Biochimica et Biophysica Acta, 1160 (1992) 67-75
67
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34352
S-100 protein binds to annexin II and pll, the heavy and light chains of calpactin I Roberta Bianchi, Grazia Pula, Paolo Ceccarelli, Ileana Giambanco and Rosario Donato Section of Anatomy, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)
Key words: S-100 protein; Annexin II; pll; Calpactin I; Calcium ion binding; Acrylodan S-100 protein, a dimeric, Ca2+-binding protein of the EF-hand type, interacts with annexin II (p36, the heavy chain of the cytoskeletal protein complex, calpactin I), with p l l (the light and regulatory chain of calpactin I) and with the hetero-tetramer annexin II2-pll 2 (calpactin I) in a Ca2+-regulated way, but not with annexins I, V and VI. The interaction of S-100 protein with the above proteins was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and acrylodan-annexin II and by cross-linking experiments using the bifunctional cross-linker disuccinimidyl suberate (DSS). S-100 protein binds with the highest affinity to annexin II (K d approx. 0.4 /xM) and with the lowest affinity to calpactin I ( K d approx. 10 /zM), with a constant stoichiometry of about 2 mol of protein/S-100 dimer. Thus, S-100 protein could substitute for p l l in regulating the activities of annexin II in cells which do not express p l l and/or act synergistically with p l l in cells expressing both p l l and S-100. The binding of S-100 protein to p l l could reflect the natural tendency of S-100 subunits and p l l to dimerize. Chimeric pll-S-100a and pll-S-100-fl proteins could therefore form in a Ca2+-regulated way. The interaction of S-100 protein with calpactin I appears of doubtful physiological importance, because of the low binding affinity, of the small extent of fluorescence changes induced by calpactin I in acrylodan-S-100 protein and of lack of DSS-induced complex formation between the two protein species.
Introduction S-100 proteins constitute a group of dimeric Ca 2+binding proteins of the EF-hand type (S-100aa, S100afl and S-100fl/3) shown to have a role in the regulation of the cytoskeleton, of protein phosphorylation, of certain enzymatic activities and of Ca2÷-in duced Ca2+-release (reviewed in Refs. 1-3). Two extracellular roles have been suggested for S-100fl/3, namely stimulation of neurite outgrowth and stimulation of glial proliferation [4,5]. In recent years, several proteins have been purified from a number of sources displaying 28 to 55% identity with the a- and fl-S-100 subunits (reviewed in Refs. 3,6). Among these is p l l , the light chain of the cytoskeleton protein complex, calpactin I [7-9]. p l l , which has a high tendency to dimerize like the S-100 subunits, does not possess conventional EF-hands and does not bind Ca 2+. p l l
Correspondence to: R. Donato, Section of Anatomy, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Cas. Post. 81 Succ. 3, 06100 Perugia, Italy. Abbreviations: DSS, disuccinimidyl suberate; DTF, dithiothreitol.
