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CRHSP-28 Regulates Ca2+-Stimulated Secretion in Permeabilized Acinar Cells.
Diana D.H. Thomas, William B. Taft, Kala M. Kaspar and Guy E. Groblewski.
Department of Nutritional Sciences, University of Wisconsin, Madison WI, 53706
Running Title: CRHSP-28 regulates secretion in acinar cells.
Correspondence:Guy E. Groblewski, Ph.D.University of WisconsinDepartment of Nutritional Sciences1415 Linden DriveMadison, WI 53706
Phone: (608) 262-0884Fax (608) 262-5830Email: [email protected]
1
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on May 30, 2001 as Manuscript M102214200 by guest on July 11, 2019
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Summary
CRHSP-28 is a Ca2+-regulated phosphoprotein, abundant in the apical cytoplasm of epithelial
cells that are specialized in exocrine protein secretion. To define a functional role for the protein
in pancreatic secretion, recombinant CRHSP-28 (rCRHSP-28) was introduced into
streptolysin-O (SLO) permeabilized acinar cells and amylase secretion in response to elevated
Ca2+ was determined. Secretion was markedly enhanced by rCRHSP-28 over a time course
that closely corresponded with the loss of the native protein from the intracellular compartment.
No effects of rCRHSP-28 were detected until approximately 50% of the native protein was lost
from the cytosol. Secretion was enhanced by rCRHSP-28 over a physiological range of Ca2+
concentrations with 2-3 fold increases in amylase release occurring in response to low
micromolar levels of free Ca2+. Further, rCRHSP-28 augmented secretion in a concentration
dependent manner with minimal and maximal effects occurring at 1 and 25 µg/ml, respectively.
Covalent crosslinking experiments demonstrated that native CRHSP-28 was present in a 60 kDa
complex in cytosolic fractions and in a high molecular mass complex in particulate fractions,
consistent with the slow leak rate of the protein from SLO permeabilized cells. Probing acinar
lysates with rCRHSP-28 in a gel-overlay assay identified two CRHSP-28 binding proteins of
35 (pp35) and 70 kDa (pp70). Interestingly, preparation of lysates in the presence of 1 mM
Ca2+ resulted in a marked redistribution of both proteins from a cytosolic to a Triton X-100
insoluble fraction, suggesting a Ca2+-sensitive interaction of these proteins with the acinar cell
cytoskeleton. In agreement with our previous study immunohistochemically localizing CRHSP-
28 around secretory granules in acinar cells, gel-overlay analysis revealed pp70 co-purified with
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acinar cell secretory granule membranes. These findings demonstrate an important cell-
physiological function for CRHSP-28 in the Ca2+-regulated secretory pathway of acinar cells.
Introduction
Exocrine cells specialized in protein secretion release a variety of factors necessary for
normal function of the digestive, urogenital, respiratory and ocular systems. Activation of these
epithelia by neural and humoral agents stimulates the exocytosis of secretory granules at the
apical plasma membrane in a process that is largely controlled by cellular Ca2+. In pancreatic
acinar cells, Ca2+ release is initiated in the apical pole and then propagates through the cell
periphery to the basal cytoplasm. The cyclic reuptake and release of Ca2+ from intracellular
stores creates an oscillatory mode of signaling with spatial and temporal characteristics that are
unique to the specific type and concentration of physiologic stimulus (reviewed in 1-3). While
the high concentrations of Ca2+ generated in the apical cytoplasm are necessary for secretory
granule trafficking and exocytosis to occur, a comprehensive understanding of the molecular
events elicited by this ion is lacking.
The secretory pathway in acinar cells is a multifactorial process beginning with the
microtubule-directed transport of newly formed zymogen granules (ZGs)1 to the apical
cytoplasm. To ultimately reach the plasma membrane, ZGs are released from microtubules and
must penetrate a prominent subapical cytoskeletal web composed of actin and intermediate
filaments (4). ZG movement along microtubules was shown to involve motor proteins from both
the microtubule minus-end directed dyneine/dynactin complex (5) as well as the plus-end
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directed kinesin family (6,7). Similarly, a potential role for myosin motor proteins in facilitating
ZG exocytosis has also been described based on the localization of myosin I to ZG membranes
(8), myosin II to the apical cytoplasm (8,9), and the acute activation of myosin light chain kinase
in response to secretogogues (10). In addition to ZG movement via cytoskeletal motor proteins, a
selective reorganization of subapical actin filaments in response to acinar cell stimulation has
been described (9,11,12). Muallem et al. (11) demonstrated that partial depolymerization of
actin filaments in permeabilized acini with the enzyme thymolysin independently stimulated
exocytosis in the absence of Ca2+. On the other hand, complete depolymerization of filaments
fully arrested agonist-induced secretion, indicating that distinct alterations in these structures are
necessary for normal secretion to occur (11). Valentijn et al. (13) recently reported that just prior
to reaching the plasma membrane, ZGs release the small GTP-binding protein Rab3D and
become coated with filamentous actin in a process that may facilitate the movement of granules
across the terminal web. Interestingly, other studies utilizing actin filament destabilizing agents
in pancreatic lobules demonstrated an integral role for the subapical web in controlling the
compensatory retrieval of ZG granule membranes back into the cell following exocytosis
(14,15). Actin filament destabilization not only inhibited membrane retrieval, but also led to the
inhibition of secretion by sequestering granule contents into large vacuolar structures that
appeared continuous with the apical plasma membrane (14).
