C). Consistent with the partial inhibition of CCK-induced dephosphorylation of CRHSP-24 following PP2B inhibition, the effects of CCK were also partially inhibited by calyculin A treatment (compare Fig. 4A and Fig. 4C), confirming that the protein is indeed regulated by multiple serine phosphatases in acinar cells.

Characterization of the cAMP-stimulated CRHSP-24 phosphatase. Mammalian cells are known to express a variety of serine/threonine phosphatases, including PP1, PP2A, PP2B, P2C, PP4, and PP5 (reviewed in Ref. 23). OA is a marine sponge toxin that inhibits PP2A and PP4 with an IC50 of 0.1–1 nM (16, 23), PP5 with an IC50 of ~3 nM (23), PP1 with an IC50 of 100 nM (23), PP2B with an IC50 >10 µM (16, 23), and has no effect on PP2C (23). Thus OA can be used to distinguish potential phosphatase activities toward various substrates. Because the permeability of OA varies in different cell types (23), acini were permeabilized with {alpha}-toxin before treatment with OA to maximize its permeability and then were stimulated with CPT-cAMP (Fig. 5A). OA inhibited CPT-cAMP-stimulated CRHSP-24 dephosphorylation in a concentration-dependent manner, with nearly complete inhibition occurring at 10 nM OA, strongly suggesting a potential role for PP2A or PP4 in regulating CRHSP-24 phosphorylation. To further confirm these findings, cells were also incubated with fostriecin, an antibiotic from Streptomyces pulveraceus that is highly selective for PP2A (IC50 = 3.2 nM) but is a weak inhibitor of PP1 (IC50 = 131 µM) (29). Similar to OA, low concentrations (1 and 3 µM) of fostriecin strongly inhibited CPT-cAMP-mediated CRHSP-24 dephosphorylation. These concentrations of fostriecin were previously shown to have minimal effects on PP1 and PP2B but did significantly inhibit PP2A and PP4 (15, 29). Collectively, these data suggest that CRHSP-24 is regulated by PP2A or PP4 in response to cAMP elevation in acinar cells.



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 5. cAMP-mediated CRHSP-24 dephosphorylation is regulated by a type 2A or type 4 serine phosphatase. Isolated acinar cells were permeabilized with {alpha}-toxin and then preincubated with okadaic acid (A) or fostriecin (B) for 10 min before stimulation of cells with 100 µM CPT-cAMP for 5 min. Cell proteins (30 µg) were separated by IEF and were immunoblotted with CRHSP-24-specific antiserum (1:1,000). CRHSP-24 phosphoisoforms ({alpha}, {beta}, and {gamma}) were quantified by densitometry and expressed as %total CRHSP-24 present in each sample. For ease in comparison, intensity of the {beta}-isoform, which did not significantly change with treatments, is not shown in the graphs. Data are means ± SE of 3 independent experiments, each performed in duplicate. *Nonspecific protein present in {alpha}-toxin that cross-reacted with the CRHSP-24 antibody.

 

Secretin stimulates CRHSP-24 dephosphorylation. The finding that CRHSP-24 was dephosphorylated after acinar treatment with cell-permeable analogs of cAMP suggested that the protein is regulated by physiological secretagogues that are coupled to adenylate cyclase activation in acini. To examine this possibility, CRHSP-24 dephosphorylation in response to various concentrations of the gastrointestinal hormone secretin was investigated (Fig. 6). Secretin evoked a concentration-dependent dephosphorylation of CRHSP-24, with a maximal response detected at 1 nM and an EC50 of ~300 pM. The effects of secretin were maximal within 2 min of acinar stimulation and were sustained for up to 15 min in the continued presence of hormone (data not shown). These results clearly demonstrate that CRHSP-24 dephosphorylation occurs in response to physiological secretagogues that elevate intracellular cAMP levels.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6. Secretin regulates CRHSP-24 phosphorylation in acini. Isolated acinar cells were treated with the indicated concentrations of secretin for 5 min, and total cell proteins (30 µg) were separated by IEF. CRHSP-24 phosphorylation was analyzed by immunoblotting. CRHSP-24 phosphoisoforms in each condition were quantified by densitometry and expressed as %total CRHSP-24. Secretin-induced changes in the {gamma}-phosphoisoform were used to calculate the concentration-dependent effects of the hormone to dephosphorylate CRHSP-24. Data are means ± SE of 4 independent experiments performed in duplicate.

