Endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by tyrosine kinases

Steven D. Freedman, Mark H. Katz, Eliza M. Parker, and Andres Gelrud

The Pancreas Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

We have shown that endocytosis at the apical plasma membrane of pancreatic acinar cells is regulated by the pH of the acinar lumen and is associated with cleavage of GP2, a glycosyl phosphatidylinositol-anchored protein. The aim of this study was to determine the transduction pathway by which endocytosis is activated. Apical endocytosis was studied in rat pancreatic acini by prestimulation with cholecystokinin followed by measurement of horseradish peroxidase (HRP) uptake. Lanthanum, staurosporine, and forskolin had no effect on HRP uptake. Cytochalasin D significantly inhibited endocytosis, indicating a dependence on actin filament integrity. Genistein and the specific tyrphostin inhibitor B42 also inhibited HRP uptake, implicating tyrosine kinases in the regulation of HRP uptake. With the use of an Src kinase-specific substrate, Src kinase activity was temporally related to activation of endocytosis. The tyrosine-dependent phosphorylation of an 85-kDa substrate in both rat and mouse pancreatic acini correlated with Src kinase activation and pH-dependent regulation of HRP uptake. These results indicate that apical endocytosis in acinar cells is associated with tyrosine kinase activation and is dependent on the actin cytoskeleton.

acini; pancreas


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

IN THE PANCREATIC ACINAR CELL, hormonal stimulation results in the insertion of large amounts of secretory granule membrane into the apical plasma membrane (APM). This membrane is postulated to remain as discrete lipid patches (6), which are then recycled back to the Golgi for reuse in the regulated secretory pathway. We have recently demonstrated that this apical endocytic process is regulated by the pH of the acinar lumen (10, 11). Specifically, endocytosis of horseradish peroxidase (HRP) was inhibited at pH values of <6.2, whereas maximal uptake occurred at pH values of 7.4-8.3. The activation of HRP uptake was associated with the cleavage of GP2, a glycosyl phosphatidylinositol (GPI)-anchored protein from the APM (12).

Endocytosis can occur at the basolateral or apical domain, potentially through different mechanisms. In renal proximal tubule cells, apical endocytosis of the ANG II receptor was dependent on microfilament and microtubule integrity, as evidenced by inhibition with cytochalasin D or colchicine, respectively (20). Similarly, actin microfilaments were found to be critical for endocytosis at the apical but not the basolateral surface of Madin-Darby canine kidney (MDCK) cells (13) as well as in Caco-2 cells (23).

Studies utilizing phorbol myristate have indicated that calcium signaling via protein kinase C is important in the regulation of apical endocytosis in both Caco-2 and MDCK cells (16, 23). In addition, activation of heterotrimeric GTP-binding proteins with mastoparan or increases in intracellular cAMP levels promoted the internalization of ricin at the apical surface of MDCK cells through a clathrin-independent pathway (8). In lymphocytes, modulation of protein kinase C or protein kinase A affected internalization of CD 59, a GPI-anchored protein (7). Taken together, these studies demonstrate that, in addition to the cytoskeletal matrix, signaling through cAMP and/or calcium pathways may play an important role in apical endocytic pathways.

Tyrosine kinases have also been implicated in endocytosis. Caveolin, an integral membrane component of caveolae, undergoes tyrosine-dependent phosphorylation both in vitro and in vivo in v-Src-transformed NIH/3T3 cells (17). Caveolae are important in potocytosis and may activate transmembrane signaling through their association with GPI-anchored proteins (1). Endocytosis of the asialoglycoprotein receptor in hepatocytes is dependent on tyrosine-induced phosphorylation (9, 15). Inhibiting phosphorylation of the receptor with genistein or tyrphostin blocks endocytosis at an early step. However, there are no data on whether nonreceptor tyrosine-dependent kinases are involved in apical endocytic events.

Given that endocytosis can be selectively activated by cleavage of GP2 from the APM, we sought to determine the signal transduction mechanism by which this process is activated in rat pancreas. The data indicate that apical endocytosis is regulated through a tyrosine kinase-dependent pathway.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

HRP, cytochalasin D, lanthanum, forskolin, and sodium orthovanadate were obtained from Sigma Chemical (St. Louis, MO); cholecystokinin (CCK-8) was from Penninsula Laboratories (Belmont, CA); genistein was from Research Biochemicals International (Natick, MA); tyrphostin A1 and B42 and staurosporine were from Calbiochem (La Jolla, CA); protein tyrosine kinase (Src family) assay kit, phosphotyrosine monoclonal antibody, and alkaline phosphatase-protein G were from Upstate Biotechnology (Lake Placid, NY); [32P]ATP was from New England Nuclear (Boston, MA); Immobilon-P was from Millipore (Bedford, MA); and phosphatidylinositol-specific phospholipase C (PI-PLC; 9.42 U/ml) was from ICN Biochemicals (Irvine, CA).