Similar to nerve and endocrine cells, the final step in ZG fusion with apical plasma
membrane is mediated by protein components of the SNARE1 apparatus (reviewed in 16).
Acinar cells possess a number of isoforms of the SNARE protein family members (16,17). Use
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of clostridial neurotoxins to selectively cleave SNARE proteins in streptolysin-O (SLO)1
permeabilzed acini (18) and in an in vitro membrane fusion system (17) indicate a direct role for
these proteins in ZG membrane fusion, however, the major ZG-associated SNARE protein that
mediates this process is currently unknown. Although it is clear from these studies that a
considerable overlap of regulatory proteins exists between acinar cells and other secretory cell
types, it is also evident that acini express unique regulatory proteins to direct the cytoskeletal and
membrane fusion events that coordinate ZG exocytosis.
Utilizing a proteomic approach to identify proteins that are functionally regulated by Ca2+ in exocrine
pancreas, we previously purified a regulated phosphoprotein, termed CRHSP-281 (19,20). Also known as D52
(21), N8 (22,23), R10 (24) and CSPP-28 (25) this protein has been identified based on its over expression in
transformed epithelial cells (21,22,24) and Ca2+-sensitive phosphorylation in gastric mucosa (25). Moreover,
Byrne et al. (26) demonstrated that CRHSP-28 belongs to a novel tumor protein D52 family (TPD52)1 comprised
of at least 3 homologous genes that may undergo alternative splicing to create different protein products (27). Using
polyclonal specific antibodies, we recently demonstrated that CRHSP-28 is abundant in digestive epithelial cells
specialized in protein secretion including acinar cells from pancreas, salivary and lacrimal glands, as well as chief
cells, paneth cells and goblet cells present throughout the gastrointestinal mucosa (20).
Despite a lack of hydrophobic membrane association motifs, CRHSP-28 partitions between soluble and
particulate fractions following cell lysis. Further, immunohistochemical localization indicates a high concentration
of CRHSP-28 surrounding secretory granules in the apical cytoplasm of acini (20). These findings together with
the acute Ca2+-sensitivity of CRHSP-28 phosphorylation suggest the protein may be involved in modulating acinar
cell secretion. To test this hypothesis, the present study takes advantage of the high solubility of purified
recombinant CRHSP-28 protein (rCRHSP-28)1, allowing its introduction into SLO-permeabilized pancreatic
acinar cells. The ability of rCRHSP-28 to partially restore the Ca2+-dependent secretory activity following the
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leakage of soluble proteins from the intracellular compartment directly demonstrates an important role for CRHSP-
28 in the later steps of the secretory pathway. Investigation of the molecular interactions of CRHSP-28 led to the
identification of a 70 kDa CRHSP-28 binding protein (pp70)1 that is present in ZG membranes. It is proposed that
CRHSP-28 functions in acinar cell secretion by modulating the delivery of secretory granules to the apical plasma
membrane via dynamic interactions with the pp70 protein.
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Methods
Materials: Soybean trypsin inhibitor, benzamidine, phenylmethylsulfonylfluoride (PMSF)1, Triton X-100
and Percol were purchased from Sigma (St. Louis, MO), and a protease cocktail containing leupeptin, ABESF, and
E64 from Cal Biochem (San Diego, CA). Bovine serum albumin and peroxidase conjugated secondary antibody
were from Amersham Corp. (Arlington Heights, IL), Bis-(sulfosuccinimidyl) suberate (BS3)1 crosslinker and
protein A beads from Pierce (Rockford, IL), SLO from Difco (Detroit, MI), Phadebas amylase assay kit from
Pharmacia Upjohn (Upsala, Sweden) and protein determination reagent from Bio Rad (Hercules, CA). The anti-rat
cysteine string protein polyclonal antibody was purchased from StressGen (Victoria, BC). Bacterial expression and
purification of recombinant human CRHSP-28 protein and characterization of the affinity purified anti-CRHSP-28
polyclonal antibodies were detailed previously (19,20).
Isolation of rat acini: Pancreatic acinar cells were isolated from adult, male Sprague-Dawley rats by
collagenase digestion as previously described (28,29). Acini were suspended in a buffer consisting of (in mM) 10
HEPES, 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 0.6 Na2HPO4, 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, 1 mg/ml bovine serum albumin, gassed with 100% O2 and adjusted to pH 7.4.