 

Type 1 and 2A serine/threonine phosphatases mediate cAMP-stimulated acinar cell secretion. Inhibition of CRHSP-24 dephosphorylation by cyclosporin A was previously shown to be closely correlated with the inhibition of stimulated amylase secretion in acini (11). Similarly, calyculin A has been shown to strongly inhibit Ca2+-stimulated digestive enzyme secretion in streptolysin-O-permeabilized cells (27). Studies were conducted to examine if type 1 and 2A serine/threonine phosphatases also altered cAMP-mediated secretion under similar conditions (Fig. 7). Treatment of cells with a combination of TPA and elevated Ca2+ to mimic the effects of secretagogues that signal through the phospholipase C-mediated pathway induced a greater than fivefold increase in amylase secretion over 30 min. In comparison, stimulation of acini with secretin or cBIMPS increased acinar cell secretory activity by greater than fourfold over basal levels. Pretreatment of acini with 30 nM calyculin A did not significantly alter the secretory response to any of the agents (data not shown). However, preincubation with 100 nM calyculin A attenuated Ca2+ plus TPA-stimulated secretion by ~40% and reduced the secretin- and cBIMPS-induced secretion by 50%. These findings support previous studies demonstrating an important role for the type 1 and 2A phosphatases in acinar cell secretion (19, 27, 28, 30).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7. Phosphatase inhibition by calyculin A inhibits cAMP-mediated amylase secretion. Acini were treated as control (Con) or preincubated with 100 nM calyculin A for 30 min and then permeabilized with streptolysin-O and stimulated for 20 min at 30°C with indicated agonists. Cells were stimulated with 10 µM free Ca2+ plus 1 µM TPA, 10 nM secretin (Sec), or 100 µM cBIMPS. Amylase secretion into the media was determined and expressed as %total cellular amylase content present at start of experiment. Values are means ± SE of 4 independent experiments, each performed in duplicate. *P < 0.01; **P < 0.001.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The acute regulation of CRHSP-24 phosphorylation by secretagogues that signal through both the phospholipase C and adenylate cyclase pathways clearly supports the pharmacological data that TPA and cAMP analogs act to dephosphorylate CRHSP-24 in acinar cells. Furthermore, the finding that cAMP-stimulated dephosphorylation was completely inhibited following phosphatase inhibition by calyculin A, OA, or fostriecin but was not affected following calcineurin inhibition with cyclosporin A indicates that CRHSP-24 is regulated by an additional serine phosphatase, likely PP2A or PP4, other than calcineurin. Comparison of the IEF patterns of CRHSP-24 seen following CCK and secretin stimulation demonstrate that CCK dephosphorylated a minimum of three and secretin a minimum of two serine sites on the protein (see Fig. 8). The enhanced dephosphorylation by CCK likely reflects the simultaneous activation of Ca2+, PKC, and cAMP pathways by the hormone (9, 31, 32). Interestingly, treatment of cells with Ca2+ ionophore produced an identical pattern of CRHSP-24 dephosphorylation to that seen for CCK (data not shown). Furthermore, identically to CCK, Ca2+-induced CRHSP-24 dephosphorylation was only partially inhibited by cyclosporin A treatment. Although elevated Ca2+ may influence other signaling pathways, including PKC, these data may also suggest that the same serine site(s) undergo overlapping regulation by different phosphatases. CRHSP-24 contains 14 serine residues; however, its precise phosphorylation sites have not yet been elucidated.



View larger version (22K):
[in this window]
[in a new window]
  		Fig. 8. Proposed scheme of CRHSP-24 regulation in acinar cells. CRHSP-24 is phosphorylated on at least 4 serine sites under basal conditions (14). A: elevation of cellular Ca2+ activates the Ca2+-calmodulin-dependent phosphatase PP2B, leading to CRHSP-24 dephosphorylation on at least 2 sites. Alternatively, agents such as secretin that elevate cAMP activate PP2A or PP4 and dephosphorylate CRHSP-24 on a minimum of 2 sites. Whether the PP2B and PP2A/PP4 regulated sites overlap is uncertain. B: stimulation by CCK activates multiple pathways, including Ca2+, cAMP, and diacylglycerol (DAG), leading to dephosphorylation of CRHSP-24 on a minimum of 3 sites.

