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Originally published In Press as doi:10.1074/jbc.M606545200 on September 19, 2006

J. Biol. Chem., Vol. 281, Issue 46, 35030-35038, November 17, 2006
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Identification of an AHNAK Binding Motif Specific for the Annexin2/S100A10 Tetramer*

Sandrine De Seranno{ddagger}§, Christelle Benaud{ddagger}§, Nicole Assard{ddagger}§, Sami Khediri{ddagger}§, Volker Gerke||, Jacques Baudier{ddagger}§1, and Christian Delphin{ddagger}§2

From the {ddagger}INSERM, EMI01-04 and the §Commissariat à l'Energie Atomique, DRDC/TS, Grenoble 38054, France, the Université Joseph Fourier, Grenoble 38041, France, and the ||Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Muenster, D-48149, Germany

Received for publication, July 10, 2006 , and in revised form, September 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Annexin2 tetramer (A2t), which consists of two Annexin2 molecules bound to a S100A10 dimer, is implicated in membrane-trafficking events. Here, we showed using a yeast triple-hybrid experiment and in vitro binding assay that Annexin2 is required for strong binding of S100A10 to the C-terminal domain of the protein Ahnak. We also revealed that this effect involves only the Annexin2 N-terminal tail, which is implicated in S100A10/Annexin2 tetramerization. The minimal A2t binding motif (A2tBP1) in Ahnak was mapped to a 20-amino acid peptide, and this peptide is highly specific for A2t. We also identified a second A2t binding motif (A2tBP2) present in the N-terminal domain of Ahnak, which binds to A2t, albeit with less affinity. When overexpressed as an EGFP fusion protein in MDCK cells, A2tBPs cofractionate in a calcium-dependent manner and co-immunoprecipitate with S100A10 and Annexin2. In living cells, A2tBPs target EGFP to the cytoplasm as does Annexin2. In response to oxidative and mechanical stress, EGFP-A2tBPs relocalize within minutes to the plasma membrane; a behavior shared with Annexin2-GFP. These results suggest that the A2t complex exists within the cytoplasm of resting living cells and that its localization at the plasma membrane relies on cellular signaling. Together, our data demonstrate that A2tBP1 is a specific A2t complex binding domain and may be a powerful tool to help elucidate A2t structure and cellular functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Annexin2 is a member of a multigene family of proteins widely expressed among multicellular organisms (1). It binds to negatively charged phospholipids in a calcium-dependent manner (2-4) and has been implicated in several membrane transport events such as endocytic and secretory pathways (5-14). Annexin2 is composed of a core domain of four 70-amino acid repeats and a short N-terminal tail of 30 residues (2). The core domain is responsible for the calcium-dependent interaction with the membrane phospholipids (15-17). The very first twelve N-terminal residues form an amphipathic {alpha}-helix that constitutes the minimal binding domain for its partner, S100A10 (15, 18-20). S100A10 belongs to the large S100 family of EF-hand calcium-binding proteins. It has the peculiarity of having lost its calcium-sensing properties and is locked in a constitutively active conformation (21). Similar to other members of the family, S100A10 homodimerizes and was found inside the cell either as a homodimer or as a heterotetramer (A2t)3 in which each of the two S100A10 subunits interacts with one Annexin2 molecule (for reviews see Refs. 22 and 23). Binding partners have been identified for each component: Annexin2 was found to interact with F-actin, caveolin, and HIV1-Gag protein (24-26), and S100A10 was reported to interact with various cell surface receptors and ion channels such as TRPV5, TRPV6, NaV1.8, TASK-1, and ASIC1 (27-32). Given the affinity of S100A10 for Annexin2 (Kd < 3 x 10-8, Ref. 19), it is generally assumed that it is indeed the A2t complex that interacts with the different substrates. However, in most cases whether both components contribute to the A2t ability to bind substrates has not been investigated. Furthermore, co-immunoprecipitation experiments failed to detect Annexin2 in association with the complex S100A10/TASK1 (30), suggesting that, at least for the S100A10 subunit, interaction with some targets occurs independently of the formation of the tetramer. Understanding the functions of the tetramer, as opposed to those of its two components, relies on the identification of substrates that specifically interact with the tetramer.

In a previous study, we have identified the giant protein Ahnak as a binding partner of A2t. In epithelial cells, A2t recruits Ahnak to cholesterol-rich microdomains of the plasma membrane. This recruitment appears to be essential for the establishment of the proper cytoarchitecture of polarized columnar epithelial cells (33). Ahnak is composed of an N-terminal 498-amino acid domain, a large central region of 4390 amino acids organized in 36 repeated units, and the C-terminal 1002 amino acids. We have shown that the S100A10 subunit mediates the interaction of A2t with the C-terminal domain of Ahnak (33).

