Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells

Guofeng Xie and Jean-Pierre Raufman

Department of Internal Medicine, Division of Gastroenterology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.

signal transduction; cAMP; A-kinase anchorin protein 150


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PEPSINOGEN SECRETION from gastric chief cells is mediated by at least two major signal transduction pathways (20). In one pathway, secretagogues like secretin, vasoactive intestinal peptide, prostaglandins, and cholera toxin activate adenylyl cyclase, thereby causing an increase in cellular cAMP and activation of cAMP-dependent protein kinase (PKA). In the other pathway, secretagogues like carbamylcholine (carbachol) and cholecystokinin cause activation of phospholipase C and subsequent increases in cellular levels of diacylglycerol (DAG) and calcium. These second messengers activate protein kinase C (PKC) and protein phosphatase 2B (PP2B), also referred to as calcineurin (17, 25, 31). Moreover, secretagogues whose actions are mediated by one mechanism potentiate the actions of secretagogues whose actions are mediated by the other (20, 23). For example, in chief cells preincubated with cholera toxin, but not in control cells, carbachol and the divalent cation ionophore A23187 increase cellular levels of cAMP and cause potentiation of pepsinogen secretion (22). This augmented response is mediated by PP2B (23). Nonetheless, the cellular mechanisms underlying so-called cross talk between secretagogues remain to be fully elucidated.

Over the past few years, it was shown that type II regulatory (RII) subunits of different PKA isoforms are physically associated with members of a family of more than 30 proteins designated A-kinase anchoring proteins (AKAPs) (4, 5). AKAPs sequester the PKA holoenzyme to particular cellular compartments in proximity to their substrates. This compartmentalization of PKA by AKAPs allows close interaction of PKA with cytoskeletal or membrane structures and confers added enzyme specificity on PKA-substrate interactions. In addition to association with PKA, AKAPs also bind other signaling molecules such as PKC and PP2B (3, 13). These AKAP-based protein complexes are thought to play a role in integrating different signaling processes, although the exact mechanisms are unknown.

The aim of the experiments reported here was to determine whether, in dispersed chief cells from guinea pig stomach, AKAP-involved compartmentalization of PKA plays a role in mediating cAMP-stimulated pepsinogen secretion and the cross talk described above. Our findings support the existence in chief cells of an AKAP-anchored protein complex that includes AKAP150, PKAalpha , PKC, and PP2B and is associated with actin. Moreover, addition of a peptide that competitively displaces PKA from the AKAP complex decreases cAMP-stimulated pepsinogen secretion and potentiated responses in cholera toxin-treated chief cells that result from cross talk between signaling pathways.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Male guinea pigs (150-175 g) were obtained from Harlan Sprague Dawley (Indianapolis, IN); collagenase (type I), BSA (fraction V), carbachol, (octylphenoxy)polyethoxyethanol (IGEPAL CA-630), 8-bromo-cAMP, cAMP-agarose beads, Ht31 and Ht31P peptides from Sigma and Promega; PKA RII subunit from Promega; cholera toxin, A23187, and PKA catalytic subunit from Calbiochem; MEM amino acids (50-times concentrated), and essential vitamin solution (100-times concentrated) from Mediatech (Herndon, VA); Percoll from Pharmacia; 125I-albumin from ICN; 125I-cAMP and gamma -32P ATP from NEN; ProLong antifade kit, Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 594 goat anti-mouse IgG from Molecular Probes (Eugene, OR). Antibodies used include cAMP from Calbiochem; AKAP150, PKAalpha cat, PKA IIalpha , PKCbeta 1, PKCbeta 2, PKCgamma , and actin from Santa Cruz Biotech; PKA IIbeta from Transduction Laboratories; PKCalpha ,beta ,gamma from Upstate Biotech; PP2B from Chemicon International; and an AKAP150 antibody kindly provided by Dr. John D. Scott (Vollum Institute, Oregon Health Sciences University).

