(Received for publication, October 28, 1994; and in revised form, July 11, 1995)
From the
Heterotetrameric annexin 2 phosphorylated ``in vitro'' by rat brain protein kinase C is purified and obtained devoid of unphosphorylated protein; it contains 2 mol of phosphate/mol of heterotetramer. The aggregative and binding properties of the phosphorylated annexin 2 toward purified chromaffin granules are compared with those of the unphosphorylated annexin 2. Annexin 2 binds to chromaffin granules with high affinity. Phosphorylation of annexin 2 decreases the affinity of this binding without affecting the maximum binding capacity. The binding curves are strongly cooperative. It is suggested that a surface oligomerization of the proteins may take place upon binding. Besides, phosphorylation of annexin 2 is followed by a dissociation of the light chains from the heavy chains in the heterotetramer. Whereas annexin 2 induces the aggregation of chromaffin granules at µM calcium concentration, the phosphorylated annexin 2 does not induce aggregation at any concentration of calcium either at pH 6 or 7. The phosphorylation of annexin 2 by protein kinase C, MgATP, and 12-O-tetradecanoylphorbol-13-acetate on chromaffin granules induces a fusion of chromaffin granules membranes observed in electron microscopy. The fusion requires the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate. Given these results and since annexin 2 is phosphorylated by protein kinase C under stimulation of chromaffin cells, it is suggested that phosphorylated annexin 2 may be implicated in the fusion step during exocytosis of chromaffin granules.
Annexin 2 is a calcium-phospholipid-binding protein of the annexin family which has been characterized in the adrenal medulla as chromobindin 8. The heterotetrameric molecule formed of two heavy chains of 36 kDa and two light chains of 11 kDa possesses the unique property among the other annexins to aggregate chromaffin granules at micromolar calcium concentration(1) .
It has been shown by
immunoelectron microscopy (2) that in chromaffin cells annexin
2 was closely associated with the inner face of the plasma membranes.
In cultured chromaffin cells, thin strands were found cross-linking the
chromaffin vesicles to the plasma membrane after stimulation with
acetylcholine. Similar thin strands were also observed between
aggregated chromaffin vesicles when they were mixed with annexin 2 in
the presence of calcium. These data strongly suggested that
conformational changes were induced in annexin 2 to cross-link the
vesicles and the plasma membrane after stimulation of cultured
chromaffin cells. When primary cultured chromaffin cells were
stimulated by nicotine, annexin 2 was phosphorylated by protein kinase
C. The phosphorylation of the protein was concomitant with the
catecholamine release. ()In streptolysin-permeabilized
cells, annexin 2 phosphorylated ``in vitro'' by
brain protein kinase C was able to reconstitute secretion of
catecholamines in cells depleted of protein kinase C
activity(3) . Taken together, all these results suggest that
annexin 2 could be involved in the exocytotic process. It was,
therefore, of interest to reinvestigate the properties of annexin 2
phosphorylated in vitro by protein kinase C.
A study of the effect of phosphorylated annexin 2 tetramer on the aggregation of lipid vesicles (4) demonstrated that phosphorylation of annexin 2 caused a loss of lipid vesicle aggregation. In order to reproduce more closely the physiological conditions found in the chromaffin cells, we focused our study on the properties of the phosphorylated annexin 2 tetramer toward the chromaffin granules in the same experimental conditions as those of Drust and Creutz (1) who observed an aggregation of granules induced by annexin 2 tetramer at micromolar calcium concentration at pH 7 and a fusion of the same granules by lowering the pH to 6 in the presence of arachidonic acid.
Our
results extended the previous studies on the properties of
phosphorylated annexin 2. We demonstrated that phosphorylated annexin 2
bound to the granules with a lower affinity than the unphosphorylated
annexin 2. This binding was strongly cooperative like that of
unphosphorylated annexin 2. When annexin 2 was added to a suspension of
chromaffin granules in the presence of protein kinase C and TPA, ()the phosphorylated annexin 2 that was produced induced a
spectacular fusion of granules. It was the first time that fusion was
induced under phosphorylation of a protein. Besides, this protein was
phosphorylated in vivo by protein kinase C under nicotine
stimulation of chromaffin cells.
Figure 1: Stoichiometry of phosphorylation of annexin 2 by protein kinase C. A-2t was phosphorylated as described under ``Experimental Procedures.'' Aliquots of reaction medium were taken at various time intervals and subjected to scintillation counting. Inset: 13% SDS-PAGE of phosphorylated A-2t after 30-min incubation. A, Coomassie Blue. B, autoradiogram. Bands 1, 2, and 3, 38, 39, and 40 kDa.
