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J. Biol. Chem., Vol. 277, Issue 28, 25217-25225, July 12, 2002

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Protein Kinase C and Guanosine Triphosphate Combine to Potentiate Calcium-dependent Membrane Fusion Driven by Annexin 7^*

Hung Caohuy and Harvey B. Pollard

From the Department of Anatomy, Physiology, and Genetics, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

Received for publication, March 13, 2002, and in revised form, May 3, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Exocytotic secretion is promoted by the concerted action of calcium, guanine nucleotide, and protein kinase C. We now show that the calcium-dependent membrane fusion activity of annexin 7 in vitro is further potentiated by the combined addition of guanine nucleotide and protein kinase C. The observed increment involves the simultaneous activation of annexin 7 by these two effectors. Guanosine triphosphate (GTP) and its non-hydrolyzable analogues optimally enhance the phosphorylation of annexin 7 by protein kinase C in vitro. Reciprocally, phosphorylation by protein kinase C significantly potentiates the binding and hydrolysis of GTP by annexin 7. Only protein kinase C-dependent phosphorylation has a significant positive effect on annexin 7 GTPase, although other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and pp60^c-src, have been shown to label the protein with high efficiency. In vivo, the ratio of bound GDP/GTP and phosphorylation of annexin 7 change in direct proportion to the extent of catecholamine release from chromaffin cells in response to stimulation by carbachol, or to inhibition by various protein kinase C inhibitors. These results thus lead us to hypothesize that annexin 7 may serve as a common site of action for calcium, guanine nucleotide, and protein kinase C in the exocytotic membrane fusion process in chromaffin cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Guanosine triphosphate (GTP)¹ and its non-hydrolyzable analogues (i.e. GTPS and GMP-P(NH)P) are known to promote Ca²⁺-dependent exocytotic secretion from chromaffin cells and many other cell types (1-10). Likewise, activation of protein kinase C (PKC) has been shown to trigger Ca²⁺-dependent secretion in these secreting cell types as well (11-24). Furthermore, many permeabilized cell studies have also supported a role for PKC in further enhancing the stimulatory action of Ca²⁺ and GTP in the exocytotic process (9, 17, 23, 25-29). These observations thus have led to the proposal of a hypothetical exocytotic model by Lillie and Gomperts (30) in which Ca²⁺, GTP, and PKC act in concert in a regulatory sequence leading to exocytosis (30). In this model, two GTP-binding proteins are involved in controlling the stimulus-secretion process. The first GTP-binding protein is the putative receptor-linked G-protein (G_P) that controls the activity of phospholipase C, thereby generating inositol 1,4,5-trisphosphate and diacylglycerol. Further downstream from the signal transduction level, a second GTP-binding protein (G_E, E for exocytosis), a putative GTPase so far undefined as a molecular entity, acts in parallel, or might be closely associated with a Ca²⁺-binding protein at the docking/fusion site of the exocytotic machinery. Subsequent phosphorylation by diacylglycerol-activated PKC triggers these proteins into mediating the exocytotic membrane fusion process. Although phenomenologically well known, the specific sites of action of Ca²⁺, GTP, and PKC in the stimulus-secretion cascade remain unknown.

Annexin 7 (ANX7; synexin) is a Ca²⁺-dependent membrane fusion protein (31-34), for which recent evidence has strongly suggested the possibility of its involvement in exocytosis. For example, we have reported that ANX7 is a Ca²⁺-activated GTPase, both in vitro and in secreting chromaffin cells, and that in vitro membrane fusion activity of ANX7 is further enhanced upon binding to GTP (35). More recently, we have reported that the heterozygous knockout Anx7(+/) mouse suffers from an insulin secretion deficit from islets of Langerhans, as well as defective Ca²⁺ signaling processes in -cells (36). Furthermore, we have reported that ANX7 is phosphorylated by PKC, both in vitro and in secreting chromaffin cells (37). Phosphorylation by PKC significantly potentiates the ability of ANX7 to fuse phospholipid vesicles, and the apparent K_1/2 of Ca²⁺ is lowered from 200 to 50 µM (37). Sequence and site-directed mutagenesis studies of ANX7 have shown that putative binding sites for GTP are located in proximity to consensus phosphorylation sites for PKC. These data thus have led us to hypothesize that these two processes may modulate the action of each other in activating ANX7-driven membrane fusion. To test this hypothesis, we have investigated the interconnections between PKC and GTP action on the Ca²⁺ dependence of ANX7-driven membrane fusion both in vitro and in vivo.

In this study, we report that GTPS and PKC both mutually enhance the binding of each other to ANX7, and also potentiate Ca²⁺-dependent membrane fusion driven by ANX7. In vitro, phosphorylation of ANX7 by PKC is optimally enhanced by GTP and its non-hydrolyzable analogues. Reciprocally, the binding and hydrolysis of GTP by ANX7 are markedly potentiated by PKC-catalyzed phosphorylation. Whereas certain other kinases label ANX7 efficiently, they do not substitute for PKC in potentiating GTP binding or membrane fusion. In vivo, we find that for ANX7, both the ratio of bound GDP/GTP as well as phosphorylation by PKC change in proportion to the extent of catecholamine release from stimulated chromaffin cells. Thus, GTP and PKC combine specifically to transform ANX7 into a highly efficient Ca²⁺-dependent membrane fusogen. We therefore conclude that the membrane fusion machinery might include ANX7 as a common site of action for Ca²⁺, GTP, and PKC in the exocytotic membrane fusion process.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation of Phosphatidylserine Lipid Vesicles-- PS lipid vesicles were prepared fresh daily by the swelling method (38). Highly purified (>99%) brain phosphatidylserine (Avanti Polar Lipids) in a 1:4 chloroform-methanol solution was dried slowly under nitrogen and then allowed to swell in 0.3 M sucrose at room temperature. The suspension was then sonicated and centrifuged at 12,000 × g. The PS lipid vesicle pellet was resuspended in sucrose solution.

Isolation and Purification of Human Recombinant ANX7-- Human recombinant ANX7 was isolated and purified as described (39). Briefly, Escherichia coli bacteria containing the ANX7-expressing vector (pTrc-FLS) were grown in 1 liter of Luria broth at 37 °C. After incubation overnight with 1 mM isopropyl--D-thio-galactopyranoside (ICN), the bacteria were harvested by centrifugation. Expressed recombinant ANX7 was then extracted from the E. coli paste, concentrated by precipitation with 0-20% (w/v) (NH₄)₂SO₄, and purified by gel filtration using Ultragel AcA54 (Biosphere). This partially purified ANX7 preparation was further purified by binding to PS lipid vesicles in the presence of Ca²⁺ and extracting with EGTA. This purification step was repeated six times to yield a highly purified ANX7 preparation (98%) determined by SDS-PAGE and silver staining.

