(Received for publication, April 9, 1996, and in revised form, November 5, 1996)
From the Cell Regulation Research Group, Department of Medical Biochemistry, Calgary, Alberta T2N 4N1, Canada
In the present article we have examined if the interaction of the Ca2+-binding protein, annexin II tetramer (AIIt) with the plasma membrane phospholipids or with the submembranous cytoskeleton, effects the accessibility of the tyrosine phosphorylation site of AIIt. In the presence of Ca2+, pp60c-src catalyzed the incorporation of 0.22 ± 0.05 mol of phosphate/mol of AIIt (mean ± S.D., n = 5). The Ca2+-dependent binding of AIIt to purified adrenal medulla plasma membrane or phosphatidylserine vesicles stimulated the pp60c-src-dependent phosphorylation of AIIt to 0.62 ± 0.04 mol of phosphate/mol of AIIt (mean ± S.D., n = 5) or 0.93 ± 0.07 mol of phosphate/mol of AIIt (mean ± S.D., n = 5), respectively. Phosphatidylserine- or phosphatidylinositol-containing vesicles but not vesicles composed of phosphatidylcholine or phosphatidylethanolamine, stimulated the phosphorylation of AIIt. In contrast, the binding of AIIt to F-actin resulted in the incorporation of only 0.04 ± 0.04 mol of phosphate/mol of AIIt (mean ± S.D., n = 5). These results suggest that the interaction of AIIt with plasma membrane and not the submembranous cytoskeleton, activates the tyrosine phosphorylation of AIIt by inducing a conformational change in the protein resulting in the enhanced exposure or accessibility of the tyrosine-phosphorylation site.
The annexins (reviewed in Refs. 1-5) are a family of Ca2+-binding proteins that bind acidic phospholipids. Members of this family contain a region of amino acid homology called the annexin fold (6). Annexin II tetramer (AIIt)1 is an abundant annexin which is composed of two Mr 36,000 annexin II subunits and two Mr 11,000 subunits. The 36-kDa subunit consists of two functional domains. The first, the amino-terminal regulatory domain contains the first 30 amino acids of the amino terminus of the heavy chain, incorporates the serine and tyrosine-phosphorylation sites and the binding site for the p11 light chain. The remaining carboxyl domain, comprises the binding sites for Ca2+, phospholipid, and F-actin (reviewed in Ref. 2). AIIt is associated with the cytosolic surface of the plasma membrane in association with the submembranous cytoskeleton of many secretory cells where the protein has been shown to form links between the plasma membrane and secretory granules (6, 7). Although the exact physiological function of AIIt is unclear, the protein is thought to play a role in Ca2+-dependent exocytosis or endocytosis (2).
AIIt has been shown to be phosphorylated in vivo by protein tyrosine kinases. For example, the expression of transforming protein tyrosine kinases in a variety of cells has been shown to correlate with the appearance of phosphotyrosine in AIIt (8, 9) and in many cells AIIt is a major in vivo substrate of pp60v-src (10-12). Activation of transmembrane protein kinase receptors, such as the platelet-derived growth factor receptor, has also been shown to result in the tyrosine phosphorylation of AIIt (13-15). The phosphorylation of AIIt in pp60v-src transformed cells or in cells activated by platelet-derived growth factor is identical to the site phosphorylated on the protein in vitro by pp60v-src, namely tyrosine 23 (13).
In previous work, we examined the consequences of the tyrosine phosphorylation of AIIt on the biological activities of the protein (16). We reported that pp60c-src-phosphorylated AIIt did not bind to heparin or bind to or bundle F-actin. Furthermore, native AIIt but not tyrosine-phosphorylated AIIt could promote the formation of a plasma membrane-AIIt-chromaffin granule complex in vitro (16). We therefore concluded that the tyrosine phosphorylation of AIIt was an inhibitory signal. Consistent with these results, we have shown that activation of adrenal chromaffin cells, with acetylcholine, results in the dephosphorylation of AIIt concomitant with the release of catecholamine (2).
In the present article we have examined the kinetics of phosphorylation of AIIt by pp60c-src. The results suggest that the binding of AIIt to the phospholipid component of the plasma membrane stimulates the phosphorylation of the protein by pp60c-src.