Cells were maintained at 37 oC for 1 hr prior to performing assays.
Acinar Cell Permeabilization: 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, 2 Mg-ATP, 0.1 mg/ml soybean trypsin inhibitor, 1 mg/ml bovine serum
albumin and 0.5 units/ml SLO. The SLO was allowed to bind to the cells on ice for 10 min and
was then removed by washing at 4 oC in the same buffer without SLO. Acini were aliquoted
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into microfuge tubes (200 µl/tube) containing the indicated amounts of rCRHSP-28. The cell
suspension was then diluted with an equal volume of the same buffer containing enough CaCl2
to create the desired final concentration of free Ca+2. The quantity of Ca2+ added to the buffer
was calculated based on dissociation constants using a computer program as described (30). Cell
suspensions were immersed in a 37 oC water bath and incubated with gentle mixing for the
indicated times. At the end of the incubation period, cells were cooled in an ice bath and then
centrifuged at 12,000 x g for 1 min. The content of amylase in the medium was determined using
a Phadabas assay kit. Data were calculated as the percent total cellular amylase present in an
equal amount of cells measured at the start of the experiment.
CRHSP-28 leakage from SLO-permeabilized cells: SLO-permeabilized acini were
exposed to basal or stimulatory concentrations of free Ca2+ and, at indicated times, cells were
pelleted in a microfuge. Proteins present in the medium were precipitated in 15% trichloroacetic
acid and then solubilized in SDS-sample buffer. Cell pellets were sonicated in a lysis buffer
containing (in mM) 50 Tris-base (pH 7.4), 25 NaFl, 10 tetrasodium pyrophosphate, 5 EDTA,
0.2% Triton-X100, 1 mM PMSF, 2 mM benzamidine and protease inhibitor cocktail. Total
protein was measured using a BioRad assay reagent. Equal amounts of cellular protein (50
µg/sample) and equal volumes of culture medium were analyzed for CRHSP-28 content by
immunobloting using enhanced chemiluminescence. The intensity of the CRHSP-28 signal was
quantified using a PDI model DNA35 scanner interfaced with the Protein and DNA Imageware
System (Huntington Station, NY).
Crosslinking studies: Acini were lysed in phosphate buffered saline containing 1 mM
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PMSF and 10 µg/ml leupeptin by repeated freeze/thawing in liquid nitrogen. The BS3
crosslinking reagent was prepared fresh in H20 and added to lysates at room temperature for 30
min. The crosslinking reaction was quenched by addition of 20 mM glycine at 4 oC for 10 min.
Soluble and particulate fractions were obtained by centrifugation at 12,000 x g for 30 min. Equal
amounts of protein were analyzed by immunoblotting following SDS-PAGE1.
Gel-overlay assays: Proteins were separated by SDS-PAGE, immobilized to
nitrocellulose membrane and blocked in Tris-buffered saline containing 0.3% Tween-20 and
3% nonfat milk for 1 hour at room temperature. Membranes were sequentially incubated with
rCRHSP-28 (2-4 µg/ml) and anti-CRHSP-28 antibody (0.5 µg/ml) for 1 hr at room temp.
CRHSP-28-binding proteins were then detected using a horseradish peroxidase conjugated
secondary antibody (1:5000).
For pp70 cell fractionation experiments, acini were sonicated in lysis buffer without
Triton X-100 and containing 1 mM CaCl2 or 5 mM EGTA. Cytosolic and particulate fractions
were prepared by centrifugation (100,000 x g). Particulate fractions were further sonicated in the
same buffer containing 0.2% Triton X-100 and centrifuged again to produce a membrane and
Triton-insoluble fraction. The detergent-insoluble proteins were dissolved directly in SDS
buffer. Equal amounts of cytosolic and membrane protein (40 µg), and equal volumes of
detergent-insoluble protein (1/10 of total volume) were separated by SDS-PAGE and analyzed
by gel-overlay or immunoblotting.
Other procedures: CRHSP-28 was immunoprecipitated from equal amounts of lysate
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(0.7 mg) from 32P-labeled acini as previously described (31). Zymogen granules, granule
membranes and granule content were prepared as detailed previously (32,33).
Results
CRHSP-28 increases Ca2+-stimulated secretion in permeabilized acini: A role for
CRHSP-28 in pancreatic secretion was examined utilizing a well-characterized SLO-
permeabilized acinar cell preparation (11,18,30,34) (Fig 1). Isolated acini were permeabilized
with SLO and exposed to basal (< 10 nM) or stimulatory (10 µM) concentrations of free Ca2+.