 

CRHSP-24 is most highly phosphorylated in the basal state, suggesting that it is controlled by a constitutively active kinase. Thus the cAMP- and TPA-stimulated dephosphorylation of CRHSP-24 may involve a decrease in kinase activity rather than an acute activation of the phosphatase. Alternatively, Feschenko et al. (7) recently demonstrated that PP2A may be acutely activated in cells by cAMP and furthermore that this pathway functions independently of PKA, because kinase inhibition does not alter phosphatase activation. Likewise, cAMP has also been shown to activate the guanine nucleotide-exchange factor Epac independently of PKA activation (6). However, the Epac-specific cAMP analog 8-(4-chlorophenylthio)-2'-O-methyl-cAMP had no effects on CRHSP-24 phosphorylation (data not shown). It is additionally possible that TPA and cAMP promote a translocation of CRHSP-24 within the cytoplasm to promote its dephosphorylation. In any case, the rapid effects of second messengers to regulate CRHSP-24 phosphorylation are clearly consistent with a close association of the protein with phosphatase enzymes and furthermore suggest that dephosphorylation provides a triggering mechanism to regulate CRHSP-24 function within the cell.

Use of serine/threonine phosphatase inhibitors in pancreatic acinar cells has clearly implicated a role for these enzymes in secretagogue-stimulated acinar cell secretion (19, 22, 27, 28, 30). Consistent with previous studies (27), treatment of acini with 30 nM calyculin A had no effect on acinar secretion, whereas 100 nM of the inhibitor markedly reduced amylase secretion in response to Ca2+, TPA, secretin, and cBIMPS. These data indicate that inhibition of amylase release by type 1 and 2A phosphatase inhibition likely occurs downstream from the generation of second messengers. The finding that 30 nM calyculin A fully abolished CRHSP-24 dephosphorylation but did not alter secretion in response to cAMP elevation argues against a role for CRHSP-24 in the exocytotic pathway of acini as previously suggested (11). However, compared with the effects of CCK, which acts through Ca2+, PKC, and cAMP, CRHSP-24 was only partially dephosphorylated in response to cAMP elevation, leaving the possibility that a full dephosphorylation of the protein by multiple phosphatases is required to induce CRHSP-24 function.

CRHSP-24 is a paralog of the brain-specific histone mRNA binding protein PIPPin (5). PIPPin has been shown to bind to H1° and H3.3 mRNAs and to prevent translation of these messages in vitro (20); however, a definitive role for the protein has not yet been described in intact cells. Consistent with mRNA binding activity, CRHSP-24 contains a cold-shock domain flanked on each side by RNA-binding motifs (amino acids 73–91), suggesting that the protein may act as a translational regulatory molecule. Pancreatic acinar cells are known undergo high rates of protein translation to support digestive enzyme secretion. Furthermore, protein translation is tightly regulated by secretagogues (reviewed in Ref. 31) and is disrupted by serine/threonine phosphatase inhibition (30). Thus it is tempting to speculate that CRHSP-24 plays an important role in acinar cell protein metabolism.

The recent findings that CRHSP-24 is present in a complex with the STYX/dead phosphatase protein in developing spermatids (35) provides an additional layer of complexity to the dynamic characteristics of CRHSP-24 phosphorylation. The combined regulation of CRHSP-24 by Ca2+, cAMP, PKC, PP2B, and PP2A or PP4 serine phosphatases suggests a centralized role for the protein in acinar cell metabolism. Further studies addressing the serine phosphorylation sites and potential translational regulatory activity of CRHSP-24 should provide valuable insight into secretagogue-regulated pancreatic function.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
G. E. Groblewski was supported by a National Science Foundation Award (MCB-0094154) and a United States Department of Agriculture Cooperative State Research Education and Extension Service Program award (WIS04221 and WIS04444). C. Schäfer was supported by a grant from the Deutsche Forschungsgemeinschaft (SCHA-766/3–1).


   ACKNOWLEDGMENTS
 
We thank A. C. C. Wagner for helpful suggestions and discussion. A very special thanks is also extended to Diana Thomas for excellent technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. E. Groblewski, Dept. of Nutritional Sciences, 1415 Linden Drive, Univ. of Wisconsin, Madison, WI 53706 (Email: groby{at}nutrisci.wisc.edu).

