©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of S-100b Binding Epitopes
IDENTIFICATION OF A NOVEL TARGET, THE ACTIN CAPPING PROTEIN, CapZ (*)

Vasily V. Ivanenkov (1), Gordon A. JamiesonJr. (2) (3), Eric Gruenstein (4), Ruth V. W. Dimlich (1) (3)(§)

From the (1)Departments of Emergency Medicine, (2)Environmental Health, (3)Cell Biology, Neurobiology, and Anatomy, and (4)Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Short amino acid sequences that interact with the Ca 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 CapZ1 and CapZ2), 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.


INTRODUCTION

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 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).

S-100 proteins are a family of acidic, dimeric, Ca 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).

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 is expressed most abundantly in heart (Kato and Kimura, 1985) and is localized specifically to cardiac myocytes (Haimoto and Kato, 1988).

S-100 proteins are thought to exert their effect in biological systems through Ca-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.

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 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.


EXPERIMENTAL PROCEDURES

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).


RESULTS

Our first experiments identified bacteriophage that bound in a Ca-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.

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).

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 ).

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 -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.




DISCUSSION

S-100 proteins are a family of small Ca 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.

Screening of a bacteriophage random peptide library for particles that bound to a S-100b affinity column in a Ca-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.

These data clearly show that the binding of S-100b to CapZ is very specific as well as Ca 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.

Additional support for co-expression of these proteins within the same cell comes from skeletal and heart muscle, where the S-100a 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).

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-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.

Despite these arguments, we have some concern about the relatively high concentration of Ca 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.

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 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

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]) 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

Inserts in bacteriophages obtained after 5 rounds of selection and the COOH-terminal sequences of -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.



FOOTNOTES

*
This study was supported in part by American Cancer Society, Ohio Division (to G. A. J.), and National Institutes of Health Grants NS-27814 (to E. G.) and NS-25635 (to R. V. W. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of Cincinnati, Dept. of Emergency Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0769. Tel.: 513-558-5281; Fax: 513-558-5791.

The abbreviations used are: CaM, calmodulin; ACP, actin capping protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

G. A. Jamieson, Jr., H. R. Bhatt, J. Minzner, J. B. Richard, M. A. Kaetzel, and J. R. Dedman, unpublished observations.


ACKNOWLEDGEMENTS

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.


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