Chief cell preparation. Dispersed chief cells from guinea pig stomach were prepared by mucosal digestion with collagenase and cell fractionation on a Percoll density gradient as described previously (24) and suspended in standard incubation solution. In this preparation, chief cells constitute more than 95% of the total cell population and trypan blue exclusion is >95% (24). Standard incubation solution contained 24.5 mM HEPES (adjusted to pH 7.4), 98 mM NaCl, 6 mM KCl, 2.5 mM KH2PO4, 1 mM MgCl2, 11.5 mM glucose, 5 mM Na pyruvate, 5 mM Na fumarate, 5 mM Na glutamate, 1.5 mM CaCl2, 2 mM glutamine, 0.1% (wt/vol) BSA, 1% (wt/vol) amino acid mixture, and 1% (wt/vol) essential vitamin mixture. The standard incubation solution was equilibrated with 100% O2, and all incubations were performed with 100% O2 as the gas phase.

Pepsinogen secretion. Pepsinogen secretion was determined as described previously (21) using 125I-albumin as substrate and expressed as the percentage of total cellular pepsinogen that was released into the medium during the incubation.

Preparation of cell lysates, immunoprecipitation, and Western analysis. SDS-soluble or IGEPAL-soluble chief cell lysates were prepared as previously described (32). Immunoprecipitation and Western blotting were performed as described in Research Applications by Santa Cruz Biotech. Briefly, for immunoprecipitations, dispersed chief cells (5 × 106 cells/ml) were lysed in lysing buffer that contained 1 × PBS, 1% IGEPAL CA-630, 0.1% SDS, 0.5% sodium deoxycholate, 100 µg/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin (Sigma), and 1 mM sodium orthovanadate. The lysates were precleared with protein A/G-agarose before incubation with specific antibodies. For SDS lysates, we used 50 µg protein. For immunoprecipitations, we used 150 µg protein to compensate for the loss occurred during the procedure.

RII overlay assay. The presence of AKAPs in chief cells was detected by a solid phase RII overlay assay as previously described (1).

Copurification of PKA and PP2B. cAMP-agarose chromatography was performed as described previously (2). Briefly, chief cell lysates were incubated overnight at 4°C with cAMP-agarose (6% beads). After extensive washing with hypotonic buffer, proteins bound to cAMP were eluted at room temperature over 2 h with 100 mM cAMP.

Immunofluorescence microscopy. Immunofluorescent staining was performed by using the protocol provided by Santa Cruz Biotech. Briefly, newly isolated chief cells were allowed to attach to chamber slides coated with type I collagen (Becton Dickinson) for 2 h in a 37°C incubator. Attached cells were stained with primary antibodies and subsequently with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes). Stained cells were mounted with ProLong antifade (Molecular Probes) and viewed under a Zeiss laser scanning confocal microscope. Chief cells comprise >95% of the attached cell population and can be distinguished easily from other gastric mucosal cells by virtue of their smaller size, polarized location of their nuclei observed by staining with 4,6-diamidino-2-phenylindole, and immunofluorescent staining with antibodies against pepsinogen (24).

Peptide delivery. Ht31 and Ht31P peptides were introduced into dispersed chief cells by use of lipofectamine. Freshly isolated chief cells were incubated with 50 µM HT31 or Ht31P peptides in standard incubation solution for 90 min at 37°C with 10 µg lipofectamine (GIBCO BRL) per 105 chief cells. After centrifugation at 800 g for 20 s, cells were resuspended in standard incubation solution, and secretagogues were added for varying times as described for individual experiments.

Measurement of cAMP. Cellular cAMP level was determined by radioimmunoassay as described previously (27). The concentration of chief cells in the incubate was adjusted to maintain cAMP on the linear portion of the standard curve.

Statistical analysis. Significance between two means was determined by Student's t-test; P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of AKAP150 in guinea pig chief cells. To determine whether AKAPs are present in guinea pig chief cells, we performed an RII overlay assay (Fig. 1A). We detected one predominant AKAP of ~150 kDa. To determine whether the 150-kDa protein is AKAP150, we performed immunoprecipitations and/or Western blots with two anti-AKAP150 antibodies, Ab1 and Ab2, that were raised against different NH2 terminal peptides of mouse AKAP150. Both antibodies detected a 150-kDa protein from SDS-soluble chief cell lysates (Fig. 1B). This protein comigrates with AKAP150 from mouse brain cell lysates and is also present in AKAP150 immunoprecipitates. A third antibody directed at the carboxy terminal portion of AKAP150 also recognized immunoprecipitated AKAP150 by Ab1, a polyclonal anti-AKAP150 antibody from Santa Cruz Biotech (data not shown). Because they were not detected with Ab1, the two prominent low molecular weight bands seen by Ab2 are probably nonspecific.