Secretion of the digestive enzyme, amylase into the medium was measured at various times over
30 min. Consistent with previous studies utilizing SLO permeabilized acini (30,34), the highest
rate of Ca2+-stimulated secretion occurred during the first 10 min of incubation when 6.1% of
total cellular amylase was released into the medium (filled squares). A considerably slower rate
of secretion occurred during the following 10 min intervals summating to 9.5 and 11.9% of total
cellular amylase at 20 and 30 min, respectively. Basal secretion in low Ca2+ represented 3.2%
of total over the entire 30 min incubation (open squares). To illustrate the diminished secretory
response caused by SLO-permeabilization of cells, secretion from intact acini stimulated with
the calcium ionophore, ionomycin is included on the graph for comparison (triangles). Intact and
SLO-treated acini showed similar rates of secretion over the first 10 min of stimulation;
however, secretion at later times was significantly diminished in the permeabilized cells.
Introduction of rCRHSP-28 into permeabilized acini had no effect on secretion during
the first 10 min of Ca2+-stimulation (filled circles). However, rCRHSP-28 augmented
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secretion by greater than 160% of untreated cells when measured at 20 and 30 min. Basal
secretion in low Ca2+ was not altered by rCRHSP-28 at any time (open circles), indicating that
the ability of the protein to modulate secretion was dependent on increased cellular Ca2+.
Addition of rCRHSP-28 to cells that had been pre-permeabilized for 10 min, washed and
resuspended in incubation buffer containing the protein had no significant effect on amylase
secretion (not shown). Rather, it was necessary for rCRHSP-28 to be present in the medium
throughout the entire incubation period to see a significant effect. For control measures,
rCRHSP-28 was introduced into the SLO-permeabilized cells in the presence of 1 mg/ml
bovine serum albumin and 0.1 mg/ml soybean trypsin inhibitor. Accordingly, rCRHSP-28
represented approximately 2% of the total protein in the incubation medium, demonstrating the
specificity of the CRHSP-28 effect.
CRHSP-28 leakage from SLO-permeabilized acini: Previous studies indicate that SLO-
permeabilization of acini is complete within 2-5 min at 37 oC and is not altered by micromolar
concentrations of free Ca2+ (11,30,34). To ensure complete permeabilization of cells in our
preparations, diffusion of the 12 kDa cytosolic protein, cyclophilin A from acini was analyzed by
immunoblotting following SLO treatment (Fig 2A, upper gel). The majority of cyclophilin A
was rapidly lost from permeabilized acini within 2 min and was completely absent from the
intracellular compartment by 5 min. Moreover, elevation of Ca2+ in the medium did not
influence the rate or extent of cyclophilin A leakage.
To examine the loss of native CRHSP-28 from permeabilized acini, the content of the
protein both inside and outside the cells was measured over time following SLO treatment (Fig
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2A-C). In contrast to the rapid and complete diffusion of cyclophilin A, a much smaller
proportion of CRHSP-28 was lost from the intracellular compartment over 15 min when cells
were incubated in basal Ca2+. With prolonged exposures of the blots, it was estimated that 50%
of CRHSP-28 remained in the cells after 15 min, consistent with the levels of the protein found
in the particulate fraction following cell lysis (see Fig 5). Interestingly, elevation of Ca2+ in the
medium significantly enhanced CRHSP-28 leakage from acini at all time points (Fig 2B).
Complimentary results were obtained when measuring CRHSP-28 levels in the extracellular
medium (Fig 2C). Again, CRHSP-28 slowly diffused from the cells over the 15 min incubation,
and its appearance in the medium was clearly enhanced at each time point when Ca2+ was
elevated.
Characterization of CRHSP-28-enhanced secretion: Corresponding with previous
studies utilizing SLO-permeabilized acini (30,34), amylase secretion in these preparations was
stimulated over 2 magnitudes of free Ca2+ concentrations, with maximal release occurring
between 3 and 10 µM Ca2+ (Fig 3). Addition of rCRHSP-28 to cells significantly enhanced
secretion over the entire range of Ca2+ concentrations. The effects of the protein were most
pronounced at low stimulatory levels of Ca2+ (0.1-1 µM) when secretion in the absence of
rCRHSP-28 was at a minimum. Indeed, rCRHSP-28 augmented secretion greater than 3 fold
when stimulating cells with 0.3 µM Ca2+. In addition, rCRHSP-28 enhanced secretion in a
concentration-dependent manner with a minimal response detected at 1 µg/ml of protein and a
maximal 210% of control increase occurring at 25 µg/ml of protein when stimulating the cells
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with 1 µM free Ca2+ (Fig. 4). Surprisingly, higher concentrations of rCRHSP-28 (>50 µg/ml)
consistently diminished, and in some cases inhibited, secretion. An identical biphasic
concentration response to rCRHSP-28 was also observed when stimulating cells with 10 µM
free Ca2+ (not shown).
