The glial-derived calcium-binding protein S100B
can be secreted to act as a neurotrophic factor or a mitogen,
stimulating proliferation of glial cells. The extracellular S100B
activities rely on the oxidation of the protein cysteine residues
(Kligman, D., and Marshak, D. R. (1985) Proc. Natl. Acad.
Sci. U. S. A. 82, 7136-7139; Winningham-Major, F., Staecker,
J. L., Barger, S. W., Coats, S., and Van Eldik, L. J. (1989) J. Cell Biol. 109, 3063-3071). Here we show that
oxidation of the S100B cysteine residues, Cys-68 and Cys-84, induces a
conformational change in the protein structure, unmasking a canonical
CKII phosphorylation site located within the typical EF-hand
calcium-binding site II
. Intrasubunit disulfide-bridged S100B
monomer and disulfide-bonded S100B dimer are phosphorylated by the
catalytic CKII-
subunit on Ser-62 with a Km of
0.5 µM and a Vmax of 10 pmol/min/100 pmol of S100B. Oxidized S100B is the best in
vitro CKII-
substrate identified so far. Next we show that
intrasubunit disulfide-bridged S100B monomer is the most potent S100B
species to stimulate [3H]thymidine uptake by C6 glial
cells in culture. In addition, the phosphorylated intrasubunit
disulfide-bridged S100B monomer retains apparent mitogenic activity
toward C6 glial cells, and hence, 32P-labeled S100B should
be a useful probe for characterizing the mechanisms by which
extracellular oxidized S100B functions. Finally, we show that formation
of intrasubunit disulfide-bridged S100B monomer is stimulated by
peroxynitrite anion, suggesting that production of mitogenic S100B
species could be enhanced in neuropathology associated with
peroxynitrite anion production.
 |
INTRODUCTION |
The S100B protein belongs to the family of EF-hand calcium-binding
proteins. In solution, S100B (Mr = 10,500) forms
noncovalent dimers (Mr = 21,000). The S100B
protein dimer binds calcium with micromolar affinity
(Kd 10-100 µM) and zinc ion with nanomolar affinity (Kd 10-100 nM)
(1-2). The S100B affinity for calcium is highly dependent on the
quaternary protein structure. Zn2+ binding or alkylation of
Cys-
84 destabilizes the S100B quaternary structure to induce an
increase in the protein's affinity for calcium (1-3). The S100B is in
highest concentration in the vertebrate nervous system. The gene for
human S100B maps to the Down's syndrome region of chromosome 21 (4),
and increased levels of S100B proteins are found in the brain of
individuals with Down's syndrome (5) and in Alzheimer brains (6),
suggesting its potential involvement in common neuropathologies
associated with these diseases. In the brain, S100B is synthesized by
glial cells, but a secreted form that appears to be an oxidized S100B
species has neurotrophic and mitogenic activity (7-9). S100B has been
detected in brain extracellular fluid and in conditioned medium from
astroglial cells (10-11). Oxidized monomeric, homodimeric, and higher
oligomeric disulfide-bonded forms of S100B were purified from bovine
brain as a protein mixture that had neurite extension activity in chick embryonic cortical neurons (7). The reduced form of S100B has no
activity in inducing neurite extension, and similarly, the activities
of the recombinant protein was dependent upon the presence of the two
cysteine residues
68 and
84 (12). S100B is also a glial mitogen,
and that activity depends on the presence of Cys-68, suggesting that
extracellular mitogenic activity of S100B also relies on the oxidation
state of its cysteine residues (8, 9). Extracellular S100B stimulates
both calcium flux in glial C6 cells (13) and inducible nitric oxide
synthase activity in rat cortical astrocytes (14). However, the
mechanism by which the extracellular form of S100B induces neurite
outgrowth and/or cell proliferation is not yet known. Taking into
account that in normal brain S100B accumulates in glial cells in a
reduced state, an important issue will be to understand the processes and pathways that regulate formation of oxidized S100B forms in vivo and the pathways implicated in S100B secretion. The second question concerns the molecular mechanisms by which extracellular S100B
elicits its effects. Is the active S100B species internalized by the
target cell or are there cell-surface receptors that transduce the
S100B signal? Hopefully these questions could be resolved if the
biologically active oxidized S100B is clearly identified and if
sufficient amounts of that species could be produced to allow more
extensive structural and functional studies.
We developed a rapid and large scale method to produce mitogenic
oxidized S100B, and we show that conformational changes induced by
intrachain or interchain disulfide bridge formation in S100B led to
unmasking a canonical CKII phosphorylation site located within the
typical EF-hand calcium binding site II
. We also show that
intrasubunit disulfide-bridged S100B monomer is the most potent S100B
mitogenic species toward rat C6 glial cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant CKII-
and holoenzyme were prepared
as described previously (15). Bovine brain S100B (
), S100A1
(
), and S100a (
) were purified as described previously
(1-2). Intrachain disulfide-bonded S100B was prepared by the method of
Mely and Gérard (16). Covalent disulfide-bonded S100B dimers were
prepared by incubation of freshly purified S100B with 5 mM
sodium tetrathionate (Sigma) in the presence of 1 mM
calcium at room temperature. The reaction was stopped by addition of 5 mM EGTA, and the protein was dialyzed against 20 mM Tris-HCl buffer, pH 7.4. After this treatment 80-90%
of S100B was disulfide-bonded S100B dimers. Oxidized S100B
concentrations were determined by using the Bio-Rad protein assay
reagent using purified reduced S100B as standard.