Lipid Vesicle Fusion Mediated by ANX7-- Simultaneous phosphorylation and phospholipid vesicle fusion reactions were carried out as described (37). The reactions in a final volume of 1 ml contained 1 µg of ANX7, 0.5 unit of PKC, 0.3 M sucrose, 40 mM histidine (pH 6.1), 2 mM MgCl₂, 100 nM PMA, 100 µM ATP, 0.5 ml of lipid vesicle suspension, and with or without 100 µM GTPS. The controls were carried out in the absence of ATP and/or GTPS, or in the presence of 500 µM GDPS. Fusion and phosphorylation were simultaneously initiated by the addition of 1 mM [Ca²⁺]_final at room temperature. Fusion was measured by the change in the turbidity in absorbance at 540 nm (A₅₄₀) over a 30-min period using a recording Hewlett-Packard spectrophotometer. The final Ca²⁺ concentration was verified using a Ca²⁺-selective electrode.

Fusion and phosphorylation reactions in the presence of other protein kinases were carried out as described for the above PKC experiments, except that no PMA was added. PKC was replaced by 2000 units of PKG (plus 10 µM cGMP), 500 units of PKA_cat, or 100 units of pp60^c-src. These conditions resulted in mole ratios of P_i to protein of 1.0 (37).

In Vitro Phosphorylation of ANX7-- Phosphorylation assays using purified rat brain PKC were performed as described (37). Rat brain PKC, with a purity of "95%" and containing isoforms , , and , was purchased from Calbiochem. To determine the effects of guanine nucleotides on ANX7 phosphorylation, 1 µg of ANX7 was incubated at 30 °C for 1 h with 0.05 unit (0.035 µg) of PKC in a final volume of 30 µl of reaction buffer. This buffer consisted of 25 mM PIPES (pH 6.8), 10 mM MgCl₂, 1 mM CaCl₂, 100 nM PMA, 400 µg/ml PS liposomes, and contained one of the following nucleotides, each with a concentration of 100 µM: GTP, GTPS, GMP-P(NH)P, or GDPS. For the time course, 1 µg of ANX7 and 0.05 unit of PKC were incubated at 30 °C for the indicated time periods in the presence or absence of 100 µM GTPS. To determine ANX7 phosphorylation as a function of GTPS concentration, 1 µg of ANX7 was incubated at 30 °C for 1 h with 0.05 unit of PKC in the presence of various concentrations of GTPS, as indicated in the figure legends. The controls were carried out in the absence of guanine nucleotides. All reactions were initiated by the addition of 100 µM [-³³P]ATP (3000-4000 cpm/pmol; Amersham Biosciences) and terminated by the addition of the SDS-PAGE sample buffer. The reaction products were analyzed by SDS-PAGE and phosphorimaging (PhosphorImager, Molecular Dynamics).

As for ANX7 phosphorylation by PKA, PKG, and pp60^c-src (37), ANX7 (1 µg) was incubated at 30 °C for 1 h with 200 units of PKG (plus 10 µM cGMP), 50 units of PKA_cat, or 10 units of pp60^c-src in 25 mM MES (pH 6.1), 10 mM MgCl₂, 1 mM CaCl₂, and 50 µM GTPS. The controls were carried out in the absence of 50 µM GTPS. All phosphorylation reactions were initiated by the addition of 100 µM [-³³P]ATP (3000-4000 cpm/pmol) and were analyzed as described above for the PKC reactions.

Extraction of Phosphorylated and Unphosphorylated ANX7-- Phosphorylation and extraction of the PKC phosphorylated and unphosphorylated protein from lipid vesicles was performed exactly as previously described (37). Phosphorylated ANX7 has a molar ratio of P_i to protein of 2.0.

GTP Binding-- GTP binding of ANX7 was determined with the photoaffinity labeling assay using 8-N₃-[-³²P]GTP as described (35), with minor modifications. ANX7 (1 µg) and PKC (0.05 unit) were simultaneously incubated at 30 °C in 25 mM PIPES (pH 6.8), 10 mM MgCl₂, 1 mM CaCl₂, 100 nM PMA, 400 µg/ml PS liposomes, 8 µM 8-N₃-[-³²P]GTP (ICN; 10 µCi/mmol), and with or without 100 µM ATP in a final volume of 30 µl. At the indicated times, 4 mM glutathione was added to each sample, and the samples were irradiated for 30 s at room temperature, followed by SDS-PAGE and phosphorimaging analysis.

GTPase Activity-- Assay of ANX7 GTPase was carried out simultaneously with the phosphorylation reaction, and the hydrolytic products were assayed as described (35). To determine the effect of phosphorylation by PKC on ANX7 GTPase activity, 1 µg of ANX7 was incubated at 30 °C for the indicated times with or without 0.05 unit of PKC in 25 mM PIPES (pH 6.8), 10 mM MgCl₂, 1 mM CaCl₂, 100 nM PMA, and 400 µg/ml PS liposomes, in a final volume of 30 µl. To determine the GTPase activity as a function of ANX7 concentration, the indicated concentrations of ANX7 were incubated at 30 °C for 1 h with or without 0.05 unit of PKC in the same reaction condition as above. The controls were carried out in the absence of PKC and ANX7, or in the presence of PKC alone. All reactions were initiated by the addition of 100 µM ATP and 50 µM [-³³P]GTP (2000-3000 cpm/pmol; Amersham Biosciences), and terminated by the addition of 10 µl of 0.5 M EDTA. The reactions (1-µl aliquot) were resolved by thin layer chromatography on polyethyleneimine-cellulose plates (Merck) in 1 M LiCl: 1 M formic acid. GTP hydrolysis was assessed by quantitating the formation of [-³³P]GDP with a PhosphorImager. The results, after subtracting the background, were calculated as total GDP formed. Background was obtained from the reactions containing PKC alone and without ANX7 and PKC.