AIIt phosphorylation
reactions were performed according to Ref. 16. AIIt at a final
concentration of 60 µg/ml, was incubated at 30 °C for 30 min in 25 mM HEPES (pH 7.5), 10 mM MgCl2, 0.5 mM EGTA, 0.6 mM CaCl2, 1.5 µg/ml
baculovirus produced human recombinant pp60c-src, and 100 µl/ml lipid vesicles which were taken from a stock containing 200 µg/ml phosphatidylserine, 200 µg/ml phosphatidylcholine, and 40 µg/ml diolein. When incubations were performed in the presence of
plasma membrane, 0.5 mM orthovanadate and 3 mg/ml
para-nitrophenyl phosphate were included as phosphatase
inhibitors. The reaction was initiated by addition of 25 µM ATP (200-2000 cpm/pmol [-32P]ATP).
To quantify the stoichiometry of phosphorylation of AIIt, 25 µl was
removed from the reaction mixture and either precipitated with 25%
trichloroacetic acid and 2% sodium pyrophosphate and subjected to
scintillation counting, or alternatively, boiled with 1 volume of
SDS-PAGE sample buffer (0.25 M Tris-HCl, pH 6.8, 10% SDS,
20% glycerol, 2 mM EGTA, 2 mM EDTA, 20 mM
-mercaptoethanol) and analyzed by SDS-PAGE (17). For
experiments testing the phospholipid specificity of phosphorylation
(Table II), phospholipid liposomes were prepared by hydrating 4 mg of
phospholipid (previously dried from a chloroform solution under
N2) with 50 mM HEPES (pH 7.5). The suspensions
were mixed in a vortex and sonicated three times for 15 s. Since
phosphatidylethanolamine did not readily form stable liposomes when
used as a pure phospholipid,
phosphatidylethanolamine/phosphatidylcholine (5:1) liposomes were used
to measure the stimulation of phosphorylation of AIIt by these
liposomes.
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AIIt was prepared from bovine lung
(18) and stored in 50 mM KCl at 70 °C. The protein was
essentially homogeneous as determined by SDS-PAGE. Adrenal medulla
plasma membranes were purified according to Ref. 16. Protein
concentration was measured with the Bradford Coomassie Blue dye binding
method using bovine serum albumin as a standard (19). Alternatively,
protein concentrations were determined spectrophotometrically using
appropriate extinction coefficients (16). Lipid vesicles for the
annexin phosphorylation reaction were prepared fresh daily according to
Ref. 20. Human pp60c-src was made using a baculovirus vector
and purified by hydroxyapatite and immunoaffinity chromatography (21).
Partially proteolyzed pp60c-src was produced by omitting
protease inhibitors during the final Mono-Q FPLC column purification
step (21). This resulted in proteolysis of the pp60c-src
preparation without any loss in enzyme activity. The partially proteolyzed pp60c-src was chromatographed on a monoclonal
antibody affinity column (utilizing Mb 203 07D10-Quality Biotech). This
monoclonal antibody recognizes an epitope at amino acids residues 2-17
of pp60c-src. The column flow-through was pooled and SDS-PAGE
analysis identified a major 51-kDa protein band. This protein reacted
with the 327 monoclonal antibody but not the 203 07D10 monoclonal
antibody. This suggested that the partially proteolyzed
pp60c-src had lost part of the NH2 terminus
including the myristate that is normally attached to Gly-2. The
proteolyzed pp60c-src was extensively
characterized2 and found to have similar
activity to the native enzyme. Calcium concentrations were
determined by Ca2+ electrode and FURA measurements
(20).
Fig. 1 presents the time
course of phosphorylation of AIIt by pp60c-src. At low
Ca2+ concentration (0.1 µM) a slow rate of
incorporation of phosphate into AIIt was observed with about 0.09 ± 0.08 mol of phosphate/mol of AIIt (mean ± S.D.,
n = 5) incorporated by pp60c-src in 60 min
(Fig. 1). When 100 µM Ca2+ was added to the
reaction, the rate of phosphorylation of AIIt by pp60c-src was
also slow and after 60 min only 0.22 ± 0.05 mol of phosphate/mol of AIIt (mean ± S.D., n = 5) was incorporated.
Increasing the Ca2+ concentration did not alter the
phosphorylation rate and at 1 mM Ca2+ about
0.21 ± 0.01 mol of phosphate/mol of AIIt (mean ± S.D., n = 5) was incorporated. The addition of phospholipid
vesicles to the reaction mixture stimulated the rate of phosphorylation of AIIt and at equilibrium, 0.84 ± 0.05 mol of phosphate/mol of AIIt (mean ± S.D., n = 5) was incorporated. In
the presence of 100 µM Ca2+ and phospholipid
vesicles, the phosphorylation of AIIt was very rapid and at equilibrium
about 0.93 ± 0.07 mol of phosphate/mol of AIIt (mean ± S.D., n = 9) was incorporated.