SDS-Polyacrylamide Gel Electrophoresis--
11% polyacrylamide
gels were used according to the method described by Schagger and Von
Jagow (17) for the separation of proteins ranging from 1 to 100 kDa.
Synthesis of Peroxynitrite and Peroxynitrite Oxidation of
S100B--
Peroxynitrite was synthesized in a quenched flow reactor,
as described previously (18-19). The concentration of peroxynitrite was determined by absorbance at 302 nm in 1 M NaOH
(
302 nm = 1670 M/cm). Peroxynitrite
oxidation of S100B was performed as follow: S100B in 20 µl of 20 mM Tris-HCl, pH 7.5, containing 1 mM
CaCl2 was first acidified by addition of 10 µl of 1 N HCl followed by addition of 10 µl of peroxynitrite in 1 N NaOH.
In-gel S100B Digestion and Matrix-assisted Laser
Desorption/Ionization Mass Spectroscopy--
S100B was run on a
SDS-PAGE,1 and the
Coomassie-stained spots were in-gel digested as described previously
(20). Briefly, the protein spots were excised and destained with 30%
ethanol. After washing with 50% acetonitrile, gel pieces were dried
and reswollen in 20 µl of 10 mM phosphate buffer, pH 6.5, containing 0.5 µg of endo-Asp-N protease (Boehringer Mannheim,
sequencing grade), and incubated at 37 °C for 3 h. A 0.4-µl
volume of the digest solution was used for mass spectrometric analysis;
it was added onto the sample probe to 0.4 µl of a saturated solution of
-cyano-4-hydroxy-trans-cinnamic acid prepared in 40%
acetonitrile, 0.1% trifluoroacetic acid. MALDI mass spectra of peptide
mixtures were obtained using a Bruker Biflex mass spectrometer
(Bruker-Franzen Analytik, Bremen, Germany) equipped with a SCOUT
multiprobe inlet and a gridless delayed extraction source. Ion
acceleration voltage was 19.5 kV, and the reflectron voltage was 20.0 kV. For delayed ion extraction, a 6.2-kV potential difference between
the probe and the extraction lens was applied. Mass spectra were
acquired as the sum of ion signals generated by irradiation of the
target with 50-100 laser pulses; they were calibrated externally.
CKII Activity Assay--
Activity (substrate phosphorylation) of
CKII-
or CKII-
2
2 was measured in a total assay volume of 60 µl, consisting of 25 mM Tris-HCl, pH 7.5, 5 mM MgCl2. The reaction was initiated by the
addition of the substrates and 40 µM
[
-32P]ATP. The reaction was stopped by addition of 30 µl of SDS sample buffer. The phosphorylated protein was separated
from [32P]ATP by SDS-Tris/Tricine-PAGE (17). The amount
of 32P incorporated into protein was quantified with a
PhosphorImager (Molecular Dynamics).
Identification of the CKII Phosphorylation Sites on
S100B--
Purified intrasubunit disulfide-bonded S100B (100 µg) in
standard reaction buffer was phosphorylated by CKII-
(0.5 µg) in the presence of [32P]ATP. The protein was precipitated in
15% trichloroacetic acid, the pellet washed twice with ethanol and
acetone, and resuspended by sonication in 200 µl of 0.1 M
Tris-HCl, pH 8.5, 0.05% SDS, 10 mM DTT. The protein was
digested with endoproteinase Lys-C (sequencing grade from Sigma) at
35 °C for 15 h with a 1:100 (protease:S100B) weight ratio. The
digested sample was adjusted to 6 M guanidinium chloride
plus 0.1% trifluoroacetic acid, incubated with 20 mM iodoacetamide for 15 min, and applied to an Octadecyl (C18), 5 µM, 4.6 × 100 mM high pressure liquid
chromatography column. The peptides were separated with a gradient of
5-70% acetonitrile in 0.1% trifluoroacetic acid in 40 min at a flow
rate of 0.5 ml/min. Fractions (0.5 ml) were collected. The
radioactivity of each fraction was determined by Cerenkov counting. The
32P-labeled peptide eluted at 30 min and was subjected to
amino acid sequence analysis. Radioactivity of the eluted fractions from each sequencing cycle was determined by liquid scintillation counting.
Cell Culture--
Rat C6 glioma cells were routinely maintained
in DMEM (Life Technologies, Inc.) supplemented with 10% fetal bovine
serum (Seromed), 20 units of penicillin per ml, and 20 µg of
streptomycin per ml (Life Technologies, Inc.). For analysis of S100B
mitogenic activity, C6 cells were placed into 35-mm tissue culture
wells at a density of 106 cells per dish and allowed to
grow for 24 h. Cells were then washed with DMEM and incubated for
36 h in DMEM in the presence of O.O5% serum. Cell continued to
grow, and at the time of stimulation they had reached confluency.
Confluent cells were stimulated by changing the medium to DMEM
containing various concentrations of S100B. After 42 h,
[3H]thymidine was added to cells, and
[3H]thymidine incorporation was determined after 2 h. All experiments were done in duplicate and repeated at least two
times with different cell batches.
 |
RESULTS |
Oxidized S100B Dimers and Oligomers Are Phosphorylated by
CKII-
--
Fig. 1, A and
B, shows an SDS-PAGE analysis and Coomassie Blue staining of
two different preparations of bovine brain S100B protein stored at
20 °C for several months (lanes 1-3). Bovine brain
calmodulin was run on the same gel (lane 2). Under
nonreducing conditions (Fig. 1A), S100B migrates mainly as a
band with an apparent Mr of 6500 corresponding
to the S100B monomer. Additional minor bands are also visible at
apparent Mr 22,000 and 33,000 (lanes 1 and 3). These bands react with S100B antibodies by
Western blot analysis and correspond to disulfide-bonded S100B dimers and larger oligomers (data not shown). Reduction of the proteins with
50 mM DTT prior to electrophoresis results in a shift of the lower mobility bands to the Mr 6500 band
(Fig. 1B). In contrast, the electrophoretic mobility of
calmodulin, which has no cysteine residues, was not sensitive to
reducing agent (lane 2).

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Fig. 1.
The phosphorylation of disulfide-bonded S100B
dimer by CKII- . A and B, S100B (lanes
1 and 3) or calmodulin (lane 2) were
phosphorylated by CKII- . Phosphorylated proteins were separated on
SDS-PAGE in the absence (A) or in the presence of 50 mM DTT (B). Gels were Coomassie Blue-stained and
processed for autoradiography as indicated. C, the
phosphorylation of oxidized S100B dimer and monomer. Different S100B
preparations were phosphorylated by CKII- . Lane 1, S100B
preparation stored for several months at 20 °C. Lane 2, alkylated S100B with iodoacetamide on Cys-68 and Cys-84. Lanes
3 and 4, oxidized S100B species generated upon
incubation with 5 mM sodium tetrathionate for 1 h and
30 min, respectively, in the presence of 1 mM
Ca2+. Lanes 5 and 6, reduced form of
S100B incubated in the presence of 2 mM EGTA with 5 mM sodium tetrathionate for 1 h and 30 min, respectively. Proteins were first dialyzed against 25 mM
Tris-HCl, pH 7.5, prior to phosphorylation. Proteins were run on
SDS-PAGE in the absence of DTT. Gels were Coomassie Blue-stained and
processed for autoradiography as indicated. D, CKII-
phosphorylated oxidized S100- but not S100- subunit.
S100B( ) (lane 1), S100A1 ( ) (lane 2),
and S100a ( ) (lane 3) were first incubated in the presence of 1 mM Ca2+ with 5 mM
sodium tetrathionate for 1 h. The dialyzed proteins were then
phosphorylated by CKII- . Proteins were run on SDS-PAGE in the
absence of DTT. Gels were Coomassie Blue-stained and processed for
autoradiography as indicated. In the left margins are the positions of molecular weight standards.
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CaM is a recognized in vitro and in vivo
substrate for CKII-
subunit (21-22). Fig. 1, A and
B, also shows a comparison of the phosphorylation of CaM and
S100B preparations by CKII-
. The data show that disulfide-bonded
S100B dimer and oligomers are much better substrates for CKII-
than
CaM and that S100B monomer is only weakly phosphorylated (Fig.
1A). As shown below, the phosphorylated S100B monomer most
likely represents S100B with intrachain disulfide bond. As expected, in
the presence of DTT, the reduced phosphorylated S100B dimer and higher
oligomers migrated at the position of S100B monomer (Fig.
1B).
To confirm the specificity of phosphorylation of the disulfide-linked
form of S100B by CKII-
, we developed a method for large scale
preparation of covalent disulfide-bonded S100B dimers. This was
achieved by incubation of freshly purified S100B with the oxidizing
agent sodium tetrathionate in the presence of calcium (Fig. 1C,
lanes 3 and 4). No free SH groups remain titratable on
the covalent disulfide-bound S100B dimers after denaturation in 6 M guanidinium chloride, suggesting that both Cys-
68 and Cys-
84 are involved in the formation of the disulfide-linked form of
S100B (not shown, see also below). In the absence of calcium, sodium
tetrathionate could not cross-link S100B as a dimer (lanes 5 and 6), and the two cysteine residues remain titratable
after denaturation in 6 M guanidinium chloride (not shown).
As expected, covalent disulfide-bound S100B dimers become
phosphorylated by CKII-
(lanes 3 and 4),
whereas the reduced protein is not (lanes 5 and
6). It is noteworthy that incubation of S100B with sodium tetrathionate in the presence of calcium (lanes 3 and
4) also resulted in phosphorylation of a population of S100B
migrating as a monomer, suggesting that sodium tetrathionate might also stimulate formation of an intrachain disulfide bond between Cys-84 and
Cys-68. Alkylation of the S100B preparation on both Cys-68 and Cys-84
by iodoacetamide after protein denaturation in guanidinium chloride
also induced phosphorylation of the alkylated S100B monomer by CKII-
(Fig. 1C, lane 2), confirming that phosphorylation depends on the oxidation of the S100B sulfhydryl groups.
We also compared the effect of sodium tetrathionate on the
Ca2+-dependent covalent dimerization and
CKII-
phosphorylation of the homodimers S100B (
) and S100A1
(
) and of the heterodimer S100a (
) (Fig. 1D).
S100B and S100A1 have a common cysteine residue, Cys-
84 and
Cys-
85, which is specifically exposed to solvent in the presence of
Ca2+ (3). Only S100B has a second cysteine residue in
position
68. Sodium tetrathionate catalyzed disulfide-linked forms
of S100B (lane 1) but was much less efficient in catalyzing
disulfide-linked forms of S100A1 and S100a (
) (lanes 2 and 3), confirming that both Cys-
84 and Cys-
68 are
probably implicated in the formation of covalent disulfide-bound S100B
dimers. Furthermore, only oxidized S100B (lane 1), and to a
lower extent the heterodimer S100a (
) (lane 3), is
phosphorylated by CKII-
indicating that phosphorylation is specific
to the S100-
subunit.
Characterization of Oxidized S100B Monomer and Phosphorylation by
CKII
--
A significant amount of the neurite extension factor
initially purified from bovine brain was shown to migrate as a monomer on SDS-PAGE, suggesting that intrachain disulfide-bridged S100B monomer
could have biological activity (7). Intrasubunit disulfide-bridged monomeric S100B species with a disulfide bond between Cys-68 and Cys-84
could be obtained by incubating the protein under denaturating conditions and gel filtration to separate intrasubunit
disulfide-bridged monomeric species from intersubunit disulfide-bridged
polymeric species (16).
Intrachain disulfide bond between Cys-68 and Cys-84 in the oxidized
S100B monomer preparation was confirmed by MALDI mass spectroscopy
analysis of the protein after digestion with endo-Asp-N protease (Fig.
2). After SDS-PAGE, the reduced and
oxidized S100B were digested with endo-Asp-N protease, and the peptide
mass maps were compared. Under our experimental conditions, both Asp
and Glu residues are potential sites of cleavage by endo-Asp-N. Hence, the major mass peaks did not fit with the masses expected in case digestion would have been restricted to Asp residues. The mass spectroscopy spectrum obtained with the reduced S100B species revealed
a series of peaks, called A, with masses of 1553.77, 1571.69, 1587,63, and 1603.66 Da that is absent in the oxidized S100B spectrum. By
comparison with peptide masses expected after cleavage at Asp or Glu
residues, the 1571.69-Da peak fit with the expected mass of peptide
72EFMAFVAMITTACH85. Oxidation of one or two
methionine residues within this peptide explains mass peaks at 1587.63 and 1603.66 Da. The mass peak at 1553.77 correspond to a dehydrated
form of this peptide. In the mass spectrum of the reduced S100B, the A
series of peaks was immediately followed by the A' series. A' series
have masses that correspond to A series incremented by 71 Da,
corresponding to acrylamide adduct on Cys-84 generated during
electrophoresis. The mass spectroscopy spectrum obtained with the
oxidized S100B species revealed another series of peaks, called B, with
two major peptide species having a molecular mass of 2772.27 Da and
2788.47 Da, respectively. That B series of peaks is not present in the
reduced S100B spectrum. The mass difference between the B series of
peaks in oxidized S100B and the A series of peaks in reduced S100B fits
exactly with the mass of peptide
61DSDGDGECDFQ71, confirming the presence of an
intramolecular disulfide bridge between Cys-68 and Cys-84 in the
oxidized S100B. Note that the Asp-61-Gln-71 peptide could not be
detected in the reduced S100 peptide mass map, most probably because
its highly acidic properties that makes its desorption under a
protonated form very unlikely. Note also the absence of cleavage at
Asp-61, Asp-63, Glu-67, and Asp-69 in the oxidized S100B, suggesting
that disulfide bond stabilizes the peptide conformation, making those acidic residues not accessible to the protease.

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Fig. 2.
MALDI mass spectrum analysis of reduced and
oxidized S100B monomers. MALDI mass spectra of reduced
(S100B) and oxidized (Ox-S100B) S100B monomers
digested with endo-Asp-N protease (left panel). The A-A' and
B series of peaks are boxed. The peak with a mass of 1723.70 is derived from endo-Asp-N. In the right panels are
enlargements of the regions corresponding to the A-A' and B series of
peaks as indicated. See text for assignment of peaks.
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The intrasubunit disulfide-bridged monomeric S100B species was also
found to be an excellent substrate for CKII-
(Fig.
3, lane 1, and Fig.
4A, lanes 1 and 2).
Incubation of the oxidized S100B monomer with 10 mM DTT
prior to incubation with CKII-
results in a total inhibition of
phosphorylation (Fig. 3, lanes 2 and 3),
confirming that intracellular disulfide bond directly contributes to
S100B phosphorylation. To determine the exact contribution of each
individual Cys residue on the phosphorylation of S100B, we compared the
phosphorylation of S100B alkylated with the thiol reagent Bimane on
Cys-84 or on both Cys-84 and Cys-68. Specific alkylation of Cys-84 with
Bimane in the presence of calcium (3) did not generate phosphorylatable
S100B protein (Fig. 4A, lane 4). Alkylation of the S100B
monomer on both Cys-68 and Cys-84 with Bimane after protein
denaturation in guanidinium chloride was required for phosphorylation
by CKII-
(Fig. 4A, lane 3), suggesting that oxidation of
Cys-68 is essential for optimum phosphorylation of S100B.

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Fig. 3.
The phosphorylation of S100B monomer by
CKII- is dependent on the oxidation state of its cysteine
residues. Oxidized S100B monomer was incubated 30 min at 30 °C
in the absence (lane 1) or in the presence of 5 mM DTT (lane 2) or 10 mM DTT
(lane 3) prior to phosphorylation by CKII- . The
phosphorylated proteins were separated on SDS-PAGE in the absence of
DTT and the gel processed for autoradiography.
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Fig. 4.
Characterization of the phosphorylation of
oxidized S100B monomer by CKII- . A, different oxidized
S100B preparations were phosphorylated by CKII- . Lane 1, oxidized S100B with an intrachain disulfide bond between Cys-68 and
Cys-84. Lane 2, reduced S100B. Lane 3, alkylated
S100B on Cys-68 and Cys-84 with Bimane. Lane 4, alkylated
S100B on Cys-84 with Bimane. B, comparison of the
phosphorylation of oxidized S100B monomer by CKII- 2 2 and CKII- . 2 µM purified reduced S100B (lane
1), alkylated S100B with iodoacetamide (lane 2), or
oxidized S100B with intrachain disulfide bonds (lane 3) were
phosphorylated with 70 nM CKII- 2 2 or 70 nM CKII- for 10 min as indicated. C, the
effect of Ca2+ and Zn2+ on S100B
phosphorylation. S100B preparation stored for several months at
20 °C (lane 1) or oxidized S100B with an intrachain disulfide bond between Cys-68 and Cys-84 (lane 2) were
phosphorylated by CKII- in the absence (EGTA) or in the
presence of 1 mM Ca2+ or 5 µM
Zn2+ as indicated. A-C, the phosphorylated
proteins were separated on SDS-PAGE in the absence of DTT. Gel were
Coomassie Blue-stained (left panels) and processed for
autoradiography (right panels). In the left
margins are the positions of molecular weight standards.
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In vitro, calmodulin is not phosphorylated by the CKII
holoenzyme
2
2. The recombinant CKII-
subunit, however, spontaneously phosphorylates calmodulin (23). As
observed with calmodulin, the
subunit of CKII was more efficient
than the holoenzyme (
2
2) in
phosphorylating alkylated S100B or disulfide-bridged monomeric S100B
(Fig. 4B, lanes 2 and 3) or disulfide-bridged
S100B dimer (not shown). The phosphorylation of monomeric and
oligomeric S100B by CKII-
was proven to rely exclusively on changes
induced upon oxidation of Cys residues. Neither calcium nor zinc ions,
two conformational effectors of the S100B (1, 2), could modulate phosphorylation of the protein (Fig. 4C).
Characterization of the CKII-
Phosphorylation of Oxidized
S100B--
The phosphorylation of oxidized monomeric and dimeric S100B
by purified CKII-
is a very rapid reaction. With 0.5 µM S100B and 80 nM CKII-
in the assay, the
phosphorylation is maximal within 1-2 min (Fig.
5A). With the S100B
preparation used in this experiment, the phosphorylation stoichiometry
of oxidized monomeric S100B was calculated to be 0.7 mol of phosphate
incorporated per mol of S100B monomer (Fig. 5A). We
confirmed these kinetic and stoichiometry procedures with two different
oxidized S100B monomer preparations and two different CKII-
preparations. The observed sub-stoichiometric phosphorylation is
probably due to overestimation of the oxidized S100B concentration that
has been determined using the Bio-Rad protein assay reagent using
purified reduced S100B as standard. To our knowledge the oxidized S100B
is the best in vitro substrate for CKII-
so far
identified with a Km of 0.5 µM and a
Vmax of 10 pmol/min/100 pmol of S100B (Fig. 5, B and C). As expected, in a competition assay,
oxidized monomeric S100B totally abolished CaM phosphorylation by
CKII-
at a S100B/CaM molar ratio of 1:10 (Fig. 5D). Note
that in this experiment 2 mM EGTA was included in the
phosphorylation buffer. Indeed, it has been shown that Ca2+
may inhibit phosphorylation of CaM in vitro (24).

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Fig. 5.
CKII- phosphorylation parameters of
oxidized S100B monomer. S100B with intrachain disulfide bond was
phosphorylated by CKII- . A, time course and stoichiometry
of S100B phosphorylation. S100B and CKII- concentrations were 0.5 µM and 80 nM, respectively. B and
C, S100B at the concentration indicated was phosphorylated with CKII- (7 nM). The reactions were terminated after 1 min. Each point represents the mean of two determinations. The plot in
B demonstrates a Vmax of 10 pmol/min/100 pmol of S100b. The double-reciprocal plot of the
phosphorylation in C demonstrates a Km of
0.5 µM. D, oxidized S100B monomer inhibits
calmodulin (CaM) phosphorylation by CKII- . CaM
(lanes 1-3) at the concentration of 10 µM was
mixed with 1 µM (lane 2) or 4 µM
(lane 3) oxidized S100B monomer in buffer containing 2 mM EGTA. The proteins were phosphorylated with CKII- (50 nM) for 1 min. Proteins were analyzed by SDS-PAGE,
Coomassie Blue staining (left panel), and autoradiography (right panel).
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To determine the CKII-
-phosphorylated residue on the monomeric and
dimeric S100B, the 32P-labeled proteins were digested by
trypsin and the resulting phosphopeptides analyzed by SDS-PAGE (Fig.
6A). In the absence of
reducing agent, the phosphopeptides obtained from digestion of the
monomeric and dimeric 32P-S100B migrate with apparent mass
of 3.4 and 6.5 kDa, respectively. In the presence of DTT, the
phosphopeptides co-migrate with the same apparent mass of 3.5 kDa. The
phosphopeptides were purified by reverse phase-high pressure liquid
chromatography as described under "Experimental Procedures" and
sequenced. The released radioactivity coincided with the identification
of Ser-62, indicating that this residue is the phosphorylated amino
acid (Fig. 6B). Ser-62 is located within the calcium binding
loop of site II
and corresponds to a canonical CKII phosphorylation
site domain (Fig. 6C) (25).

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Fig. 6.
Determination of the CKII- phosphorylation
site on oxidized S100B. A, SDS-PAGE analysis of
phosphorylated S100B with intrachain disulfide bond (lane 1)
and S100B dimer with interchain disulfide bond (lane 2). The
phosphorylated proteins were digested with trypsin, and the
phosphopeptides were analyzed in the absence or in the presence of DTT
as indicated. Lane 3, phosphopeptide derived from S100B with
intrachain disulfide bond. Lane 4, phosphopeptide derived
from S100B dimer with interchain disulfide bond. In the left
margins are position of molecular mass standards. B,
radiosequencing of the tryptic phosphopeptide derived from S100B with
intrachain disulfide bond. The sequence of the first 10 amino acids of
the S100B peptide and the corresponding release of radioactivity are shown. C, the amino acid sequence of the C-terminal region
of S100B. The C-terminal EF-hand calcium binding site II is
indicated. The amino acids that coordinate Ca2+ are shown
by asterisks. The disulfide bridge between Cys-68 and Cys-84
in the intrachain disulfide bounded S100B monomer is shown as well as
phosphorylated Ser-62 by CKII- .
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Peroxynitrite Anion Stimulates S100B Oxidation and
Phosphorylation--
We next investigated if a more physiological
oxidant than sodium tetrathionate could catalyze the formation of
disulfide bridges and CKII-
phosphorylation of S100B. NO as a second
messenger reacts readily with other free radicals such as superoxide
anion (O
2) to form peroxynitrite (ONOO
). NO and
products of NO oxidation are capable of reaction with thiols to give
further products with biological activities (26, 27). We tested the
effect of various NO donors (DEA, SIN-1), O
2 precursor
(pyrogalol), and peroxynitrite anion (OONO
) on the
oxidation of S100B. S100B oxidation was analyzed by phosphorylation of
the S100B species with CKII-
(Fig.
7A). The most potent oxidant we found is peroxynitrite anion (OONO
) (lane
3). In contrast to sodium tetrathionate which mostly oxidized S100B as dimer, OONO
stimulated both formation of
intrachain and interchain disulfide bonds in S100B. If
OONO
is first decomposed into NO2 and
·OH, formation of the intrachain disulfide bond within S100B
monomer was inhibited (lane 4). Note that S100B oxidation
into covalent disulfide bound dimer still occurred after
OONO
decomposition with an intensity comparable to that
of nitric oxide (NO) precursors (DEA; SIN-1) confirming the specificity of OONO
in generating intrachain disulfide bond within
S100-
subunit. A dose-dependent experiment showed that
ONOO
at a micromolar concentration was able to mediate
S100B phosphorylation (Fig. 7B). This observation, together
with the fact that in our experimental condition ONOO
has
a very short half-life in the second range (28), supports a
physiological relevance of ONOO
-mediated S100B oxidation.
Incubation of the oxidized S100B protein samples with 10 mM
DTT prior to incubation with CKII-
resulted in a total inhibition of
phosphorylation, confirming that phosphorylation is dependent on
OONO
-mediated disulfide bond formation (not shown).

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Fig. 7.
S100B monomer oxidation by peroxynitrite.
A, 10 µM S100B in 50 µl of 20 mM
Tris-Cl, pH 7.5, 0.5 mM CaCl2 was incubated 30 min with 0.1 mM each of DEANO (lane 1), SIN-1
(lane 2), peroxynitrite (lane 3), decomposed
peroxynitrite (lane 4), pyrogalol (lane 5), and a
mixture of pyrogalol and DEANO (lane 6). The protein was then phosphorylated by CKII- . The phosphorylated proteins were separated on SDS-PAGE in the absence of DTT, and the gel was proceeded for autoradiography. , oxidized S100B monomer; 2,
disulfide-bonded S100B dimer. B, 10 µM S100B
was incubated for 30 min with increasing concentrations of
peroxynitrite. The proteins were phosphorylated by CKII- and
separated on SDS-PAGE in the absence of DTT and the gel proceeded for
autoradiography.
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Oxidized S100B Monomer Stimulates [3H]Thymidine
Uptake by Rat Glial C6 Cells--
It has been shown that neurotrophic
S100B stimulates [3H]thymidine uptake by rat C6 glial
cell (8) and also stimulates calcium fluxes and nitric oxide synthase
activity in astrocytes (13, 14). All these activities have been
reported to be dependent on the presence of Cys-68 or Cys-84 and on the
oxidation state of these residues. However, considerable variability in
specific activity between S100B preparations has been reported (8,
13-14) that could rely on the heterogeneity of the S100B preparation. Here we compared the effect of the oxidized monomeric and dimeric S100B
species and of the reduced S100B on [3H]thymidine uptake
by confluent rat C6 glial cells in serum-free medium (Fig.
8A). As described previously
by Selinfreund et al. (8), the reduced S100B preparation had
no effect on [3H]thymidine uptake by rat C6 cells. The
oxidized S100B dimer stimulated [3H]thymidine
incorporation to a low extent, whereas the monomeric oxidized S100B
species was the most potent S100B species in stimulating [3H]thymidine uptake by glial C6 cells. Half-maximal
stimulation was obtained with 0.1-0.2 nM S100B.
Phosphorylation by CKII-
had no effect on stimulation of
[3H]thymidine uptake by the monomeric oxidized S100B
species (Fig. 8B).

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Fig. 8.
Oxidized S100B monomer stimulates glial C6
cell proliferation. A, subconfluent rat C6 glioma cells were
kept in DMEM containing low serum. After 48 h cells reached
confluence and were stimulated by changing media to DMEM containing
0.05% fetal bovine serum alone (control) or plus various
concentrations of reduced S100B preparations ( ), intrachain
disulfide-bonded S100B monomer ( ), or interchain disulfide-bonded
S100B dimer ( ). After 24 h, [3H]thymidine was
added, and cells were solubilized 2 h later, and incorporated
counts/min were determined. B, effect of CKII-
phosphorylation on S100B mitogenic activity. Subconfluent C6 grown in
low serum were stimulated or not stimulated with reduced S100B
(control, C) or stimulated with 1 nM
dephosphorylated intrachain disulfide-bonded S100B monomer
(S-S) or 1 nM phosphorylated intrachain
disulfide-bonded S100B monomer (S-S-P). After 24 h,
[3H]thymidine was added, and cells were solubilized
2 h later, and incorporated counts/min were determined.
A and B, data represent the mean of two
experiments in duplicate.
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DISCUSSION |
Intracellular S100B exists mainly as a noncovalent protein
homodimer with free reduced sulfhydryl groups. However, oxidized S100B
with a neurite extension activity has been purified from bovine brain
(7). This oxidized S100B migrates with molecular weight of 6500, 21,000, 30,000, and 40,000 in nonreducing conditions. The neurite
extension activity of the oxidized S100B preparation is dependent on
the absence of reducing agent in the preparation, suggesting that
neurite extension activity is associated with an oxidized form of
S100B. Subsequently it was shown that oxidized S100B is also a glial
mitogen (8) and that S100B stimulates both calcium flux in glial C6
cells (13) and nitric oxide synthase activity in rat cortical
astrocytes with accumulation of the NO metabolite in the conditioned
medium (14). Transgenic mice expressing elevated levels of S100B also
show abnormal astrocytosis and neurite proliferation consistent with
alterations observed in vitro on cell culture (29). It is
commonly accepted that the neurotrophic and mitogenic activities of
S100B depend on a disulfide-linked dimeric form of the protein (for
review see Ref. 9). However, we demonstrate here that the monomeric
S100B species with intrachain disulfide bond also has a mitogenic
activity that is even stronger than a preparation containing
essentially interchain disulfide-bonded S100B (Fig. 8).
The human S100B gene is found on a region of chromosome 21 that is
triplicated in individuals with Down's syndrome (4), and it has been
suggested that the dosage imbalance of this gene is a major contributor
to the abnormalities of brain development and function that occur
invariably in Down's syndrome individuals and Alzheimer brains (5). In
Alzheimer brains a correlation exists between S100B protein synthesis
and neurotrophic activity (6). The S100B protein in Alzheimer brain
migrates essentially as a monomer on SDS-PAGE, strongly supporting the
notion that neurotrophic active S100B species could be oxidized S100B
monomer (6). Because only oxidized S100B is capable of mitogenic and neurotrophic activities (7-8), one has to envision that not only overproduction but also alteration of the redox status of the cysteine
residues in S100B are implicated in pathogenicity. We have shown that
the formation of oxidized monomeric S100B can be selectively catalyzed
by peroxynitrite anion (ONOO
). ONOO
production could be enhanced by pathological processes such as hypoxia,
neurodegenerative disorders, and aging when O
2 is generated in
excess, and abnormal ONOO
production underlies the
pathogenesis of familial Alzheimer's (30). We would like to suggest
that ONOO
could be implicated in S100B oxidation in brain
pathologies such as Alzheimer's disease and Down's syndrome. The
presence of S100B in cerebrospinal fluid of patient with Down's
syndrome and other neurological disorders (31) could reflect enhanced
secretion of the oxidized protein, and oxidized extracellular S100B
could directly participate in development of the pathologies (6). Finally, because extracellular S100B stimulates NO synthase activity and mRNA levels in rat astrocytes (14), and that potentially toxic
levels of ONOO
can be achieved by nitric oxide synthase
(26, 31), it is possible that extracellular S100B could stimulate its
own oxidation within the glial cells in a positive feedback loop.
The second major observation reported in this study is the specific
phosphorylation of the oxidized S100B conformation by CKII-
on
Ser-62 located within the typical Ca2+ binding loop of site
II-
. It is likely that the phosphorylation of Ser-62 upon cysteine
oxidation results from conformational changes that are induced by the
oxidation of S100B (3, 16). These conformational changes may lead to
the exposure of the Ca2+-binding site II-
to solvent and
favor the phosphorylation. This hypothesis is consistent with our
previous data showing that alkylation of Cys-84 induced changes in the
protein structure that resulted in a drastic increase of the affinity
of site II-
for Ca2+ (3). Whether or not phosphorylation
on Ser-62, which is not directly involved in Ca2+
coordination, could regulate site II-
affinity for calcium remains to be investigated.
Casein kinase II is a second messenger-independent, spontaneously
active protein kinase that consists of a heterotetramer containing two
catalytic (CKII-
) and two regulatory (CKII-
) subunits (25). There
is, however, evidence that CKII-
might sometimes function
independently of CKII-
. Phosphorylation of certain physiological
substrates such as calmodulin and ornithine decarboxylase is in fact
inhibited by CKII-
(21). Several cell types seem to contain a
separate pool of CKII-
in addition to the holoenzyme (32-33). It is
hardly conceivable that a pleiotropic protein kinase such as CKII,
involved in a variety of cellular functions, be devoid of any kind of
regulatory device. Binding of CKII-
markedly enhances CKII-
activity toward most substrates (15). We show here for the first time
that the conformation of the protein substrate rather than kinase
activation is the limiting step for the phosphorylation reaction, thus
revealing another putative regulatory mode of CKII-
activity. The
reduced state of the S100B protein is detrimental to the
phosphorylation reaction, but oxidized S100B is, to our knowledge, the
best in vitro CKII-
substrate so far identified. It
remains essential to establish whether or not oxidized S100B is also an
in vivo substrate for CKII-
and the meaning of that mode
of phosphorylation. Several attempts to demonstrate an in
vivo or in vitro phosphorylation of S100B in glial C6
were unsuccessful. Glial C6 cells express an endogenous S100B that
migrates as a monomer when analyzed by Western blot or
immunoprecipitation after metabolic labeling with [35S]methionine. We have not yet been able to observe an
in vivo phosphorylation of S100B in glial cells after
metabolic labeling of cells with 32P, and we have not been
able to detect any in vitro phosphorylation of
immunoprecipitated S100B with purified CKII-
. It could be that in
glial C6 cells, intracellular soluble S100B exists mainly in a reduced
and nonphosphorylatable state and that alteration of the redox status
of the cysteine residues in S100B is tightly regulated and may concern
only a minor S100B population. As stated above, oxidized S100B might be
produced only under certain circumstances, particularly in pathologies
when S100B is overproduced and when the redox status and calcium
homeostasis of the cells are perturbed, a situation that could take
place in neurodegenerative disorders (31). Because the extracellular
functions of the oxidized S100B species depend on its secretion, it
could also be that CKII-
phosphorylation of oxidized S100B is
implicated in the processes of S100B secretion, thus complicating
further in vivo phosphorylation studies on S100B. A role of
CKII in the regulation of intracellular trafficking and sorting
mechanisms of protein substrate has recently been reported (34). We
believe that the role of CKII-
phosphorylation on Ser-62 of S100B
in vivo should be more easily resolved by mutagenesis studies. These studies are under way.
Finally, the specificity of CKII-
phosphorylation of oxidized S100B
opens up new strategies for characterizing the mechanisms by which
extracellular oxidized S100B functions. The in vitro phosphorylation of monomeric S100B by CKII-
has no effect on the
S100B mitogenic activity on C6 glial cells; thus
32P-labeled S100B might serve as a useful tool to identify
putative S100B receptors or target proteins. Phosphorylation of S100B
by CKII-
might also be valuable to investigate the oxidation state of the S100B protein in brain pathologies where alteration of the redox
status of S100B is thought to be linked with alteration of S100B
functions (6).
We thank Dr. A. M. Chinn for critical
reading of the manuscript.