CRHSP-28 binds to a high molecular mass complex in acinar cells: CRHSP-28 has a
bipolar charge distribution composed of a basic middle region flanked by acidic amino and
carboxyl termini (Fig 5A). In addition, the protein contains a putative leucine zipper motif
between amino acids 21-72, suggesting it may form homo- and/or heteromeric complexes
through coiled-coil interactions (35). To begin to identify CRHSP-28 protein interactions in
acini, covalent cross-linking experiments were conducted using the homobifunctional amine
crosslinker BS3. The BS3 reagent, which crosslinks charged amino groups positioned within
11.4 angstroms, was selected due to the abundance of lysine and arginine residues (13% by
frequency) in CRHSP-28. Other than the start site, CRHSP-28 lacks methionine and cysteine
precluding the use of other sulfhydryl reacting crosslinking reagents.
Immunoblot analysis demonstrated a single 28 kDa protein corresponding to a CRHSP-
28 monomer that was equally partitioned between soluble and particulate fractions (Fig 5B, lanes
1 and 2). BS3 crosslinking of lysates nearly abolished the 28 kDa signal in both subcellular
fractions (lanes 3 and 4). The soluble form of crosslinked CRHSP-28 migrated at approximately
60 kDa, consistent with a CRHSP-28 homodimer (lane 3). In contrast, two highly
immunoreactive large molecular mass complexes were detected in the particulate fraction, just
below the loading well and at the interface of the stacking and separating gels (lane 4). Absence
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of a laddering effect of the signal suggested CRHSP-28 was in a complex with additional acinar
cell proteins rather than simply aggregates of CRHSP-28 alone. The same pattern of
crosslinking occurred over a range of BS3 concentrations, with minimal and maximal
crosslinking detected at 0.2 and 2 mM of BS3, respectively (not shown). No significant change
in the distribution of CRHSP-28 in either subcellular fraction was noted in lysates from cells
that had been stimulated with the secretagogue cholecystokinin (not shown).
Detection of CRHSP-28 binding proteins in acini: Immunoprecipitation of CRHSP-28
under non-denaturing conditions from 32P-labeled acini demonstrated a large increase in
CRHSP-28 phosphorylation following treatment with cholecystokinin (Fig 6A). Two additional
phosphoproteins of approximately 35 and 70 kDa (pp35 and pp70, respectively)1 were also
reproducibly precipitated with the CRHSP-28 from the lysate. Neither protein was detected by
immunoblotting, with the same antibody, suggesting they were bound to CRHSP-28 in the
lysate. To verify these results, a gel-overlay analysis was conducted by probing acinar cell
proteins immobilized on a nitrocellulose filter with the rCRHSP-28 protein (Fig 6B). Binding
proteins were then detected using the CRHSP-28 antibody. In comparison to the single 28 kDa
signal seen when immunoblotting acinar proteins, gel-overlay analysis revealed 2 additional
bands at 35 and 70 kDa, consistent with the co-immunoprecipitation experiments. The band at
approximately 45 kDa did not remain upon further washing of the membrane. These same results
were obtained when probing with radiolabled rCRHSP-28 protein (not shown).
Localization of CRHSP-28 binding proteins to membrane and detergent-insoluble
fractions in acini. Gel-overlay analysis of sub-cellular fractions indicated pp 35 was a mostly
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soluble protein, whereas pp70 was equally partitioned between cytosolic and membrane (Triton
X-100 soluble) fractions when isolated in the absence of Ca2+ (Fig 7). Interestingly,
preparation of lysates in the presence of 1 mM Ca2+ resulted in a striking redistribution of both
proteins from the cytosolic to membrane and Triton-insoluble fractions. Due to the difficulty of
measuring the protein content of detergent-insoluble fractions, these samples were dissolved
directly in 2% SDS. Coomassie staining was conducted to ensure equal loading of each sample,
and also demonstrated that Ca2+-treatment of lysates did not cause a large redistribution of total
cellular protein between fractions. Although CRHSP-28 also partitioned between the soluble and
particulate fractions, no consistent redistribution of the protein was detected in multiple
experiments when Ca2+ was included in the lysis buffer (Fig 7B).
pp70 localizes to purified zymogen granules. Previous studies indicated that although
CRHSP-28 immunolocalized to the cytoplasm immediately surrounding zymogen granules
(ZG), the protein was not detected when immunoblotting intact ZGs or ZG membranes (20). The
possibility that the CRHSP-28 binding proteins were localized to these secretory organelles was
therefore investigated (Fig 8). Gel-overlay analysis demonstrated pp70 was highly abundant on
purified ZG membranes, but was not detected in intact ZGs or ZG contents. The absence of a
signal in intact ZGs was due to the high concentration of secretory enzymes compared to the
small amount of granule membrane proteins. This was supported by the lack of signal seen in
ZG contents following granule lysis. The high purity of the ZGs prepared under these conditions
was previously documented using electron microscopy (33). To ensure the purity of our
preparations, the same fractions were probed with antiserum against cysteine string protein.