The results presented in Fig. 1 suggested that the addition of
phospholipid vesicles to the reaction mixture stimulated the rate and
extent of phosphorylation of AIIt by pp60c-src. In order to
determine if the stimulation of AIIt phosphorylation was due to the
interaction of phospholipid vesicles with AIIt or pp60c-src,
the effect of phospholipid vesicles on pp60c-src activity was
examined. The pp60c-src activity in these experiments was
measured using a peptide to the phosphorylation site of AIIt
(KLSLEGDHSTPPSAYGSVKAYT). This peptide exhibited the following
enzymatic parameters: Km = 198.4 ± 25.5 µM (mean ± S.D., n = 5);
Vmax = 41.3 ± 2.7 pmol of
phosphate·min1·pmol
1 (mean ± S.D., n = 5) which compared favorably with the
pp60c-src-dependent phosphorylation of the CDC 2 peptide (residues 6-20) (Km = 245.5 ± 26.6 µM; mean ± S.D., n = 5;
Vmax = 61.8 ± 4.1 pmol of
phosphate·min
1·pmol; mean ± S.D.,
n = 5) (22). As shown in Table I,
pp60c-src exhibited a maximal initial rate of phosphorylation
of the AIIt-phosphorylation site peptide in the absence of added
Ca2+ or phospholipid vesicles. The addition of
Ca2+, phospholipid vesicles or both, resulted in a small
decrease in the initial phosphorylation rate. In contrast, the initial rate of phosphorylation of AIIt was maximal in the presence of both
Ca2+ and phospholipid vesicles. The phosphorylation rate in
the presence of Ca2+ was only 12% of the rate of
phosphorylation of AIIt in the presence of both Ca2+ and
phospholipid vesicles. Furthermore, at a Ca2+ concentration
of 0.1 µM, the initial rate of phosphorylation of AIIt
was stimulated almost 13-fold by the addition of phospholipid vesicles.
These results therefore suggest that the stimulation of phosphorylation
of AIIt observed in the presence of phospholipid vesicles was not due
to the stimulation of pp60c-src activity by the phospholipid
vesicles but due to a phospholipid-induced increased exposure of the
tyrosine-phosphorylation site of AIIt.
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As shown in Fig.
2, at equilibrium, about 0.62 ± 0.04 mol of
phosphate (mean ± S.D., n = 5) were incorporated
into AIIt by pp60c-src in the presence of purified adrenal
medulla plasma membrane (Fig. 2, inset). This compared to
0.22 ± 0.05 mol of phosphate/mol of AIIt (mean ± S.D.,
n = 5) incorporated in the absence of plasma membrane.
The stimulation of the phosphorylation of the
pp60c-src-dependent phosphorylation of AIIt by
plasma membrane could be due to the interaction of AIIt with
phospholipid or F-actin components of the plasma membrane. However, as
shown in Fig. 2, the interaction of AIIt with F-actin did not activate
the phosphorylation of AIIt by pp60c-src. In contrast, the
interaction of AIIt with phospholipid stimulated the rate (Table I) and
extent (Fig. 2) of phosphorylation of AIIt. These results therefore
suggest that the binding of AIIt to the phospholipid component of the
plasma membrane is responsible for stimulation of the phosphorylation
of AIIt.
Phosphorylation of AIIt by Partially Proteolyzed pp60c-src
Both AIIt and pp60c-src are
membrane-associated proteins that are known to bind acidic
phospholipids (23-26). Therefore, it was possible that the
phospholipid-dependent activation of phosphorylation of
AIIt could be due to recruitment of AIIt and pp60c-src to the
phospholipid vesicles, thereby increasing the proximity of
pp60c-src and AIIt. This would result in an increase in the
effective concentration of AIIt and pp60c-src in the
phosphorylation reaction and could produce an enhanced phosphorylation
rate. To examine this possibility, we prepared a partially proteolyzed
pp60c-src. The partial proteolysis of the pp60c-src
results in the loss of a portion of the NH2-terminal domain
of the enzyme and the loss of the NH2-terminal myristic
acid. It was therefore expected that the loss of this domain of
pp60c-src would inhibit the binding of the enzyme to
phospholipid liposomes. Therefore, native and partially proteolyzed
pp60c-src were incubated with phosphatidylserine liposomes and
following centrifugation, the pellets were examined for
pp60c-src activity. As shown in Fig. 3, the
partially proteolyzed pp60c-src did not bind to
phosphatidylserine liposomes. In contrast, the native pp60c-src
bound to the phospholipid liposomes.