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Fig. 1.   Identification of A-kinase anchoring protein (AKAP)150 in guinea pig gastric chief cells. Identified AKAP150 proteins are indicated by arrowheads. A: a chief cell SDS-soluble extract was probed with 32P-RII subunit in an RII overlay assay. Lower band is the result of dimerization between the probe and endogenous protein kinase A (PKA) RII subunits. B: Western blots of SDS-soluble cell extracts from chief cells, mouse brain cells (Transduction Lab), AKAP150, and PKA IIalpha immunoprecipitates were probed with antibodies against AKAP150 as indicated. Ab1 is a goat polyclonal antibody against AKAP150 from Santa Cruz Biotech; Ab2 is a rabbit polyclonal anti-AKAP150 antibody provided by Dr. John Scott. AKAP150 was immunoprecipitated from (octylphenoxy)polyethoxyethanol (IGEPAL)-soluble cell lysates prepared from dispersed chief cells with Ab1 and probed with Ab2 on the Western blot. C: the regulatory PKA subunit IIalpha was immunoprecipitated from IGEPAL-soluble cell lysates prepared from dispersed chief cells and probed with Ab1, a goat polyclonal antibody against AKAP150 from Santa Cruz Biotech.

Chief cell AKAP150 binds PKA IIalpha and IIbeta . To identify the specific RII PKA isoforms that bind AKAP150, we performed immunoprecipitations with antibodies against PKA IIalpha and IIbeta from IGEPAL-soluble chief cell extracts. From Western blots, we determined that AKAP150 was associated with both PKA IIalpha (Fig. 1C) and IIbeta (data not shown).

AKAP150 binds PKC and PP2B. Human neuronal AKAP79 binds not only PKA, but also PKC and PP2B. Guinea pig chief cells have been reported to express PKCalpha and -zeta (19). To determine whether AKAP150 in chief cells is also associated with PKC and/or PP2B, we performed immunoprecipitation with an anti-AKAP150 antibody (Santa Cruz Biotech) and affinity chromatography with cAMP-agarose. Using a polyclonal antibody that recognizes the conventional PKC isoforms (alpha , beta , gamma ) (Upstate Biotechnology), we detected PKC in AKAP150 immunoprecipitates (Fig. 2). Because previous experiments (19) and current data (not shown) indicate that the alpha  isoform is the only conventional PKC isoform expressed in guinea pig chief cells, the present data indicate that PCKalpha is associated with AKAP150.


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Fig. 2.   Association of AKAP150 and protein kinase C (PKC). AKAP150 was immunoprecipitated with Ab1 and probed with an anti-conventional PKC rabbit polyclonal antibody from Upstate Biotechnology.

Because the PP2B catalytic A and regulatory B subunits migrated closely with immunoglobulin heavy chain and light chain, respectively, and were difficult to observe in immunoprecipitates on Western blots, we used biochemical methods to determine whether PKA and PP2B are associated in chief cells. PKA were isolated from chief cells with cAMP-agarose beads (Fig. 3A). After extensive washing (see MATERIALS AND METHODS), we found both the catalytic A and regulatory B subunits of PP2B in cAMP eluates (Fig. 3B). AKAP150 was detected in the same eluates (Fig. 3C). These findings indicate that AKAP150, PKA, and PP2B are in the same protein complex.


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Fig. 3.   Copurification of the alpha -catalytic subunit of PKA (PKAalpha cat), protein phosphatase 2B (PP2B), and AKAP150 by affinity chromatography with cAMP-agarose. PKA was isolated from chief cells by cAMP chromatography, and the copurifying proteins were eluted with cAMP after extensive washing and were immunoblotted as described in MATERIALS AND METHODS. Immunoblots were probed with a rabbit polyclonal antibody against PKAalpha cat (A), a rabbit polyclonal antibody against both the catalytic A and regulatory B subunits of PP2B from Chemicon International (B), and Ab1 against AKAP150 (C). Positions of molecular weight markers are indicated on left (in kDa).