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


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Arbuzova A, Schmitz AA, and Vergeres G. Cross-talk unfolded: MARCKS proteins. Biochem J 362: 1–12, 2002.[ISI][Medline]
  2. Bragado MJ, Groblewski GE, and Williams JA. Regulation of protein synthesis by cholecystokinin in rat pancreatic acini involves PHAS-I and the p70 S6 kinase pathway. Gastroenterology 115: 733–742, 1998.[ISI][Medline]
  3. Burnham DB, Munowitz P, Hootmann SR, and Williams JA. Regulation of protein phosphorylation in pancreatic acini. Biochem J 235: 125–131, 1986.[ISI][Medline]
  4. Burnham DB and Williams JA. Effects of carbachol, cholecystokinin, and insulin on protein phosphorylation in isolated pancreatic acini. J Biol Chem 257: 10523–10528, 1982.[Abstract/Free Full Text]
  5. Castiglia D, Scaturro M, Nastasi T, Cestelli A, and DiLeigro I. PIPPin, a putative RNA-binding protein specifically expressed in the rat brain. Biochem Biophys Res Commun 218: 390–394, 1996.[ISI][Medline]
  6. Enserink JM, Christensen AE, de Rooij J, van Triest M, Schwede F, Genieser HG, Doskeland SO, Blank JL, and Bos JL. A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nat Cell Biol 4: 901–906, 2002.[ISI][Medline]
  7. Feschenko MS, Stevenson E, Nairn AC, and Sweadner J. A novel cAMP-stimulated pathway in protein phosphatase 2A activation. J Pharmacol Exp Ther 302: 111–118, 2002.[Abstract/Free Full Text]
  8. Freedman SD and Jamieson JD. Hormone-induced protein phosphorylation. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid. J Cell Biol 95: 903–908, 1982.[Abstract/Free Full Text]
  9. Gardner JD and Jensen RT. Receptors for secretagogues on pancreatic acinar cells. In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by Go VWL, Di Magno EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA. New York: Raven, 1993, p. 151–166.
  10. Groblewski GE, Grady T, Mehta N, Lambert H, Logsdon CD, Landry J, and Williams JA. Cholecystokinin stimulates heat shock protein-27 phosphorylation in rat pancreas both in vivo and in vitro. Gastroenterology 112: 1354–1361, 1997.[ISI][Medline]
  11. Groblewski GE, Wagner ACC, and Williams JA. Cyclosporin A inhibits Ca2+/calmodulin dependent protein phosphatase and secretion in pancreatic acinar cells. J Biol Chem 269: 1511–1517, 1994.
  12. Groblewski GE, Wang Y, Ernst SA, Kent C, and Williams JA. Cholecystokinin stimulates the down-regulation of CTP-phosphocholine cytidylytransferase in pancreatic acinar cells. J Biol Chem 270: 1437–1442, 1995.[Abstract/Free Full Text]
  13. Groblewski GE, Wishart MJ, Yoshida M, and Williams JA. Purification and identification of a novel calcium-regulated heat-stable protein. J Biol Chem 271: 31502–31507, 1996.[Abstract/Free Full Text]
  14. Groblewski GE, Yoshida M, Bragado JM, Leykam J, and Williams JA. Purification and characterization of a novel calcineurin substrate in mammalian cells. J Biol Chem 273: 22738–22744, 1998.[Abstract/Free Full Text]
  15. Hastie CJ and Cohen PTW. Purification of protein phosphatase 4 catalytic subunit: inhibition by the antitumor drug fostriecin and other tumor suppressers and promoters. FEBS Lett 431: 357–361, 1998.[ISI][Medline]
  16. Ishihara H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato Y, Fusetani N, Watabe S, Hashimoto K, Uemura D, and Hartshorne DJ. Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 159: 871–877, 1989.[ISI][Medline]
  17. Kitagawa M, Williams JA, and DeLisle RC. Interactions of intracellular mediators of amylase secretion in permeabilized pancreatic acini. Biochim Biophys Acta 1073: 129–135, 1991.[Medline]
  18. Knight SA, Kohr W, and Korc M. Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2. Biochim Biophys Acta 1092: 196–204, 1991.[Medline]
  19. Meyer-Alber A, Höcker M, Fetz I, Fornefeld H, Waschulewski HI, Fölsch UR, and Schmidt WE. Differential inhibitory effects of serine/threonine phosphatase inhibitors and a calmodulin antagonist on phosphoinositol/calcium- and cyclic adenosine monophosphate-mediated pancreatic amylase secretion. Scand J Gastroenterol 30: 384–391, 1995.[ISI][Medline]