binds with high affinity in a Ca2+-independent way to the N-terminal tail of annexin II (p36, the heavy chain of calpactin I) [10,11]. In this way, one dimer of p l l would cross-bridge two molecules of annexin II to generate a p362-p112 heterotetramer, i.e., calpactin I [12-14]. In so doing, p l l behaves as the regulatory chain of calpactin I, in that it regulates the ability of annexin II to aggregate and fuse artificial membranes and to promote aggregation and fusion of chromaffin granules [15,16], the interaction with F-actin and the F-actin-bundling activity of annexin II in the presence of physiological free Ca 2+ levels [13,17] and the level of phosphorylation of annexin II by pp60 ..... and protein kinase C [18,19]. Within cells, p l l exists as a complex with annexin II in the submembrane cytoskeleton, whereas uncomplexed annexin II exists soluble in the cytoplasm [14,20,21]. Bovine brain S-100 protein was shown to inhibit the phosphorylation of annexin II in a Ca2+-independent way [22] like p l l . This effect was attributed to direct binding of S-100 protein to annexin II on the basis of the sequence homology of S-100 subunits with p l l [22] and on the basis of the observation that the regulatory effects of S-100 proteins on protein phosphorylation
68 reported thus far were shown to be dependent on interaction with the substrates rather than with the kinases [3]. However, no direct evidence of S-100 protein binding to annexin II has been offered thus far. We have investigated this possibility by a number of experimental approaches. The results indicate that: (1), S-100 protein binds to annexin II; (2), Ca 2÷ regulates the S-100 protein binding to annexin II; (3), S-100 protein also binds to p l l and to the heterotetramer, calpactin I, although with much lower affinity than to annexin II and (4), S-100 protein does not bind to annexins I, V, and VI, whereas it binds to annexin IV to some extent. Materials and Methods
Acrylodan was obtained from Molecular Probes, dithiothreitol (DTT) from Sigma, disuccinimidyl suberate (DSS) from Pierce, AcA54 and CM Trysacryl M from IBF, DEAE-Sephacryl and Sephacryl S-200 from Pharmacia. All other reagents were analytical grade reagents from Sigma, Fluka, BDH, Bio-Rad, or Carlo Erba. Purification of proteins. S-100 protein was purified from bovine brain as described [23]. The two annexin V isoforms and annexin VI were purified from bovine lung as reported [24]. The two bovine annexin V isoforms were separated from one another as described [25]. Bovine lung annexin IV was purified by the same procedure used to purify annexins V and VI [24], being recovered in the protein peak eluting from the DEAE-Sephacel column between 0 and 0.1 M NaCI. Calpactin I, monomeric annexin II, and annexin I were purified from bovine lung as reported [17], with some modifications. The EGTA-extract from Ca2+-precipi tated proteins was concentrated by pressure filtration and loaded onto a column of DEAE-Sephacel (1 x 5 cm) equilibrated with 20 mM Tris-HC1 (pH 7.5), 1 mM E D T A and 5 mM 2-mercaptoethanol. The flow-through fractions were collected, concentrated, and loaded onto a column of AcA54 (2 x 90 cm) to separate calpactin I from the mixture of monomeric annexin II and annexin I. Annexin I was separated from annexin II by filtration on CM Trysacryl M (1 X 5 cm) equilibrated with 25 mM 2-(N-morpholino)ethanesulfonic acid, (pH 6.0), 5 mM 2-meracptoethanol and 1 mM EDTA. The flowthrough fractions contained annexin I, whereas the absorbed fraction, which was eluted with a linear 0-0.3 M NaCI in the same buffer, contained annexin II. Calpactin I was separated into annexin II and p l l by dialysis against 20 mM Tris-HC1 (pH 7.5), 0.1 M NaC1, 1 mM EDTA, 1 mM DT-F, 9 M Urea, followed by chromatography on Sephacryl S-200 (1 x 90 cm) equilibrated with the same buffer [26]. Individual annexin II and p l l were renatured by dialysis against 20 mM Tris-HCl (pH 7.5), 0.1 M NaC1, 0.2 mM MgCle, 0.5
1
2
3
4
Fig. 1. Electrophoretic characterization of annexin II, calpactin I, p l l and S-100 protein. Purified annexin II (lane 1), calpactin I (lane 2), p l l (lane 3) and S-100 protein (lane 4) were subjected to SDSPAGE. Gels were stained with Coomassie blue. The arrow points to the positions of p l l and S-100 protein and the arrowhead to the position of annexin II.
mM D T T [26]. Calmodulin was purified from bovine brain by CaZ+-dependent affinity chromatography using the same procedure employed to purify the annexin V isoforms [27]. The purity of individual proteins was checked by SDS-PAGE [28]. Fig. 1 shows SDS gels (7.5% acrylamide) of annexin II, calpactin I, p l l and S-100 protein.
Fluorescence experiments with acrylodan-S-lO0 protein and acrylodan-annexin II. S-100 protein and annexin II were labelled with acrylodan as described, respectively [29,30]. Briefly, S-100 protein was incubated at 4°C for 2 h in 25 mM imidazole-HC1 (pH 7.1), 0.1 M NaC1, 0.2 mM E G T A (buffer A) containing 2.5 mM CaCI2, with 5-fold molar equivalent of acrylodan, a thiol-reactive derivative of Prodan [31]. Annexin II was labelled with acrylodan exactly as described [30]. The reaction was terminated by the addition of 5 mM DTT, in both cases. In the Case of S-100 protein, 2.5 mM E G T A was added to the mixture 30 min after the addition of DTT. Uncoupled dye was removed by gel filtration on AcA54 equilibrated with buffer A plus 1 mM D T T (buffer B). The labelling ratio (dye/protein), which was determined as reported [31], was 1.8 for S-100 protein and 0.75 for annexin II. Fluorescence emission spectra were recorded at 25°C on a Shimadzu RF-5000 spectrofluorophotometer with excitation at 380 nm and emission collected between 400 and 650 nm. Emission data were corrected for dilution which never exceeded 5%. Experiments were done in buffer B in the presence or absence of 1 mM CaC1 e. The following molecular masses were used: 21 kDa for S-100 protein, 36 kDa for annexin II, 11 kDa for p l l and 95 kDa for calpactin I. Cross-linking experiments. S-100 protein (50/zg) was incubated in a final volume of 0.2 ml of buffer B in the presence or absence of 1 mM CaC12 at 37°C for 30 min
69 in the absence or presence of equimolar amounts of annexin II, p l l , or calpactin I, after which the bifunctional cross-linker DSS was added to a final concentration of 0.5 mM. After 5 min at room temperature, the reaction was terminated by the addition of SDS and 2-mercaptoethanol (2% (w/v)). Samples were then subjected to SDS-PAGE (7.5% acrylamide). Electrophoretically separated polypeptides were transblotted onto nitrocellulose paper [32] for immunochemical analysis with a polyclonal anti-S-100 protein antiserum raised in rabbits and characterized as described [33]. Results
Fluorescence experiments with acrylodan-S-lO0 protein The emission spectrum of acrylodan-S-100 protein (0.8 /zM) was characterized by a maximum at 492 nm in the absence of Ca 2÷ (0.2 mM E G T A ) (Fig. 2A, trace 1), which shifted to 500 nm on addition of 1 mM CaCI 2 (Fig. 2A, trace 2). This change was accompanied by a 4% decrease in the emission spectrum a r e a (Fig. 2A, traces 1 and 2). These data were in accordance with previous observations [29]. The Ca2+-dependent redshift of the fluorescence maximum indicated that in the presence of Ca 2+ the fluorophore was in a more polar environment, a finding which agrees with the known notion that in the presence of Ca 2÷ some of the -SH groups in S-100 protein become exposed to the solvent [1-3]. The addition of a 3-fold molar excess of annexin II to acrylodan-S-100 protein produced no significant changes in the fluorescence spectrum in the absence of Ca 2÷ (Table I), whereas a 23% increase in the emission spectrum area and a 12-nm blue-shift of the emission maximum were registered in the presence of Ca 2+ (Fig. 2A, trace 3, Table I). The final addition of E G T A to 2.5 mM completely reverted the annexin II effects on acrylodan-S-100 protein (not shown). These data suggest that S-100 protein binds to annexin II in a Ca2+-dependent way, that the interaction is promptly off-set upon decreasing the free Ca 2 ÷ concentration to subnanomolar amounts and that the annexin II binding induces the fluorophore in S-100 protein to be located in a more hydrophobic environment. The above changes in the emission spectrum of acrylodan-S-100 protein in the presence of Ca 2+ were linearly related to the annexin II concentration up to an annexin II/S-100 protein molar ratio of approx. 2, suggesting that two mol of annexin II bound to one mol of S-100 dimer (Fig. 2C,D), as is the case with p l l binding to annexin II [30]. Above a molar ratio of 2, a slightly f u r t h e r increase in the emission spectrum area (Fig. 2B) and a slightly further blue-shift of the emission maximum (Fig. 2C) were registered. Neither unlabeled S-100 protein nor calmodulin at a 100-fold molar excess with respect to acrylodan-S-100 protein (0.1 txM) produced changes in the emission spectrum, irrespective of the
B
120
,co ~'*e
° o"
;[//, \ 400
500 )~,nm
e" P
100
600
I 1
I 2
AnnexinIllS-lO0 100 -tAD % o%
50Ok,,C
/%;;.
E '
67
© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34352
S-100 protein binds to annexin II and pll, the heavy and light chains of calpactin I Roberta Bianchi, Grazia Pula, Paolo Ceccarelli, Ileana Giambanco and Rosario Donato Section of Anatomy, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)
Key words: S-100 protein; Annexin II; pll; Calpactin I; Calcium ion binding; Acrylodan S-100 protein, a dimeric, Ca2+-binding protein of the EF-hand type, interacts with annexin II (p36, the heavy chain of the cytoskeletal protein complex, calpactin I), with p l l (the light and regulatory chain of calpactin I) and with the hetero-tetramer annexin II2-pll 2 (calpactin I) in a Ca2+-regulated way, but not with annexins I, V and VI. The interaction of S-100 protein with the above proteins was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and acrylodan-annexin II and by cross-linking experiments using the bifunctional cross-linker disuccinimidyl suberate (DSS). S-100 protein binds with the highest affinity to annexin II (K d approx. 0.4 /xM) and with the lowest affinity to calpactin I ( K d approx. 10 /zM), with a constant stoichiometry of about 2 mol of protein/S-100 dimer. Thus, S-100 protein could substitute for p l l in regulating the activities of annexin II in cells which do not express p l l and/or act synergistically with p l l in cells expressing both p l l and S-100. The binding of S-100 protein to p l l could reflect the natural tendency of S-100 subunits and p l l to dimerize. Chimeric pll-S-100a and pll-S-100-fl proteins could therefore form in a Ca2+-regulated way. The interaction of S-100 protein with calpactin I appears of doubtful physiological importance, because of the low binding affinity, of the small extent of fluorescence changes induced by calpactin I in acrylodan-S-100 protein and of lack of DSS-induced complex formation between the two protein species.
Introduction S-100 proteins constitute a group of dimeric Ca 2+binding proteins of the EF-hand type (S-100aa, S100afl and S-100fl/3) shown to have a role in the regulation of the cytoskeleton, of protein phosphorylation, of certain enzymatic activities and of Ca2÷-in duced Ca2+-release (reviewed in Refs. 1-3). Two extracellular roles have been suggested for S-100fl/3, namely stimulation of neurite outgrowth and stimulation of glial proliferation [4,5]. In recent years, several proteins have been purified from a number of sources displaying 28 to 55% identity with the a- and fl-S-100 subunits (reviewed in Refs. 3,6). Among these is p l l , the light chain of the cytoskeleton protein complex, calpactin I [7-9]. p l l , which has a high tendency to dimerize like the S-100 subunits, does not possess conventional EF-hands and does not bind Ca 2+. p l l
Correspondence to: R. Donato, Section of Anatomy, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Cas. Post. 81 Succ. 3, 06100 Perugia, Italy. Abbreviations: DSS, disuccinimidyl suberate; DTF, dithiothreitol.
binds with high affinity in a Ca2+-independent way to the N-terminal tail of annexin II (p36, the heavy chain of calpactin I) [10,11]. In this way, one dimer of p l l would cross-bridge two molecules of annexin II to generate a p362-p112 heterotetramer, i.e., calpactin I [12-14]. In so doing, p l l behaves as the regulatory chain of calpactin I, in that it regulates the ability of annexin II to aggregate and fuse artificial membranes and to promote aggregation and fusion of chromaffin granules [15,16], the interaction with F-actin and the F-actin-bundling activity of annexin II in the presence of physiological free Ca 2+ levels [13,17] and the level of phosphorylation of annexin II by pp60 ..... and protein kinase C [18,19]. Within cells, p l l exists as a complex with annexin II in the submembrane cytoskeleton, whereas uncomplexed annexin II exists soluble in the cytoplasm [14,20,21]. Bovine brain S-100 protein was shown to inhibit the phosphorylation of annexin II in a Ca2+-independent way [22] like p l l . This effect was attributed to direct binding of S-100 protein to annexin II on the basis of the sequence homology of S-100 subunits with p l l [22] and on the basis of the observation that the regulatory effects of S-100 proteins on protein phosphorylation