Figure 2:
Effect
of phosphorylation of A-2t on association of light chain p11 to the
heavy chain p36. A, sucrose gradient analysis. 24 µg of
A-2t or P-A-2t and 6 µg of bovine serum albumin in 30
µl of 20 mM imidazole, pH 7.4, 100 mM NaCl, 1
mM EGTA, 2 mM NaN
, 0.5 mM DTT
were layered on a 5-40% sucrose gradient in the same buffer
medium and centrifuged for 15 h at 48,000 rpm in a Beckman SW56 rotor
at 4 °C. Fractions (0.15 ml) were collected and analyzed by 10 and
15% SDS-PAGE and Coomassie Blue staining. Heavy chain p36 was
quantified by densitometry and PhosphorImager analysis from 10%
SDS-PAGE; light chain p11 was quantified by densitometry from 15%
SDS-PAGE. B, chromatography on a Superose 12 (Pharmacia
Biotech Inc.) column of 100 µg of
P-A-2t. The column
was developed with 25 mM Tris-HCl pH 7.4, 150 mM NaCl. The column fractions (0.5 ml) were analyzed as described
above.
, monophosphorylated heavy chain p36;
,
diphosphorylated heavy chain p36;
, light chain p11;
, heterotetramer A-2t;
, monomer A-2;
, bovine serum
albumin.
Figure 3: Binding of annexin 2 to chromaffin granules. A, the binding conditions were 40 mM MES, pH 6, 30 mM KCl, 0.24 M saccharose, 10 µM calcium, and 50 µg of granule protein at room temperature. B, the binding conditions were 40 mM Hepes, pH 7, 30 mM KCl, 0.24 M saccharose, 10 µM calcium, and 50 µg of granule protein at room temperature. The binding was sigmoidal (upper part). Scatchard analysis of the A-2t binding data (lower part). The downward concave curve indicated the positively cooperative nature of this interaction. Inset, Hill plot of the binding data. The slope of the regression curve was 1.9 at pH 6 and 1.4 at pH 7. The log(y/(I - y)) was plotted on the ordinate; log(free (nM)) was plotted on the abscissa. y represented the fractional saturation: bound/maximal binding.
Figure 4: Binding of phosphorylated annexin 2 to chromaffin granules. The binding conditions were those described in the legend to Fig. 3. A, pH 6. The binding curve was sigmoidal (upper part). The Scatchard analysis gave a downward concave curve (lower part). B, pH 7. The binding curve was sigmoidal. The Scatchard analysis gave a downward concave curve.
The concentrations of I-A-2t used were in a range of 0.17-8 µg for 50
µg of granule protein.
For I-labeled A-2t, the
Hill coefficient was 2.0 ± 0.5 at pH 6.0 and 1.7 ± 0.1 at
pH 7.0, the apparent K
of these binding at pH 6.0
and 7.0 was 5.7 ± 1.3 and 13.5 ± 1.4 nM,
respectively. The Scatchard plot of these bindings showed that the
binding of A-2t to granule membranes was strongly cooperative (Fig. 3, A and B), with a K
of 7.8 ± 4 and 34 ± 4 nM, a Hill
coefficient of 1.9 ± 0.1 and 1.3 ± 0.1 at pH 6.0 and 7.0,
respectively. At saturation, 1.7 ± 0.3 and 2.2 ± 0.6 nmol
of A-2t bound per mg of granule proteins at pH 6.0 and 7.0 (n:10) (Table 1). The concentration of A-2t necessary to
obtain saturation was 8 µg for 50 µg of granule protein.
The phospholipid content determined on the chromaffin granule membranes was 0.48 µmol/mg of proteins. Assuming that 10.4% phosphatidylserine and phosphatidylinositol were found in total lipids of the granule membranes (18, 19, 20) and taking these data into account, we obtained 1 mol of A-2t bound per 27 and 33 mol of phosphatidylserine at pH 6.0 and 7.0, respectively (Table 1).
For studying binding of P-A-2t, the protein
concentrations used were in a range of 0.19-30 µg for 50
µg of granule protein.
The nonlinear regression of the binding
curve gave a Hill coefficient of 2.3 ± 0.5 at pH 6.0 and 1.7
± 0.4 at pH 7.0. The apparent K for these
bindings at pH 6.0 and 7.0 was 54 nM ± 6 and 99 nM ± 23 (Fig. 4, A and B). The
Scatchard plot for these bindings demonstrated that the binding of
P-A-2t was strongly cooperative (Fig. 4, A and B) with a K
70 and 60 nM and a Hill coefficient of 1.6 and 1.3 at pH 6.0 and 7.0,
respectively. At saturation, 1.4 and 3.2 nmol of
P-A-2t
bound per mg of protein i.e. 1 mol of
P-A-2t
binds 40 and 17 mol of phosphatidylserine at pH 6.0 and 7.0,
respectively (n:5) (Table 1).
The concentration of P-A-2t available to reach the saturation was of the order
of 30 µg/50 µg of granule protein.
On the basis of this result we studied the
aggregation of granules by A-2t at pH 7.0 and 6.0. Fig. 5shows
that the half-maximum aggregation of A-2t at pH 7.0 occurred at 5
µM Ca similar to the previous report (1) and at pH 6.0 at 2.5 µM Ca
. Fig. 5shows clearly that the phosphorylated A-2t did not promote
granule aggregation at any concentration of Ca
from 0
to 1 mM either at pH 7.0 or 6.0.
Figure 5:
Ca-dependent aggregation
of chromaffin granules by annexin 2. The reaction mixture contained a
granule suspension approximately at 0.1 mg/ml (absorbance
0.3-0.35 at 540 nm) and the indicated Ca
concentrations in buffer pH 7.0 or pH 6.0, as described under
``Experimental Procedures.'' The aggregation was initiated by
adding 10-12 µg/ml of unphosphorylated or phosphorylated
A-2t. The change in absorbance was monitored for 5 min at 540 nm.
, A-2t, pH 6;
, A-2t, pH 7;
,
P-A-2t pH
6 and pH 7.
In order to test the dynamic of the phosphorylation of annexin 2 on granules, we studied directly the phosphorylation by adding protein kinase C to a suspension of granules containing in addition to the components of the aggregation medium, MgATP and TPA. In these conditions (Fig. 6), we observed in the first 2 min of the incubation an increase of absorbance indicative of an aggregation of granules, then, a slow decrease of the absorbance. After 12-15 min, the starting base line was attained. If we suppressed protein kinase C in the reaction mixture, we observed only an increase of absorbance at 540 nm that reached a plateau after 5 min. If we suppressed TPA or MgATP instead of protein kinase C, we observed the same effect. These results suggested that MgATP, TPA, or protein kinase C alone were unable to induce the decrease of the aggregation (Table 2). The decrease of absorbance observed with all the components cited above might reflect either a reversibility of the aggregation, a lysis, or a fusion of granules. Given these results, we fixed and examined the different assays for electron microscopy.
Figure 6:
Effect of protein kinase C on chromaffin
granules aggregated by annexin 2. The reaction mixture contained a
granule suspension of 0.1 mg of protein/ml, 100 µM Ca, 50 µM ATP or
[
P]ATP, 5 mM MgCl
50
µM TPA in buffer, pH 6, as described under
``Experimental Procedures.'' The aggregation and the
phosphorylation reactions were simultaneously initiated by 0.4 µg
of protein kinase C and 5 µg of A-2t added together in the mixture.
The change in absorbance was monitored for 12 min at 540 nm in the
absence (
) or presence (
) of protein kinase C. Inset: 10% SDS-PAGE of granules pelleted after 12 min of
aggregation in the absence (lanes 1) or presence (lanes
2) of protein kinase C. a, Coomassie Blue. b,
PhosphorImager. The two
P-labeled bands were respectively
mono (38 kDa)- and di (40 kDa)-phosphorylated
proteins.
Figure 7: Electron micrographs of chromaffin granules incubated with unphosphorylated or phosphorylated annexin 2. Granules were incubated with unphosphorylated or phosphorylated annexin 2 at 10 µM calcium, pH 6.0, for 5 min. Bars: 0.3 µm; inset bars: 0.1 µm. a, chromaffin granules control; b, aggregation of chromaffin granules induced by A-2t; c, chromaffin granules incubated with phosphorylated A-2t.
Figure 8: Electron micrographs of chromaffin granules incubated with unphosphorylated annexin 2 and protein kinase C. Granules were incubated at 100 µM calcium, pH 6.0, for 12 min with MgATP, TPA, and A-2t (a) or with MgATP, TPA, A-2t, and protein kinase C (b) Bar: 1 µm.
In order to understand how phosphorylation could regulate secretion, we carried out a comparative study of the respective biochemical properties of unphosphorylated and phosphorylated annexins 2.