In other GTPase assays, phosphorylated ANX7 and its unphosphorylated forms, at protein ratios of 3:0, 2:1, 1.5:1.5, 1:2, or 0:3, respectively, were incubated at 30 °C for 1 h in the phosphorylation buffer (without ATP, PMA, and PS liposomes) containing 50 µM [-³³P]GTP (2000-3000 cpm/pmol). Each reaction contained the same amount of total ANX7 protein (0.75 µg/30-µl reaction). GTP hydrolysis was assessed by chromatography on polyethyleneimine plates as described above.

To determine ANX7 GTPase in the presence of other kinases, ANX7 (1 µg) was incubated at 30 °C for 1 h with or without 200 units of PKG (plus 10 µM cGMP), 50 units of PKA_cat, or 10 units of pp60^c-src in 25 mM MES (pH 6.1), 10 mM MgCl₂, 1 mM CaCl₂, 100 µM ATP, and 50 µM [-³³P]GTP. The controls were carried out in the absence of the respective kinases and ANX7, or in the presence of the kinase alone. GTP hydrolysis was analyzed as described above.

Isolation and Culture of Chromaffin Cells-- Chromaffin cells were isolated from bovine adrenal glands by collagenase digestion and purified on Percoll gradient, as described (37). Isolated cells were further purified by a selective plating method (40) and maintained in a CO₂ incubator under 5% CO₂, 95% air.

[³³P]Orthophosphoric Acid Labeling and Treatment of Chromaffin Cells with Carbachol and PKC Inhibitors-- Cultured chromaffin cells (5 × 10⁶/dish, Falcon, 35 mm) were labeled with [³³P]orthophosphoric acid (0.2 mCi/ml; Amersham Biosciences) in phosphate-free Eagle's minimal essential medium containing 10% dialyzed fetal calf serum for 10 h at 37 °C (37). The cells were washed once with Ca²⁺-free extracellular buffer A (118 mM NaCl, 4.2 mM KCl, 10 mM NaHCO₃, 10 mM glucose, 25 mM Hepes (pH 7.2), 0.1% bovine serum albumin, and 1.2 mM MgCl₂). The cells were pretreated for 1 h at 37 °C with or without 50 nM calphostine C (Calbiochem) or 0.7 µM chelerythrine chloride (Calbiochem) in buffer A and then stimulated to secrete by incubation for 30 min at 37 °C in the presence or absence of 100 µM carbachol (Sigma) in extracellular buffer B (buffer A with 2.2 mM CaCl₂ added). The control experiments were performed using cells incubated with buffer B or with buffer B containing Me₂SO₄. The latter solvent was a necessary control for drugs such as calphostine C and chelerythrine chloride, which are soluble in Me₂SO₄. After incubation, the media were collected for measuring catecholamine secretion. The cells were rapidly washed twice and then lysed in lysis buffer for immunoprecipitation as described below.

Immunoprecipitation of ³³P-Labeled ANX7-- The cells were lysed in 1 ml of ice-cold lysis buffer as described (37). After clarification by centrifugation, the resulting lysates were precleared by incubation for 30 min with 50 µl of a 10% (v/v) suspension of protein G-Sepharose (Zymed Laboratories Inc.), followed by centrifugation. The final lysates, with equal protein amounts determined by the BCA method (Pierce), were incubated with 10 µg of anti-ANX7 monoclonal antibody 10E7 for 6 h at 4 °C. The immunoprecipitates were divided into two equal halves, one for determining ANX7 phosphorylation levels and one for assaying GTP/GDP bound to ANX7. All immunoprecipitates were collected on protein G-Sepharose and washed four times by pelleting in ice-cold lysis buffer.

Determination of ANX7 Phosphorylation and Bound GDP/GTP to ANX7-- In the assay to determine the extent of ANX7 phosphorylation, the immunoprecipitates were subjected to SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes. Radioactively labeled ANX7 was analyzed by phosphorimaging. To determine the amounts of ANX7 present in the immunoprecipitates, the same membranes were blotted with a polyclonal antibody against ANX7. The bound primary antibody was detected using a peroxidase-conjugated secondary antibody and visualized chromographically using 4-chloro-1-naphthol. In the assay to determine the bound GDP/GTP to ANX7 (35, 41), the immunoprecipitates were incubated for 20 min at 68 °C in elution buffer (25 mM Tris-HCl (pH 7.5), 2 mM EGTA, 2 mM DTT, 0.2% SDS, 0.5 mM GTP, 0.5 mM GDP), followed by centrifugation at 12,000 × g for 15 min at 4 °C. The supernatants were collected, concentrated by lyophilization, and then resuspended in distilled water to a final volume of 5 µl. The entire samples were spotted on a thin layer cellulose plate (Merck), followed by chromatography in 1 M LiCl, 1 M formic acid buffer. Radioactively labeled nucleotides were analyzed by phosphorimaging.

Measurement of Catecholamine Release-- The assay for catecholamine release from chromaffin cells was performed exactly as described previously (37). The release of catecholamines was expressed as a total amount released into the medium.

Statistical Analysis-- Data are presented as mean ± S.D. A relationship between catecholamine secretion and ANX7 phosphorylation and its guanine nucleotide binding profile was assessed by a linear regression analysis (left and right y axes, mean values of the ratio of ANX7-bound GDP/GTP and ANX7 phosphorylation, respectively, induced by carbachol and inhibited by PKC inhibitors; x-axis, mean value of catecholamine secretion under similar conditions as above). The statistical significant values (p) were determined by Student's t test, and a p value less than 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effects of GTPS and PKC-dependent Phosphorylation on ANX7-driven Membrane Fusion Activity-- Several studies have shown that Ca²⁺-dependent secretion is further enhanced by the combined action of guanine nucleotides and PKC in vivo (9, 17, 23, 25-29). In addition, we have previously reported that GTP (35) and PKC (37) independently activate the Ca²⁺-dependent membrane fusion activity of ANX7 in vitro. We therefore tested the hypothesis that the ANX7 membrane fusion activity in vitro might be further potentiated by the combined addition of GTP and PKC. To test this possibility, we examined the ANX7-driven lipid vesicle fusion reaction during simultaneous activation of both the phosphorylation of ANX7 by PKC and the presence of GTPS on ANX7-induced fusion of lipid vesicles. As the fusion reaction progresses in the presence of 1 mM Ca²⁺, the lipid vesicle fusion activity of ANX7 is indeed activated further by GTPS plus PKC (Fig. 1A). The rate and extent of lipid vesicle fusion induced by ANX7 under this condition is significantly increased over that of the control, which contains neither GTPS nor ATP (Fig. 1, A and D, bars 1 versus 2; p < 0.005). In addition, the increasing ANX7 activity stimulated by the combination of individually optimal concentrations of GTPS and PKC can be distinguished by a simple additive model when comparing activation by either GTPS or PKC alone (Fig. 1, A and D).