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Biochemical and immuno-electron microscopic analyses demonstrated that cysteine string
protein is localized exclusively on ZG membranes in pancreatic acini (36,37). Corresponding
with the presence of pp70 in membrane fractions of cells isolated under Ca2+ free conditions
(see Fig 7), ZGs were isolated in buffer lacking added Ca2+. These data demonstrate a Ca2+-
independent association of pp70 with ZG membranes, and clearly support an important
regulatory role for CRHSP-28 and its associated proteins in acinar cell exocytosis.
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Discussion
In rat (34) and mouse (30) acini the highest rates of Ca2+-stimulated secretion occur
during the first 10 min following SLO-permeabilization. The diminished secretory activity at
later times was shown to be due to the loss of soluble proteins diffusing from the cells, as
addition of cytosolic extracts from lacrimal gland or brain were able to restore Ca2+-stimulated
secretion (34). Introduction of rCRHSP-28 into SLO-permeabilized acini markedly enhanced
Ca2+-stimulated amylase release with a time course that corresponded with the loss of the native
protein from the intracellular compartment (compare Fig 1 and 2). No effects of rCRHSP-28
were detected until approximately 50% of the native protein had diffused from the cytoplasm.
Further, it was necessary that rCRHSP-28 be added to the cells during the initial incubation
period. Addition of protein to cells that had been pre-permeabilized and “run down” for 10 min
had little effect, indicating that CRHSP-28 must function in concert with other soluble
regulatory proteins in modulating secretion. Because the secretory pathway in acini
encompasses multiple regulatory steps coordinating cytoskeletal and membrane fusion events,
the specific point at which CRHSP-28 modulates this pathway is uncertain. However, the
inability of CRHSP-28 alone to reconstitute acinar secretion presents a likelihood that the
protein acts at a step preceding the final stage of ZG fusion with the plasma membrane.
Secretion from acini was augmented by rCRHSP-28 over a micromolar range of Ca2+
concentrations, well within the physiological levels detected in secretagogue-stimulated acini
using digital-imaging and microfluorimetry (1-3). Interestingly, a biphasic concentration
response for rCRHSP-28 was seen, with high levels of the protein (> 50 µg/ml, ~2.6 µM) having
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a markedly diminished effect on secretion. Morgan and Burgoyne (38) reported almost identical
effects of the soluble NSF-attachment protein α-SNAP on catacholamine secretion from
digitonin-permeabilized chromafin cells. In regulating secretion, α-SNAP is transiently
associated with the SNARE complex and released during membrane fusion. Subsequent studies
showed that excess levels of the yeast homologue of α-SNAP (Sec17p) arrested membrane
fusion in vitro by stabilizing SNARE complexes and preventing α-SNAP dissociation (39). Our
results may support an analogous on/off role for CRHSP-28 protein interactions in mediating
secretion. Cell fractionation and crosslinking demonstrated CRHSP-28 was bound to a large
protein complex in acini, in agreement with the slow release of the protein from SLO-
permeabilized acini. The Ca2+-enhanced release of CRHSP-28 from permeabilized acini
suggests that cell stimulation increased the dissociation of CRHSP-28 from this complex.
Presumably, flooding the cells with high concentrations of recombinant protein may saturate
CRHSP-28 binding sites leading to a loss of the regulatory activity of the protein.
By expressing the 3 known members of the TPD52 family in the yeast 2-hybrid system,
Byrne et al. (35) demonstrated both homo- and hetromeric interactions between the D52 proteins
that were dependent on an intact coiled-coil motif. Similarly, crosslinking of CRHSP-28 in
acinar lysates produced a 60 kDa protein in soluble fractions consistent with a CRHSP-28
dimer. Gel-overlay analysis also identified an interaction between rCRHSP-28 and the native
protein on the blot (Fig 6). Whether or not other members of the TPD52 family or alternatively
spliced isoforms of CRHSP-28 are expressed in acinar cells is unknown. TPD52 mRNAs were
shown to be present alone and together in different carcinoma cell lines indicating that CRHSP-
28 may be expressed independently of other family members (26). The polyclonal antibody used
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in the present study was raised against full length CRHSP-28 and did not cross react with any
other acinar proteins present on 1 or 2 dimensional SDS gels, suggesting no alternatively spliced
isoforms of CRHSP-28 were present.
Similar to CRHSP-28, the 70 kDa binding protein, pp70, also partitions between
cytosolic and membrane fractions of acinar cells prepared in the absence of Ca2+, indicating it is
unlikely to be an integral membrane protein. Translocation of pp70 to detergent insoluble-
fractions occurs following the addition of Ca2+ to cell lysates, suggesting the ion directly
interacts with pp70 in promoting this effect. Identically, pp35, which was found only in soluble
fractions, also translocated to detergent insoluble fractions in the presence of Ca2+. These
results may suggest that pp70 is a dimer of the 35 kDa protein. However, the distribution of
pp35 and pp70 was unchanged when cell lysates were prepared under reducing conditions in 2%
SDS (+ boiling) or 8 M urea. Alternatively, the large difference in molecular mass between the 2
proteins may indicate that pp35 represents a proteolytic fragment of pp70 rather than a
homologous form of the same molecule.