68 reported thus far were shown to be dependent on interaction with the substrates rather than with the kinases [3]. However, no direct evidence of S-100 protein binding to annexin II has been offered thus far. We have investigated this possibility by a number of experimental approaches. The results indicate that: (1), S-100 protein binds to annexin II; (2), Ca 2÷ regulates the S-100 protein binding to annexin II; (3), S-100 protein also binds to p l l and to the heterotetramer, calpactin I, although with much lower affinity than to annexin II and (4), S-100 protein does not bind to annexins I, V, and VI, whereas it binds to annexin IV to some extent. Materials and Methods
Acrylodan was obtained from Molecular Probes, dithiothreitol (DTT) from Sigma, disuccinimidyl suberate (DSS) from Pierce, AcA54 and CM Trysacryl M from IBF, DEAE-Sephacryl and Sephacryl S-200 from Pharmacia. All other reagents were analytical grade reagents from Sigma, Fluka, BDH, Bio-Rad, or Carlo Erba. Purification of proteins. S-100 protein was purified from bovine brain as described [23]. The two annexin V isoforms and annexin VI were purified from bovine lung as reported [24]. The two bovine annexin V isoforms were separated from one another as described [25]. Bovine lung annexin IV was purified by the same procedure used to purify annexins V and VI [24], being recovered in the protein peak eluting from the DEAE-Sephacel column between 0 and 0.1 M NaCI. Calpactin I, monomeric annexin II, and annexin I were purified from bovine lung as reported [17], with some modifications. The EGTA-extract from Ca2+-precipi tated proteins was concentrated by pressure filtration and loaded onto a column of DEAE-Sephacel (1 x 5 cm) equilibrated with 20 mM Tris-HC1 (pH 7.5), 1 mM E D T A and 5 mM 2-mercaptoethanol. The flow-through fractions were collected, concentrated, and loaded onto a column of AcA54 (2 x 90 cm) to separate calpactin I from the mixture of monomeric annexin II and annexin I. Annexin I was separated from annexin II by filtration on CM Trysacryl M (1 X 5 cm) equilibrated with 25 mM 2-(N-morpholino)ethanesulfonic acid, (pH 6.0), 5 mM 2-meracptoethanol and 1 mM EDTA. The flowthrough fractions contained annexin I, whereas the absorbed fraction, which was eluted with a linear 0-0.3 M NaCI in the same buffer, contained annexin II. Calpactin I was separated into annexin II and p l l by dialysis against 20 mM Tris-HC1 (pH 7.5), 0.1 M NaC1, 1 mM EDTA, 1 mM DT-F, 9 M Urea, followed by chromatography on Sephacryl S-200 (1 x 90 cm) equilibrated with the same buffer [26]. Individual annexin II and p l l were renatured by dialysis against 20 mM Tris-HCl (pH 7.5), 0.1 M NaC1, 0.2 mM MgCle, 0.5
1
2
3
4
Fig. 1. Electrophoretic characterization of annexin II, calpactin I, p l l and S-100 protein. Purified annexin II (lane 1), calpactin I (lane 2), p l l (lane 3) and S-100 protein (lane 4) were subjected to SDSPAGE. Gels were stained with Coomassie blue. The arrow points to the positions of p l l and S-100 protein and the arrowhead to the position of annexin II.
mM D T T [26]. Calmodulin was purified from bovine brain by CaZ+-dependent affinity chromatography using the same procedure employed to purify the annexin V isoforms [27]. The purity of individual proteins was checked by SDS-PAGE [28]. Fig. 1 shows SDS gels (7.5% acrylamide) of annexin II, calpactin I, p l l and S-100 protein.