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Effects of guanine nucleotides and phosphorylation by protein kinases on ANX7-driven lipid vesicle fusion. A, ANX7 (1 µg) and PKC (0.5 unit) were added to a fusion-phosphorylation reaction in the presence (filled squares) or absence (filled circles) of 100 µM GTPS, or in the presence of GTPS but no ATP added (empty circles). B, ANX7 and PKC were added to a reaction mixture containing both 100 µM GTPS and 500 µM GDPS (filled circles). C, ANX7 (1 µg) and PKG (2000 units; filled triangles), PKAcat (500 units; filled circles), or pp60c-src (100 units; empty circles) were added to a fusion-phosphorylation reaction containing 100 µM GTPS. D, the rates of lipid vesicle fusion driven by ANX7 obtained in A-C are summarized. Bars 1, control; 2, PKC plus GTPS; 3, PKC minus GTPS; 4, PKC plus GTPS, no ATP added; 5, PKC plus GTPS and GDPS; 6, PKG plus GTPS; 7, PKA plus GTPS; and 8, pp60c-src plus GTPS. *, p < 0.005 and **, p < 0.05 compared with the control. In A-C the reaction containing neither ATP nor GTPS was used as the control (empty squares). In all panels, the phosphorylation and fusion reactions were simultaneously initiated by the addition of 1 mM Ca2+ at room temperature. Fusion was measured by the change in absorbance at 540 nm for 30 min. All data are the mean ± S.D. (n = 4).

Using the above method developed to study the GTPS plus PKC effect, two additional experiments were carried out to test the specificity of this effect on ANX7 membrane fusion activity. In the first set of experiments, an excess molar concentration of the GDP non-hydrolyzable analogue, GDPS, was added to the phosphorylation-fusion reaction in the presence of both PKC and 100 µM GTPS. As shown in Fig. 1B, the addition of 500 µM GDPS markedly reduces the lipid vesicle fusion activity of ANX7 stimulated by GTPS plus PKC, and even abolishes the stimulatory effect of PKC on fusion of lipid vesicles driven by ANX7 under this optimal condition. These data are comparable with the finding of inhibition of Ca²⁺-dependent secretion by GDPS from various secretory cell types (9, 29, 42-46).

In the second set of experiments, we tested the consequences of phosphorylation by PKA, PKG, and p60^c-src for ANX7-driven membrane fusion, in the presence of 100 µM GTPS. As shown in Fig. 1C, the relative rates of lipid vesicle fusion driven by ANX7 under these conditions increase moderately, as observed in the case of PKA or PKG, or show no change, as seen in the case of pp60^c-src. As compared with the control activity (Fig. 1D, bar 1), the increasing membrane fusion activities of ANX7 observed here are induced by GTPS, but not by the phosphorylation catalyzed by these various protein kinases (Fig. 1D, bars 6-8). The latter conclusion is consistent with our previous findings that GTPS enhances ANX7-driven lipid vesicle fusion activity (35), and phosphorylation by either PKA or PKG does not significantly affect this activity, or may even decrease it, as in the case of pp60^c-src (37). These results thus show that the binding of GTPS to ANX7, and selective phosphorylation of ANX7 by PKC mutually activate the membrane fusion activity of ANX7 in vitro.

Effect of Guanine Nucleotides on Phosphorylation of ANX7 by PKC-- To further investigate the mechanism of the additive effect of GTPS and PKC on ANX7 membrane fusion activity, we examined the effect of GTP and its non-hydrolyzable analogues on the in vitro ANX7 phosphorylation reaction. As shown in Fig. 2A, the levels of phosphorylation of ANX7 by PKC are optimally enhanced by GTP and its non-hydrolyzable analogues. At 100 µM, GTPS significantly increases the level of ANX7 phosphorylation with a stoichiometry of 1.83 ± 0.22 (Fig. 2A, bar 2, p < 0.005) after 1 h. By contrast, the molar ratio of ANX7 phosphorylation achieved in the absence of GTPS is 1.27 ± 0.27 (Fig. 2A, bar 1), which is in accord with previously published data (37). Similarly, GMP-P(NH)P and GTP significantly enhance ANX7 phosphorylation (Fig. 2A, bars 3 and 4, p < 0.005). However, their effects are less potent than that of GTPS. At the same concentration as GTPS, both GMP-P(NH)P and GTP increase the levels of ANX7 phosphorylation with stoichiometries of 1.75 ± 0.2 and 1.64 ± 0.21, respectively, after 1 h. Strikingly, the order of efficacy, GTPS > GMP-PNP > GTP, for enhancement of PKC phosphorylation of ANX7 is comparable with that for the stimulation of exocytosis (2, 3, 8, 9, 43). These results thus suggest that upon binding to GTP and these non-hydrolyzable GTP analogues, ANX7 is configured into a highly susceptible target for phosphorylation by PKC.

View larger version (42K): [in this window] [in a new window]   Fig. 2.   Effect of guanine nucleotides on phosphorylation of ANX7 by PKC. A, the effect of GTPS, GMP-PNP, GTP, and GDPS (100 µM each) on ANX7 phosphorylation by PKC. *, p < 0.005. B, the time course of ANX7 phosphorylation by PKC in the presence (filled circles) or absence (empty circles) of 100 µM GTPS. C, PKC-dependent phosphorylation of ANX7 as a function of GTPS concentration. In all panels, the phosphorylation reaction mixtures, as described under "Experimental Procedures," were carried out at 30 °C for 1 h (A and C), or various indicated times (B), and then analyzed by SDS-PAGE and phosphorimaging. The reaction containing no guanine nucleotide was used as the control. Data are the mean ± S.D. (n = 4). Upper panels (A and B) and the inset in C show the representative phosphorimaging data.

As a more definitive test for the positive modulatory role of GTP and its analogues, we determined whether GDPS could modulate ANX7 phosphorylation by PKC. As shown in Fig. 2A, GDPS does not significantly enhance ANX7 phosphorylation by PKC. The molar ratio of ANX7 phosphorylation achieved in the presence of 100 µM GDPS is 1.32 ± 0.22 (Fig. 2A, bar 5), which is equivalent to that of the control (Fig. 2A, bar 1). This finding suggests that although GDPS binds to ANX7, this type of guanine nucleotide is incapable of configuring the molecular structure of ANX7. Thus, ANX7 is left in the control state.