A potential cytoskeletal role for CRHSP-28 in epithelial cell function was previously
described based on the finding that its expression is dramatically induced following activation of
an epithelial gene program in mesenchymal cells by the adenoviral E1a protein (23). The E1a
protein induces the expression of a number of epithelial specific cytoskeletal regulatory proteins
including desmoplakin, desmogelin, desmocolin, E-cadherin and the cytokeritins K8/K18 (40).
Similarly, ectopic expression of CRHSP-28 in NIH3T3 cells was shown to convert them to a
spheroid shape under low serum conditions (23) suggesting CRHSP-28 modulates cytoskeletal
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elements related to epithelial cell shape and function. In addition, CRHSP-28 shares some
limited homology throughout its coiled-coil region with cytoskeleton-related proteins including
myosin heavy chain and CLIP-170/arrestin (41). In particular, CRHSP-28 is ~30% identical
and ~45% similar to the Drosophila cytoplasmic linker protein, CLIP-190 (41) over a 107 amino
acid region. CLIP-190 was reported to coordinate vesicle interactions between microtubule and
actin filaments by binding to both intact microtubules and the class VI unconventional myosin
protein. In preliminary experiments we have been unable to demonstrate CRHSP-28 binding to
taxol-stabilized microtubules in vitro. Further, the microtubule binding motif in CLIP-190 is
absent in CRHSP-28. A potential interaction between CRHSP-28 and nonmuscle myosin is
currently being investigated.
Contrasting a potential association of CRHSP-28 with actin filaments in acini, we
previously reported that although the protein is localized around ZGs in the apical cytoplasm, it
was absent in the subapical actin web (20). Further, little or no CRHSP-28 was detected in
actin-rich detergent insoluble fractions following cell lysis, suggesting, at best, a weak
association with intact filaments. One possibility is that under conditions of basal Ca2+,
CRHSP-28 is associated with ZGs via an interaction with pp70. Upon an increase in cell Ca2+,
CRHSP-28 dissociates from this complex promoting the translocation of the ZG-bound pp70 to
actin filaments. This would explain the Ca2+-enhanced leakage of CRHSP-28 from SLO-
permeabilized acini and Ca2+-sensitive translocation of pp70 to cytoskleton-rich detergent
insoluble fractions. The importance of Ca2+ in coordinating these events may entail the acute
phosphorylation of CRHSP-28, which occurs on serine residues within seconds of acinar cell
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stimulation (19). Parente, et al. (25) reported that CRHSP-28 is a substrate for the
multifunctional Ca2+/calmodulin dependent protein kinase II in vitro. Interestingly, CaM kinase
II was recently localized to the sub-apical region of acinar cells by immunofluorescence
microscopy (42), indicating that the kinase is positioned precisely at the site of ZG entry into the
terminal web.
Collectively, the a) Ca2+-dependent secretory effects of rCRHSP-28 on digestive
enzyme secretion, b) Ca2+-sensitive translocation of the CRHSP-28 binding proteins to
detergent insoluble fractions of cell lysates, c) localization of pp70 to ZG membranes, and d) the
acute Ca2+-regulated phosphorylation of CRHSP-28 clearly implicate this protein as a major
regulatory factor in the acinar cell secretory pathway. Identification of the molecular identity of
pp70 and characterization of its interactions with CRHSP-28 will likely provide key insight into
the biochemical mechanisms by which these molecules regulate ZG exocytosis.
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Acknowledgements: We extend special thanks to J. A. Williams for his valuable comments and
advice on this project.
This work was supported by a grant from USDA Cooperative State Research Education and
Extension Service (CSREES) Program #WISO4221 and WIS04444 and an American Cancer
Society Institutional Research Grant IRG-58-011-42-2 to G.E. Groblewski.
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1Footnotes: ZGs, zymogen granules; SNARE, soluble N-ethylmaleimide-sensitive fusion
protein attachment protein receptor; SLO, streptolysin-O; CRHSP-28, calcium regulated heat
stable protein; rCRHSP-28, recombinant CRHSP-28 protein; TPD52, tumor protein D52
family; BS3, Bis-(sulfosuccinimidyl) suberate; PMSF, phenylmethylsulfonylfluoride; PAGE,
polyacrylamide gel electrophoresis; pp35 and pp70, 35 and 70 kDa CRHSP-28 binding proteins,
respectively.
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Figure Legends
Figure 1. CRHSP-28 augments Ca2+-stimulated amylase secretion in permeabilized acini.