Cellular association and colocalization of AKAP150 and PKC. Subcellular location of AKAP150 and further evidence for the association of PKC and AKAP150 were provided by double-immunofluorescence staining of chief cells. In chief cells, AKAP150 and PKC exhibited a predominantly colocalized staining pattern at the cell periphery (Fig. 4, B-D). AKAP150 has no predicted transmembrane domain, and >80% of AKAP was detected from the IGEPAL-soluble cytoplasmic fraction compared with that of the cell membrane pellet (data not shown). This evidence suggests that, like its mammalian counterpart AKAP75 (16), chief cell AKAP150 is located in the cytoskeleton near the cytoplasmic surface of the plasma membrane. Collectively, the immunoprecipitation and immunocytochemistry indicate that chief cell AKAP150 binds PKC and that this complex occurs in vivo.


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Fig. 4.   Colocalization of AKAP150 and PKC at the periphery of guinea pig gastric chief cells. Cells were viewed by light microscopy (A), stained with antibodies to AKAP150 (Ab1) (B) and PKC (Upstate Biotech) (C), and analyzed by confocal microscopy (magnification, ×400, see MATERIALS AND METHODS). Double staining for AKAP150 and PKC is displayed by superimposing the images from B and C (D).

AKAP150 is associated with actin cytoskeleton. In nonneuronal cells, mammalian AKAP75 accumulates in the actin-rich, cortical cytoskeleton, in close proximity to the plasma membrane (16). Moreover, in rat pancreatic acinar cells, it has been reported that the subapical actin cytoskeleton plays a regulatory role in carbachol-stimulated exocytosis and membrane retrieval (29). Hence, to determine whether guinea pig chief cell AKAP150 is associated with actin, we performed immunoprecipitations and Western blots using Ab1 against AKAP150 and an anti-actin antibody. This experiment demonstrated coimmunoprecipitation of AKAP150 and actin (Fig. 5). Nevertheless, at present, the structural basis of this interaction is not clear, nor do we know which actin isoform is involved.


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Fig. 5.   Coimmunoprecipitation of AKAP150 and actin. AKAP150 was immunoprecipitated from IGEPAL-soluble chief cell lysates and probed with an antibody against actin on Western blots.

The association of PKA and AKAP150 is necessary for maximal cAMP-induced pepsinogen secretion. To determine whether the cellular compartmentalization of PKA provided by AKAP150 anchoring is important in mediating pepsinogen secretion, we examined the effects of the anchoring inhibitor peptide Ht31 on secretagogue-induced pepsinogen secretion. Ht31 is a 24-amino acid peptide containing the minimal region of the PKA RII subunit required for AKAP binding (1). Ht31 can block the interaction between AKAPs and RII subunits, thus dissociating PKA from its anchoring sites and uncoupling cAMP-responsive events (1). Ht31P is a peptide derived from Ht31 with two prolines replacing isoleucine residues (1). Because Ht31P is unable to disrupt the interaction between PKA RII and AKAPs, it was used as a negative control. Lipofectamine, used as a delivery agent to introduce Ht31 and Ht31P into dispersed chief cells, did not alter basal pepsinogen secretion (data not shown). Likewise, the Ht31 and Ht31P peptides did not alter basal secretion (Fig. 6). However, Ht31 caused a 32% reduction in 8-Br-cAMP-stimulated pepsinogen secretion (P < 0.01), whereas Ht31P did not alter secretion (Fig. 6). Secretion stimulated by carbachol and the ionophore A23187, agents whose actions are not mediated by cAMP, was not altered by addition of either Ht31 or Ht31P (Fig. 6).


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Fig. 6.   Inhibitory effect of anchoring inhibitor peptide Ht31 on secretagogue-induced pepsinogen secretion. Cells were transfected (lipofectamine) at 37°C for 90 min with 50 µM Ht31 or Ht31P peptides (see MATERIALS AND METHODS). 8-Br-cAMP (1 mM), carbachol (1 mM), or A23187 (100 nM) was added, and the cells were incubated at 37°C for an additional 15 min. Pepsinogen secretion was calculated as the percentage of pepsinogen that was released into the medium during the 15-min incubation after the initial 90-min incubation with lipofectamine. In each experiment, each value was determined in duplicate, and results given are means ± SE from 5 separate experiments. *Values obtained with Ht31 that are significantly less (P < 0.01, Student's t-test) than those obtained with Ht31P or secretagogues alone.