Furthermore, GTPS markedly increases the rate and the extent of ANX7 phosphorylation over those of the control (Fig. 2B). In the presence of 100 µM GTPS, phosphorylation of ANX7 catalyzed by PKC is complete after 90 min with a stoichiometry of 1.94 ± 0.10 (n = 3). By contrast, in the absence of GTPS, the optimal level of ANX7 phosphorylation is achieved after 120 min, similar to previously published data (37). Moreover, the extent of ANX7 phosphorylation modulated by GTPS is varied depending on the concentrations of this nucleotide. As shown in Fig. 2C, the molar ratio of ANX7 phosphorylation is markedly increased in a dose-dependent manner. In the presence of 200 µM GTPS the level of ANX7 phosphorylation is maximally attained with a stoichiometry of 1.94 ± 0.05 after 1 h.

In contrast to the increased levels of ANX7 phosphorylation stimulated by GTP and its analogues, the levels of autophosphorylation of PKC are relatively constant under the conditions described above (Fig. 2, A and C). Collectively, these results suggest that GTP and its non-hydrolyzable analogues, but not GDPS, are highly efficient activators of PKC-dependent phosphorylation of ANX7, and that their effects appear to be on the susceptibility of ANX7 to PKC, not on the activity of PKC, per se.

Effect of PKC Phosphorylation on Photoaffinity Binding of 8-N₃-[-³²P]GTP to ANX7 and Its GTPase Activity-- We next turned our attention to the question of whether PKC phosphorylation of ANX7 alters guanine nucleotide binding and hydrolyzing activities of ANX7. Previously, we have reported that ANX7 exhibits the ability to bind and hydrolyze intrinsically the bound nucleotides (35). Therefore, we chose to examine both the photoaffinity binding of 8-N₃-[-³²P]GTP and the intrinsic hydrolysis of [-³³P]GTP by PKC-phosphorylated ANX7 and its unphosphorylated form. For 8-N₃-[-³²P]GTP binding assays, ANX7 and PKC were simultaneously incubated at 30 °C in phosphorylation reactions containing 8-N₃-[-³²P]GTP in the presence or absence of 100 µM ATP, followed by irradiation at the indicated times. Fig. 3 shows that incubation of ANX7 with PKC in the presence of ATP significantly stimulates the ability of ANX7 to bind 8-N₃-[-³²P]GTP in a time-dependent manner. We calculated from the PhosphorImager data that the binding affinity of PKC-phosphorylated ANX7 for 8-N₃-[-³²P]GTP is increased 3-fold over that for the unphosphorylated form of ANX7. Although this finding clearly shows that ANX7 phosphorylation by PKC significantly enhances the guanine nucleotide binding activity of ANX7, we anticipated that 8-N₃-[-³²P]GTP could be rapidly hydrolyzed by ANX7 during the reaction. To avoid this hydrolysis problem, we performed a membrane filter binding assay using [³⁵S]GTPS as a substrate. Consistently, this latter result also shows a substantial increase in the binding of [³⁵S]GTPS to PKC-phosphorylated ANX7, yielding a binding ratio of 0.081 pmol of GTPS/min/pmol of ANX7 (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of PKC phosphorylation on photoaffinity binding of 8-N3-[-32P]GTP to ANX7. ANX7 (1 µg) and PKC (0.05 unit) were incubated at 30 °C in a phosphorylation reaction containing 8 µM 8-N3-[-32P]GTP in the presence (filled circles) or absence (empty circles) of 100 µM ATP. At the indicated times, the reactions were irradiated for 30 s at room temperature and subjected to SDS-PAGE analysis. 32P incorporation was analyzed by phosphorimaging. Results are the mean of two independent experiments. The inset shows representative phosphorimaging data.

For GTP hydrolysis assays, two parallel experimental strategies were employed. In the first experimental strategy, both ANX7 phosphorylation by PKC and GTP hydrolysis by ANX7 were initiated simultaneously by the addition of 100 µM ATP and 50 µM [-³³P]GTP. Fig. 4 shows the time course of the hydrolysis of [-³³P]GTP by ANX7 in the presence or absence of added PKC. In the presence of added PKC, the intrinsic GTPase activity of ANX7 is significantly increased in a time-dependent manner. Ultimately, the overall rate of GTP hydrolysis catalyzed by PKC-phosphorylated ANX7 is ~7-fold faster than that determined for the native form of ANX7. We calculated that the molar turnover number of the steady-state GTPase reaction mediated by phosphorylated ANX7 is 0.086 ± 0.006 pmol of GDP/min/pmol of ANX7. By contrast, the equivalent value for the native form of ANX7 is 0.012 ± .001 pmol of GDP/min/pmol of ANX7. Thus, phosphorylation by PKC also modulates the intrinsic GTPase activity of ANX7, as it does on the guanine nucleotide binding activity of ANX7 (Fig. 3).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Effect of PKC phosphorylation on GTPase activity of ANX7. ANX7 GTPase activity was measured simultaneously with ANX7 phosphorylation by PKC as described under "Experimental Procedures." ANX7 (1 µg) was incubated at 30 °C for the various indicated times in the presence (filled circles) or absence (empty circles) of 0.05 unit of PKC. The reactions were initiated by the addition of ATP and [-33P]GTP, and the products were analyzed by polyethyleneimine-cellulose thin layer chromatography and phosphorimaging. The positions of GTP, GDP, and origin (ORI) are indicated. Data are the mean ± S.D. (n = 4). The levels of GTP hydrolysis produced in the reactions containing PKC alone and the buffer alone were subtracted from the values presented. The upper panel shows representative phosphorimaging data. Lane 1, ANX7 plus PKC; lane 2, ANX7 alone; lane 3, PKC alone; lane 4, buffer.

