From the Department of Anatomy and Cell Biology, Uniformed Services University School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
Received for publication, September 15, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Annexin 7, a Ca2+/GTP-activated
membrane fusion protein, is preferentially phosphorylated in intact
chromaffin cells, and the levels of annexin 7 phosphorylation increase
quantitatively in proportion to the extent of catecholamine secretion.
Consistently, various protein kinase C inhibitors proportionately
reduce both secretion and phosphorylation of annexin 7 in these cells.
In vitro, annexin 7 is quantitatively phosphorylated by
protein kinase C to a mole ratio of 2.0, and phosphorylation is
extraordinarily sensitive to variables such as pH, calcium,
phospholipid, phorbol ester, and annexin 7 concentration.
Phosphorylation of annexin 7 by protein kinase C significantly
potentiates the ability of the protein to fuse phospholipid vesicles
and lowers the half-maximal concentration of calcium needed for this
fusion process. Furthermore, other protein kinases, including
cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine
kinase pp60c-src, also label annexin
7 with high efficiency but do not have this effect on membrane fusion.
In the case of pp60c-src, we note that this kinase,
if anything, modestly suppresses the membrane fusion activity of
annexin 7. These results thus lead us to hypothesize that annexin 7 may
be a positive mediator for protein kinase C action in the exocytotic
membrane fusion reaction in chromaffin cells.
Protein kinase C (PKC)1
and possibly other protein kinase activators are believed to play a
regulatory role in exocytotic secretion of hormones and
neurotransmitters. Indeed, activation of PKC in bovine chromaffin
cells, for example, with tumor-promoting phorbol esters (1, 2), or
other secretagogues (3, 4), causes an increase in catecholamine
secretion in a Ca2+-dependent manner. By
contrast, secretion is reduced when PKC activity is down-regulated by
24-h pretreatment with phorbol esters (5) or inhibited using various
PKC inhibitors (6-8). The stimulatory effect of PKC activation on
exocytosis has also been reported in various other cell types,
including platelets (9), neutrophils (10), pituitary cells (11),
insulin-secreting cells (12, 13), and mast cells (14, 15). Although
phenomenologically well known, the specific sites of action of PKC in
the stimulus-secretion cascade remain unknown.
The SNARE hypothesis has been proposed to explain the interactions
between vesicle and plasma membranes during the period preceding
exocytosis (16). In this model, a Ca2+-independent core
complex is formed between plasma membrane protein syntaxin and SNAP-25
and the synaptic vesicle protein synaptobrevin/VAMP. Vesicular
synaptotagmin is identified as a low affinity Ca2+ sensor
for subsequent exocytosis (17). Additional evidence suggests that
trans-SNARE pairing may precede membrane fusion but is not
be required during fusion (18-21). In addition, the preceding
interaction of SNAP-25 with syntaxin is found to enhance the
interaction between syntaxin and synaptobrevin/VAMP, suggesting that
SNAP-25 regulates the formation of the SNARE complex (22). However, it
has recently been reported that the phosphorylation of SNAP-25 by PKC
actually decreases the interaction between syntaxin and SNAP-25. Thus,
PKC makes the formation of the SNARE complex less likely. These data
therefore suggest that the positive action of PKC on exocytosis is not
likely to be mediated by SNARE proteins (23).
Alternatively, annexins have also been considered as possible mediators
of exocytosis. Annexin 1 (ANX1), annexin 2 (ANX2), and annexin 7 (ANX7), which are members of the annexin family, have the ability to
aggregate and fuse lipid vesicles (24). Such a result has been
interpreted to suggest that they might play a role in regulating
membrane fusion. Indeed, both ANX1 and ANX2 are found to be
phosphorylated by PKC both in vivo (25, 26) and in
vitro (27-29). However, phosphorylation of these proteins by PKC
markedly inhibits their aggregation and fusion activities in
vitro (27-29), indicating that they are also unlikely to mediate the positive action of PKC on exocytosis.
Annexin 7 (ANX7; synexin), which fuses membranes in a
Ca2+-dependent manner (30-33), has properties
that have led us to give fuller credence to the possibility of its
involvement in exocytosis. We have recently reported that ANX7 is a
Ca2+-activated GTPase, both in vitro and
in vivo, and that its GTPase activity is increased in
secreting chromaffin cells (34). More recently, we have reported that
the heterozygous knockout anx7 (+/ In this study, we report that ANX7 is phosphorylated in stimulated
bovine chromaffin cells, and the level of ANX 7 phosphorylation is well
correlated with the release of catecholamines. ANX7 is also
phosphorylated in vitro by various kinases, including PKC, cAMP-dependent protein kinase (PKA),
cGMP-dependent protein kinase (PKG), and protein-tyrosine
kinase pp60c-src. Significantly, only
PKC-dependent phosphorylation of ANX7 enhances the membrane
fusion activity of the protein, whereas phosphorylation by other
kinases does not affect this activity, or may even decrease it, as in
the case of pp60c-src. Thus, the selective
activation by PKC on exocytosis in vivo and the activation
of ANX7 membrane fusion in vitro suggests that ANX7 may act
as a positive mediator of PKC for the exocytotic membrane fusion
reaction in chromaffin cells.
Isolation and Culture of Chromaffin Cells--
Chromaffin cells
were isolated from bovine adrenal glands by collagenase digestion and
purified on a Percoll gradient as described previously (37). Isolated
cells were further purified by a selective plating method (38) and
maintained in a CO2 incubator under 5%
CO2/95% air.
[33P]Orthophosphoric Acid Labeling and Treatment of
Chromaffin Cells with Secretagogues, PKC Activator, and
Inhibitors--
Cultured chromaffin cells (5 × 106/dish, Falcon, 35 mm) were labeled with
[33P]Pi (0.1 mCi/ml; Amersham Pharmacia
Biotech) in phosphate-free Eagle's minimal essential medium containing
10% dialyzed fetal calf serum for 8 h at 37 °C (39). Then, the
cells were washed once with Ca2+-free extracellular buffer
A (buffer B without 2.2 mM CaCl2 added). The
cells were stimulated with extracellular buffer B (118 mM NaCl, 4.2 mM KCl, 10 mM NaHCO3, 10 mM glucose, 25 mM Hepes (pH 7.2), 0.1% bovine
serum albumin, 1.2 mM MgCl2, and 2.2 mM CaCl2) containing 100 nM phorbol
12-myristate 13-acetate (PMA; ICN), 100 µM carbachol
(Sigma) or 10 µM nicotine (Sigma) for 30 min at 37 °C.
After incubation, the cells were rapidly washed twice with buffer A and
then solubilized in lysis buffer for immunoprecipitation. For
experiments with PKC inhibitors, 33P-labeled cells were
preincubated for 60 min at 37 °C with or without staurosporine (50, 100, and 200 nM, Calbiochem), calphostine C (50 and 500 nM; Calbiochem), or chelerythrine (0.7 and 1.0 µM, Calbiochem) in buffer A. The cells were then
stimulated for 30 min with 100 nM PMA, 100 µM
carbachol, or 10 µM nicotine, followed by cell lysis for
immunoprecipitation. Control experiments were performed using cells
incubated with buffer B or buffer B containing Me2SO
(for experiments using Me2SO-soluble compounds,
e.g. PMA, staurosporine, calphostine C, and chelerythrine).
For determining catecholamine secretion under the above conditions,
parallel experiments were carried out using unlabeled chromaffin cells,
and the media were then collected for measuring catecholamine concentrations.
Immunoprecipitation of 33P-Labeled ANX7--
The
cells were lysed in 1 ml of ice-cold lysis buffer (150 mM
NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 5 mM EGTA, 0.2 mM Na3VO4,
1 mM 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 percentage of total cellular catecholamines.
Preparation of Phosphatidylserine Lipid Vesicles--
PS lipid
vesicles were prepared fresh daily by the swelling method (40).
Briefly, 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 0.3 M sucrose solution.
Isolation and Purification of Human Recombinant ANX7--
Human
recombinant ANX7 was isolated and purified as described previously
(41). Briefly, Escherichia coli bacteria containing the
anx7-expressing vector (pTrc-FLS) were grown in 1 liter of Luria broth
at 37 °C. After reaching an A540 level
of 0.6, the culture was incubated overnight in the presence of 1 mM isopropyl- In Vitro Phosphorylation of ANX7--
All phosphorylation assays
using purified rat brain PKC were performed at 30 °C in a final
volume of 30 µl as described elsewhere (42). Rat brain PKC with a
purity of
As for ANX7 phosphorylation by PKA, PKG, and
pp60c-src, the assays were carried out at 30 °C
in a final volume of 30 µl as described (44-46). Purified human
recombinant ANX7 (0.25 µg) was incubated for 30 min with 10 units of
pp60c-src (Calbiochem (44)), 50 units of catalytic
subunit of PKA (Promega (45)) or 200 units of PKG (Promega (46)) plus
10 µM cGMP in 25 mM MES (pH 6.1), 10 mM MgCl2, and 1 mM
CaCl2. All phosphorylation reactions were initiated and
analyzed as described above for the PKC reactions.
Phosphoamino Acid Analysis--
The phosphoamino acid analysis
was performed as described (47). After autoradiography, labeled ANX7
bands were excised from the SDS-PAGE gel and then electroeluted
according to the manufacturer's instructions (Bio-Rad). The eluate was
dialyzed overnight in water to remove SDS. The dialysate was
concentrated by lyophilization and was then resuspended in 6 N HCl, followed by incubation at 110 °C for 2 h.
O-Phosphoserine, O-phosphothreonine, and
O-phosphotyrosine (Sigma Chemical Co.) at 5 mM
each were added to the sample, and a 5-µl aliquot was spotted on a
thin layer cellulose plate (Merck), followed by electrophoresis at 1 kV
for 30 min in a pH 3.5 buffer (pyridine/acetic acid/water, 1:33:40,
v/v). Unlabeled phosphoamino acids were stained with ninhydrin (0.2%
in acetone), and labeled phosphoamino acids were detected using the PhosphorImager.
Extraction of Phosphorylated and Unphosphorylated
ANX7--
Extraction of the phosphorylated and unphosphorylated
protein from lipid vesicles was performed as described (29). In each of
30 reactions, 1 µg of ANX7 was incubated for 2 h at 30 °C
with 0.05 unit of PKC in 25 mM PIPES (pH 6.8), 10 mM MgCl2, 1 mM CaCl2, 100 nM PMA, 400 µg/ml PS liposomes, and 0.1 mM ATP in a final volume of 30 µl. Unphosphorylated ANX7
was treated in a similar manner as described for phosphorylated ANX7,
except ATP was omitted. All reactions were pooled and centrifuged at
100,000 × g for 10 min, and the pellet, containing
lipid and lipid-associated ANX7, was resuspended in 25 mM
Tris-HCl (pH 7.5), 20 mM EGTA, and 20 mM EDTA.
The mixture was sonicated in a bath type sonicator and then incubated
for 30 min on ice, followed by centrifugation at 100,000 × g. The supernatant, containing ANX7, was collected and recentrifuged twice to remove residual lipid vesicles. After removing EGTA and EDTA, the concentration of phosphorylated and unphosphorylated ANX7 was determined by immunoblotting using 125I-labeled
secondary antibody and a known amount of ANX7 as a standard, and
quantitated with the PhosphorImager. A parallel experiment using
[ Lipid Vesicle Fusion Mediated by ANX7--
The PS lipid vesicle
fusion assay was performed as previously described (37). Lipid vesicles
were first diluted to an A540 of 0.6 in fusion
reaction buffer (0.3 M sucrose, 40 mM histidine (pH 6.1), 0.5 mM MgCl2, and 0.1 mM
EGTA). Phosphorylated or unphosphorylated ANX7 (0.5 µg) was incubated
with 0.5 ml of lipid vesicle suspension in a final volume of 1 ml of
fusion reaction buffer. Fusion was initiated by the addition of 1 mM [Ca2+]final and then measured
by the change in the turbidity at absorbance of 540 nm
(A540) using a recording Hewlett-Packard
spectrophotometer for 20 min at room temperature. For the
Ca2+-dependent lipid vesicle fusion reaction
mediated by phosphorylated or unphosphorylated ANX7, similar reactions
were carried out as described above. Fusion was initiated by the
addition of the indicated final Ca2+ concentrations (0.01, 0.05, 0.4, and 1 mM) and then monitored spectrophotometrically for 20 min. Free Ca2+ concentrations
were determined as described elsewhere (43) and verified using a
Ca2+-selective electrode.
Phosphorylation and Fusion Reaction--
Simultaneous
phosphorylation and lipid vesicle fusion reactions were carried out as
described elsewhere (29). The reaction 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,
and 0.5 ml of lipid vesicle suspension. Controls were carried out in
the absence of added ATP. Fusion and phosphorylation were
simultaneously initiated by the addition of 1 mM
[Ca2+]final at room temperature. Fusion was
measured for 30 min as described above. To confirm that ANX7
phosphorylation occurred during fusion, parallel experiments were
carried out in the presence of [
Fusion and phosphorylation reactions in the presence of other kinases
were carried out as described for PKC experiments above, except no PMA
was added and PKC was replaced by 2000 units of PKG (plus 10 µM cGMP), 500 units of PKAcat, or 100 units
of pp60c-src.
Statistical Analysis--
Data are presented as means ± S.D. A relationship between ANX7 phosphorylation and catecholamine
secretion was assessed by a linear regression analysis (y
axis, mean value of ANX7 phosphorylation induced by PMA, carbachol, or
nicotine, and inhibited by various PKC inhibitors; x axis,
mean value of catecholamine secretion under similar conditions as in
phosphorylation). The statistical significant values (p)
were determined by using Student's t test.
In Vivo Phosphorylation of ANX7 and Stimulation of Catecholamine
Release from Chromaffin Cells--
Using intact bovine adrenal
chromaffin cells, we investigated whether ANX7 is phosphorylated under
a variety of pro-secretory conditions, including treatment with PMA,
carbachol, and nicotine. In these experiments, 33P-labeled
cells were stimulated for 30 min with 100 nM PMA, 100 µM carbachol, 10 µM nicotine, or
extracellular buffer B (control), and labeled endogenous ANX7 was
immunoprecipitated with monoclonal antibody 10E7, followed by SDS-PAGE
and PhosphorImager analysis. As shown in Fig.
1 (AI and BI),
stimulation of cells with buffer B (control) results in a small amount
of 33P incorporation into ANX7. In contrast, labeling of
ANX7 is markedly increased by about 3- to 5-fold for all agonists
tested (Fig. 1, AI (bar 2) and BI (bars
2 and 5)).
As a further test for the involvement of PKC in the phosphorylation
process, we examined whether the in vivo phosphorylation of
ANX7 could be inhibited by various PKC inhibitors prior to stimulation
with PMA or with other secretagogues. For these experiments we chose
not only the relatively nonselective staurosporine (48) but also the
more selective calphostine C (49) and chelerythrine (50). As shown in
Fig. 1AI, all three inhibitors substantially reduce labeling
of immunoprecipitated ANX7 from cells stimulated with 100 nM PMA. Staurosporine, at concentrations of 50, 100, and
200 nM, causes 35 ± 5, 54 ± 3, and 64 ± 7% inhibition of ANX7 labeling (mean ± S.D., n = 3), respectively (bars 3-5). In addition, calphostine C, at
concentrations of 50 and 500 nM, causes 48 ± 7 and
64 ± 3% inhibition of ANX7 labeling, respectively (bars 6 and 7), whereas chelerythrine, at concentrations of
0.7 and 1 µM, also causes 42 ± 11 and 58 ± 16% inhibition of ANX7 labeling, respectively (bars 8 and
9). Furthermore, calphostine C and chelerythrine both also
cause a substantial reduction in labeling of immuno-precipitated ANX7
from cells stimulated with carbachol or nicotine (Fig. 1BI). In these experiments, 50 nM calphostine C and 0.7 µM chelerythrine cause 44 ± 2 and 45 ± 7%
inhibition in carbachol-induced labeling of ANX7, respectively
(bars 3 and 4), and these two inhibitors at the
same concentrations also cause 46 ± 6 and 49 ± 4%
inhibition in nicotine-induced labeling of ANX7, respectively
(bars 6 and 7).
We further examined whether phosphorylation of ANX7 in vivo
could be correlated with catecholamine secretion under the above conditions. Unlabeled cells were preincubated for 1 h in the
presence or absence of 100 nM staurosporine, 50 nM calphostine C, or 0.7 µM chelerythrine.
The cells were then stimulated with or without 100 nM PMA,
100 µM, or 10 µM nicotine for 30 min. After
incubation, the medium from each well was collected and assayed for
secreted catecholamines. As shown in Fig. 1AII and
1BII, incubation with PMA, carbachol, or nicotine results in
a 3.5-, 5.3-, or 6.0-fold increase in catecholamine secretion,
respectively (Fig. 1, AII (bar 2) and
BII (bars 2 and 5). By contrast,
preincubation with staurosporine, calphostine C, or chelerythrine only
results in a 1.5-, 1.6-, or 1.4-fold increase in PMA-induced secretion,
respectively (Fig. 1AII, bars 3-5). Likewise,
calphostine C and chelerythrine also allow 2.7- and 2.9-fold increases
in carbachol-induced secretion, respectively (Fig. 1BII,
bars 3 and 4), and 3.1- and 3.2-fold increases in
nicotine-induced secretion, respectively (Fig. 1BII, bars 6 and 7). Moreover, as shown in Fig.
1C, there appears to be a good correlation between the two
processes, secretion and ANX7 phosphorylation
(R2 = 0.9622).
In Vitro Phosphorylation of ANX7--
To determine whether ANX7
might be a substrate for PKC in vitro, we used purified rat
brain PKC to phosphorylate purified human recombinant ANX7 and analyzed
the products by SDS-PAGE and PhosphorImager. As shown in Fig.
2, PKC indeed phosphorylates ANX7 in a
highly efficient manner and is affected by a variety of extensive
variables. ANX7 phosphorylation by PKC is somewhat dependent on pH
between pH 6.1 and 7.5 (Fig. 2A). After 30 min of incubation
at 30 °C, an apparent maximal level of ANX7 phosphorylation by PKC
at pH 6.8 is achieved with a stoichiometry of 1.61 ± 0.13 mol of
Pi/mol of ANX7 (mean ± S.D., n = 5).
As compared with the pH 6.8 condition, the stoichiometries of ANX7
phosphorylation at pH 6.1 and 7.5 are 1.35 ± 0.17 and 1.17 ± 0.16 mol of Pi/mol of ANX7, respectively. By contrast,
the level of autophosphorylation of PKC is relatively unchanged under
these pH conditions (inset of Fig. 2A). Thus, the
effect of pH appears to be on the susceptibility of ANX7 to PKC not on
the activity of the PKC, per se.
At pH 6.8, phosphorylation of ANX7 is dependent on the presence of PKC,
Ca2+, phospholipid, and the PKC activator PMA (Fig.
2B). No phosphorylation of ANX7 is detected when PKC is
omitted from the reaction mixture (bar 9). Similar negative
results are found when both Ca2+ and phospholipid are
omitted from the reaction mixture containing PKC (bar 1).
Furthermore, the presence of 1 mM Ca2+ alone,
or phospholipid alone, is unable to support an optimal level of
phosphorylation (bars 2 and 3). However, when
both are present, the level of phosphorylation is greatly enhanced with a stoichiometry of 1.52 ± 0.02 mol of Pi/mol of ANX7
for 60 min (bar 4). Moreover, 100 nM PMA
significantly enhances the level of PKC-catalyzed phosphorylation of
ANX7 under the various conditions (compared bars 5-8 with
bars 1-4, respectively).
Because we could vary the mole fraction of phosphorylation between 1 and 2, we then examined the kinetics of the process in greater detail
(Fig. 2C). Under optimal experimental conditions, phosphorylation of ANX7 is complete after 60 min with a stoichiometry of 2.01 ± 0.01 mol of Pi/mol of ANX7 (mean ± S.D., n = 5). The rate of the phosphorylation reaction
is also dependent on the ANX7 concentration. As shown in Fig.
2D, the efficiency of phosphorylation is decreased as the
ANX7 concentration increases. At a higher ANX7 concentration
(e.g. 1 µg/30 µl), however, the optimal level of
phosphorylation is achieved only if the incubation time is extended
(inset).
The fact that Ca2+ is absolutely required for ANX7
phosphorylation (see Fig. 2B) suggests either that
Ca2+ is needed to activate only the
Ca2+/phospholipid-dependent PKC activity, or
that Ca2+ binding to ANX7 is a prerequisite for
PKC-dependent phosphorylation. To distinguish between these
two possibilities, we examined the Ca2+ dependence of ANX7
phosphorylation by PKC at both pH 6.1 and 6.8 (Fig. 2E). As
shown in the inset of Fig. 2E, the
autophosphorylation level of PKC is essentially the same throughout the
range of final Ca2+ concentrations tested (0.01-1.0
mM), at both pH conditions. By contrast, the mole ratio of
ANX7 phosphorylation is increased as the Ca2+ concentration
increases. At pH 6.8, the Ca2+ titration curve for
PKC-dependent ANX7 phosphorylation is biphasic. A minimal
saturated phosphorylation level with a stoichiometry of 0.5 mol of
Pi/mol of ANX7 is observed throughout the lower range of
Ca2+ concentrations (0.01-0.20 mM), and this
level is eventually increased as the free Ca2+
concentration increases from 0.20 to 1.0 mM. On the other
hand, the curve obtained at pH 6.1 is more sigmoidal. Under this pH condition, no phosphorylation of ANX7 is observed at any
Ca2+ concentration below 0.05 mM, and ANX7
phosphorylation eventually increases as the Ca2+
concentration increases beyond 0.05 mM. Thus, the action of
Ca2+ on ANX7 labeling efficiency appears to be on ANX7
rather than PKC.
Phosphoamino Acid Analysis--
To further analyze the PKC
reaction both in vivo and in vitro, we performed
phosphoamino acid analysis of ANX7 immunoprecipitated from
33P-labeled chromaffin cells. We also examined ANX7
phosphorylated in vitro by purified rat brain PKC. After
carbachol or nicotine stimulation, only labeled phosphoserine and
phosphothreonine, but not phosphotyrosine, are detected in the
immunoprecipitate (Fig. 3, A
and B). Similarly, acid hydrolysis of ANX7 phosphorylated in vitro by PKC only yields labeled phosphoserine and
phosphothreonine (Fig. 3C). These results thus support the
hypothesis that phosphorylation of ANX7 in stimulated chromaffin cells
is likely to be mediated by PKC.
Lipid Vesicle Fusion by Phosphorylated ANX7--
To study the
effect of PKC-dependent phosphorylation on a relevant
in vitro activity of ANX7, we chose to examine the lipid vesicle fusion reaction mediated by this protein. Two parallel experimental strategies were employed. In one experiment, ANX7 was
prephosphorylated with PKC in the presence or absence of ATP, followed
by extraction from the reaction mixture, and these phosphorylated and
unphosphorylated forms were used to initiate the membrane fusion
reaction (Fig. 4A). Fig.
4A shows a time course of the fusion of lipid vesicles
catalyzed by either phosphorylated or unphosphorylated ANX7. When the
fusion reaction is initiated with 1 mM Ca2+,
the rate and the extent of lipid vesicle fusion induced by
phosphorylated ANX7 is markedly enhanced over that of the
unphosphorylated ANX7.
In the second experiment, PKC was added simultaneously with ANX7 in the
presence or absence of added ATP. As shown in Fig. 4B, as
the reaction progresses, the relative rate of the fusion reaction
containing ATP is greatly enhanced, as compared with that of the
control (minus ATP). In parallel control experiments, in which ANX7 has
been omitted from the reaction mixture containing either ATP or no ATP,
the addition of PKC is unable to induce fusion of lipid vesicles (data
not shown). Similarly, the addition of PMA is also unable to alter the
fusion reaction induced by ANX7 (data not shown). These two results
(Fig. 4, A and B) are thus consistent with each
other and suggest that access to phosphorylated ANX7 is a rate-limiting
step for efficient activation of membrane fusion.
Ca2+ Dependence of Fusion Reactions Induced by
Phosphorylated and Unphosphorylated ANX7--
Because the fusion of
lipid vesicles by ANX7 is dependent on Ca2+, we then
examined the effect of phosphorylation by PKC on the Ca2+
dependence of this fusion process. In these experiments, phosphorylated and unphosphorylated ANX7 were prepared as described above (see Fig.
4A), and their fusion activities were examined at different final Ca2+ concentrations, ranging from 0.01 to 1.0 mM. As shown in Fig. 5A, the rate of lipid vesicle
fusion induced by phosphorylated ANX7 is markedly increased at lower
Ca2+ concentrations, as compared with the same process
induced by unphosphorylated ANX7 (p < 0.005). In
addition, not only is the potency of the reaction increased by PKC
phosphorylation of ANX7, but the efficacy is also increased. Thus,
there is a significant difference between the fusion processes mediated
by phosphorylated ANX7 and its unphosphorylated form with the specific
consequence of phosphorylation increasing the efficacy and extent of
Ca2+ activation.
As shown in Fig. 5B, the Ca2+ concentration
required to induce half-maximal fusion activity (50% of
Fmax) is 200 µM for the unphosphorylated protein, which is in accord with previous reports (34,
51). By contrast, when ANX7 is phosphorylated by PKC, at the same
protein concentration, this value is lowered to ~50 µM.
Thus, Ca2+ not only potentiates the susceptibility of ANX7
to labeling by PKC (see Fig. 2E), but PKC action also raises
the affinity of ANX7 for Ca2+.
It was also possible that the enhancement of fusion by PKC might be due
to an increase in lipid binding activity. To test this hypothesis we
examined the lipid binding properties of phosphorylated and
unphosphorylated ANX7. Following a 20-min fusion reaction, the mixture
was centrifuged at 100,000 × g, and the protein bound to lipid vesicles was quantified by SDS-PAGE. The inset of
Fig. 5A shows the recovery of both phosphorylated and
unphosphorylated forms of ANX7 from the lipid pellets incubated at
different Ca2+ concentrations. As shown by the figure,
phosphorylation did not increase the amount of protein recovered with
lipid vesicles. Thus, binding of ANX7 to membranes depends exclusively
on Ca2+; however, the efficiency by which the
Ca2+-induced membrane binding step of ANX7 is converted
into membrane fusion depends on PKC.
In Vitro Phosphorylation of ANX7 by Other Kinases and Their Effects
on ANX7-driven Lipid Vesicle Fusion--
As a control for the
specificity of PKC on ANX7 activity, we also examined the
phosphorylation of ANX7 by other kinases, including purified PKG, PKA,
and pp60c-src, each with an optimal enzymatic
activity (Fig. 6A). Based on the calculation of ANX7 phosphorylation by these kinases, the amounts
of phosphate incorporated into ANX7 were 0.7, 1.0, and 0.9 mol/mol of
ANX7 when incubated for 60 min with PKG, PKA, and pp60c-src, respectively (data not shown).
Based on these conditions, we then tested the consequences of
phosphorylation by PKA, PKG, or pp60c-src for ANX7
activity on membrane fusion. Using the methods developed to study the
PKC effect (see Fig. 4B), ANX7 was incubated in the presence
of lipid vesicles and PKA, PKG plus cGMP, or
pp60c-src, with or without added ATP. As shown in
Fig. 6B, PKA or PKG phosphorylation has no effect on the
membrane fusion activity of ANX7. In contrast to the PKC-mediated
effect, we observed a modest decrease in the rate of fusion activity in
the reaction containing pp60c-src and ATP.
In this study, we present, for the first time, evidence that
stimulation of intact bovine chromaffin cells with phorbol ester PMA,
carbachol, or nicotine markedly increases the phosphorylation of
endogenous ANX7 (Fig. 1). Furthermore, using PKC inhibitors with both
selective and relatively nonselective properties, we also show that the
levels of PKC-dependent labeling of endogenous ANX7 are
closely correlated with the levels of catecholamine secretion. These
results indicate that ANX7 phosphorylation in vivo appears to be mediated by this kinase. Equivalent studies in vitro
show that ANX7 is a quantitative substrate for PKC (Fig. 2) and that PKC phosphorylation enhances the Ca2+-dependent
membrane fusion reaction driven by ANX7 (Figs. 4 and 5). These findings
strongly imply that ANX7 is one of the potential phosphoproteins
involved in the exocytotic machinery in chromaffin cells and possibly
in other cell types. These conclusions are further supported by our
recent report that a nullizygous ( Ca2+ and pH Action on ANX7 Control the Efficiency of
Phosphorylation by PKC--
The limiting factor for the ANX7
phosphorylation event appears to be the structural conformation of the
ANX7 protein itself. The efficiency of in vitro
phosphorylation of ANX7 by PKC is somewhat dependent on pH with an
optimal pH of pH 6.8 (Fig. 2A). This effect of pH on ANX7
phosphorylation is not attributed to the pH-dependent activity of PKC itself, because the optimal pH of PKC activation is
known to be at pH 7.5 (52). Rather, it is likely that ANX7 phosphorylation site(s) become more accessible to PKC at this pH range.
Circular dichroism studies of recombinant ANX7 have indicated
substantial conformational flexibility over the pH interval of
6.5-7.5.2 The in
vitro pH condition (pH 6.8) used to yield an optimal ANX7 phosphorylation appears to be in accord with the cytosolic pH of the
chromaffin cell. For instance, several previous studies have shown that
the Ca2+-dependent catecholamine secretion is
increased at low pH with an optimal pH around pH 6.6 (53) and that the
cytosolic pH of the chromaffin cell is transiently acidified upon
stimulation by acetylcholine or nicotine (54).
Our data also support the concept that Ca2+ modifies the
conformation of ANX7 to permit enhanced labeling by PKC (see Fig.
2E). The evidence is that, although autophosphorylation of
PKC remains unchanged, ANX7 phosphorylation is increased significantly
as the free Ca2+ concentration elevates from 10 µM to 1 mM. The pH of the medium also
dictates the pattern of Ca2+-dependent
phosphorylation of ANX7, either having a biphasic dose-response curve
(at pH 6.8) or a sigmoidal curve (at pH 6.1). Thus, the elevated
Ca2+ concentration and the slightly acidic pH, both of
which are observed to change coincidentally in the cell during
stimulation, appear synergistically to induce the structural
conformations of ANX7 that enhance the in vitro
phosphorylation reaction.
PKC Activates ANX7-driven Membrane Fusion in Vitro--
The
ANX7-driven membrane fusion reaction is a well-established in
vitro model for exocytosis (30-33). The results shown in Figs. 4
and 5 suggest that the lipid binding and fusion activities of ANX7 are
separable functions and that only the fusion activity of the protein is
regulated by PKC. Phosphorylation of ANX7 by PKC markedly increases the
lipid vesicle fusion activity, and significantly lowers the
half-maximal Ca2+ concentration needed for ANX7-induced
lipid vesicle fusion. PKC confers a
K1/2(app) of 50 µM
for phosphorylated ANX7 as opposed to 200 µM for the
unphosphorylated form. However, both phosphorylated and
unphosphorylated ANX7 are found to bind to lipid vesicles with
equivalent affinities as a function of free Ca2+
concentration (inset of Fig. 5). Only the phosphorylated
protein, however, is able to induce lipid vesicle fusion at lower
Ca2+ concentrations ( Other Kinases Phosphorylate ANX7 but Do Not Activate Membrane
Fusion--
Despite ANX7 phosphorylation occurring during a
secretion-related phosphorylation event, we have not concluded that PKC
is solely responsible for ANX7 phosphorylation in stimulated chromaffin cells. In this report, we show that ANX7 also serves as a good substrate for PKG, PKA, and pp60c-src in
vitro (Fig. 6A). However, phosphorylation by these
kinases either have no significant impact on ANX7-induced lipid vesicle fusion, as seen in the cases of PKA and PKG, or even decrease the rate
of fusion of lipid vesicles by ANX7, as seen in the case of
pp60c-src. These results suggest that, in the
presence of a secretion-related phosphorylation event, the fusion
activity of ANX7 may be not activated by any of these kinases. This
suggestion is supported by previously published reports. For example,
in permeabilized chromaffin cells, cAMP (59, 60) and cGMP (60, 61) have little or no effect on Ca2+-dependent
catecholamine secretion. Thus, the direct involvement of PKA or PKG in
exocytosis would appear to be ruled out. Similarly, pp60c-src appears not to be directly involved in
exocytosis, because its activity is found to decrease following
stimulation of the intact cell (62). Thus, although ANX7 is an
efficient substrate for these kinases, we can only conclude that these
kinases may be involved in regulating other as yet unidentified
activities of ANX7. Recently, ANX7 has been shown to be phosphorylated
by Src kinase, in vitro (63) and to be a tumor suppressor
gene for prostate cancer (64).
In conclusion, we have demonstrated that ANX7 serves as the substrate
for PKC and certain other kinases. Only PKC-dependent phosphorylation has a positive effect on the in vitro
membrane fusion model of exocytosis. The specific action involves
lowering the K1/2(app) for
Ca2+ from 200 µM to 50 µM.
Consistently, stimulation of chromaffin cells with PKC activators
indeed results in phosphorylation of endogenous ANX7 concomitantly with
the release of catecholamines. These results thus support the
hypothesis that ANX7 is a site of action for PKC activation during exocytosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) mouse suffers from an insulin
secretion deficit from islets of Langerhans, as well as defective
Ca2+ signaling processes in
-cells (35). In addition, a
homology analysis of anx7 has suggested to us the likelihood that this protein might be a target for PKC (36) and, therefore, a candidate for
mediation of PKC action during exocytosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerolphosphate, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 50 mM
Tris-HCl (pH 7.5)), followed by incubation for 20 min on ice. The
lysates were clarified by centrifugation at 12,000 × g
for 15 min at 4 °C, and 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 a BCA kit (Pierce), were then incubated with 10 µg of anti-ANX7 monoclonal antibody 10E7 for 6 h at 4 °C.
Immunoprecipitates were collected on protein G-Sepharose, washed four
times by pelleting in cold lysis buffer, separated by SDS-PAGE, and
analyzed by phosphorimaging (PhosphorImager, Molecular Dynamics) or autoradiography.
-D-thiogalactopyranoside (ICN).
After incubation, 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). Partial purified ANX7 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 finally yield a highly purified (
98%) ANX7
preparation, determined by SDS-PAGE and silver staining.
95% was purchased from Calbiochem and contained the PKC
isoforms
,
, and
. Phosphorylation of ANX7 by PKC was examined
at different pH values, and the reaction mixture contained the
following: 25 mM Tris-HCl (pH 7.5), 25 mM PIPES
(pH 6.8) or 25 mM MES (pH 6.1), 10 mM
MgCl2, 1 mM CaCl2, 100 nM PMA, 400 µg/ml PS liposomes, 0.05 unit (0.035 µg) of
PKC, and 0.25 µg of purified human recombinant ANX7. All reactions were then incubated for 30 min. In other assays to determine the conditions of ANX7 phosphorylation, 0.25 µg of ANX7 was incubated for
60 min with or without 0.05 unit of PKC in 25 mM PIPES (pH 6.8), 10 mM MgCl2, and the following
conditions: no lipid or Ca2+ added; 1 mM
Ca2+ added, without lipid; lipid added, without
Ca2+; or both Ca2+ and lipid added. These
conditions were also examined in the presence of 100 nM
PMA. To determine the mole ratio of ANX7 phosphorylation by PKC, ANX7
(0.25 µg) was incubated for the indicated time periods with 0.05 unit
of PKC under the optimal phosphorylation condition (25 mM
PIPES (pH 6.8), 10 mM MgCl2, 1 mM
CaCl2, 100 nM PMA, and 400 µg/ml PS
liposomes). In the assay to determine the optimal ANX7 concentration
for phosphorylation, ANX7 (0.05, 0.1, 0.25, 0.5, and 1 µg) was
incubated for 60 min with 0.05 unit of PKC under the optimal
phosphorylation condition. The reactions containing 1 µg ANX7 were
further incubated for 90, 120, and 160 min. In the assay to determine
ANX7 phosphorylation as a function of Ca2+ concentration,
ANX7 (0.25 µg) was incubated for 30 min with 0.05 unit of PKC in 25 mM MES (pH 6.1) or 25 mM PIPES (pH 6.8), 10 mM MgCl2, 100 nM PMA, 400 µg/ml
PS liposomes, and the different free Ca2+ concentrations
(0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, and 1 mM). Free
Ca2+ concentrations were determined as described (43). The
above reactions were initiated by the addition of 2 µCi of
[
-33P]ATP in a final concentration of 0.1 mM (3000-4000 cpm/pmol; Amersham Pharmacia Biotech) and
stopped by the addition of the SDS-PAGE sample buffer. The
phosphorylation products were analyzed by SDS-PAGE and PhosphorImager
analysis or autoradiography.