In numerous secretory models, potentiating interactions between secretagogues have been used to demonstrate cross talk between signaling pathways (20). In gastric chief cells, preincubation of chief cells with 100 nM cholera toxin and subsequent incubation with A23187 or carbachol results in augmented levels of cellular cAMP and potentiation of pepsinogen secretion (22). These findings indicate interaction between the phospholipase C-mediated pathway, involving calcium- and DAG-activated kinases (PKC and calcium/calmodulin kinase-II) and phosphatases (PP2B), and the cAMP-PKA pathway (20). These interactions and other observations in chief cells suggest compartmentalization of signaling systems within the cell. Nevertheless, the physicochemical nature of this compartmentalization has remained obscure.

We designed experiments to determine whether AKAP150, by localizing involved kinases and phosphatases in close proximity, plays a role in mediating potentiating interactions between chief cell signaling pathways. In cholera toxin-treated chief cells, Ht31, but not Ht31P, inhibited augmentation of carbachol- and A23187-stimulated pepsinogen secretion by 36 and 29%, respectively (Fig. 7). These observations suggest that, in cholera toxin-treated cells, PKA anchoring by AKAP is necessary for maximal potentiation of secretion by carbachol and A23187.


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Fig. 7.   Inhibitory effect of Ht31 on carbachol- and A23187-induced pepsinogen secretion from chief cells preincubated with cholera toxin. Chief cells were preincubated at 37°C for 90 min with 100 nM cholera toxin and lipofectamine, alone or with 50 µM Ht31 or Ht31P peptides. Carbachol (1 mM) or A23187 (100 nM) was added for an additional 15-min incubation at 37°C. Pepsinogen secretion was calculated as the percentage of pepsinogen after the 90-min incubation with or without cholera toxin that was released into the medium during the subsequent 15-min incubation. In each experiment, each value was determined in duplicate, and results given are means ± SE from 4 separate experiments. *Values that are significantly less (P < 0.05) than those obtained with secretagogues alone or secretagogues plus Ht31P.

To localize these effects to the PKA portion of the signaling pathway and exclude possible effects of Ht31 on adenylyl cyclase and cAMP production, we determined whether, in the presence of cholera toxin, Ht31 affects cellular levels of cAMP. Neither Ht31 nor Ht31P altered stimulated levels of cAMP (Table 1). These findings confirm that the inhibitory actions of Ht31 are caused by disruption of the PKA-AKAP interaction, not by actions of the peptide on adenylyl cyclase.

                              
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Table 1.   Lack of effect of Ht31 on carbachol- and A23187-induced augmentation of cAMP levels in cholera toxin-treated chief cells


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Association with anchoring proteins and cellular compartmentalization of protein kinases and phosphatases results in greater efficiency of action (closer proximity of enzyme and substrate) and specificity (decreased potential for interaction with other protein substrates). Subcellular targeting of PKA action by association with AKAPs has emerged as an important mechanism for mediating PKA-dependent signal transduction. Functional studies by Scott and others (6, 10, 11, 14, 15, 30) that examined the consequences of disrupting AKAP-PKA association have proved that anchored PKA is required for maximal expression of various cellular processes including gene transcription, ion-channel regulation, and hormone-mediated insulin secretion. Some AKAPs, like AKAP75/79/150, representing the bovine, human, and rodent AKAP orthologs, respectively, bind PKA and other protein kinases and phosphatases, including PKC and PP2B. Moreover, association of PKC and PP2B with AKAP75/79/150 may inhibit their enzymatic activity, thereby serving an additional regulatory function (8, 9, 12).