In the second experimental strategy, ANX7 was incubated at 30 °C for 3 h with PKC in the presence or absence of added ATP, and affinity isolated on PS liposomes. After eluting from the liposomes, the different ANX7 forms were assayed for GTPase activity. Similar to the results obtained from the first experiment (Fig. 4), pre-phosphorylated ANX7 exhibits higher detectable GTPase activity than the unphosphorylated form of ANX7 (Fig. 5, fifth bar versus first). In another experiment, to further examine the relationship between ANX7 phosphorylation by PKC and GTPase activity of ANX7, we mixed pre-phosphorylated ANX7 and its unphosphorylated form at protein ratios of 2:1, 1.5:1.5, or 1:2, respectively. These reactions were then assayed for GTPase activity. Strikingly, the amounts of GTP hydrolysis was increased linearly with increasing amounts of pre-phosphorylated ANX7 (Fig. 5, second to fourth bars). As shown in the lower panel of Fig. 5, immunoblotting analysis using an antibody against ANX7 clearly shows that each GTPase reaction contains approximately the same amount of ANX7 protein. These findings thus are consistent with the results in the first experiment, indicating that phosphorylation by PKC indeed enhances the hydrolysis of GTP by ANX7.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   GTPase activity of PKC-phosphorylated and unphosphorylated forms of ANX7. PKC-phosphorylated ANX7 and its unphosphorylated form were prepared as described under "Experimental Procedures." Mixtures of unphosphorylated ANX7 alone (first bar), phosphorylated ANX7 alone (fifth bar), or unphosphorylated and phosphorylated forms at protein ratios of 2:1, 1.5:1.5, or 1:2 (second to fourth bars, respectively) were incubated at 30 °C for 1 h. Each mixture contained the same amount of total ANX7 protein (0.75 µg/30 µl of reaction). The GTPase reaction products were analyzed as described in the legend to Fig. 4. The top panel shows representative phosphorimaging data. The value of GTP hydrolysis produced in the reaction containing the buffer alone was subtracted from the values presented (mean ± S.D., n = 3). The bottom panel shows the immunoblot of the above reactions using an ANX7 monoclonal antibody.

In a parallel control experiment, we tested the possibility that the increasing GTPase activity might be because of a contaminant of the PKC preparation. As shown in the upper panel of Fig. 4, addition of PKC alone has no significant effect on GTP hydrolysis over the entire time course. Furthermore, the results shown in Fig. 5 may also eliminate the possibility of a contaminant from the ANX7 sample that might contribute to the increasing GTPase activity of ANX7. Collectively, these findings thus clearly indicate that PKC-catalyzed phosphorylation stimulates both guanine nucleotide binding and hydrolysis activities of ANX7.

Effects of Phosphorylation by Other Protein Kinases on ANX7 GTPase Activity-- To determine whether the enhancement of the ANX7 GTPase activity was specific for PKC-mediated phosphorylation or was a more general effect of the phosphorylation process, we examined the effects of phosphorylation by other protein kinases on ANX7 GTPase activity. Using the methods developed to study the PKC effect (see Fig. 4), ANX7 was incubated for 1 h with or without PKC, PKA, PKG, or pp60^c-src in a phosphorylation reaction containing 50 µM [-³³P]GTP. In contrast to the enhanced effect of PKC phosphorylation on ANX7 GTPase activity, simultaneous phosphorylation of ANX7 by PKA, PKG, and pp60^c-src does not yield a significant increase above the basal GTPase activity (Fig. 6). Thus, the nature of the phosphorylation process does indeed regulate the specificity of the action of a particular kinase on the ANX7 guanine nucleotide binding/hydrolysis activity.

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Effects of phosphorylation by various protein kinases on ANX7 GTPase activity. The effects of phosphorylation by various protein kinases on ANX7 GTPase activity were determined and analyzed as described in the legend to Fig. 4. ANX7 (1 µg) was incubated at 30 °C for 1 h with or without 200 units of PKG, 50 units of PKAcat, or 10 units of pp60c-src. The values of GTP hydrolysis produced in the reactions containing kinase alone and the buffer alone were subtracted from the values presented. Data are the mean ± S.D. (n = 4). The upper panel shows representative phosphorimaging data. First lane, ANX7 plus kinase; second lane, ANX7 alone; third lane, kinase alone; fourth lane, buffer. The right panel shows representative phosphorimaging results of ANX7 phosphorylation by PKG, PKAcat, or pp60c-src in the presence or absence of 50 µM GTPS. These conditions resulted in mole ratios of Pi to ANX7 of 1.0.

Catecholamine Secretion, ANX7 Phosphorylation, and ANX7-bound GDP/GTP in Stimulated Chromaffin Cells-- To correlate the in vitro data with events in cells, we examined the biochemical profile of endogenous ANX7 in secreting chromaffin cells. In these experiments, intact bovine adrenal chromaffin cells were metabolically labeled with [³³P]orthophosphoric acid, and then stimulated with or without 100 µM carbachol. Following cell lysis and immunoprecipitation, both ANX7 phosphorylation and binding of GDP/GTP to ANX7 were analyzed and compared simultaneously with the release of catecholamines into the medium. As shown in Fig. 7C, catecholamine secretion in response to carbachol is increased concomitantly with the increasing levels of both the ANX7 phosphorylation and the ratio of ANX7-bound GDP/GTP (Fig. 7, A and B, respectively). By contrast, stimulation of cells with buffer B (control) results in small changes in secretion, ANX7 phosphorylation, and ANX7-bound GDP/GTP.

View larger version (37K): [in this window] [in a new window]   Fig. 7.   Effects of PKC inhibitors and carbachol on catecholamine secretion, ANX7 phosphorylation, and ANX7-bound nucleotides in cultured chromaffin cells. The 33P-labeled chromaffin cells were stimulated with or without 100 µM carbachol at 37 °C for 30 min, or preincubated with or without the indicated PKC inhibitors, followed by stimulation with carbachol. Following cell lysis, the lysates were immunoprecipitated with an anti-ANX7 monoclonal antibody. The immunoprecipitates were divided into two equal halves. The first half of the immunoprecipitate was then analyzed by SDS-PAGE, followed by electrotransfer to a polyvinylidene difluoride membrane, for the analysis of protein phosphorylation, whereas the second half was analyzed by thin layer chromatography for guanine nucleotide binding. 33P incorporation into ANX7 and labeled GDP/GTP bound to ANX7 were analyzed by phosphorimaging, and the phosphorimaging data represent one of three different experiments. A, the levels of 33P incorporation into immunoprecipitated ANX7. After phosphorimaging, the membrane was immunoblotted with another anti-ANX7 polyclonal antibody, and the immunoreactive ANX7 bands were detected chromogenically (top panel). B, the ratio of bound GDP/GTP to immunoprecipitated ANX7. The positions of GDP, GTP, and origin (ORI) are indicated. C, catecholamines secreted into the medium, from the same cells, were measured and expressed as micrograms of epinephrine plus norepinephrine (mean ± S.D., n = 3). The abbreviations used are: carb, carbachol; calph, calphostine C; and chele, chelerythrine chloride. D, correlation between catecholamine secretion and ANX7 phosphorylation (empty circles) and ANX7-bound GDP/GTP (filled circles) in response to stimulation by carbachol, or to inhibition by various PKC inhibitors. Correlation coefficient (R2) and the computer-fitted line for all data points were obtained from the results described in A-C.