Since the partially proteolyzed pp60c-src did not bind to phospholipid liposomes it was possible to directly examine the role of phospholipid in the phosphorylation of AIIt without the potential complication of the binding of both pp60c-src and AIIt to the phospholipid liposomes. We therefore examined the specificity of the phospholipid stimulation of phosphorylation of AIIt by partially proteolyzed pp60c-src. As shown in Table II, phosphatidylserine and phosphatidylinositol liposomes stimulate the phosphorylation of AIIt by partially proteolyzed pp60c-src. In contrast, phosphatidylcholine or phosphatidylethanolamine liposomes do not stimulate the phosphorylation of AIIt. Therefore, this data provides direct evidence that phospholipid-induced conformational change in AIIt stimulates the tyrosine phosphorylation of AIIt by pp60c-src. Furthermore, since AIIt binds phosphatidylserine and phosphatidylinositol but not phosphatidylcholine or phosphatidylethanolamine (23), these results suggest that phospholipid binding and not a nonspecific effect of phospholipid is responsible for the phospholipid stimulation of phosphorylation of AIIt by pp60c-src.
Dephosphorylation of AIIt by Plasma Membrane PhosphatasesOur
results suggest that the binding of AIIt to plasma membrane activates a
conformational change in AIIt which enhances exposure of the
tyrosine-phosphorylation site of the protein, resulting in the
stimulation of the phosphorylation of the protein by pp60c-src.
Previous work from our laboratory has shown that the tyrosine phosphorylation of AIIt induces a conformational change in AIIt which
results in the inability of the protein to bind F-actin or heparin or
to bridge biological membranes (16). Therefore, it was unclear from
these studies if the tyrosine-phosphorylation site of plasma
membrane-bound, tyrosine-phosphorylated AIIt, was accessible to plasma
membrane-associated protein tyrosine phosphatases. Since the binding of
AIIt to plasma membrane stimulated the phosphorylation of AIIt by
pp60c-src, it was important to ascertain if the plasma membrane
bound AIIt was a substrate for plasma membrane associated
phosphotyrosine phosphatases. As shown in Fig. 4, the
incubation of pp60c-src-phosphorylated AIIt with plasma
membrane resulted in the dephosphorylation of AIIt. The
dephosphorylation was very rapid and was essentially complete after
10 s of incubation of tyrosine-phosphorylated AIIt with
plasma membrane. This indicated that the tyrosine-phosphorylation site of plasma membrane-bound AIIt was accessible to membrane phosphatases and could be regulated by cycles of tyrosine
phosphorylation and dephosphorylation.
Studies of the x-ray crystallographic analysis of annexin V have provided valuable information on the possible structure of all members of the annexin family of Ca2+-binding proteins (27-29). These studies support the prediction that the p36 heavy chain is a planar molecule composed of opposing concave and convex surfaces. The convex surface is predicted to lie along the plane of the phospholipid membrane and contain the Ca2+-binding sites. The concave surface of the protein faces the cytosol and contains the amino-terminal and carboxyl-terminal domains. The exposure of the amino-terminal domain of the p36 heavy chain to the cytosol results in the accessibility of the Tyr-23 and Ser-25 phosphorylation sites to pp60src and protein kinase C, respectively, and also allows binding of the p11 light chains to the first 12 amino acids of the amino-terminal domain. These studies have not addressed the issue of whether or not the accessibility of the Tyr-23 and Ser-25 phosphorylation sites are influenced by the binding of AIIt to Ca2+ or plasma membrane.
Predictions based on the x-ray crystallographic structure of annexin V
as well as fluorescence spectroscopic analysis of AIIt have suggested
that AIIt can exist in several distinct conformations. AIIt binds
Ca2+ with a Kd (Ca2+) of
about 0.5 mM Ca2+. Ca2+ binding
results in a decrease in the -helical content of AIIt (30) and the
exposure of Trp-212 to a more hydrophobic environment (30-32). In the
presence of phosphatidylserine, AIIt binds 15 mol of
Ca2+/mol of AIIt with a Kd
(Ca2+) of 1.3 µM (33, 34). X-ray
crystallographic analysis has suggested that phosphatidylserine binding
will cause a substantial conformational change in AIIt. The
phosphatidylserine-induced conformation change results from
phosphatidylserine linking two Ca2+-binding sites that are
about 8.8 Å apart and from the interaction of phosphatidylserine with
amide group of Gly-206 and the hydroxyl group of Thr-207. The
phosphoryl oxygen of phosphatidylserine is involved in the coordination
of AB site Ca2+ while the serine carboxylate
oxygen of phosphatidylserine coordinates the AB
Ca2+. However, there are significant differences between
the Ca2+-induced conformational change that occurs when
Ca2+ is added to AIIt at millimolar concentration in the
absence of phosphatidylserine and the conformational change that occurs
when AIIt binds Ca2+ at micromolar Ca2+ in the
presence of phosphatidylserine (34). Therefore, it has been concluded
that the conformation of AIIt, induced by binding of Ca2+
to its Ca2+-binding sites on AIIt differs from the
conformation of AIIt induced by the binding of Ca2+ to its
Ca2+-binding sites in the presence of
phosphatidylserine.