As illustrated by the cartoon in Fig. 8, in chief cells from guinea pig stomach, we propose the existence of an AKAP150-anchored protein complex that includes AKAP150, PKAalpha , PKC, and PP2B. We acknowledge the limitation that none of our experiments proves that these components are associated with AKAP150 concurrently. Nevertheless, the individual experiments support the presence in chief cells of such an AKAP-anchored complex of signaling molecules. To our knowledge, the present communication represents the first report of a functional AKAP complex in a gastrointestinal secretory cell. Although several novel AKAPs have been detected in gastric parietal cells, these have not been shown necessary for the acid secretory function of these cells (7). In chief cells, dissociation of PKA from the AKAP150-based complex reduces pepsinogen secretion in response to stimulation by agents whose actions are mediated by cAMP, but not secretion that is stimulated by agents that increase cellular calcium concentration or activate PKC. In the cartoon (Fig. 8), the calcium ionophore A23187 and the muscarinic (M3) receptor are shown as examples of signaling mechanisms that increase cellular calcium and DAG levels (17). Both of these mediators are known to activate conventional forms of PKC (17). Moreover, calcium is an activator of PP2B (25, 31). The failure of the Ht31 peptide, as well as the Ht31P control peptide, to alter A23187- and carbachol-stimulated secretion serves as further evidence that the actions of the peptide are specific to cAMP-mediated events.


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Fig. 8.   Cartoon illustrating elements of the AKAP150-associated protein complex in gastric chief cells. Examples of the 3 major signaling pathways that mediate pepsinogen secretion are shown. Cholera toxin interaction with cholera toxin receptor (GM1) ganglioside receptors results in activation of adenylyl cyclase and production of cAMP. The divalent cation ionophore A23187 promotes influx of extracellular calcium, thereby increasing cellular levels of calcium. Cholinergic agonists interact with M3 muscarinic receptors to activate phospholipase C, thereby increasing cellular levels of calcium and diacylglycerol (DAG). cAMP activates PKA. Calcium activates PKC and PP2B. DAG activates PKC. The approximate locations of PKA, PP2B, and PKC sites of association with AKAP150 are derived from previous publications (4, 5). R, regulatory component of PKA; C, catalytic component of PKA; S, PKA substrate; S*, phosphorylated PKA substrate.

Our experiments show that, in terms of pepsinogen secretion, maximal expression of PKA action requires association with AKAP. Disruption of this association by incubation with the Ht31 peptide reduces cAMP-stimulated pepsinogen secretion by ~30%. Because, in these experiments, we were unable to determine the percentage of PKA dissociated from AKAP150, we do not know whether PKA association with AKAP150 is absolutely necessary for mediation of cAMP-induced secretion. That is, we do not know whether complete dissociation of all molecules of PKA from AKAP150 would completely abolish the ability of increases in cAMP to stimulate pepsinogen secretion.

Previous observations from our laboratory have shown that in guinea pig chief cells potentiating interactions exist between agents that increase cellular cAMP levels and those that increase cellular calcium levels or directly activate PKC (22). Moreover, PP2B activity is required for these potentiating interactions to occur (23). As shown in the cartoon (Fig. 8), the presence of an AKAP150-based complex including PKA, PKC, and PP2B facilitates conceptualization of how these effectors are compartmentalized. Further, it suggests how these kinases and PP2B can interact in sequence to alter the phosphorylation status of substrates that are important for stimulating exocytosis of pepsinogen-containing zymogen granules. When cells were treated with cholera toxin, an activator of adenylyl cyclase, and then with A23187 or carbachol, the addition of the Ht31 peptide reduced potentiation of pepsinogen secretion by ~30%. We measured cAMP levels to confirm that the inhibitory actions of the Ht31 peptide are mediated by disruption of the PKA-AKAP150 association, not by inhibitory effects on the levels of cAMP produced by activation of adenylyl cyclase. These observations confirm that the AKAP150 complex plays an important role in facilitating potentiating interactions between PKA, PKC, and PP2B.