Furthermore, we examined whether both the in vivo phosphorylation of ANX7 and the binding of GDP/GTP to ANX7, along with catecholamine secretion, could be inhibited by the selective PKC inhibitors. For these experiments, labeled chromaffin cells were pretreated with either PKC inhibitor calphostine C (50 nM) or chelerythrine chloride (0.7 µM) prior to incubation with 100 µM carbachol. As shown in Fig. 7, A-C, both PKC inhibitors markedly reduce the levels of catecholamine secretion, ANX7 phosphorylation, and the ratio of ANX7-bound GDP/GTP from cells stimulated with carbachol. These in vivo findings thus clearly show a close relationship between catecholamine secretion and ANX7 phosphorylation and its guanine nucleotide binding profile, with correlation coefficient (R²) of 0.9698 (Fig. 7D). These data thus complement the in vitro data. Together, the present findings further support the hypothesis that ANX7 functions as a Ca²⁺/GTP-binding protein/PKC substrate, very close to the exocytotic membrane fusion site in the stimulus-secretion cascade.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GTP and PKC, in concert with Ca²⁺, are known to constitute a highly potent intracellular effector system for exocytosis in a variety of secreting cell types (1-29). In addition, Lillie and Gomperts (30) have suggested that these effectors may exert their positive actions either directly on a common site, or on putative target proteins that are closely associated with each other in the exocytotic machinery. Based on our previous studies (35, 37), we have proposed that ANX7 might function as a common site for these effectors in the exocytotic machinery. To further support this hypothesis, we demonstrate here that the Ca²⁺-dependent lipid vesicle fusion activity of ANX7 in vitro is significantly amplified by the combination of GTPS and PKC (Fig. 1). Furthermore, the in vitro data on ANX7 membrane fusion activity appear to be well correlated with what we have observed in vivo. In these in vivo studies with ANX7, the ratio of bound GDP/GTP and phosphorylation by PKC change in direct proportion to the extent of catecholamine release from ³³P-labeled chromaffin cells in response to stimulation by carbachol, or to inhibition by various PKC inhibitors. This close correlation between the in vivo and in vitro data implies that ANX7 functionally behaves like a G_E (G-protein for exocytosis; Ref. 30), and transduces the intracellular signals for exocytosis by simultaneously binding GTP and being phosphorylated by PKC in a Ca²⁺-dependent manner.

Based on these findings, the simplest explanation for the observed additive effects of PKC and GTPS on ANX7 membrane fusion activity in vitro involves the same mechanisms of activation induced by these two agents in vitro. Indeed, our further in vitro analyses have shown that the combined presence of guanine nucleotides and PKC in the reaction mixture simultaneously increases the sensitivity to the action of each other on ANX7.

Guanine Nucleotides Stimulate ANX7 Phosphorylation by PKC-- With regard to the PKC action on ANX7, we have found that the efficiency of ANX7 phosphorylation by PKC in vitro is further enhanced by GTP and its non-hydrolyzable analogues, but not by GDPS (Fig. 2). Significantly, the concentrations of added guanine nucleotides that activate this ANX7 phosphorylation event are relevant to the physiological GTP concentration range (47, 48). These data clearly imply that the binding of GTP and its non-hydrolyzable analogues to ANX7 can confer conformational flexibility that makes ANX7 phosphorylation sites more accessible to PKC. Because ANX7 is a Ca²⁺-dependent GTPase (35), this implication appears to be relevant because increased conformational flexibility of GTP binding is a common feature of most GTPases. Such flexibility enables GTPase proteins to function as molecular switches in which GTP- and GDP-bound forms have different conformations, and therefore significantly different activities (49). To further support this concept, we have found that activation of ANX7 phosphorylation by guanine nucleotides is not attributed to changes in PKC activity. The evidence is that the levels of autophosphorylation of PKC, which are proportional to the activity of the kinase (50), remain relatively constant under all experimental conditions tested (Fig. 2, A and C). Furthermore, we have found that the rank order of effectiveness for ANX7 phosphorylation by PKC is GTPS > GMP-P(NH)P > GTP GDPS (Fig. 2A). This finding indicates that the binding of GTP rather than its hydrolysis is of critical importance for the phosphorylation process. In addition, the finding indicates that this modification is specifically sensitive to the activated, GTP-bound form of ANX7. Together with the data from our previously published study (37), the present data strongly suggest that GTP further enhances the synergistic action of the elevated Ca²⁺ concentration and the slightly acidic pH (i.e. pH 6.8) in transforming ANX7 into a highly susceptible substrate for phosphorylation by PKC. Significantly, this optimal condition for the in vitro ANX7 phosphorylation by PKC appears to be physiologically relevant, because all of these factors are observed to be localized endogenously, as in the case of GTP, or to change coincidently, as in the case of Ca²⁺ concentration and pH, in the cell during stimulation.

Phosphorylation by PKC Stimulates the Ability of ANX7 to Bind and Hydrolyze GTP-- Reciprocally, the ANX7 phosphorylation by PKC substantially stimulates the basal levels of GTP binding and GTP hydrolysis by ANX7 (Figs. 3-5). The significance of these results is that upon phosphorylation by PKC, the turnover number for ANX7 is now relatively equivalent to those of some known G-proteins, including EF-G, EF-Tu, tubulin, and the G components of adenylate cyclase and transducin, with turnover numbers of 0.012-0.25 mol/min/mol of protein (51-55). Furthermore, the rate of GTP binding for phosphorylated ANX7 is quite similar to that of GTP hydrolysis, indicating that the hydrolytic/exchange reaction is rapid and is limited by GDP dissociation. Such an indication is supported by the present data (Fig. 1B) showing that the addition of excess GDPS markedly inhibits the additive effect of PKC and GTPS on ANX7 membrane fusion activity. This result strongly suggests that the exchange reaction of GDPS for GTPS is blocked by the excess molar concentration of GDPS. Thus, it appears from these data that once ANX7-bound GTP is hydrolyzed, the newly formed GDP is released rapidly, and the empty nucleotide-binding pocket of ANX7 is ready to accommodate a new GTP molecule. ANX7 has a higher affinity for GTP than it has for GDP, thus indicating that the exchange is supported by energetic properties (35).