For the first time, annexin 2 phosphorylated in vitro by
brain protein kinase C has been purified devoid of protein kinase C and
unphosphorylated annexin 2. P labeling of the protein has
been quantified. The phosphorylated protein has been obtained in
amounts (0.5 mg) that allowed a careful study of its properties.
The purified phosphorylated protein contains up to 2.5 mol of phosphate/mol of heterotetramer. During the phosphorylation process we observe that the rate of the phosphorylation of one serine residue is faster than the phosphorylation of one another, since, in most cases, the phosphorylation experiments give 85% of monophosphorylated and 15% of diphosphorylated heavy chain. If we refer to the in vitro phosphorylation of annexin 2 (calpactin 1) by protein kinase C described by Johnsson et al.(21) and Gould et al.(22) , the major phosphorylation site involves serine 25, located near tyrosine 23, a relatively short region of the p36 heavy chain known to provide contact with the p11 light chain but likely accessible to protein kinase C and tyrosine kinase v-Src(21, 22) . The second phosphorylation site must involve another serine residue of the amino-terminal domain, likely residue 11 which is close to a lysine residue(21) . The fact that the diphosphorylated form appears concomitantly with the monophosphorylated form suggests that the phosphorylation on serine 25 could induce the exposure of serine 11. Powell and Glenney (23) have reported that phosphorylation of tyrosine 23 by tyrosine kinase v-Src decreased the affinity of light chain to the heavy chain. This is in accord with our data which show that a fraction of monophosphorylated form and the totality of the diphosphorylated form of annexin 2 have lost their light chains. However, the two phosphorylated heavy chains do not seem to be dissociated in monomers but rather to exist as homodimers. This is surprising, since until now, it was admitted that the small subunits p11 induced p36 dimerization and that annexin 2 existed either as an heterotetramer or as a monomer(24) . Johnsson et al.(21) , however, noticed that the minor phosphorylated p36 resulting from kinase C treatment, following reconstitution with p11 presented a small shift in the elution profile from Superose S12. They attributed this shift to a weak remaining affinity between p36 and p11. The formation of an homodimer devoid of p11 would suggest that the phosphorylation of the heavy chains could induce a head-to-head association. Such a dimerization has been observed on a monomeric annexin, annexin 1, in placenta (25) and in brain(26) .
When chromaffin
cells in primary culture labeled with P are stimulated by
nicotine, the immunoprecipitated p36 heavy chain shows a labeled band
which migrates slower than p36, at the same distance as
monophosphorylated p36 obtained by the in vitro phosphorylation by protein kinase C.
Hence, in
stimulated cells, protein kinase C phosphorylates preferentially one
site on the 36-kDa heavy chain.
Phosphorylation of annexin 2 by
protein kinase C reduces its affinity to granule membranes with no
change in its binding capacity. An average B values of 2.2 nmol of annexin 2/mg of granule protein (19%, w/w)
is obtained either with unphosphorylated or phosphorylated annexin 2.
If one assumes that 1 mg of granule protein correspond to 5
10
granules, 2.5
10
mol of annexin 2
bind to one granule. These values are in agreement with a binding of
annexin 2 to phospholipids, since the amount of the more represented
integral membrane protein, cytochrome b
is 1.75
10
mol in the granule membrane(18) .
It
is likely that annexin 2 binds to phosphatidylserine heads, since it
has been demonstrated that it binds in a Ca-dependent
manner specifically to phosphatidylserine. If we calculate the binding
of 1 mol of unphosphorylated or phosphorylated annexin 2 in relation to
the content of phosphatidylserine/mg of granule protein, we find that 1
mol of annexin 2 would bind 28-30 mol of phosphatidylserine, i.e. 14-15 mol/36-kDa heavy chain. This number is close
to that found for protein kinase C which binds 12 mol of
phosphatidylserine/mol (27) but far less than the fairly low
stoichiometry of the binding of synapsin I, 1 mol/900 mol of acidic
phospholipids(28) . Finally, this protein-phospholipid
interaction could be a major factor in the high affinity binding.
The binding of annexin 2 to granules is strongly cooperative. It is unknown whether this positive cooperativity of unphosphorylated annexin 2 is a consequence of enhanced ligand presentation once membrane aggregation begins (zipper mechanism) or if it arises from a surface oligomerization along the membrane, since phosphorylated annexin 2 does not aggregate granules.