-33P]ATP was also carried out to determine the
stoichiometry of phosphorylation.
-33P]ATP, and the
stoichiometry was measured as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of secretagogues, PKC activator and
inhibitors on ANX7 phosphorylation and secretion of catecholamines from
chromaffin cells. AI and BI,
33P-labeled cells were stimulated for 30 min at 37 °C
with 100 nM PMA, 100 µM carbachol, 10 µM nicotine, or buffer B (control). In experiments using
PKC inhibitors, labeled cells were preincubated for 60 min with or
without staurosporine, calphostine C, or chelerythrine at indicated
concentrations, followed by stimulation with PMA, carbachol, or
nicotine. The cells were lysed in lysis buffer. The lysates, with equal
amounts of total protein, were then incubated with anti-ANX7 antibody
for 6 h at 4 °C. By Western blot analysis, equal amounts of the
protein were found to be present on the blots under the various
experimental conditions. Immunoprecipitation of ANX7 was analyzed by
SDS-PAGE and PhosphorImager. The level of 33P incorporation
into ANX7 is reported as the arbitrary unit (mean ± S.D.,
n = 3-4; open bars). The inset
shows a representative PhosphorImager data. AII and
BII, to determine catecholamine secretion under the above
conditions, parallel experiments were carried out using unlabeled
chromaffin cells, and the media were collected for measuring
catecholamine concentrations. The release is expressed as % of the
total catecholamine content (mean ± S.D., n = 4;
speckled bars). C, correlation between ANX7
phosphorylation and catecholamine secretion from chromaffin cells in
response to various PKC inhibitors and PMA (empty squares),
carbachol (filled squares), or nicotine (empty
triangles) is shown. Correlation coefficient
(R2) and the computer-fitted line for
all data points were obtained from the results described in
A and B.
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Fig. 2.
In vitro phosphorylation of ANX7
by protein kinase C. A, pH dependence. ANX7 (0.25 µg)
was incubated at 30 °C with 0.05 unit of PKC in 25 mM
Tris-HCl (pH 7.5), 25 mM PIPES (pH 6.8), or 25 mM MES (pH 6.1), 10 mM MgCl2, 1 mM CaCl2, 100 nM PMA, 400 µg/ml
PS liposomes, and 100 µM [ -33P]ATP. *,
p < 0.05, compared with pH 6.1;
, p < 0.005, compared with pH 7.5. B, ANX7 (0.25 µg) was
incubated with 0.05 unit of PKC in 25 mM PIPES (pH 6.8), 10 mM MgCl2, 100 µM
[
-33P]ATP, and various conditions as described in the
text. C, ANX7 (0.25 µg) was incubated with 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. The reactions were
stopped at the indicated time intervals after the addition of 100 µM [
-33P]ATP. D, various
indicated ANX7 concentrations were incubated for 60 min with 0.05 unit
of PKC under the optimal phosphorylation condition (pH 6.8) in a final
volume of 30 µl. The inset shows the extended time course
of phosphorylation of ANX7 at 1.0 µg. E, ANX7 (0.25 µg)
and PKC (0.05 unit) were incubated for 30 min in the pH 6.1 (filled circles) or pH 6.8 (empty circles)
phosphorylation buffer containing the indicated free Ca2+
concentrations. In A, B, C, and
E, the inset shows a representative
PhosphorImager data of three to five different experiments. All data
are the mean ± S.D. (n = 3-5) and are expressed
in mole ratios.
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Fig. 3.
One-dimensional phosphoamino acid analysis of
ANX7. After autoradiography, the bands of ANX7 immunoprecipitated
from stimulated chromaffin cells (A and B) or
phosphorylated in vitro by PKC (C) were excised
from the gel, and the phosphoamino acid analysis was carried out as
described under "Experimental Procedures". A,
stimulation with 100 µM carbachol. B,
stimulation with 10 µM nicotine. C, in
vitro phosphorylation by PKC. The positions of the standard
phosphoamino acids are outlined by the broken ovals:
phosphoserine (P-Ser), phosphothreonine (P-Thr),
and phosphotyrosine (P-Tyr). O marks the position
of the sample origin.
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Fig. 4.
Effect of PKC-dependent
phosphorylation on ANX7-induced lipid vesicle fusion.
A, phosphorylated (pANX7; empty circles) or
unphosphorylated (nANX7; filled circles) ANX7 (0.5 µg),
prepared as described under "Experimental Procedures," was added to
a 1-ml reaction mixture containing 0.3 M sucrose, 40 mM histidine (pH 6.1), 0.5 mM
MgCl2, 0.1 mM EGTA, and 0.5 ml of lipid vesicle
suspension. Fusion was initiated by the addition of 1 mM
Ca2+ and measured by the change in absorbance at 540 nm
after 20 min. The inset shows the equivalent amounts of the
protein present in both reactions. B, ANX7 (1 µg) and PKC
(0.5 unit) were added to a 1-ml reaction mixture containing 0.3 M sucrose, 40 mM histidine (pH 6.1), 2 mM MgCl2, 100 nM PMA, 100 µM ATP, and 0.5 ml of lipid vesicle suspension. The
control was carried out in the absence of added ATP. 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 after 30 min in the
presence (empty circles) or absence (filled
circles) of ATP. All data are the mean ± S.D.
(n = 3).
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Fig. 5.
Ca2+ dependence of fusion of
lipid vesicles by phosphorylated and unphosphorylated ANX7.
A, phosphorylated (empty circles) or
unphosphorylated (filled circles) ANX7 (0.5 µg), prepared
as described in Fig. 4A, was added to a 1-ml reaction
mixture containing 0.3 M sucrose, 40 mM
histidine (pH 6.1), 0.5 mM MgCl2, 0.1 mM EGTA, and 0.5 ml of lipid vesicle suspension. Fusion was
initiated by the addition of the indicated final Ca2+
concentrations and measured by the change in absorbance at 540 nm after
20 min. Data are the mean ± S.D. (n = 3). *,
p < 0.005, compared with the control (nANX7). To
determine lipid binding by ANX7, the lipid vesicles were centrifuged
after the fusion reaction was complete, and the lipid-associated
protein was analyzed by SDS-PAGE. The inset shows
phosphorylated ANX7 (pANX7; ) and its unphosphorylated
form (nANX7;
) that cosedimented with the lipid vesicles
at indicated free Ca2+ concentrations. B, data
from A were replotted to highlight the increasing affinity
of ANX7 for Ca2+ by PKC phosphorylation. The
Vmax of each curve was determined by a
Lineweaver-Burk plot and then used as 100% maximal fusion activity.
