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RECEPTORS AND SIGNAL TRANSDUCTION

Role of protein phosphatase 2A in calcium-dependent chloride secretion by human colonic epithelial cells

Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, California

Submitted 25 January 2006 ; accepted in final form 3 August 2006

	ABSTRACT

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EGF inhibits carbachol-induced chloride secretion by regulating a basolateral potassium channel via phosphatidylinositol 3-kinase (PI 3-kinase) and PKC activation. Although both EGF and carbachol cause tyrosine phosphorylation of p85 of PI 3-kinase, only EGF activates the enzyme. Serine phosphorylation of p85 is thought to suppress the lipid kinase of PI 3-kinase. Our present study examined whether the differential effects of carbachol and EGF on PI 3-kinase activity correspond to varying phosphorylation of p85, and the mechanisms and consequences. T₈₄ colonic epithelial cells were treated with either EGF or carbachol. Cell lysates were immunoprecipitated with p85 antibody and blotted with either phosphotyrosine or phosphoserine antibodies. Protein phosphatase (PP) 1 and 2A activities were also measured. Both tyrosine and serine residues of p85 were phosphorylated by carbachol, whereas EGF induced only tyrosine phosphorylation. Moreover, EGF abolished carbachol-induced serine phosphorylation of p85 and activated PP2A without affecting PP1. Carbachol did not affect either phosphatase. Calyculin A or okadaic acid pretreatment reversed the inhibitory action of EGF on carbachol-induced chloride secretion and restored serine phosphorylation of p85. Although carbachol recruits p85, it phosphorylates both serine and tyrosine residues so that the lipid kinase of PI 3-kinase is inhibited. EGF results in p85 tyrosine phosphorylation as well as dephosphorylation of serine residues via the activation of PP2A. This explains the differential induction of PI 3-kinase enzyme activity in response to EGF and/or carbachol and has functional implications. Our data provide further insights into negative signals that regulate chloride secretion and into the molecular basis of signaling diversification in the intestinal epithelium.

epithelial secretion; PI 3-kinase; EGF

The ion transport pathways comprising the chloride secretory mechanism of T₈₄ cells, a line of human colonic epithelial cells with a crypt cell phenotype, have been well defined (12, 13). Chloride is taken up across the basolateral membrane of the cells via a Na⁺-K⁺-2Cl^– cotransporter and exits the cell across the apical membrane via chloride channels. An important member of such chloride channels is the cystic fibrosis transmembrane conductance regulator. Basolateral potassium channels support chloride secretion by allowing for potassium recycling, whereas energy for the process is supplied by the activity of a basolateral Na⁺,K⁺-ATPase. Primary control for the overall transport process occurs at the level of apical chloride channels and basolateral potassium channels, in response to agonists that elevate positive second messengers for chloride secretion, namely, cyclic nucleotides and cytosolic calcium. Signaling mechanisms intrinsic to the epithelium can also inhibit secretion. Thus the muscarinic agonist carbachol initially activates secretion in a calcium-dependent fashion and then renders cells refractory to additional stimulation. Furthermore, growth factors such as epidermal growth factor (EGF) inhibit secretion induced by calcium-dependent agonists, including carbachol, without themselves serving as positive effectors of secretion.

Previously, work in our laboratory (35) has shown that the inhibitory effect of EGF on chloride secretion in T₈₄ cells is due to its ability to stimulate phosphatidylinositol (PI) 3-kinase and the production of 3-phosphorylated lipids. However, whereas both carbachol and EGF recruit PI 3-kinase, only EGF increases the activity of this enzyme. Nuclide efflux studies showed that EGF reduced calcium-stimulated basolateral ⁸⁶Rb⁺ efflux, but not apical ¹²⁵I^– efflux, suggesting that a basolateral potassium channel constitutes the target of the PI 3-kinase-dependent negative signaling pathway. PI 3-kinase is a ubiquitous lipid kinase that phosphorylates the 3-position of the inositol ring of inositol phospholipids to generate such lipid messengers as phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. The exact role and molecular targets of these lipid products have not been fully elucidated, although increasing evidence suggests that they may serve as intracellular second messengers (14, 29). PI 3-kinase is a heterodimer consisting of a p85 regulatory subunit with SH2 domains and a p110 catalytic subunit (34). Regulation of the p85/p110 PI 3-kinase is complex. Membrane recruitment of p85 can be stimulated by growth factors or other stimuli that activate tyrosine kinase activity, presumably via SH2 binding. p85 likewise has been shown to bind p110 via the inter-SH2 region (10) once p85 is activated. Activation of the p85 subunit requires tyrosine phosphorylation to permit binding and activation of p110. However, Dhand et al. (11) additionally identified serine 608 in the inter-SH2 domain of p85 as playing a critical role as an inhibitory regulatory site. Phosphorylation of this site apparently converts p110 into a protein kinase.

In this study, we hypothesized that carbachol and EGF exhibit different effects on PI 3-kinase activity because they differentially alter the balance of serine and tyrosine phosphorylation of the p85 subunit. Furthermore, we predicted that this would alter chloride secretion. We have determined that EGF reduces serine phosphorylation of p85, likely by activating a protein phosphatase that in turn regulates PI 3-kinase activity and its functional consequences.

	MATERIALS AND METHODS

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Materials. The following materials were purchased from the sources indicated: okadaic acid and calyculin A from Alexis (San Diego, CA); [³²P]ATP from NEN New England Biolabs (Beverly, MA); EGF from Genzyme (Cambridge, MA); Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) from JRH (Lenexa, KS); monoclonal antibodies directed against the p85 subunit of PI 3-kinase and positive controls (Jurkat, human fibroblast, and macrophage cell extracts) and goat anti-mouse horseradish peroxidase secondary antibody from Transduction Laboratories (Lexington, KY); Hybond ECL polyvinylidene difluoride (PVDF) membrane, Kodak X-ray films, and ECL Plus detection kit from Amersham Pharmacia Biotech (Piscataway, NJ); cell culture membrane inserts (Millicell, 0.45-µm-pore size mixed cellulose ester) from Millipore (Bedford, MA); Me₂SO, Tween-20 (EIA grade), dithiothreitol, glycine, Trizma, SDS, glycerol, bromphenol blue, sterile deionized water, leupeptin, phenylmethylsulfonyl fluoride, sodium orthovanadate, and carbachol from Sigma (St. Louis, MO); blotting grade nonfat dry milk from Upstate Biotechnology (Lake Placid, NY); protein phosphatase assay systems from Life Technologies GIBCO BRL (Gaithersburg, MD); D_c protein assay kit, 7.5% polyacrylamide gels, and molecular mass markers from Bio-Rad (Hercules, CA); trypsin from Irvine Scientific (Santa Ana, CA); and newborn calf serum from Hyclone (Logan, UT). All other chemicals were of at least reagent grade and were obtained commercially.

Cell culture. Methods for the maintenance of T₈₄ cells for use in transepithelial electrolyte transport studies have been described previously (13). In brief, T₈₄ cells were grown in DMEM/F12 medium with 5% newborn calf serum. For experiments involving Western blotting, 10⁶ cells were seeded onto 30-mm inserts. Cells were cultured for 7–10 days to develop confluent monolayers before use.

Chloride secretion. Chloride secretion was measured as short-circuit current (I_sc) across monolayers of T₈₄ cells, mounted in Ussing chambers (window area = 0.6 cm²) modified for use with cultured cells (13). I_sc (normalized to µA/cm²) was used to quantitate both basal transepithelial chloride secretion and that induced by calcium-dependent secretagogues. T₈₄ cells secrete chloride in response to various agonists, and the resulting changes in I_sc are wholly reflective of chloride secretion (4). I_sc measurements were carried out in Ringer solution containing (in mM) 140 Na⁺, 5.2 K⁺, 1.2 Ca²⁺, 0.8 Mg²⁺, 119.8 Cl^–, 25 HCO₃^–, 2.4 H₂PO₄^–, 0.4 HPO₄^2–, and 10 glucose.

Immunoprecipitation and Western analysis. T₈₄ cells were washed three times with Ringer solution and allowed to equilibrate for 30 min at 37°C. Cells were then stimulated as noted. The reaction was stopped by three washes with ice-cold phosphate-buffered saline (PBS), and the cells were lysed in ice-cold lysis buffer (1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, and 1 nM EDTA in PBS) for 45 min. Cells were then scraped into microcentrifuge tubes and spun at 12,000 rpm for 10 min, and the pellet was discarded. Samples were analyzed for protein content (Bio-Rad protein assay kit) and adjusted so that each sample contained an equal amount of protein. For immunoprecipitation studies, lysates were incubated with immunoprecipitating antibody, per the manufacturer's instructions, for 1 h at 4°C followed by another 1-h incubation at 4°C with protein A-Sepharose. Lysates were then centrifuged for 3 min at 15,000 rpm, and the supernatant was discarded. The pellets were washed in PBS (3x) and resuspended in 2x gel loading buffer (50 mM Tris, pH 6.8, 2% SDS, 100 mM dithiothreitol, 0.2% bromphenol blue, and 20% glycerol), boiled for 5 min, and then loaded onto a polyacrylamide gel for separation. Resolved proteins were transferred overnight at 4°C onto a PVDF membrane. After transfer, the membrane was preblocked with a 1% solution of blocking buffer for 30 min followed by a 1-h incubation with the appropriate concentration of primary antibody in 1% blocking buffer. After washing (4 x 10 min) in Tris-buffered saline with 1% Tween (TBST), membranes were incubated for 30 min in horseradish peroxidase-conjugated secondary antibody in 1% blocking buffer. After washing in TBST (4 x 10 min), immunoreactive proteins were detected using an enhanced chemiluminescent detection kit. In all studies, blots were stripped and reprobed with the immunoprecipitating antibody to ensure equal loading of the protein of interest in each lane. Densitometric analysis of the Western blots was performed using NIH Image digital imaging software.

Protein phosphatase activity assay. The procedures were described in the manufacturer's instruction manual. Briefly, a kinase reaction was initiated by adding 0.5 mCi of [-³²P]ATP to the phosphorylation reaction buffer that contained phosphorylase A (a substrate of protein phosphatases). The reaction was incubated at 30°C for 1 h and was terminated by addition of diluted ammonium sulfate solution. The precipitated protein was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was discarded, and the protein pellet was washed five times with ice-cold diluted ammonium sulfate solution. The protein pellet was then dissolved in solubilization buffer (50 mM Tris·HCl, 0.1 mM EDTA, 15 mM caffeine, and 1% -mercaptoethanol). The solution was then applied to a concentrator and purified by centrifugation at 5,000 g for 30 min at 20°C. The retentate containing radiolabeled phosphorylase A was then stored at 4°C until use in the protein phosphatase assay. Twenty microliters of protein extract were mixed with 20 µl of protein phosphatase assay buffer (2 mM EDTA, 20 mg/ml BSA, 400 mM imidazole-HCl, and 2% -mercaptoethanol), and the reaction was initiated by addition of 20 µl of radioactive phosphorylase A. The reaction was allowed to proceed at 30°C for 10 min and was terminated by addition of TCA solution. The mixture was centrifuged at 12,000 g for 3 min at 4°C, and the supernatant was used to determined the amount of radioactivity released as free ³²P_i. Protein phosphatases 1 and 2A are the two most prominent types of protein phosphatases found in mammalian cells and can be distinguished from type 2B and 2C phosphatases by their preference for phosphorylase A as a substrate (8, 17). Protein phosphatase 1 was distinguished from protein phosphatase 2A by its susceptibility to okadaic acid added to the crude protein extract (0.1 nM) before the reaction was started.

Data analysis. All data are expressed as means ± SE for a series of n experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc test or Student's t-tests for unpaired samples using GraphPad Prism 2.0 (San Diego, CA). P < 0.05 was considered statistically significant.

	RESULTS

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Effects of EGF or carbachol on tyrosine phosphorylation of p85. EGF is able to inhibit carbachol-induced chloride secretion via the activation of PI 3-kinase that involves tyrosine phosphorylation of p85, the regulatory subunit of the enzyme (35). To confirm this finding, we first pretreated T₈₄ cells with either EGF and/or carbachol, followed by immunoprecipitation of the cell lysates with an anti-p85 antibody and Western blotting with an anti-phosphotyrosine antibody. As shown in Fig. 1, EGF stimulated tyrosine phosphorylation of p85 as early as 1 min after addition, with the level of phosphorylation sustained at least for 5 min. Similarly, carbachol also possessed the ability to stimulate tyrosine phosphorylation of p85 at 1 min after its addition. However, unlike EGF, the effect of carbachol on tyrosine phosphorylation of p85 appeared somewhat more transient (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effect of EGF on tyrosine phosphorylation of the p85 subunit of phosphatidylinositol (PI) 3-kinase in T84 cells. Monolayers were treated with buffer alone (control, 0 min) or EGF (100 ng/ml, basolateral) for 1 or 5 min. Top panel depicts a representative blot, and bottom panel shows summary data, quantitated by densitometry and expressed as percentages of the untreated control. Data are means ± SE for 6 experiments. *P < 0.05, response that differs significantly from the unstimulated control. Statistical effects were determined by 1-way ANOVA with Student-Newman-Keuls post hoc test.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effect of carbachol on tyrosine phosphorylation of the p85 subunit of PI 3-kinase in T84 cells. Monolayers were treated with buffer alone (control, 0 min) or carbachol (100 µM, basolateral) for 1 or 5 min. Top panel depicts a representative blot, and bottom panel shows summary data, quantitated by densitometry and expressed as percentages of the untreated control. Data are means ± SE for 7 experiments. *P < 0.05, response that differs significantly from the unstimulated control. Statistical effects were determined by 1-way ANOVA with Student-Newman-Keuls post hoc test.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of carbachol on serine phosphorylation of the p85 subunit of PI 3-kinase in T84 cells. Monolayers were treated with buffer alone (control, 0 min) or carbachol (100 µM, basolateral) for 1 or 5 min. Top panel depicts a representative blot, and bottom panel shows summary data, quantitated by densitometry and expressed as percentages of the untreated control. Data are means ± SE for 11 experiments. *P < 0.05, response that differs significantly from the unstimulated control. Statistical effects were determined by 1-way ANOVA with Student-Newman-Keuls post hoc test.

View larger version (28K): [in this window] [in a new window]   Fig. 4. Effect of treatment with both EGF and carbachol on serine phosphorylation of the p85 subunit of PI 3-kinase in T84 cells. Monolayers on inserts were pretreated basolaterally with EGF (100 ng/ml) for 15 min, followed by buffer alone or carbachol (100 µM) basolaterally for the times indicated. Top panel depicts a representative blot, and bottom panel shows summary data, quantitated by densitometry and expressed as percentages of the untreated control. Data are means ± SE for 6 experiments.

View larger version (7K): [in this window] [in a new window]   Fig. 5. Effect of carbachol on protein phosphatase 2A (PP2A) activity in T84 cells. Monolayers were treated with carbachol (100 µM) basolaterally for the times indicated. Monolayers were then lysed as described in MATERIALS AND METHODS, and cell lysates were then used in the protein phosphatase activity assay. Data are means ± SE for 4 experiments.

View larger version (7K): [in this window] [in a new window]   Fig. 6. Effect of EGF on PP2A activity in T84 cells. Monolayers were treated with EGF (100 ng/ml) basolaterally for the times indicated. Monolayers were then lysed as described in MATERIALS AND METHODS, and cell lysates were then used in the protein phosphatase activity assay. Data are means ± SE for 9 experiments. *P < 0.05, significant increase in phosphatase activity. Statistical effects were determined by 1-way ANOVA with Student-Newman-Keuls post hoc test.