Analysis of HRP uptake in pancreatic acini. Rat (male Wistar, 75 g) or mouse (C57, adult males) pancreatic acini were prepared by collagenase and mechanical dissociation (10). Endocytosis at the APM was examined using our previously described two-step protocol (10), in which, in step one, acini are incubated for 1 h at 37°C with 0.5 nM CCK-8 in Krebs-Henseleit buffer (KHB) to maximize exocytosis and the insertion of zymogen granule membrane into the APM. Cells are then washed in buffer alone, and then, in step two, endocytosis is assessed by incubation of acini with HRP (5 mg/ml final concentration) in KHB at pH 7.4. For studies examining the effects of various agents on the inhibition of HRP uptake, acini prestimulated with CCK were incubated with the various agents at 4°C for 10 min in KHB-containing HRP followed by incubation for 30 min at 37°C. HRP uptake was measured using a colorimetric assay (3). Statistical analyses were performed using Student's t-test, and results were expressed as means ± SD.

Src kinase assay. Using the protocol supplied with the Src assay kit, 5 µl of the substrate peptide were added to 5 µl of reaction buffer containing (in mM) 100 Tris (pH 7.2), 125 MgCl2, 25 MnCl2, 2 EGTA, 0.25 sodium orthovanadate, and 2 dithiothreitol. Sample (10 µl) was added at 4°C, and the reaction initiated by addition of 10 µl of a solution containing 75 mM MnCl2 and 0.5 mM ATP plus 10 µCi of [32P]ATP (3,000 Ci/mmol). After incubation for various times at 30°C, 20 µl of 40% TCA were added to precipitate the peptide. After incubating at room temperature for 5 min, 25 µl were spotted on 2 × 2 cm P81 filter paper. The filters were washed three times with 0.75% phosphoric acid and once with acetone and then were transferred to scintillation vials. Radioactivity was measured in a Wallack model 1409 liquid scintillation counter.

Western blot analysis. With the use of the same protocol for the analysis of HRP uptake, these same samples were subjected to SDS-PAGE using 10% polyacrylamide gels. This was followed by transfer to Immobilon-P membranes. The blot was then incubated for 2 h at room temperature with a 1:2,000 dilution of a mouse monoclonal phosphotyrosine antibody. The blot was then washed and incubated for 1 h in protein G conjugated to alkaline phosphatase. Bound antibody was detected by reacting with alkaline phosphatase. Phosphorylated polypeptides on Western blots were scanned with a Hewlett-Packard 4C scanner, and the intensity of the bands was quantitated using NIH Image software.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Determination of the signal transduction process in apical endocytosis. To determine the signal transduction process by which endocytosis is regulated, pancreatic acini prestimulated with CCK were incubated with 20 µM cytochalasin D (microfilament inhibitor), 100 µM lanthanum (calcium uptake inhibitor), 100 nM staurosporine (protein kinase C inhibitor), 10 µM forskolin (protein kinase A activator), 50 µM genistein (tyrosine kinase inhibitor), or 200 µM orthovanadate (tryosine phosphatase inhibitor). As shown in Fig. 1, there was ~90% inhibition of HRP uptake by cytochalasin D, confirming previous studies showing that actin filaments are required for endocytosis at the apical surface of polarized epithelial cells (13). Lanthanum was without significant effect as well as staurosporine, consistent with our prior data on calcium signaling, demonstrating that HRP uptake could not be activated with a phorbol ester or diacylglycerol (12). Similarly, activation of protein kinase A with forskolin had no significant effect compared with the control condition. In contrast, genistein inhibited HRP uptake by ~50%. If HRP uptake is regulated by tyrosine kinases, inhibition of tyrosine phosphatase would be expected to enhance HRP uptake. The 62% increase in HRP uptake observed with orthovanadate compared with the control condition is consistent with tyrosine-dependent regulation of HRP uptake.


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Fig. 1.   Effects of modulators of signal transduction on horseradish peroxidase (HRP) uptake. Rat pancreatic acini were stimulated with cholecystokinin (CCK), washed free of hormone, and then incubated for 30 min at 37°C in Krebs-Henseleit buffer (KHB) containing HRP alone (control) or one of the above agents. HRP uptake is expressed as ng HRP/mg cell protein. Means ± SD are shown (n = 3). * P < 0.005 compared with control condition.