Although the structure of AIIt-F-actin complex is unknown, it is reasonable to suggest that this conformation of AIIt will be distinct from other AIIt conformations. Within the context of our studies, our data suggests that the Tyr-23 phosphorylation site of AIIt is not fully exposed or accessible to pp60c-src when AIIt is in the Ca2+-induced conformation or in the F-actin-induced conformation. In contrast, when AIIt is in the phosphatidylserine-induced conformation, the tyrosine-phosphorylation site is accessible to pp60c-src. The binding of Ca2+ and phosphatidylserine to AIIt, which induces the Ca2+- and phosphatidylserine-induced conformation of AIIt, results in only a modest increased exposure of the tyrosine-phosphorylation site compared to the phosphatidylserine-induced conformation of AIIt.
We have also examined the possibility that the stimulation of the pp60c-src-dependent AIIt phosphorylation by phosphatidylserine or plasma membrane is not due to a phospholipid-induced conformational change in AIIt but is due to the binding and therefore the enhanced concentration of pp60c-src and AIIt on the phosphatidylserine liposomes. However, as shown in Table II, the phospholipid-dependent stimulation of phosphorylation of AIIt is also observed in the presence of a partially proteolyzed pp60c-src. Since this truncated enzyme does not bind to phospholipid liposomes (Fig. 3), the phospholipid-stimulation of phosphorylation of AIIt must be due to a direct effect of phospholipid on AIIt. Furthermore, since the phosphorylation of AIIt is stimulated only by phospholipids that bind to AIIt (Table II), such as phosphatidylserine and phosphatidylinositol, our data suggest that a phospholipid-dependent conformational change in AIIt is responsible for stimulation of the phosphorylation of AIIt by pp60c-src.
The phosphorylation of other substrates of pp60src have also been shown to be accelerated upon plasma membrane binding. Vinculin binds phosphatidylserine, phosphatidylglycerol, and phosphatidic acid and the binding of these phospholipids to vinculin correlates with a increase in the phosphorylation of vinculin by pp60v-src (35). The stimulation of vinculin phosphorylation was shown to be due to a phospholipid-induced conformational change in the protein and these investigators concluded that phospholipid binding resulted in the increased accessibility of a phosphorylation site of vinculin to pp60v-src.
Within the cell, AIIt has been shown to be localized to the cytoplasmic surface of the plasma membrane in the submembranous cytoskeleton (34). Furthermore, AIIt has been shown to interact with endosomes and transfection of Madin-Darby canine kidney cells with a dominant negative mutant form of AIIt causes translocation of AIIt and early endosomes to the cytoplasm (36, 37). Similarly, pp60c-src has also been shown to be localized at the inner surface of the plasma membrane (38-40) and more recently this enzyme has been localized to the endosomes of fibroblasts (41). Therefore, AIIt and pp60c-src exist in the same intracellular compartment. The intracellular localization of AIIt has been suggested to be due to two properties of AIIt observed in vitro, namely phospholipid binding and F-actin binding. Although it is possible that the binding of AIIt to biological membranes could utilize plasma membrane-associated F-actin as the AIIt-binding site, our results (Fig. 2) suggest that AIIt bound to F-actin is not appreciably phosphorylated by pp60c-src. This result therefore suggests that AIIt bound to the phospholipid component of the plasma membrane is regulated by tyrosine phosphorylation.
Previous results from our laboratory (2, 16) have established that the tyrosine phosphorylation of AIIt produces a large conformational change in AIIt which results in the alteration or inhibition of many of the biological activities of AIIt. It was unclear from these previous studies if the phosphorylation site of the plasma membrane-associated tyrosine-phosphorylated AIIt was accessible to phosphotyrosine phosphatases. The results presented in Fig. 4 suggest that the plasma membrane-bound form of AIIt is readily dephosphorylated by plasma membrane-associated phosphotyrosine phosphatases. Therefore, considering the established association of pp60c-src with the plasma membrane, it appears that the key elements for the regulation of AIIt by phosphorylation and dephosphorylation are present at the plasma membrane.