View larger version (6K): [in this window] [in a new window]   Fig. 7. Effect of EGF on protein phosphatase 1 (PP1) activity in T84 cells. Monolayers were treated with EGF (100 ng/ml) basolaterally for the times indicated. Monolayers were then lysed as described in MATERIALS AND METHODS, and cell lysates were then used in the protein phosphatase activity assay. Data are means ± SE for 5 experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effects of calyculin A on carbachol-induced chloride secretion and the inhibitory effect of EGF on carbachol-induced chloride secretion in T84 cells. A: monolayers on inserts were pretreated bilaterally with or without calyculin A at the concentrations shown, followed 30 min later by basolateral addition of carbachol (100 µM). Control monolayers received carbachol alone. Calyculin A at 0.5 and 5 nM, but not 50 nM, did not affect carbachol-induced chloride secretion. Therefore, 0.5 and 5 nM concentrations were selected for further study. B: monolayers were treated with basolateral EGF (100 ng/ml) 15 min after calyculin A pretreatment at 0.5 or 5 nM, followed 30 min later by basolateral addition of carbachol (100 µM). CA, calyculin A; CCh, carbachol. Data are presented as peak increases in short-circuit current (Isc) induced by carbachol. Data are means ± SE for 5 (A) and 7 (B) experiments. *P < 0.05; ** P < 0.01, response that differs significantly from that induced by carbachol alone. +P < 0.05, response that differs significantly from that to carbachol in the presence of EGF. All statistical effects were determined by ANOVA with Student-Newman-Keuls post hoc test.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Effects of okadaic acid (a specific inhibitor of PP2A) on carbachol-induced chloride secretion and the inhibitory effect of EGF on carbachol-induced chloride secretion in T84 cells. A: monolayers on inserts were pretreated bilaterally with okadaic acid at the concentrations shown, followed 30 min later by basolateral addition of carbachol (100 µM). Control monolayers received carbachol alone. Okadaic acid at 0.1 and 1 nM, but not 50 and 100 nM, did not affect carbachol-induced chloride secretion. Therefore, 0.1 and 1 nM concentrations were selected for further study. B: monolayers were treated with basolateral EGF (100 ng/ml) 15 min after okadaic acid pretreatment at 0.1 or 1 nM, followed 30 min later by basolateral addition of carbachol (100 µM). OK, okadaic acid. Data are presented as Isc induced by carbachol. Data are means ± SE for 4 (A) or 6 (B) experiments. **P < 0.01, response that differs significantly from that induced by carbachol alone. +P < 0.05; ++P < 0.01, response that differs significantly from that to carbachol in the presence of EGF. All statistical effects were determined by ANOVA with Student-Newman-Keuls post hoc test.

View larger version (20K): [in this window] [in a new window]   Fig. 10. Effects of okadaic acid on EGF-induced downregulation of carbachol-induced p85 serine phosphorylation. T84 cell monolayers grown on inserts were pretreated with or without basolateral EGF (100 ng/ml) 15 min after bilateral treatment with okadaic acid (1 nM), followed 30 min later by basolateral addition of carbachol (100 µM). Top panel depicts a representative blot, and bottom panel shows summary data, quantitated by densitometry and expressed as percentages of the untreated control. Data are means ± SE for 3 experiments. *P < 0.05, response that differs significantly from control. +P < 0.05, response that differs significantly from that induced by EGF and carbachol without pretreatment with okadaic acid. All statistical effects were determined by ANOVA with Student-Newman-Keuls post hoc test.

	DISCUSSION

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Significant information is available regarding the regulation of PI 3-kinase activity in cells stimulated with a variety of agonists. The enzyme is a heterodimer consisting of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. p85 is capable of interacting with a variety of proteins by virtue of the fact that it contains SH2, SH3, and proline-rich domains. It has been proposed to act as an adaptor protein, in a manner analogous to Shc, and also presumably serves to recruit the p110 subunit to its substrates in the plasma membrane following activation (15). The regulatory subunit p85 is activated by tyrosine phosphorylation (19).

The PI 3-kinase holoenzyme is a lipid kinase responsible for the production of 3-phosphorylated lipids and has been implicated in cell proliferation (36), cell movement (37), and glucose transport (28). Its activation also has been shown to be involved in regulating the Na⁺/H⁺ exchanger (23) and in the activation of sodium chloride absorption (22). PI 3-kinase can also act as a bifunctional enzyme. In addition to its lipid kinase activity, p110 also contains a serine kinase activity that can act on p85 (10, 5). In fact, activation of PI 3-kinase requires dimerization of p85 and p110, and the binding of p85 to p110 is extremely stable (38). The basis for activation could lie in phosphopeptide-induced conformational changes, which have been seen in the NH₂-terminal SH2 domain of p85 (30). However, when p85 is serine-phosphorylated, the lipid kinase activity of the holoenzyme is dramatically reduced (11). We have shown that both carbachol and EGF recruit p85 to anti-phosphotyrosine immunoprecipitates, but only EGF increased PI 3-kinase activity in colonic epithelial cells (35). Moreover, the inhibitory effect of EGF, but not that of carbachol, on chloride secretion was reversed by inhibitors of PI 3-kinase (35). We hypothesize that the p85 subunit of the PI 3-kinase could serve as a switch on the basis of serine vs. tyrosine phosphorylation.