Acini were treated with 0.5 U/ml streptolysin-O (SLO) at 4 oC for 10 min. Cells were washed to
remove excess unbound SLO and incubated in the presence or absence of recombinant CRHSP-
28 protein (25 µg/ml). The amount of amylase secreted into the media over time was measured
in response to basal (< 10 nM) or stimulatory (10 µM) concentrations of free Ca2+. Squares,
control cells; Circles, CRHSP-28 treated cells; Open symbols, basal Ca2+; Closed symbols, 10
µM free Ca2+. Triangles, amylase secretion from intact cells stimulated with ionomycin (2 µM),
included for comparison. Secretion is expressed as a % of total cellular amylase measured at the
start of the experiment. Data are the mean and standard error of 3 independent experiments, each
performed in duplicate.
Figure 2. Diffusion of native CRHSP-28 from SLO-permeabilized acini . Acini were
permeabilized in the presence of a basal (<10 nM) or stimulatory concentration (10 µM)
of free Ca2+ and equal amounts of cell lysates (50 µg/lane), and extracellular fluid volume were
analyzed by immunoblotting. A) Upper gel: immunoblot of intracellular levels of cyclophilin A
following SLO-permeabilization. Lower gels: representative immunoblot of CRHSP-28 content
in cell lysates and extacellular medium following SLO-permeabilization. B and C)
Densitometric analysis of CRHSP-28 content in intra- and extracellular compartments
following SLO-treatment of cells. Data are the mean and standard error of 3 independent
experiments performed in duplicate.
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Figure 3. CRHSP-28 increases amylase secretion over a range of calcium concentrations . SLO-permeabilized acini were
stimulated with the indicated concentrations of Ca2+ and the effect of CRHSP-28 (25 µg/ml) on amylase secretion was
determined after 30 min. Data are the mean and SE of 5 experiments performed in duplicate.
Basal secretion in low Ca2+ was less than 3% of total cellular amylase in each experiment and
was therefore subtracted from the stimulated values.
Figure 4. . B: Concentration dependent effects of recombinant CRHSP-28 on Ca2+-stimulated
secretion. SLO-permeabilized acini were incubated with the indicated concentrations of
recombinant CRHSP-28 and stimulated with 1 µM free Ca2+. Control cells received no
rCRHSP-28. Data are the mean and standard deviation of 2 independent experiments performed
in duplicate.
Figure 5. Chemical cross-linking identifies CRHSP-28 protein interactions. Acinar cell lysates
were incubated with Bis-(sulfosuccinimidyl) suberate (BS3) crosslinker, and soluble (S) and
particulate (P) fractions were separated by centrifugation. CRHSP-28 content of each fraction
(50 µg/lane) was analyzed by immunoblotting following 8% SDS-PAGE. This experiment was
performed 3 times with identical results.
Figure 6. CRHSP-28 binds to 35 and 70 kDa proteins in acini. A) CRHSP-28 was
immunoprecipitated from 32P-labeled acini treated as control or with 100 pM cholecystokinin
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(CCK) for 2 min and proteins were detected by autoradiography. B) Acinar cell lysates (50
µg/lane) were separated by SDS-PAGE and transferred to nitrocellulose. Gel-overlay analysis was
conducted by probing the membrane with recombinant CRHSP-28 protein (2 µg/ml) followed
by anti-CRHSP-28 antibody (0.5 µg/ml). A CRHSP-28 immunoblot is shown on the right for
comparison.
Figure 7. Ca 2+-sensitive translocation of CRHSP-28 binding proteins to
membrane/cytoskeletal fractions in acini. Acini were sonicated in lysis buffer without
Triton X-100 and containing either 2 mM EGTA or 1 mM CaCl2. Cytosolic (Cyto) and
particulate fractions were prepared by centrifugation. Particulate fractions were further
sonicated in the same buffer containing 0.2% Triton X-100. Detergent-soluble or
membrane (Mem) fractions and Triton-insoluble (TX100-insol) fractions were obtained
by a second centrifugation. Triton-insoluble proteins were dissolved directly in SDS
buffer. Equal amounts of cytosolic and membrane protein (40 µg/lane), and equal volumes
of Triton-insoluble proteins (1/10 of total volume) were separated by SDS-PAGE. A) Upper
panel: coomassie stained proteins demonstrating the relative distribution of cellular protein
isolated under each condition. Lower panels: gel-overlay assay showing CRHSP-28 binding
proteins. B) CRHSP-28 immunoblot.
Figure 8. The CRHSP-28 binding protein pp70 copurifies with zymogen granule (ZG)
membranes. ZGs were purified by Percoll gradient centrifugation. The granules were lysed by
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nigericin treatment in hypotonic buffer and ZG content (ZGC) was separated from membranes
(ZGM) by centrifugation. Proteins (30 µg/lane for acini, ZG and ZGC and 10µg/lane for ZGM)
were analyzed by gel-overlay analysis. Bottom panel: The same fractions were also probed with
anti-rat cysteine string protein antibody (CSP) (1:1000) as a control.
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Diana D.H. Thomas, William B. Taft, Kala M. Kaspar and Guy E. Groblewski+-stimulated secretion in permeabilized acinar cells2CRHSP-28 regulates Ca
published online May 30, 2001J. Biol. Chem.
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