  20. Nastasi T, Scaturro M, Bellafiore M, Raimondi L, Beccari S, Cestelli A, and DiLeigro I. PIPPin is a brain-specific protein that contains a cold-shock domain and binds specifically to H1 degrees and H3.3 mRNAs. J Biol Chem 274: 24087–24093, 1999.[Abstract/Free Full Text]
  21. Schaefer C, Ross SE, Bragado JM, Groblewski GE, and Williams JA. A role for the p38 MAPkinase/HSP 27 pathway in cholecystokinin-induced changes in actin cytoskeleton in rat pancreatic acini. J Biol Chem 273: 24173–24180, 1998.[Abstract/Free Full Text]
  22. Schäfer C, Steffen H, Printz H, and Göke B. Effects of synthetic cyclic AMP analogs on amylase exocytosis from rat pancreatic acini. Can J Physiol Pharmacol 72: 1138–1147, 1994.[ISI][Medline]
  23. Sheppeck JE, Gauss CM, and Chamberlin R. Inhibition of the Ser-Thr phosphatases PP1 and PP2A by naturally occurring toxins. Bioorg Med Chem 5: 1739–1750, 1997.[Medline]
  24. Thomas DH, Kaspar KM, Taft WB, Weng N, and Groblewski GE. Identification of annexin VI as a CRHSP-28 binding protein in pancreatic acinar cells. J Biol Chem 277: 39456–39502, 2002.[Abstract/Free Full Text]
  25. Thomas DH, Taft WB, Kaspar KM, and Groblewski GE. CRHSP-28 regulates Ca2+-stimulated secretion in permeabilized acinar cells. J Biol Chem 276: 28866–28872, 2001.[Abstract/Free Full Text]
  26. Vandermeers A, Vandermeers-Piret MC, Delange JP, Wieland J, and Christophe J. Phosphorylation of three particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin, and cholecystokinin (CCK). Regul Pept 5: 359–365, 1984.
  27. Wagner ACC, Schäfer C, and Williams JA. Effects of calyculin A on amylase release in streptolysin-O permeabilized acinar cells. Biochem Biophys Res Commun 189: 1606–1612, 1992.[ISI][Medline]
  28. Wagner ACC, Wishart MJ, Yule DI, and Williams JA. Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling. Am J Physiol Cell Physiol 263: C1172–C1180, 1992.[Abstract/Free Full Text]
  29. Walsh AM, Cheng A, and Honkanen RE. Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 and 2A is highly selective for PP2A. FEBS Lett 416: 230–234 1997.[ISI][Medline]
  30. Waschulewski IH, Kruse ML, Agricola B, Kern HF, and Schmidt WE. Okadaic acid disrupts Golgi structure and impairs enzyme synthesis and secretion in the rat pancreas. Am J Physiol Gastrointest Liver Physiol 270: G939–G947, 1996.[Abstract/Free Full Text]
  31. Williams JA. Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis, and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 63: 77–97, 2001.[ISI][Medline]
  32. Williams JA and Yule DI. Stimulus-secretion coupling in pancreatic acinar cells. In: The Pancreas: Biology, Pathobiology, and Disease (2nd ed.), edited by Go VWL, Di Magno EP, Gardner JD, Lebenthal E, Reber HA, and Scheele GA. New York: Raven, 1993, p. 167–188.
  33. Wishart MJ, Denu JM, Williams JA, and Dixon JE. A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase. J Biol Chem 270: 26782–26785, 1995.[Abstract/Free Full Text]
  34. Wishart MJ and Dixon JE. Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem Sci 23: 301–306, 1998.[ISI][Medline]
  35. Wishart MJ and Dixon JE. The archetype STYX/dead-phosphatase complexes with a spermatid mRNA-binding protein and is essential for normal sperm production. Proc Natl Acad Sci USA 99: 2112–2117, 2002.[Abstract/Free Full Text]
  36. Wishart MJ, Groblewski GE, Göke B, Wagner ACC, and Williams JA. Secretagogue regulation of pancreatic acinar cell protein phosphorylation shown by two-dimensional electrophoresis. Am J Physiol Gastrointest Liver Physiol 267: G676–G686, 1994.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
S. Z. Husain, W. M. Grant, F. S. Gorelick, M. H. Nathanson, and A. U. Shah
Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin
Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1594 - G1599.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
S. J. Crozier, M. D. Sans, L. Guo, L. G. D'Alecy, and J. A. Williams
Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice
J. Physiol., June 15, 2006; 573(3): 775 - 786.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
285/4/G726    most recent
00111.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schäfer, C.
Right arrow Articles by Groblewski, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schäfer, C.
Right arrow Articles by Groblewski, G. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.