At present, the mechanisms by which both GTP binding and GTP hydrolysis by ANX7 are enhanced by PKC phosphorylation remain to be fully elucidated. Nonetheless, it is plausible to speculate that the ANX7 conformational change induced by the PKC phosphorylation event is instrumental for both the stimulated GTPase activity and the rapid GDP/GTP exchange.

No Effects of Phosphorylation by Other Protein Kinases on ANX7 GTPase Activity-- The in vitro studies have shown that, in a simultaneous GTPase-phosphorylation reaction, PKA-, PKG-, and pp60^c-src-catalyzed phosphorylation do not significantly alter the molar turnover number of the GTPase reaction mediated by ANX7 (Fig. 6). These results strongly suggest that, unlike PKC, these kinases may phosphorylate ANX7 on sites distant to the GTP-binding site, and are incapable of influencing the binding and hydrolysis of GTP of ANX7. Thus, the lack of stimulation by PKA, PKG, and pp60^c-src on both the guanine nucleotide binding/hydrolysis property and on the membrane fusion activity (Fig. 1C) of ANX7 coincides with other observations showing that these kinases are not directly involved in regulated exocytosis (8, 56-60). In addition, the lack of effects of these phosphorylation events, compared with the consequences of PKC, serves as an important control for emphasizing the importance of PKC-induced changes in ANX7 function.

Membrane Fusion Cycle of ANX7-- Based on all of the present observations and of those in previously published reports (35, 37), we propose the following ANX7-driven membrane fusion cycle (see Fig. 8). Under a resting, low-Ca²⁺ condition, ANX7 exists in an inactive state (ANX7-Mg²⁺-GDP), which is formed by a process of constitutive Mg²⁺-dependent hydrolysis of GTP (Fig. 8, (1)). Upon elevation of Ca²⁺, ANX7 binds Ca²⁺, transforming into a moderately active form (ANX7-Ca²⁺/Mg²⁺-GDP), which can drive membrane fusion (Fig. 8, (2)). GDP bound to this form can be replaced by GTP, and in this (ANX7-Ca²⁺/Mg²⁺-GTP) form, membrane fusion activity of ANX7 is further activated (Fig. 8, (3)). Upon hydrolysis of GTP to GDP, a transient (ANX7-Ca²⁺/Mg²⁺-GDP) complex is formed (Fig. 8, (4, 5)). Under a suitable phosphorylation condition, either the GDP- or the GTP-bound forms of ANX7 can be phosphorylated by PKC, and PKC phosphorylation oscillates ANX7 between two phosphorylated states, (P-(ANX7-Ca²⁺/Mg²⁺-GTP)) and (P-(ANX7-Ca²⁺/Mg²⁺-GDP)), by stimulating the intrinsic GTPase and GTP/GDP exchange activities of ANX7. As a result, the membrane fusion activity of ANX7, with an order of efficiency (P-(ANX7-Ca²⁺/Mg²⁺-GTP)) > (P-(ANX7-Ca²⁺/Mg²⁺-GDP)), is at the optimal level, even operating at lower Ca²⁺ concentrations (Fig. 8, (6)). The GDP-bound, phosphorylated ANX7 is subsequently dephosphorylated by the action of a serine/threonine-protein phosphatase which, we have now learned, is calcineurin.² Then, with the reduction of free Ca²⁺ concentration, the GDP-bound, unphosphorylated ANX7 releases Ca²⁺ and returns to the inactive form, and the cycle can recur.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   ANX7 membrane fusion cycle regulated by Ca2+, GTP, and PKC. This model schematically illustrates that ANX7 oscillates between two major transitional states. In the "off" state and low Ca2+ concentrations, ANX7 is inactive and exists in the favored GDP-bound form. Upon elevation of Ca2+, GDP bound to ANX7 can be replaced with GTP and this GTP-bound form is optimally phosphorylated by PKC, activating ANX7 (the "on" state), which drives membrane fusion much more efficiently. Then, with the reduction in the free Ca2+ concentration, the off state complex is reformed, and the cycle can recur. See "Discussion" for a detailed explanation.

In summary, the present observations on ANX7 are remarkably congruent with the original exocytotic model of Gomperts (30). The ANX7 data are consistent with the concept that the stimulatory actions of Ca²⁺, GTP, and PKC converge on ANX7 to drive membrane fusion activity occurring during exocytosis. To further support such an inference, we have recently found that botulinum neurotoxin type C, which is a zinc-dependent protease and a specific inhibitor of exocytosis (61), efficiently cleaves ANX7 both in vitro and in permeabilized chromaffin cells. This proteolytic activity is concurrent with botulinum neurotoxin type C-dependent inhibition of ANX7 membrane fusion activity in vitro, and with inhibition of catecholamine secretion in vivo, respectively (62). These recent findings significantly parallel the proteolytic effect of this toxin on syntaxin (63) and SNAP-25 (64), which are protein components of the SNARE hypothesis (65). Inasmuch as the identification of SNARE proteins as targets for botulinum neurotoxins has been taken as prima facie evidence favoring the SNARE hypothesis for exocytotic membrane fusion, the apparent role of ANX7 in the exocytotic membrane fusion process thus cannot be excluded.

	ACKNOWLEDGEMENT

We thank Dr. Cathy Jozwik for preliminary reading of the manuscript.

	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. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, Uniformed Services University School of Medicine, USUHS, Bethesda, MD 20814. Tel.: 301-295-3200; Fax: 301-295-1715; E-mail: hpollard@usuhs.mil.

Published, JBC Papers in Press, May 6, 2002, DOI 10.1074/jbc.M202452200

² H. Caohuy and H. B. Pollard, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GTP, guanosine 5'-triphosphate; ANX7, annexin 7; cGMP, cyclic guanosine monophosphate; GDPS, guanosine 5'-O-(2-thiodiphosphate); GMP-P(NH)P, guanyl-5'-yl imidodiphosphate; GTPS, guanosine 5'-O-(3-thiotriphosphate); MES, 2-(N-morpholino)ethanesulfonic acid; PKC, protein kinase C; PS, phosphatidylserine; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); PKA_cat, catalytic subunit of cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PMA, phorbol 12-myristate 13-acetate; SNARE, soluble NSF attachment protein receptors.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES