From the
Short amino acid sequences that interact with the Ca
Calcium functions as an important regulator of numerous
intracellular processes (Kemp et al., 1987; Klee and Vanaman,
1982; Dedman, 1984; Klee, 1988; Means et al., 1991). Commonly,
this regulatory role is mediated via the interaction of calcium ions
with a variety of Ca
S-100 proteins
are a family of acidic, dimeric, Ca
S-100 proteins are expressed in a cell- and tissue-specific
fashion, which may provide clues to the functional specificity of
different S-100 family members. Classical S-100 proteins were isolated
initially from bovine brain and were considered to be unique to nervous
tissues (Moore, 1965), although subsequent studies have proven this
concept to be incorrect (see review, Van Eldik and Zimmer(1988)). The
richest source of S-100b is the brain, where expression is almost
exclusively restricted to astrocytes (Dyck et al., 1993). On
the other hand, S-100a
S-100 proteins are thought to
exert their effect in biological systems through
Ca
Recognition at the molecular level depends upon the ability of two
or more structural motifs to interact in a specific fashion. Biological
systems have developed numerous mechanisms that allow for these types
of interactions to occur. In particular, many high affinity
intracellular targeting mechanisms are based upon the recognition of
short peptide sequences, including the chelation of calcium by
Ca
Our first experiments identified bacteriophage that bound in
a Ca
Four isolates (1, 13,
14, and 32) were obtained multiple times, suggesting they may represent
preferential S-100b binding peptides. The most frequently obtained
bacteriophage isolates (1 and 13) were used to test the specificity of
peptide interaction for S-100b. Both of these bacteriophage isolates
bound to S-100b-Sepharose 4B and neither bound to CaM-Sepharose.
Similarly, bacteriophage expressing known CaM binding peptides bound
CaM-Sepharose but not S-100b-Sepharose, demonstrating, at least
initially, the specificity of our peptides for S-100b. We fully
expected this result because, as noted above, the S-100b binding
peptides we have obtained are structurally distinct from those Jamieson
and co-workers previously identified as CaM binding peptides (Dedman et al., 1993).
A review of our data determined that the most
highly enriched bacteriophage, i.e. those that had survived
five rounds of selection, exhibited highest homology to a COOH-terminal
domain in the
S-100 proteins are a family of small Ca
Screening of a bacteriophage random peptide library for particles
that bound to a S-100b affinity column in a
Ca
These data clearly show that the binding
of S-100b to CapZ is very specific as well as Ca
Additional support for co-expression of these
proteins within the same cell comes from skeletal and heart muscle,
where the S-100a
As an
ACP, CapZ appears to be a primary regulator of microfilament
polymerization. The specific interaction of S-100b and CapZ suggests
S-100b may participate in regulating microfilament organization in a
Ca
Despite these arguments, we have some
concern about the relatively high concentration of Ca
Our results identify CapZ as a potential
target for S-100b binding in vivo and designate a specific
amino acid sequence within the COOH terminus of CapZ as the probable
site of S-100b binding. These results suggest that S-100b may play a
role in the regulation of actin nucleation and microfilament elongation
at sites of local high intracellular Ca
Conventional single letter amino acid code was used. The
bacteriophage library contains a random sequence of 15 amino acids,
which is flanked by [AE] at the amino terminus, and six
consecutive proline residues ([P
Inserts in bacteriophages obtained after
5 rounds of selection and the COOH-terminal sequences of
We express our appreciation to Dr. W. David Behnke for
expert advice and assistance in performing the fluorescence
spectrophotometric analysis of interaction of TRTK-12 with S-100b and
to Dr. John R. Dedman for initial advise and continued encouragement.
We are extremely appreciative of the assistance provided by Dr. John A.
Cooper for providing reagents and sequence information for several ACPs
and for critical reading of the manuscript. We also thank Hetal Bhatt
for technical assistance in the bacteriophage selection procedures and
initial assistance during the sequencing of bacteriophage inserts and
Elena Y. Kupert for technical assistance in the processing of gel
overlays.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
binding protein S-100b were identified by screening a
bacteriophage random peptide display library. S-100b binding
bacteriophages were selected by Ca
-dependent affinity
chromatography, and the sequence of the random peptide insert contained
in 51 clones was determined. Alignment of the sequence of 44 unique
S-100b binding peptides identified a common motif of eight amino acids.
A subgroup of peptides that contained sequences with the highest degree
of similarity had the consensus motif
(K/R)(L/I)XWXXIL, in which predominantly P, S, and N
were found in position 3, and S and D were found in position 5.
Analysis of sequence databanks identified a similar sequence in the
COOH-terminal region of the
-subunit of actin capping proteins.
The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of
greatest homology within this region of the subunit of actin capping
proteins (e.g. amino acids 265-276 in CapZ
1 and
CapZ
2), was synthesized and shown by fluorescence
spectrophotometry to bind S-100b in a Ca
-dependent
manner. Gel overlay and cross-linking experiments demonstrated the
interaction of S-100b with CapZ to be Ca
dependent.
Moreover, this interaction was blocked by addition of TRTK-12 peptide.
These results identify Ca
-dependent S-100b target
sequence epitopes and designate the carboxyl terminus of the
-subunit of actin capping proteins, like CapZ, to be a target of
S-100b activity. The high level of conservation within this region of
actin capping proteins and the apparent high affinity of this
interaction strongly suggest that the interaction between S-100b and
the
-subunit of actin capping proteins is biologically
significant.
binding proteins. Occupation of
the Ca
binding sites of these proteins induces a
conformational change that promotes the interaction of Ca
sensor proteins with specific intracellular target proteins
(LaPorte et al., 1980; Klevit et al., 1985). While
some Ca
mediators, for example calmodulin
(CaM),
(
)are expressed ubiquitously in eukaryotic
cells, most Ca
sensors are expressed in a limited
number of tissues and/or cell types. The S-100 proteins represent one
such group of Ca
sensors, which are expressed in a
cell type-specific fashion (Hilt and Kligman, 1991).
binding proteins
that range in molecular mass from 10 to 12 kDa (for reviews, see
Donato(1986, 1991); Kligman and Hilt(1988); Persechini et
al.(1989); Hilt and Kligman(1991)). Classical S-100 proteins
contain two subunits (
and
) combined in homodimers or
heterodimers (S-100a
(
), S-100a (
), and
S-100b (
)) and share extensive primary sequence homology that
extends beyond the two EF-hand Ca
binding domains
contained in each subunit (Isobe et al., 1981, 1983; Masure et al., 1984). The two Ca
binding domains
differ significantly in their affinity for Ca
,
10-50 µM for the COOH-terminal site and
200-500 µM for the amino-terminal site (Baudier and
Cole, 1989). The high degree of conservation of primary amino acid
sequences in both the Ca
and non-Ca
binding domains and the extensive conservation of hydrophobicity
and hydrophilicity within S-100 proteins suggest that S-100 proteins
regulate an important set of cellular processes (Kligman and Hilt,
1988).
is expressed most abundantly in
heart (Kato and Kimura, 1985) and is localized specifically to cardiac
myocytes (Haimoto and Kato, 1988).
-regulated interactions with specific intracellular
target proteins. Both extracellular as well as intracellular functions
have been ascribed to S-100 (for reviews, see Donato(1986, 1991);
Kligman and Hilt(1988); Van Eldik and Zimmer (1988); Hilt and
Kligman(1991)). Relative to extracellular function, S-100b is secreted
from cells within the brain as well as from rat C6 glioma cells, and
extracellular S-100b has been demonstrated to promote neurite
outgrowth, induce prolactin secretion, and stimulate proliferation of
glia in the central nervous system. Intracellular functions for S-100
include alteration of enzyme activity, regulation of cell cycle events,
alteration of the phosphorylation of specific substrates, and
regulation of the polymerization state of a variety of cytoskeletal
elements. Many specific cellular targets for S-100 have been described,
including a number which suggest S-100 interacts with cytoskeletal
components. These putative targets are myosin (Burgess et al.,
1984), junctional membrane proteins (Van Eldik et al., 1985),
tubulin (Donato, 1984, 1987; Donato et al., 1989),
microtubule-associated
proteins (Baudier and Cole, 1989),
caldesmon (Skripnikova and Gusev, 1989), and glial fibrillary acidic
protein (Bianchi et al., 1993). Although these reports enhance
our knowledge of S-100 proteins in general, they provide only limited
information relevant to understanding S-100 function(s) at the
molecular level. In particular, despite decades of investigation, a
clear understanding of the structural determinants underlying the
interaction of S-100 proteins with their targets remains unclear.
binding proteins (CaM, S-100) as well as their
subsequent interaction with specific intracellular targets (Baudier and
Gerard, 1983, 1986; Moore, 1988). In addition to being both time and
labor intensive, deciphering the molecular code, which enables these
specific interactions to occur, can prove experimentally difficult. For
example, subsequent to the proteolytic fragmentation of various S-100
target proteins, isolation and characterization of S-100 binding
peptides from these proteolytic soups might permit the identification
of consensus S-100 binding epitopes within different S-100 targets,
similar to the analysis of CaM target binding sequences that has been
performed over the last few decades (Charbonneau et al., 1991;
Blumenthal et al., 1988; Roth et al., 1991). As an
alternative to this arduous process, over the past 3 years using a
bacteriophage random sequence library (Devlin et al., 1990),
Jamieson and co-workers
(
)have developed a
procedure that permits the facile selection and identification of
peptides that bind specifically to particular biological targets
(Dedman et al., 1993). This procedure provides a rapid and
direct approach to the identification of selective, high affinity
peptide ligands for designated proteins, which can facilitate
investigation of their mechanism of action at both the cellular and
molecular levels. In the current study, we have successfully employed
this approach to identify S-100b target epitopes.
Materials
M13 bacteriophage random
peptide library (Devlin et al., 1990) was obtained from Chiron
Corp. Bovine brain S-100a, S-100b, and 3`:5`-cyclic
nucleotide phosphodiesterase (cNMP) as well as rabbit muscle aldolase
and glycogen synthase were from Sigma. Molecular weight standards for
SDS-PAGE were from Bio-Rad, and 10-kDa protein ladder was from Life
Technologies, Inc. Bovine brain CaM was prepared as described (Jamieson
and Vanaman, 1979).
protein was purified from frozen calf brain
(Pel-Freez) as described (Lindwall and Cole, 1984). The actin capping
protein, CapZ, from chicken skeletal muscle (subunits
and
),
glutathione S-transferase CapZ
and
fusion
proteins, affinity-purified goat anti-CapZ antibody, and yeast actin
capping protein were generously provided by Dr. John Cooper (Washington
University, St. Louis). Cap Z and its fusion proteins were purified as
described (Hug et al., 1992). Rabbit anti-bovine brain S-100
was from Sigma. Alkaline phosphatase-labeled goat anti-rabbit and
rabbit anti-goat immunoglobulins were from Southern Biotechnology
Associates. In some studies, an unrelated peptide APAT-14
(APATPWPLSSFVPS) was used. TRTK-12 and APAT-14 were custom synthesized
by Chiron Mimotopes. Rat C6 glioma cells were obtained from the
American Type Culture collection and grown in
-minimal essential
medium (Life Technologies, Inc.) supplemented with 2.5% (v/v) fetal
bovine serum (Hyclone), 50 units/ml penicillin, and 50 µg/ml
streptomycin (Life Technologies, Inc.).
Selection of the S-100b Binding Phages by Affinity
Chromatography
S-100b was coupled to CNBr-activated
Sepharose 4B (1-2 mg/ml gel volume), according to the
manufacturer's instructions. An aliquot of random peptide library
(5 10
plaque-forming units) was diluted in buffer
A (50 mM Tris, 150 mM NaCl, pH 7.5) containing 1
mM CaCl
and 1 mg/ml bovine serum albumin, combined
with S-100b Sepharose (1 ml), and mixed by inversion at room
temperature for 2 h. The mixture was poured into a mini-column
(Spectrum) and washed exhaustively, and the bacteriophage that bound in
a Ca
-dependent manner was eluted with buffer
containing EGTA essentially as described (Dedman et al.,
1993). Fractions eluted with EGTA-containing buffer were neutralized
immediately by addition of CaCl
to 1 mM free
Ca
. The column was washed with glycine buffer (125
mM glycine, pH 2.2), re-equilibrated with buffer A containing
0.1 mM CaCl
and 0.05% sodium azide, and reused
several times. The number of plaque-forming units contained in
fractions was determined by standard microbiological titering
procedures using XL1-Blue Escherichia coli (Stratagene) as
host. Bacteriophage contained in selected fractions were amplified on
solid media at a density not exceeding 400 plaque-forming
units/cm
, harvested, and subjected to two to four
additional rounds of selection. The sequence of the peptide inserts
contained within individual bacteriophage isolates was determined by
dideoxy termination DNA sequencing (Sequenase Version 2.0, U. S.
Biochemical Corp.).
Gel Electrophoresis and
Immunoblotting
12% SDS-PAGE was performed using minislab
gel apparatus (Life Technologies, Inc.) according to Laemmli(1970). The
gels were stained with Coomassie Blue or used for immunoblotting
(Towbin et al., 1979) or gel overlay analysis. Immunoblots
were processed using a Bio-Rad ImmunoBlot assay kit.
Gel Overlay Analysis
S-100b and CaM were
radioiodinated by lactoperoxidase method using Enzymobeads (Bio-Rad).
Reactions were performed with 100 µg of protein and 1 mCi of
NaI (DuPont NEN) for 30 min. In some experiments, S-100b
was iodinated using the Bolton-Hunter reagent as described (Chafouleas et al., 1979). Radiolabeled proteins were separated from free
iodine by gel filtration on Sephadex G-15 column, aliquoted, and stored
at -80 °C until use. Specific activity of S-100b and CaM was
approximately 1 and 20 Ci/mmol, respectively. Gel overlay analyses were
performed essentially as described (Burgess et al., 1984;
Hertzberg and Van Eldik, 1987). After staining, destaining, and drying,
the radiolabeled S-100b and CaM specifically bound by gel
matrix-immobilized proteins was detected by exposing gels to Kodak
X-Omat film using an intensifying screen at -80 °C.
Slot Blot Analysis
Analyses were
essentially as described for gel overlays, except CapZ (1 µg) was
applied to nitrocellulose using a slot blot apparatus and blocked (2%
bovine serum albumin), and the blot subdivided into pieces containing
one slot each. Pieces were incubated in wells of a 12-well plate with I-S-100b (2-4
10
cpm/ml) and
increasing concentrations of cold S-100b (0.001-1 µm).
Cross-linking Experiments
Proteins were
dialyzed against buffer (50 mM HEPES, 150 mM NaCl, pH
7.5) and preincubated for 20 min at room temperature (final volume of
24 µl) with additives as indicated. Reactions were initiated by
addition of 3 µl of freshly prepared stock solutions of
bifunctional cross-linking reagent bis(sulfosuccinimidyl)
suberate (Pierce) in the buffer. Reactions were terminated by addition
of 3 µl of 100 mM 2-aminoethanol/HCl, and samples were
processed for SDS-PAGE. Gels were stained with Coomassie Blue or
electroblotted onto nitrocellulose and analyzed immunochemically with
anti-S-100 or anti-CapZ antibodies. Protein concentration was
determined by the method of Bradford(1976) or using a BCA kit (Pierce).
-dependent manner to S-100b (Fig. 1). The
sequence of the random peptide insert of 51 individual bacteriophage
contained within the final EGTA eluate pool was determined by dideoxy
sequencing. Sequence analysis of 51 independent clones identified 44
unique peptide inserts (). Analysis of these inserts
revealed several important features of the S-100b binding peptides
isolated by this technique. First, although about 45% of the
bacteriophage contained within the initial library aliquot had no
insert sequence, all of the phage analyzed contained a 15-amino acid
insert, indicating that our selection procedure is highly specific for
insert-containing phage. Secondly, of the 44 unique inserts obtained,
none of the peptides contained cysteine (C), while glycine (G) was
found at a frequency of 1.3%. Conversely, polar amino acids serine (S)
and threonine (T) were over-represented (total 19.2.%). In addition,
positively charged amino acids (lysine (K) and arginine (R)) were
present twice as frequently (11.9%) as negatively charged residues
(aspartate (D) and glutamate (E), 5.1%).
Figure 1:
Selection of S-100b binding
bacteriophage by Ca2+-dependent affinity chromatography.
Chromatography was as described. Bacteriophage expressing sequences,
which bound S-100b-Sepharose in a Ca-dependent
manner, were eluted by EGTA containing buffer (see arrow).
S-100b binding peptides
were aligned by a combination of computer-assisted analysis (MACAW)
(Schuler et al., 1991) together with visual inspection. This
sequence alignment is presented in . 35 unique sequences,
representing 80% of all inserts analyzed, contained a common motif of
eight amino acids. This length is significantly shorter than that
required for interaction of CaM binding peptides with CaM (Dedman et al., 1993). 31 inserts contained a positively charged
residue in position one. A charged or aminated residue occupied
position minus one or minus two in 4 additional isolates and 18
isolates overall. The second, fourth, seventh and eighth positions were
occupied by a hydrophobic residue, while the fifth position was
commonly occupied by a hydrophilic amino acid. Furthermore, aligned
sequences could be arranged within several subgroups, which possessed a
higher degree of similarity. The tryptophan-proline-serine (PWS) motif
in positions three to five and a proline (P) in position one were
characteristic of group I and is distinct from the WP motif found in a
majority of CaM binding peptides (Dedman et al., 1993).
Tryptophan (W) in the fourth position and proline in position three
were specific for groups II and III, respectively. Group IV represents
more heterogenous sequences. Group V contains isolates that could not
be readily placed within any particular group.
Identification of Novel S-100b Target
Proteins
In addition to aligning sequences of individual
S-100b binding peptides with each other, we investigated whether
naturally occurring proteins possessed sequences similar to our
consensus motif of S-100b binding peptides. We compared the binding
peptide isolates () with structures residing in GenBank
using the BLAST server at National Center for Biotechnology Information
(Altschul et al., 1990). While no proteins demonstrated
complete identity with our S-100b binding peptide isolates, many
proteins possessed five to six residues identical or homologous to
individual peptide isolates. To reduce the complexity of our analysis
for potential S-100b targets, several additional criteria were applied:
(i) conservation or conservative replacement of the most highly
conserved portions of the consensus sequence, (ii) presence of this
motif in homologous proteins isolated from different species, and (iii)
localization of the homologous sequence at either the NH or
COOH terminus of the putative S-100b binding proteins, positions that
are more accessible in many proteins for interaction with other
macromolecules. This approach, together with a further decision to
restrict, at least initially, our focus to identification of peptides
contained within molecules known to participate in regulating
cytoskeletal interactions, allowed us to identify a novel S-100b target
protein, the
-subunit of the actin capping protein (ACP), CapZ
(see ).
-subunit of ACPs. This sequence alignment is
presented in . A consensus motif was identified based on
the structural homology between our S-100b binding peptide isolates and
the
-subunit of ACPs, and a twelve-amino acid peptide encompassing
the putative S-100b binding domain of the chicken skeletal muscle ACP,
CapZ
(residues 265-276), termed TRTK-12, was chemically
synthesized. This peptide, which corresponds also to the sequence in
the
-subunit of ACPs in toads, dogs, and humans (),
was used in studies designed to analyze S-100b interactions with S-100b
binding peptides and proteins.
Interaction of S-100b with TRTK-12
The
interaction of S-100b with TRTK-12 was evaluated initially by measuring
changes in the fluorescence of tryptophan, a residue contained in
TRTK-12 but absent in S-100b (Isobe and Okuyama, 1978). TRTK-12 by
itself exhibited a significant fluorescence when excited at 295 nm (Fig. 2A). Subsequent to the addition of S-100b in the
presence of limiting Ca concentrations (0.1
mM), fluorescence yield was unchanged. Upon adjusting the
Ca
concentration to 1 mM, the intensity of
fluorescence emission increased approximately 2-fold and exhibited a
blue shift (Fig. 2A), indicating a switch of the
tryptophan residue into a less polar environment upon interaction with
S-100b. EGTA completely reversed this change in tryptophan
fluorescence. These studies demonstrate the
Ca
-dependent interaction of S-100b with TRTK-12.
Additional studies determined that the interaction of peptide with
S-100b became significant at 400 µM CaCl
and
reached maximum at 2.0 mM CaCl
(Fig. 2B). Addition of increasing amounts of
peptide to S-100b (0.5 µM) determined the
Ca
-dependent increase in fluorescence to be maximal
at about 1 µM of peptide (data not shown).
Figure 2:
TRTK-12 peptide binding to S-100b. A, Ca-dependent change of tryptophan
fluorescence. TRTK-12 (1.4 µM in 50 mM Tris-HCl,
pH 7.5, 150 mM NaCl) in the presence of S-100b (2.2
µM) was excited at 295 nm, and emission was recorded on a
fluorescence spectrophotometer (trace1). The same
sample after successive addition of CaCl
(0.1, 0.2, 0.5, 1,
2, 5, and 10 mM), (traces2-8,
respectively). In another experiment, 5 mM EGTA completely
reversed the shift in fluorescence (data not shown). B, the
change in tryptophan fluorescence (excitation at 295 nm, emission at
the peak maximum wavelength) of TRTK-12 (1.4 µM) plus
S-100b (2.2 µM) as a function of Ca
concentration measured as described above. Results are
representative of two experiments.
In
experiments with CaM, which also lacks tryptophan, the peptide
demonstrated 15% increase in tryptophan fluorescence upon addition
of 0.1 mM CaCl
; fluorescence intensity remained
the same at 1 mM CaCl
and was completely reverted
to the original level upon addition of 5 mM EGTA, indicating
some interaction of TRTK-12 with CaM (data not shown). The
15%
increase in fluorescence displayed upon interaction of TRTK-12 is
markedly less than the
200% observed upon interaction of TRTK-12
with S-100b.
Gel Overlay Analysis of S-100b (Target
Interaction)
To further evaluate the potential interaction
of S-100b with ACPs, we examined the ability of labeled S-100b to bind
in a Ca-dependent manner to a chicken skeletal muscle
ACP, CapZ (Casella et al., 1987). Our studies used a gel
overlay assay, essentially as previously described for the
characterization of both CaM and S-100 target proteins in a variety of
tissues and cell types (Burgess et al., 1984; Hertzberg and
Van Eldik, 1987). Purified CapZ and bacterial expressed glutathione S-transferase (GST) CapZ fusion proteins were separated by
SDS-PAGE, and the gel was equilibrated with physiological buffers in
the presence (1 mM CaCl
) or absence (5 mM EGTA) of free Ca
ions and incubated with
radiolabeled S-100b (2-20
10
cpm/ml).
Subsequent to extensive washing, gels were dried, and bound S-100 was
detected by autoradiography. Both chicken CapZ
and the
GST-CapZ
fusion proteins bound S-100b in the presence of
Ca
(Fig. 3, A-C). By contrast,
the
-subunit of CapZ did not bind, and its GST-fusion product
showed weak if any binding with radiolabeled S-100b in this assay when
compared with the interaction of S-100b with CapZ
and its fusion
protein. S-100b interaction with all forms of CapZ was clearly
Ca
dependent, as no detectable binding was observed
in the presence of 5 mM EGTA. Competition binding analysis of
S-100b interaction with CapZ determined half-maximal inhibition of
S-100b binding to occur at about 40 nM (Fig. 3D).
Figure 3:
Gel overlay and slot blot analyses of
S-100b interaction with CapZ. CapZ was subjected to SDS-PAGE (about
1-2 µg/band), and their interaction with I-S100b (in 50 mM Tris-HCl, pH 7.5, 150
mM KCl, 1 mM MgCl
) was analyzed by gel
overlay. A, Coomassie Blue-stained gel. B and C, corresponding autoradiograms of gels incubated with
I-S100b in presence of 1 mM CaCl
(B), or 5 mM EGTA (C). Lane1, CapZ from chicken muscle consisting of two subunits,
CapZ
(36 kDa) and CapZ
(32 kDa). Lane2,
CapZ
fused with glutathione S-transferase
(GST-CapZ
), M
about 62,000. Lane3, CapZ
fusion protein (GST-CapZ
), M
about 58,000. For D, pieces of
nitrocellulose containing CapZ (1 µg) were incubated with
I-S-100b (2-4
10
cpm/ml) and
increasing concentrations of cold S-100b (0.001-1
µM). Relative levels of binding were determined by
densitometry of the resultant autoradiograms. Results representative of
three experiments.
Next, the effect of Ca concentration and TRTK-12 peptide on
I-S100b-CapZ
interaction was examined (Fig. 4). Binding of
I-S-100b to CapZ was Ca
dose dependent (Fig. 4A), and TRTK-12 significantly reduced that
binding in a dose and Ca
-dependent manner (Fig. 4B). Based on quantification of those results,
half-maximal inhibition of S-100b binding to CapZ would be expected to
occur at 7 µM TRTK-12. There was no effect of the
unrelated peptide, APAT-14, on binding. These results indicate that
binding of
I-S100b with CapZ
is saturable, dose,
Ca
, and site dependent.
Figure 4:
Effects of Ca and
TRTK-12 on S-100b interaction with CapZ. Mixture of GST-CapZ
plus
GST-CapZ
was subjected to SDS-PAGE (about 1-2 µg/band),
and their interaction with
I-S100b (in 50 mM
Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl
)
was analyzed by gel overlay. A, effect of Ca
on
I-S100b binding. Lane1,
Coomassie Blue-stained gel. Lanes2-7,
corresponding autoradiograms of gels incubated with
I-S100b in the presence of CaCl
(1
mM, 200, 60, and 20 µM) (lanes2-5), no CaCl
(lane6), and 5 mM EGTA (lane7). B, effect of TRTK-12 on
I-S100b binding. Lane1, Coomassie Blue-stained gel. Lanes2-12, corresponding autoradiograms of gels
incubated with Bolton-Hunter-labeled
I-S100b in the
presence of 2 mM CaCl
(lane2),
2 mM CaCl
plus 1, 3, 10, 30, and 100
µM TRTK-12 (lanes3-7), 5
mM EGTA (lane8), and 2 mM
CaCl
plus 0, 10, 30, and 100 µM of an
unrelated peptide APAT-14 (lanes9-12). Results
are representative of three experiments.
In additional studies, we
examined the ability of S-100b protein to bind a number of putative
S-100 target proteins, i.e. (Baudier et al.,
1987), aldolase (Zimmer and Van Eldik, 1986), glycogen phosphorylase
(Zimmer and Dubuisson, 1993), and myosin (Burgess et al.,
1984) (Fig. 5). We also investigated whether S-100b would bind a
yeast ACP that contained a sequence homologous to TRTK-12 or to
glycogen synthase, which was determined to contain a region homologous
to some of our S-100b binding epitopes. S-100b bound weakly to yeast
ACP but strongly to all other non-ACP proteins examined. However,
unlike its interaction with ACP, binding to these other proteins was
Ca
independent. For comparison the binding of CaM to
the same set of target proteins was studied. cNMP was used as a
positive control. Among the ACP proteins, CaM bound weakly to
GST-CapZ
but not to CapZ
and relatively strongly to yeast
ACP. CaM also bound all other proteins examined, but unlike S-100b
interaction with these molecules, CaM binding was Ca
dependent. These results indicate that CapZ
is a specific
target of S-100b and that this interaction is Ca
dependent and distinct from the interactions between CaM and its
target proteins.
Figure 5:
Ca2+ dependence of iodinated S-100b
and CaM interaction with target proteins by gel overlay. Purified
target proteins were subjected to SDS-PAGE (about 1-2
µg/band), and their interaction with iodinated S-100b or CaM was
studied in the presence of either 1 mM CaCl or 5
mM EGTA. Buffer A was used in the assay. NS indicates
not studied.
Cross-linking Analysis of CapZ-S100
Interaction
As the overlay studies employed partially
renatured CapZ protein, we next investigated whether S-100b protein
interacts in a Ca-dependent fashion with native CapZ.
Chemical cross-linking experiments were performed, and resultant
conjugates were analyzed by SDS-PAGE and immunoblotting using anti-CapZ
and anti-S-100 antibodies ( Fig. 6and 7). Optimal cross-linker
concentration for use in these experiments was determined
experimentally. Cross-linking of the mixture of CapZ (0.7
µM) and S-100b (10 µM) gave rise to multiple
conjugates containing both CapZ and S-100b molecules, as detected by
immunoblotting with anti-CapZ and S-100 antibodies (Fig. 6). In
the presence of 1 mM CaCl
, but not in the absence
of Ca
(2 mM EGTA), multiple conjugates
containing S-100b were detected in the range of M
75,000-150,000. Appearance of these S-100-containing
conjugates was Ca
dependent (Figs. 6B and
7). Addition of TRTK-12 peptide completely abolished conjugate
formation in the presence of 1 mM CaCl
, confirming
that S-100b interaction with CapZ was via the site on the CapZ
subunit (Fig. 6).
Figure 6:
Chemical cross-linking analysis of native
CapZ binding to S-100b. Proteins were subjected to cross-linking under
conditions indicated. Resultant conjugates were analyzed by
immunoblotting using either anti-CapZ (A) or anti-S-100 (B) antibodies. Arrow (A) indicates location
of CapZ/S-100b immunoreactive materials. Asterisk (B)
indicates S-100b dimers; S-100b monomers migrated at the dye front
under the conditions employed and are not shown. Molecular mass
standards (kDa) are indicated. Results are representative of three
experiments.
Detection of S100b Binding Proteins in Glial
Cells
Because S-100b is most abundant in glial cells
(Donato, 1991), we examined whether ACPs like CapZ also are expressed
in these cells. In C6 rat glioma cells, anti-CapZ antibody revealed
cross-reacting proteins with M similar to those of
- and
-subunits of CapZ (Fig. 8). These data suggest
that CapZ or a similar ACP is expressed in glial cells and, thus, could
potentially interact with S-100b.
Figure 8:
Cross-reaction of anti-CapZ antibody with
proteins from C6 glioma cells. C6 rat glioma cells were lysed in
SDS-sample buffer and subjected to SDS-PAGE. Lane1,
total homogenate of C6 cells stained with Coomassie Blue. Lane2, C6 homogenate probed with anti-CapZ antibody. Lane3, CapZ protein probed with anti-CapZ antibody. Lanes4 and 5, same as lanes3 and 4, except primary goat anti-CapZ antibody was omitted.
Position of molecular mass standards (kDa) are indicated. Results are
representative of two experiments.
binding proteins that have been reported to participate in the
regulation of a variety of cellular responses. Many of these reports
describe the effects of S-100 on cytoskeletal elements; however, the
significance of these interactions has yet to be elucidated. In the
current study, our goal was to define the molecular targets of S-100
action within cytoskeletal elements so that we might begin to develop
an integrated understanding of the role of S-100 in regulating
cytoskeletal architecture. Our approach to dissecting the molecular
interactions of S-100 with various intracellular targets was to
initially characterize S-100b isoform binding epitopes using an
approach similar to that used recently by Jamieson and co-workers
(Dedman et al., 1993) to identify novel CaM binding peptides.
-dependent fashion allowed us to identify S-100b
binding peptides. The 44 distinct peptides obtained were subdivided
into 5 groups, and from the group possessing the highest degree of
internal similarity, the consensus sequence
(K/R)(L/I)XWXXIL was derived. A search of GenBank
revealed a large number of proteins containing closely homologous
peptides, so additional criteria were applied to restrict the number of
candidate S-100b binding proteins: conservation amongst species,
location near the COOH or amino terminus, and prior demonstration of
their involvement as structural or regulatory components of the
cytoskeleton. Application of these criteria permitted us to identify
the
-subunit of the ACP, CapZ (specifically, a 12-amino acid
sequence contained within the COOH terminus of CapZ, TRTK-12) as a
potential S-100b binding site. This prediction was verified on the
basis of the Ca
-dependent binding of S-100b to
denatured CapZ
in gel overlay experiments and to native CapZ in
cross-linking experiments. We believe these data to be the first to
demonstrate that S-100b is a CapZ binding protein as well as being the
first to identify a specific S-100b binding domain within S-100b target
proteins. These results demonstrate the ability of our approach to
rapidly identify target epitopes for a specific protein (i.e. S-100b), for which there is only limited information regarding its
interactions, and raises the possibility that this approach may be a
broadly applicable method.
dependent and suggest that this interaction occurs with high
affinity. However, is the binding of S-100b to CapZ a physiologically
relevant phenomenon? Although direct evidence for this interaction
within living cells is not available, several observations suggest that
it does occur. An absolute requirement for biological relevance is that
the two proteins must coexist with the same cells. As S-100b is found
in greatest abundance in the astrocytes of the central nervous system
(Zimmer and Van Eldik, 1988), we determined whether CapZ was present in
these cells. Using a rat astrocytoma cell line, C6, known to express
S-100b, two polypeptides that cross-reacted to a polyclonal CapZ
antibody were identified. Since both had M
consistent with the known molecular weights of the CapZ subunits
and bound S-100b, it seems reasonable to conclude that both S-100b and
CapZ are present in these cells. This result is not surprising as
Schafer et al. (1994) have recently demonstrated ACPs, like
CapZ to be present in all embryonic cells and tissues, including the
chicken brain.
isoform is predominantly expressed (Kato
and Kimura, 1985; Haimoto and Kato, 1988; Casella et al.,
1986, 1987). At the ultrastructural level, S-100a
is
localized to the Z-lines and fascia adherens of the intercalated discs
in mouse myocardial cells (Haimoto and Kato, 1988). Similarly ACPs,
such as CapZ, localize to the Z-lines of skeletal muscles (Casella et al., 1987) and the intercalated disc and intracellular
junctions of cardiac myocytes (Schafer et al., 1994).
-dependent manner. Supportive of a role for S-100b
in microfilament organization are the results of Selinfreund and
co-workers(1990). Their introduction of S-100b antisense mRNA in C6
cells induced a flattened cell morphology that is associated with the
appearance of more highly organized microfilament bundles. Together
with our results, this strongly suggests S-100b may regulate
microfilament assembly, further supporting our belief that the
interaction we have characterized between S-100b and CapZ is
biologically significant.
that is required to promote this interaction. Whereas cytoplasmic
free Ca
is rarely reported at concentrations of more
than a few micromolar, substantial binding of S-100b to CapZ does not
occur below 60 µM, and binding to TRTK-12 does not occur
until 200 µM. There are several possible explanations for
this apparent discrepancy between available Ca
and
the concentrations required to promote these interactions. With
TRTK-12, it is possible that we have not identified the complete S-100b
binding domain, while in the gel overlay experiments partial
renaturation of target proteins may be insufficient to provide the
native S-100b binding site. This reduction in affinity of peptide and
target proteins for their binding site(s) on S-100b might be expected
to reduce the affinity of the Ca
binding sites, as
cooperative effects between substrate and the Ca
binding sites on S-100b do occur. On the other hand, the reason
why high concentrations of Ca
were required to
demonstrate S-100b:CapZ interactions in cross-linking experiments,
where both proteins are present in their native configuration, is more
difficult to explain. One interesting possibility is that
concentrations of cytoplasmic free Ca
may actually
rise into the tens or even hundreds of µM in localized
regions of the cell. This possibility has been a matter of common
conjecture for some time, and evidence has been obtained that this is
actually the case in the neighborhood of open plasma membrane
Ca
channels (Augustine and Neher, 1992; Llinas et
al., 1981). If these reports are correct, then one consequence
might be that S-100b and CapZ should preferentially bind to each other
at or near cell membranes.
concentration. The high degree of conservation across species of
this region in the
-subunits of all ACPs suggests that S-100b
interaction with these proteins represents an important and
biologically significant aspect of S-100b function.
Table: Amino acid sequence alignment of S-100b binding
peptides
]) at the
carboxyl terminus (Devlin et al., 1990). Position of the eight
amino acids contained within the common motif is numbered at the top.
The residues in the inserts satisfying this consensus motif are
highlighted and shown by capital letters, and the motif common for the
sequences in groups (I-IV) is shown schematically at the bottom
of the group IV (+, positively charged residue; O, hydrophobic
residue;
, hydrophilic residue; X, variable residue). Sequences
in brackets indicate invariable sequences present in all random peptide
pIII-fusion protein isolates. If greater than one, the number of
occurrences of a particular insert is indicated in parentheses. Dashes
have been inserted for spacing and do not indicate position of unnamed
amino acids.
Table: Sequence alignment of actin capping proteins
and S-100b binding peptides
-subunits
of actin capping proteins from yeast, Saccharomyces cerevisiae (Amatruda and Cooper, 1992); protozoan, Dictyostelium
discoideum (Hartmann et al., 1989); nematode Caenorhabditis elegans (J. A. Cooper, personal communication);
toad, Xenopus laevis (Ankenbauer et al., 1989);
human, Homo sapiens (J. A. Cooper, personal communication);
dog, Canis familiaris (Armatruda et al., 1992); and
chicken, Gallus gallus
-1 and
-2 (Casella et
al., 1989; Cooper et al., 1991). Dashes have been
inserted for the purpose of maximizing alignment and do not indicate
the position of unnamed amino acids. The sequence R-K- -W is conserved
in all species, including yeast. This motif is also present within the
peptide insert of many bacteriophage isolates.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.