Based on these Vmax values, the original data
were transformed, expressed as a percentage of maximum of fusion
activity, and replotted as shown.
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Fig. 6.
In vitro phosphorylation of ANX7
by various kinases and their effects on the fusion activity of
ANX7. A, ANX7 (0.25 µg) was incubated for 30 min at
30 °C with 200 units of PKG plus 10 µM cGMP, 50 units
of catalytic subunit of PKA, or 10 units of
pp60c-src in 25 mM MES (pH 6.1), 10 mM MgCl2, 1 mM CaCl2,
and 100 µM [ -33P]ATP. The result shown
is a representative PhosphorImager data of four different experiments.
Arrows indicate the position of ANX7. Other labeled bands
shown are PKG (¶), PKAcat (*), and
pp60c-src (
). B, ANX7 (1 µg) and
2000 units of PKG plus 10 µM cGMP (empty
triangles), 500 units PKAcat (empty
squares), or 100 units pp60c-src (empty
circles) were added to a 1-ml reaction mixture containing 0.3 M sucrose, 40 mM histidine (pH 6.1), 2 mM MgCl2, 100 µM ATP, and 0.5 ml
of lipid vesicle suspension. Controls were carried out in the absence
of added ATP (filled circles). Phosphorylation and fusion
reactions were initiated simultaneously by the addition of 1 mM Ca2+ at room temperature. Fusion was
measured by the change in absorbance at 540 nm after 30 min. Data are
the mean ± S.D. (n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
) knockout of the anx7 gene in
mouse is lethal and that insulin secretion from islets of Langerhans of
the heterozygous knockout anx7 (+/
) mouse is defective (35).
50 µM). At present,
the mechanism by which the fusion activity of ANX7 is enhanced by PKC
remains to be fully elucidated. Several studies have suggested that
annexin self-association, after binding to the membrane, may be
required to allow the annexins to aggregate and fuse lipid vesicles
(55, 56). Therefore, it is reasonable to anticipate that
phosphorylation of ANX7 by PKC may potentiate the intermolecular
interactions occurring between ANX7 molecules, resulting in the
enhancement of membrane fusion. ANX7 can therefore be usefully
hypothesized as part of the Ca2+ control site in the
exocytotic machinery. In support of this hypothesis we recall that more
than one factor may be involved in mediating the Ca2+
signal for exocytosis. For example, although synaptotagmin has been
considered as a putative Ca2+ receptor for exocytosis (21),
the knockout mutation of the synaptotagmin gene does not completely
abolish Ca2+-evoked secretion (57, 58). By contrast, the
anx7 (
/
) knockout is lethal, whereas the anx7 (+/
) heterozygote
expresses only low amounts of ANX7 protein and defectively secretes
insulin (35). The lethality of the anx7 (
/
) nullizygous knockout
thus serves to emphasize how critical ANX7 is to survival. The
secretory defect of the heterozygote anx7 (+/
) animal serves to
emphasize that the anx7 gene is critical for the secretory process in
some tissues.
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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, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. Tel.: 301-295-3200; Fax: 301-295-1715; E-mail: hpollard@helix.nih.gov.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M008482200
2 H. B. Pollard, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: PKC, protein kinase C; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; MES, 2-(N-morpholino)ethanesulfonic acid; Pi, inorganic phosphate; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; SNAP-25, 25-kDa synaptosome-associated protein; VAMP, vesicle-associated membrane protein; SNARE, soluble NSF-attachment protein receptor.
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1. | Pocotte, S. L., Frye, R. A., Senter, R. A., TerBush, D. R., Lee, S. A., and Holz, R. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 930-934[Abstract] |
2. | Brocklehurst, K. W., Morita, K., and Pollard, H. B. (1985) Biochem. J. 228, 35-42[Medline] [Order article via Infotrieve] |
3. |
TerBush, D. R.,
and Holz, R. W.
(1986)
J. Biol. Chem.
261,
17099-17106 |
4. |
TerBush, D. R.,
Bittner, M. A.,
and Holz, R. W.
(1988)
J. Biol. Chem.
263,
18873-18879 |
5. | Burgoyne, R. D., Morgan, A., and O'Sullivan, A. J. (1988) FEBS Lett. 238, 151-155[CrossRef][Medline] [Order article via Infotrieve] |
6. | Isosaki, M., Minami, N., and Nakashima, T. (1994) Jpn. J. Pharmacol. 64, 217-219[Medline] [Order article via Infotrieve] |
7. |
TerBush, D. R.,
and Holz, R. W.
(1990)
J. Biol. Chem.
265,
21179-21184 |
8. | Tachikawa, E., Takahashi, S., Kashimoto, T., and Kondo, Y. (1990) Biochem. Pharmacol. 40, 1505-1513[CrossRef][Medline] [Order article via Infotrieve] |
9. | Coorssen, J. R., Davidson, M. L., and Haslam, R. J. (1990) Cell Regul. 1, 1027-1041[Medline] [Order article via Infotrieve] |
10. | Smolen, J. E., Stoehr, S. J., and Bartone, D. (1989) Cell Signal. 1, 471-481[Medline] [Order article via Infotrieve] |
11. |
Stojikovic, S. S.,
Iida, T.,
Merelli, F.,
Torsello, A.,
Krsmanovic, L. Z.,
and Catt, K. J.
(1991)
J. Biol. Chem.
266,
10377-10384 |
12. | Persaud, S. J., Jones, P. M., Sugden, D., and Howell, S. L. (1989) Biochem. J. 264, 753-758[Medline] [Order article via Infotrieve] |
13. | Wollheim, C. B., and Regazzi, R. (1990) FEBS Lett. 268, 376-378[CrossRef][Medline] [Order article via Infotrieve] |
14. | Howell, T. W., Kramer, I. M., and Gomperts, B. D. (1990) Cell Signal. 1, 157-163 |
15. | Churcher, Y., and Gomperts, B. D. (1990) Cell Regul. 1, 523-530[Medline] [Order article via Infotrieve] |
16. | Rothman, J. E. (1994) Nature 372, 55-63[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Chapman, E. R.,
Hansen, P. I.,
An, S.,
and Jahn, R.
(1995)
J. Biol. Chem.
270,
23667-23671 |
18. | Ungermann, C., Sato, K., and Wickner, W. (1998) Nature 396, 543-548[CrossRef][Medline] [Order article via Infotrieve] |
19. | Peters, C., and Mayer, A. (1998) Nature 396, 575-580[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Coorssen, J. R.,
Blank, P. S.,
Tahara, M.,
and Zimmerberg, J.
(1998)
J. Cell Biol.
143,
1845-1857 |
21. |
Tahara, M.,
Coorssen, J. R.,
Timmers, K.,
Blank, P. S.,
Whalley, T.,
Scheller, R.,
and Zimmerberg, J.
(1998)
J. Biol. Chem.
273,
33667-33673 |
22. | Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Sudhof, T. C., and Niemann, H. (1994) EMBO J. 13, 5051-5061[Abstract] |
23. |
Shimazaki, Y.,
Nishiki, T.,
Omori, A.,
Sekiguchi, M.,
Kamata, Y.,
Kozaki, S.,
and Takahashi, M.