In Fig. 2, the effect of different concentrations of tyrphostin A1, an inactive analog of the tyrphostins, is shown. No significant inhibition of HRP uptake was observed. In contrast, the active analog tyrphostin B42 resulted in ~70% inhibition of HRP uptake at concentrations of 50-100 µM. This is similar to the degree of inhibition observed by these agents as well as genistein in the tyrosine-dependent epidermal growth factor regulation of rabbit intestinal smooth muscle (14). These results demonstrate that HRP uptake is regulated through tyrosine kinase activation.


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Fig. 2.   HRP uptake is inhibited selectively by the active analog tyrphostin B42. To determine specificity of genistein's inhibition of HRP uptake, effects of 10, 50, and 100 µM tyrphostin A1 and tyrphostin B42 on HRP uptake were analyzed using same protocol described in Fig. 1. Results are expressed as mean HRP uptake ± SD from 3 experiments. Compared with control values, only tyrphostin B42 showed a statistically significant difference at 50 and 100 µM (P < 0.005).

Src kinase activity correlates with activation of HRP uptake. To determine whether the tyrosine-dependent regulation of HRP uptake may be mediated through Src kinases, acini prestimulated with CCK were incubated for various times at 37°C in KHB buffered at either pH 6.0 or 7.4. Src kinase activity was measured by incubating cell lysates with an Src-specific peptide substrate in vitro and measuring labeled phosphate incorporation in the presence of [gamma -32P]ATP. As shown in Fig. 3, Src kinase activity in acini incubated in KHB at pH 7.4 peaked at 15 min and then decreased over the subsequent 30 min examined. Src kinase activity did not increase in acini incubated in KHB at pH 6.0. No significant activity was detected using an Src nonspecific peptide substrate or a calcium-dependent phosphoserine-specific substrate under these conditions.


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Fig. 3.   Src kinase activity is associated with the activation of HRP uptake. To determine whether tyrosine-dependent regulation of HRP uptake may be mediated through Src kinases, Src kinase activity was measured as a function of time by analysis of 32P incorporation into an Src-specific peptide substrate. Acini incubated with CCK were washed and then incubated in KHB at pH 7.4 (maximal endocytosis) or pH 6.0 (no endocytosis) for times indicated. Results from 5 experiments are expressed as mean percent Src kinase activity ± SD, where 100% is defined as Src kinase activity in acini at the end of the prestimulation with CCK (before incubating in KHB at either pH 6.0 or 7.4).

The tyrosine-dependent phosphorylation of an 85-kDa polypeptide correlates with HRP uptake. To examine the substrates phosphorylated by tyrosine-dependent kinases, acini incubated under the same conditions used to examine HRP uptake were subjected to Western blot analysis using a monoclonal antibody that binds to tyrosine phosphoproteins. We have previously shown that HRP uptake is abolished when acini are incubated in KHB buffered at pH 6.0, whereas maximal stimulation occurs at pH 7.4 (10). This block in endocytosis of HRP at pH 6.0 could be completely overcome by addition of PI-PLC. The effect of PI-PLC on restoring HRP uptake at pH 6.0 was due to the cleavage of GPI-anchored proteins, specifically GP2 (12). To determine whether phosphorylation of specific proteins correlates with HRP uptake, acini preincubated for 1 h with CCK were subsequently incubated in KHB buffered at pH 6.0 or 7.4 for 30 min at 37°C. Western blot analysis of these fractions is shown in Fig. 4A. With the use of rat pancreatic acini, the phosphorylation of an 85-kDa protein was specifically increased when cells were incubated at pH 7.4 compared with 6.0. The addition of PI-PLC in KHB buffered at pH 6.0 also increased the phosphorylation of this protein. No other phosphorylated substrates reproducibly showed this correlation with endocytosis.


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Fig. 4.   Western blot analysis of acini for tyrosine-phosphorylated proteins. Acini incubated under conditions described in Fig. 1 were subjected to Western blot analysis using a monoclonal antibody to tyrosine phosphopeptides. A: tyrosine phosphorylation in rat acini is shown following incubation in KHB buffered at pH 6 (inhibition of endocytosis), pH 7.4 (maximal endocytosis), and in KHB at pH 6 + phosphatidylinositol-specific phospholipase C (PLC; 0.02 U/400 µl reaction vol). Arrow, position of 85-kDa phosphoprotein. B: same conditions, except that mouse pancreatic acini were utilized. C: rat pancreatic acini from A (pH 7.4 condition) were homogenized in 0.3 M sucrose containing 0.1 mg/ml soybean trypsin inhibitor and then centrifuged at 100,000 g for 1 h at 4°C in a Beckman TL-100 centrifuge. Pellet (total particulate fraction) is shown in lane 1. Remaining supernatant is shown in lane 2. The 85-kDa protein indicated by the arrow was exclusively localized to the particulate fraction. Mr, relative molecular mass.

A similar phosphoprotein should be demonstrable in other species, given the universal nature of apical endocytosis. Therefore, mouse pancreatic acini were examined using the same protocol utilized for rat. As shown in Fig. 4B, an 80- to 85-kDa phosphoprotein also demonstrated an increase in phosphorylation as the pH of the medium was increased from 6.0 to 7.4. Similarly, PI-PLC increased the phosphorylation of this protein in cells incubated in KHB buffered at pH 6.0. This phosphoprotein localizes to a 100,000-g particulate fraction (Fig. 4C). This indicates that, in two species, phosphorylation of a membrane-associated 85-kDa polypeptide shows a tight correlation with endocytosis as assessed by HRP uptake.

To examine the relevance to endocytosis, the effects of genistein and orthovanadate on the tyrosine-dependent phosphorylation of the 85-kDa protein was examined. As shown in Fig. 5, 50 µM genistein inhibited phosphorylation of this substrate in intact acini by 56% compared with the control condition. In contrast, addition of orthovanadate resulted in a 76% increase in the phosphorylation of the 85-kDa protein over control values. This is similar to the results observed for genistein and orthovanadate on HRP uptake (Fig. 1).


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Fig. 5.   Effect of genistein and orthovanadate on tyrosine-dependent phosphorylation of the 85-kDa protein. Using same protocol described in Fig. 4, acini prestimulated with CCK were incubated in absence (control condition) or presence of 50 µM genistein or 200 µM sodium orthovanadate for 30 min at 37°C in KHB at pH 7.4. Western blot analysis using a monoclonal antibody to tyrosine phosphopeptides was performed. Phosphorylation of the 85-kDa protein was quantitated and data from 3 experiments expressed as percent control condition (means ± SD).

The phosphorylation of the 85-kDa protein in rat pancreatic acini as a function of pH of the medium is shown quantitatively in Fig. 6. After incubation in KHB buffered at pH 7.4, phosphorylation of the 85-kDa protein was maximal at 15 min. Dephosphorylation was observed over the subsequent 30 min to levels approaching that observed for the pH 6.0 condition. The increase in phosphorylation of the 85-kDa protein at pH 7.4 compared with pH 6.0 is similar to the results observed for Src kinase activity (Fig. 3). Note that some increase in the phosphorylation of this substrate occurs in acini incubated in KHB buffered at pH 6.0 over the initial 20 min of incubation. Assuming there are multiple sites of phosphorylation as is typical for many phosphorylated proteins, the explanation for this phenomenon may be as follows. Phosphorylation at specific sites on the 85-kDa protein under conditions of incubation in KHB at pH 6.0 may be unrelated to endocytosis, or, alternatively, phosphorylation at certain sites on this molecule may inhibit endocytosis in a manner similar to activation or inhibition of Src kinase, depending on which sites are phosphorylated.


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Fig. 6.   Time course of phosphorylation of 85-kDa protein as a function of pH of incubation medium. Acini incubated under the same conditions shown in Fig. 4 were subjected to Western blot analysis. The 85-kDa protein was then quantitated and expressed as the percent maximum phosphate incorporation under conditions of incubation in KHB at pH 7.4 or pH 6.0. Means ± SD are shown from 4 experiments.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The results of this study demonstrate that endocytosis at the APM of the pancreatic acinar cell is regulated through activation of tyrosine kinase(s). The tyrosine-dependent phosphorylation of principally one substrate correlated with the activation of HRP uptake. This protein, with an apparent relative molecular mass of 85 kDa, was present in both mouse and rat pancreas. Although this protein was localized to the particulate fraction, its identity remains unknown.

Does phosphorylation of the 85-kDa protein mediate apical endocytosis and is this process regulated through Src kinase activation? Our data are correlative, but the fact that genistein inhibited and orthovanadate stimulated phosphorylation of the 85-kDa protein to an extent similar to that observed for HRP uptake suggests a relationship between these two processes. Activation of Src kinase activity was maximal within 15 min, correlating with peak phosphorylation of the 85-kDa protein. Src kinase activity returned to pH 6.0 levels within 30 min of incubation. Coincident with this, the 85-kDa protein dephosphorylated, although with slower kinetics that are perhaps related to the regulation of phosphatase activity.

Other signaling pathways were not identified. We have previously shown that modulation of calcium-dependent pathways by measurement of fura 2 or the addition of phorbol esters or diacylglycerol had no effect on the activation of HRP uptake in this system (12). Similarly, increasing intracellular cAMP had no effect on HRP uptake. The inhibition of HRP uptake by cytochalasin D is consistent with the finding in other polarized epithelial cell systems that actin filaments are critical in the endocytic process (7, 13, 20, 23). Of note, the inhibition of HRP uptake by cytochalasin D in our study was much greater than that observed for genistein. This is most likely explained by the fact that genistein only partially inhibits tyrosine kinase activity at this dose (14), although we cannot exclude that HRP uptake may occur through other constitutive pathways.

Because the cleavage of GP2 from the APM appears to be the signal that activates HRP uptake in these cells (12), activation of tyrosine kinases may be the transduction pathway through which GP2 mediates this endocytic process. Tyrosine kinase activity has been shown to colocalize with immunoprecipitates of GPI-anchored proteins. In human T lymphocytes, CD59, CD55, and CD48 coimmunoprecipitate with p56lck (25). However, the mechanism by which a GPI-anchored protein, which is confined to the exoplasmic leaflet of the lipid bilayer, can lead to activation of Src kinases on the endoleaflet remains unknown. A likely explanation is that a transmembrane protein also exists, which transduces the signal across the lipid bilayer. Solomon et al. (24) have found that heterotrimeric GTP binding proteins colocalize to these complexes. These authors postulate that clustering of the GPI-anchored proteins on the ectoleaflet of the membrane leads to aggregation of heterotrimeric GTP binding proteins with the tyrosine kinases p59fyn and p56lck on the endoleaflet of the membrane, resulting ultimately in cell proliferation and cytokine production.

Shenoy-Scaria et al. (21) have examined the mechanism by which cross-linking the GPI-linked protein decay-accelerating factor (DAF) leads to lymphocyte proliferation. EL-34 murine thymoma cells were transfected with the cDNA encoding either DAF or a transmembrane form of DAF (DAF-TM). Only the GPI-linked form led to cellular proliferation, activation of tyrosine kinases (p59fyn and p56lck), and phosphorylation of substrates, including an 85-kDa protein. In one study, palmitoylation of an amino-terminal cysteine motif of p59fyn and p56lck was required for colocalization with immunoprecipitates of DAF (22), although another study found palmitoylation was not required (18). However, this cysteine residue at position 3 appears to be critical in forming a complex with GPI-linked proteins. This is absent in p60src, which does not coimmunoprecipitate with DAF.

GPI-anchored proteins are generally clustered within specific lipid domains enriched in glycosphingolipids on the cell surface (4, 5). The cholesterol within this lipid domain is essential for the clustering of GPI-anchored proteins (19) and may contain caveolin (1). Because of the glycosphingolipid composition, these complexes can be isolated as Triton X-100-insoluble complexes from cell lysates. Colocalization of the nonreceptor tyrosine kinase p62yes to these detergent-insoluble membrane complexes has been demonstrated in MDCK cells (2).

Taken together, GPI-anchored proteins appear to exist in a complex with nonreceptor tyrosine kinases in lymphocytes as well as polarized epithelial cells such as MDCK cells. These lipid microdomains on the cell surface, by virtue of other components such as heterotrimeric GTP binding proteins or caveolin, would contain the machinery necessary for endocytosis. In addition, GPI-anchored complexes are associated with patches of polymerized actin that are critical in the early events in endocytosis (7). The current findings that 1) apical endocytosis in the pancreatic acinar cell is regulated by tyrosine kinase activation and 2) our previous studies showing that GPI anchor cleavage is the signal that activates this endocytic process suggest that GP2 may be complexed with tyrosine kinases such as Src. The cleavage of GP2 from the membrane would appear to be the signal that activates membrane internalization through tyrosine kinase signaling, a process that subsequently is dependent on actin filaments.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-52765 (to S. D. Freedman).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: S. D. Freedman, Beth Israel Deaconess Medical Center, Dana 501, 330 Brookline Ave., Boston, MA 02215.

Received 19 March 1998; accepted in final form 13 October 1998.


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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Cell Physiol 276(2):C306-C311
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