It is of interest to compare our findings to those observed in RINm5F insulin-secreting cells (15). In RINm5F cells, addition of the Ht31 peptide reduced hormone- and cAMP-stimulated insulin secretion. Moreover, in RINm5F cells transfected with plasmids encoding a soluble Ht31 fragment, there is an inverse relationship between the amount of peptide expressed and cAMP-stimulated insulin secretion (15). Hence, as in the above discussion of the reduction in pepsinogen secretion observed with addition of Ht31 to chief cells, without measuring intracellular levels of Ht31 or the degree of PKA-AKAP dissociation, it is not possible to determine more than a limited quantitative relation between dissociation of the PKA-AKAP complex and reduced secretion. Nevertheless, in both chief and RINm5F cells, addition of Ht31 has no effect on cAMP production but reduces the regulated release of the secretory products (insulin and pepsinogen), indicating that PKA-AKAP association is necessary for maximal cAMP-mediated secretion.

Consistent with the report that, in nonneuronal cells, mammalian AKAP75 targets PKA to the cortical actin cytoskeleton (16), we have demonstrated that chief cell AKAP150 is associated with actin. In polarized pancreatic acinar cells, which have cellular machinery similar to that of gastric chief cells (20), the subapical actin cytoskeleton is involved in regulated exocytosis, both as a negative regulator of membrane fusion and as a facilitator of the movement of secretory granules to the sites of fusion (29). In pancreatic acinar cells, zymogen granules engaged in exocytosis are coated with actin before fusion with the plasma membrane. This coating process is tightly coupled to the release of the zymogen granule marker, rab3D, a low-molecular-weight GTP-binding protein (28). The following observations suggest that the actin cytoskeleton plays a role in regulated pepsinogen secretion from chief cells. First, rab3D is also present in guinea pig gastric chief cells (18). Second, disruption of rab3D expression results in defective secretagogue-stimulated pepsinogen secretion (26). Finally, in the present study, we demonstrate that AKAP150 is associated with actin. It remains to be determined whether actin is a substrate for any AKAP150-associated kinases or phosphatases, or whether actin simply acts to place the AKAP-anchored protein complex in close proximity to protein substrates such as adenylyl cyclase and other actin-associated cytoskeletal proteins that regulate exocytosis.


    ACKNOWLEDGEMENTS

We thank Dr. John D. Scott (Vollum Institute, Oregon Health Science University) for providing a rabbit polyclonal anti-AKAP150 antibody and Meshelle Helms for assistance in preparing the manuscript.


    FOOTNOTES

Address for reprint requests and other correspondence: J.-P. Raufman, Univ. of Arkansas for Medical Sciences, Mail Slot 567, 4301 West Markham St., Little Rock, AR 72205-7199 (E-mail: raufmanjeanpierre{at}uams.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.

Received 18 February 2001; accepted in final form 11 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Carr, DW, Stofko-Hahn RE, Fraser ID, Bishop SM, Acott TS, Brennan RG, and Scott JD. Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J Biol Chem 266: 14188-14192, 1991[Abstract/Free Full Text].

2.   Coghlan, VM, Langeberg LK, Fernandez A, Lamb NJ, and Scott JD. Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase. J Biol Chem 269: 7658-7665, 1994[Abstract/Free Full Text].

3.   Coghlan, VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, and Scott JD. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 267: 108-111, 1995[ISI][Medline].

4.   Colledge, M, and Scott JD. AKAPs: from structure to function. Trends Cell Biol 9: 216-221, 1999[ISI][Medline].

5.   Dodge, K, and Scott JD. AKAP79 and the evolution of the AKAP model. FEBS Lett 476: 58-61, 2000[ISI][Medline].

6.   Dodge, KL, Carr DW, Yue C, and Sanborn BM. A role for AKAP (A kinase anchoring protein) scaffolding in the loss of a cyclic adenosine 3',5'-monophosphate inhibitory response in late pregnant rat myometrium. Mol Endocrinol 13: 1977-1987, 1999[Abstract/Free Full Text].

7.   Dransfield, DT, Bradford AJ, and Goldenring JR. Distribution of A-kinase anchoring proteins in parietal cells. Biochim Biophys Acta 1269: 215-220, 1995[ISI][Medline].

8.   Faux, MC, Rollins EN, Edwards AS, Langeberg LK, Newton AC, and Scott JD. Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction. Biochem J 343: 443-452, 1999[ISI][Medline].

9.   Faux, MC, and Scott JD. Regulation of the AKAP79-protein kinase C interaction by Ca2+/calmodulin. J Biol Chem 272: 17038-17044, 1997[Abstract/Free Full Text].

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