Fluorescence experiments with acrylodan-S-lO0 protein and acrylodan-annexin II. S-100 protein and annexin II were labelled with acrylodan as described, respectively [29,30]. Briefly, S-100 protein was incubated at 4°C for 2 h in 25 mM imidazole-HC1 (pH 7.1), 0.1 M NaC1, 0.2 mM E G T A (buffer A) containing 2.5 mM CaCI2, with 5-fold molar equivalent of acrylodan, a thiol-reactive derivative of Prodan [31]. Annexin II was labelled with acrylodan exactly as described [30]. The reaction was terminated by the addition of 5 mM DTT, in both cases. In the Case of S-100 protein, 2.5 mM E G T A was added to the mixture 30 min after the addition of DTT. Uncoupled dye was removed by gel filtration on AcA54 equilibrated with buffer A plus 1 mM D T T (buffer B). The labelling ratio (dye/protein), which was determined as reported [31], was 1.8 for S-100 protein and 0.75 for annexin II. Fluorescence emission spectra were recorded at 25°C on a Shimadzu RF-5000 spectrofluorophotometer with excitation at 380 nm and emission collected between 400 and 650 nm. Emission data were corrected for dilution which never exceeded 5%. Experiments were done in buffer B in the presence or absence of 1 mM CaC1 e. The following molecular masses were used: 21 kDa for S-100 protein, 36 kDa for annexin II, 11 kDa for p l l and 95 kDa for calpactin I. Cross-linking experiments. S-100 protein (50/zg) was incubated in a final volume of 0.2 ml of buffer B in the presence or absence of 1 mM CaC12 at 37°C for 30 min
69 in the absence or presence of equimolar amounts of annexin II, p l l , or calpactin I, after which the bifunctional cross-linker DSS was added to a final concentration of 0.5 mM. After 5 min at room temperature, the reaction was terminated by the addition of SDS and 2-mercaptoethanol (2% (w/v)). Samples were then subjected to SDS-PAGE (7.5% acrylamide). Electrophoretically separated polypeptides were transblotted onto nitrocellulose paper [32] for immunochemical analysis with a polyclonal anti-S-100 protein antiserum raised in rabbits and characterized as described [33]. Results
Fluorescence experiments with acrylodan-S-lO0 protein The emission spectrum of acrylodan-S-100 protein (0.8 /zM) was characterized by a maximum at 492 nm in the absence of Ca 2÷ (0.2 mM E G T A ) (Fig. 2A, trace 1), which shifted to 500 nm on addition of 1 mM CaCI 2 (Fig. 2A, trace 2). This change was accompanied by a 4% decrease in the emission spectrum a r e a (Fig. 2A, traces 1 and 2). These data were in accordance with previous observations [29]. The Ca2+-dependent redshift of the fluorescence maximum indicated that in the presence of Ca 2+ the fluorophore was in a more polar environment, a finding which agrees with the known notion that in the presence of Ca 2÷ some of the -SH groups in S-100 protein become exposed to the solvent [1-3]. The addition of a 3-fold molar excess of annexin II to acrylodan-S-100 protein produced no significant changes in the fluorescence spectrum in the absence of Ca 2÷ (Table I), whereas a 23% increase in the emission spectrum area and a 12-nm blue-shift of the emission maximum were registered in the presence of Ca 2+ (Fig. 2A, trace 3, Table I). The final addition of E G T A to 2.5 mM completely reverted the annexin II effects on acrylodan-S-100 protein (not shown). These data suggest that S-100 protein binds to annexin II in a Ca2+-dependent way, that the interaction is promptly off-set upon decreasing the free Ca 2 ÷ concentration to subnanomolar amounts and that the annexin II binding induces the fluorophore in S-100 protein to be located in a more hydrophobic environment. The above changes in the emission spectrum of acrylodan-S-100 protein in the presence of Ca 2+ were linearly related to the annexin II concentration up to an annexin II/S-100 protein molar ratio of approx. 2, suggesting that two mol of annexin II bound to one mol of S-100 dimer (Fig. 2C,D), as is the case with p l l binding to annexin II [30]. Above a molar ratio of 2, a slightly f u r t h e r increase in the emission spectrum area (Fig. 2B) and a slightly further blue-shift of the emission maximum (Fig. 2C) were registered. Neither unlabeled S-100 protein nor calmodulin at a 100-fold molar excess with respect to acrylodan-S-100 protein (0.1 txM) produced changes in the emission spectrum, irrespective of the
B
120
,co ~'*e
° o"
;[//, \ 400
500 )~,nm
e" P
100
600
I 1
I 2
AnnexinIllS-lO0 100 -tAD % o%
50Ok,,C
/%;;.
E '