We have observed that annexin 2 induces the
aggregation of chromaffin granules at micromolar calcium concentration
either at pH 7 or 6. The electron microscopy data corroborate these
results. The presence of numerous aggregated granules observed at pH 6
and at micromolar Ca concentration demonstrates that
annexin 2 joins tightly the granule membranes at micromolar
Ca
concentration, pH 6, but is unable to fusion the
membranes at this pH. The phosphorylated annexin 2, tested at the same
protein concentration as unphosphorylated protein, does not aggregate
chromaffin granules from 0.1 µM to 1 µM Ca
concentration. These results are in accord
with those obtained by Johnstone et al.(4) who
obtained no aggregation of phosphatidylserine liposomes with
phosphorylated annexin 2. Electron microscopy shows that the granules
brought together with phosphorylated annexin 2 are not aggregated or
fused; however, their membranes appear fluffy. This appearance does not
result from some traces of detergent, since Triton X-100 used for the
preservation of purified protein kinase C has been eliminated on a
detergent removing gel.
This effect on the morphology of the membrane may result from a conformational change of the phosphorylated protein which perturbs the phospholipid bilayer upon binding of the protein. Indeed, when protein kinase C is added in the presence of its activator TPA and annexin 2 in a suspension of granules, phosphorylation of annexin 2 induces the fusion of granules. The fusion is dependent on the activation of protein kinase C, since in the absence of TPA, the enzyme is unable to decrease the aggregation induced by unphosphorylated annexin 2. These results demonstrate that if unphosphorylated annexin 2 promotes the formation of contacts between membranes, the fusion effect is obtained only when the membrane structure is perturbed by a change of conformation likely induced by the phosphorylation of the molecule of annexin 2. This fusion effect is comparable with that described by Drust and Creutz (1) on chromaffin granules and by Mayorga et al.(29) on endosomes. In these two cases, annexin 2 is unable to fuse the membranes unless arachidonic acid is added. In our hands, phosphorylated annexin 2 is able by itself to fuse the aggregated membranes, but only if the phosphorylation by protein kinase C is effected on annexin 2 cross-linked membranes.
In conclusion, we have demonstrated that the binding of unphosphorylated as phosphorylated annexin 2 to the granule membranes is a positive cooperative mechanism that expresses likely an oligomerization of the annexin molecules at the surface of the membranes. This oligomerization may encircle a small patch of phospholipids, since each mole of annexin 2 may bind 28-30 mol of phosphatidylserine. This notion of cluster is in agreement with the lipidic fusion pore model of Monck and Fernandez(30) . If we refer to the electron microscopic studies from Nakata et al.(2) which show that annexin 2 forms fine strands associating closely the granule membrane to the plasma membrane under stimulation of the cell, the close interaction of unphosphorylated annexin 2 we have observed with chromaffin granule membranes alone may be similar with that of granule membrane and plasma membrane, since the cytoplasmic face of the plasma membrane of chromaffin cells may have a similar lipid composition and one could predict a similar interaction(31) .
In addition, in response
to stimulation of primary chromaffin cells annexin 2 is phosphorylated
by protein kinase C. The translocation of protein kinase C
at sites adjacent to those of annexin 2 on the membrane and the
activation of the enzyme by diacylglycerol, a second messenger mediated
by the activation of phospholipase C, are absolutely required for the
phosphorylation of annexin 2. Upon phosphorylation, a conformational
change of annexin 2 may occur in the NH
-terminal domain of
the molecule that leads to a decrease in affinity of the phosphoprotein
affecting calcium and phospholipid binding on the two heavy chains at
the opposite face of the molecule. This conformational change would
explain the perturbation of the phospholipid bilayer and the fusion
induced by phosphorylation of annexin 2. The results obtained strongly
support our study on phosphorylation of annexin 2 by protein kinase C
in primary cultures of streptolysin permeabilized chromaffin cells
stimulated by 4 µM calcium, where the activators of
protein kinase C, TPA and diacyglycerol, activate in the same time the
phosphorylation of annexin 2 and the secretion of catecholamines. This
activation of secretion concomitant with an increase of phosphorylation
of annexin 2 is not in accord with the suggestion of Creutz that
phosphorylation of annexin in stimulated chromaffin cells might play a
role in down-regulating exocytosis, since an inhibitory effect of the
phosphorylated protein was observed on the granule aggregating
activity(7) . In fact, in vivo, the fusogenic effect
of the phosphorylated protein may be the major effect, the inability of
the phosphorylated protein to aggregate granules being effective only
when the protein dissociates from the fused membranes.