(1996)
J. Biol. Chem.
271,
14548-14553 |
24. | Raynal, P., and Pollard, H. B. (1994) Biochim. Biophys. Acta 1197, 63-93[Medline] [Order article via Infotrieve] |
25. |
Michener, M. L.,
Dawson, W. B.,
and Creutz, C. E.
(1986)
J. Biol. Chem.
261,
6548-6555 |
26. | Gould, K. L., Woodgett, J. R., Isacke, C. M., and Hunter, T. (1986) Mol. Cell. Biol. 6, 2738-2744[Medline] [Order article via Infotrieve] |
27. | Wang, W., and Creutz, C. E. (1992) Biochemistry 31, 9934-9939[Medline] [Order article via Infotrieve] |
28. | Johnstone, S. A., Hubaishy, I., and Waisman, D. M. (1993) Biochem. J. 294, 801-807[Medline] [Order article via Infotrieve] |
29. |
Johnstone, S. A.,
Hubaishy, I.,
and Waisman, D. M.
(1992)
J. Biol. Chem.
267,
25976-25981 |
30. | Creutz, C. E., Scott, J. H., Pazoles, C. J., and Pollard, H. B. (1982) J. Cell. Biochem. 18, 87-97[Medline] [Order article via Infotrieve] |
31. | Hong, K., Duzgunes, N., Ekerdt, R., and Papahadjopoulos, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4642-4644[Abstract] |
32. | Nir, S., Stutzin, A., and Pollard, H. B. (1987) Biochim. Biophys. Acta 903, 309-318[Medline] [Order article via Infotrieve] |
33. | Pollard, H. B., Rojas, E., and Burns, A. L. (1992) Prog. Brain Res. 92, 247-255[Medline] [Order article via Infotrieve] |
34. |
Caohuy, H.,
Srivastava, M.,
and Pollard, H. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10797-10802 |
35. |
Srivastava, M.,
Atwater, I.,
Glasman, M.,
Leighton, X.,
Goping, G.,
Caohuy, H.,
Miller, G.,
Pichel, J.,
Westphal, H.,
Mears, D.,
Rojas, E.,
and Pollard, H. B.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
13783-13788 |
36. | Hauptmann, R., Maurerfogy, I., Krystek, E., Bodo, G., Andree, H., and Reutelingsperger, C. P. M. (1989) Eur. J. Biochem. 185, 63-71[Abstract] |
37. | Brocklehurst, K. W., and Pollard, H. B. (1990) in Peptide Hormones: A Practical Approach (Hutton, J. C. , and Siddle, K., eds) , pp. 233-255, IRL Press, Oxford, UK |
38. | Yanagihara, N., Isosaki, M., Ohuchi, T., and Oka, M. (1979) FEBS Lett. 105, 296-298[CrossRef][Medline] [Order article via Infotrieve] |
39. | Tsutsui, M., Yanagihara, N., Miyamoto, E., Kuroiwa, A., and Izumi, F. (1994) Mol. Pharmacol. 46, 1041-1047[Abstract] |
40. | Reeves, J., and Dowben, R. (1968) Cell. Physiol. 78, 49-60 |
41. | Burns, A. L., Magendzo, K., Srivastava, M., Rojas, E., Parra, C., de la Fuente, M., Cultraro, C., Shirvan, A., Vogel, T., Heldman, J., Caohuy, H., Tombaccini, D., and Pollard, H. B. (1990) Biochem. Soc. Trans. 18, 1118-1121[Medline] [Order article via Infotrieve] |
42. |
Uchida, T.,
and Filburn, C. R.
(1984)
J. Biol. Chem.
259,
12311-12314 |
43. | Fabiato, A., and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-465[Medline] [Order article via Infotrieve] |
44. | Hubaishy, I., Jones, P. G., Bjore, J., Bellagamba, C., Fitzpatrick, S., Fujita, D. J., and Waisman, D. M. (1995) Biochemistry 34, 14527-14534[Medline] [Order article via Infotrieve] |
45. | Trautwein, C., van der Geer, P., Karin, M., Hunter, T., and Chojkier, M. (1994) J. Clin. Invest. 93, 2554-2561[Medline] [Order article via Infotrieve] |
46. |
Komalavilas, P.,
and Lincoln, T. M.
(1994)
J. Biol. Chem.
269,
8701-8707 |
47. | Hunter, T., and Sefton, B. M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1311-1315[Abstract] |
48. | Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and Tomita, F. (1986) Biochem. Biophys. Res. Commun. 135, 397-402[Medline] [Order article via Infotrieve] |
49. | Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. (1989) Biochem. Biophys. Res. Commun. 159, 548-553[Medline] [Order article via Infotrieve] |
50. | Herbert, J. M., Augereau, J. M., Gleye, J., and Maffrand, J. P. (1990) Biochem. Biophys. Res. Commun. 172, 993-999[Medline] [Order article via Infotrieve] |
51. | Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1978) J. Biol. Chem. 253, 2858-2866[Medline] [Order article via Infotrieve] |
52. | Ferrari, S., Marchiori, F., Marin, O., and Pinna, L. A. (1987) Eur. J. Biochem. 163, 481-487[Abstract] |
53. | Knight, D. E., and Baker, P. F. (1985) J. Membr. Biol. 83, 147-156[Medline] [Order article via Infotrieve] |
54. | Rosario, L. M., Stutzin, A., Cragoe, E. J., and Pollard, H. B. (1991) Neuroscience 41, 269-276[Medline] [Order article via Infotrieve] |
55. | Zaks, W. J., and Creutz, C. E. (1991) Biochemistry 30, 9607-9615[Medline] [Order article via Infotrieve] |
56. | Creutz, C. E., Pazoles, C. J., and Pollard, H. B. (1979) J. Biol. Chem. 254, 553-558[Abstract] |
57. | Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., and Sudhof, T. C. (1994) Cell 79, 717-727[Medline] [Order article via Infotrieve] |
58. | DeBello, W. M., Betz, H., and Augustine, G. J. (1993) Cell 74, 947-950[Medline] [Order article via Infotrieve] |
59. |
Bittner, M. A.,
Holz, R. W.,
and Neubig, R. R.
(1986)
J. Biol. Chem.
261,
10182-10188 |
60. | Knight, D. E., and Baker, P. F. (1982) J. Membr. Biol. 68, 107-140[Medline] [Order article via Infotrieve] |
61. | O'Sullivan, A. J., and Burgoyne, R. D. (1990) J. Neurochem. 54, 1805-1808[Medline] [Order article via Infotrieve] |
62. | Ely, C. M., Oddie, K. M., Litz, J. S., Rossomando, A. J., Kanner, S. B., Sturgill, T. W., and Parson, S. J. (1990) J. Cell Biol. 110, 731-742[Abstract] |
63. |
Furge, L. L.,
Chen, K.,
and Cohen, S.
(1999)
J. Biol. Chem.
274,
33504-33509 |
64. | Srivastava, M., Bubendorf, L., Srikantan, V., Fossom, L., Nolan, L., Glasman, M., Leighton, X., Fehle, W., Pittaluga, S., Raffeld, M., Koivisto, P., Willi, N., Gasser, T. J., Kononen, J., Sauter, G., Kallioniemi, O. P., Srivastava, S., and Pollard, H. B. (2001) Proc. Natl. Acad. Sci. U. S. A., in press |