Protein phosphorylation plays a key role in many cellular processes. The phosphorylation state of a target protein is regulated by opposing kinases and phosphatases (16). Although both EGF and carbachol induced tyrosine phosphorylation of p85, carbachol also induced serine phosphorylation of this protein. EGF failed to induce an increase in the level of serine phosphorylation, and interestingly, EGF pretreatment markedly reduced carbachol-induced serine phosphorylation of p85. These data strongly suggest that the differential effect of carbachol or EGF on PI 3-kinase activation, as well as on chloride secretion, may stem from their differential effect on p85 serine phosphorylation. Although it has been postulated that p110 could serve as a protein serine kinase that acts on p85, it was not clear how serine on p85 becomes dephosphorylated after cells are treated with EGF.

Protein phosphatase 2A is a major cytoplasmic serine/threonine phosphatase that plays an important role in the regulation of cell growth and a diverse set of cellular proteins, including metabolic enzymes, ion channels, hormone receptors, and kinase cascades (9, 27). There is presently little information on the regulation of protein phosphatase 2A. It has been postulated that phosphorylation of protein phosphatase 2A tyrosine 307 leads to its inactivation in A431 cells (6). We, however, did not observe tyrosine phosphorylation of protein phosphatase 2A in response to EGF in our system (data not shown). Conversely, several studies have also shown that a major function of protein phosphatase 2A is downregulation of the RAS/ERK MAP kinase pathway (2, 3). However, the mechanisms of this effect are controversial (1, 33). In EGF-treated adipocytes or PC12 chromaffin cells, protein phosphatase 2A has been shown to be responsible for ERK inactivation, suggesting that induction of protein phosphatase 2A activity by the growth factor may directly dephosphorylate ERK (2). These disparate results can be accounted by the fact that there are multiple targeting B subunits for protein phosphatase 2A. Different forms of this trimeric protein may be responsible for its multiple actions, because each B subunit has different activity and substrate specificity and displays tissue- and cell type-specific distribution (18, 24, 26, 31, 32).

PI 3-kinase activation is an important mechanism downstream of EGF receptor phosphorylation that is responsible for the suppression of carbachol-induced chloride secretion (35). The results of our studies illustrate a more complex mechanism whereby activation of PI 3-kinase is induced. Namely, the balance of tyrosine and serine phosphorylation of the p85 subunit plays a critical role in deciding whether the holoenzyme will display lipid kinase or serine protein kinase properties, as summarized diagrammatically in Fig. 11. If T₈₄ cells are treated with carbachol alone, both tyrosine (a condition that is required for activation of PI 3-kinase) and serine are phosphorylated. This likely inhibits association of p110 and p85, and therefore the lipid kinase activity of PI 3-kinase is not activated. EGF treatment, on the other hand, causes protein phosphatase 2A activation and thus serine dephosphorylation on p85, and as a result, p110 associates with p85 and the lipid kinase activity of the holoenzyme is activated. Our data likewise suggest that these signaling events have fundamental implications for the extent of chloride secretion. They further underscore the complex mechanisms that set the level of chloride (and fluid) secretion in colonic, and perhaps other, epithelia.

View larger version (18K): [in this window] [in a new window]   Fig. 11. A schematic showing the hypothesized effect of EGF or carbachol treatment on tyrosine or serine phosphorylation on the p85 subunit of PI 3-kinase as well as the role of PP2A. Carbachol treatment induces both tyrosine and serine phosphorylation of the p85 protein, reducing association with the p110 subunit. It is hypothesized that p110, when not combined with the p85 subunit, displays serine kinase activity. Therefore, serine residues on p85 will be constitutively phosphorylated. On the other hand, EGF treatment does not cause serine phosphorylation, secondary to its ability to activate PP2A, which can also reverse p85 serine phosphorylation induced by carbachol.

	GRANTS

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	ACKNOWLEDGMENTS

 
We thank Glenda Wheeler for assistance with manuscript submission.

This work was presented in part at the annual meeting of the American Gastroenterological Association in San Francisco, CA, May 2001, and published in abstract form (Chow JYC and Barrett KE. Gastroenterology 120, 5 Suppl 1: A22, 2001).

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. E. Barrett, Univ. of California, San Diego, Division of Gastroenterology, 0063, Univ. Center 303, 9500 Gilman Drive, La Jolla, CA 92093-0063 (e-mail: kbarrett{at}ucsd.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.

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