In the present work, we have extended the molecular and functional characterization of the A2t/Ahnak interaction. We have shown that the C-terminal domain of Ahnak constitutes a highly specific substrate of A2t and does not bind significantly to S100A10 or Annexin2 individually. We have delineated the Annexin2 contribution in the A2t-Ahnak complex formation to its S100A10 binding site and have mapped down the A2t binding site in the Ahnak C-terminal domain to a 20-amino acid peptide (A2tBP1). We have shown that in vitro A2tBP1 binding to A2t is characterized by a high affinity and specificity. We have also demonstrated that EGFP-A2tBP1 expressed in MDCK cells co-fractionates and co-immunoprecipitates with S100A10 and Annexin2. Furthermore, we have shown, that through its specific binding, EGFP-A2tBP1 allows us to follow stress-induced relocalization of Annexin-2 in living cells. Thus, A2tBP1 is the first specific A2t complex binding motif, which might be a powerful tool to study the structure of A2t and to dissect functions strictly involving the complex A2t, not its individual subunits.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—MDCK epithelial cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA), 10% fetal bovine serum (Sigma Aldrich), and 1% penicillin/streptomycin (Invitrogen). For metabolic labeling, cells were labeled in methionine-free MEM, 5% fetal calf serum supplemented with [35S]Met/Cys mix (5 µCi/ml) for 4 days.

Production of MDCK Stable Transformants—Annexin2-EGFP plasmid was a generous gift from Dr. Moss. EGFP-A2tBP1 and 2 were obtained by subcloning of the BamHI/XbaI A2tBP fragments from the pGEX-KG vector to pEGFP-C1 vector. Plasmids were purified using the Qiagen kit and used for transformation of MDCK cells with FuGENE6 transfection reagent following the manufacturer's instructions (Roche Applied Science). Transfected cells were selected and maintained in culture medium containing 0.6 mg/ml of geneticin. Because aminoglycoside antibiotics have been shown to bind to and interfere with phospholipid-dependent cellular functions (34), geneticin was removed from the culture medium at least a week before the experiment was performed.

Cellular Fractionation and Immunoprecipitation—MDCK cells grown to confluence in 10-cm culture plates were washed with phosphate-buffered saline and collected in 0.5 ml of Buffer L (Tris-HCl 20 mM, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.3% Triton X-100 plus 10 µg of each protease inhibitor (AEBSF, leupeptin, aprotinin)) in the presence of 0.3 mM CaCl2 or 1 mM EGTA. Cells were passed through a 26-gauge needle and centrifuged at 20,000 x g for 30 min at 4 °C. Supernatants were removed (S), and pellets (P) were resuspended by sonication in one volume of Buffer L. For Western blot analysis, 20 µl of each sample were separated by 11% Tris-Tricine polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane, proteins were detected using anti-Annexin2 mAb, anti-S100A10 mAb (both Transduction Laboratories), and anti-EGFP pAb (AbCam, Paris, France). For immunoprecipitation, the calcium concentration of 100-µl samples was adjusted to 0.3 mM and incubated with 1.5 µg of anti-EGFP mAb (AbCam) for 1 h at 4°C. 10 µl of protein A-Sepharose were then added and incubated at 4 °C for 1 h. Beads were spun down at 2000 x g, washed three times with buffer L, and resuspended in 100 µl of Laemmli buffer. Western blot analysis was carried out as described above.

Purification of S100A10 and Annexin2—Native A2t was purified from porcine intestine as previously described (35). S100A10 and Annexin2 were dissociated from the complex with 9 M urea. Each subunit was purified by gel filtration in 20 mM Tris-HCl, pH 7.4, 9 M urea, 100 mM NaCl, 2 mM DTT, 1 mM EGTA, 1 mM NaN3 and stored in aliquots at -80 °C. Prior to the binding experiment, S100A10 and Annexin2 were renatured by dialyzing against 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3% Triton X-100, and 1 mM DTT.

In Vitro Protein-Protein Interaction/GST Pull-down Assays— For production of GST fusion proteins, Ahnak C-ter cDNA was cloned into pGEX-KG (GE Health Care) as previously described (33). Shorter Ahnak fragments were obtained either by deleting Ahnak-Cter by restriction digestion, blunting, and re-ligation or by PCR cloning. Detailed cloning information for specific GST-Ahnak fragment expression is available upon request. Numbering of Ahnak residues is from the human Ahnak sequence (GenBankTM accession number NM001620). The constructs were introduced in Escherichia coli BL21(DE3)pLysS strain. Bacteria were grown to an A600 of 0.6-0.8 and treated with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 2-3 h to induce protein expression. All subsequent steps were carried out at 4 °C. Bacteria were lysed in buffer A (40 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM DTT) complemented with 1 mg/ml lysozyme, 1% Triton X-100, 5 mM EDTA, and protease inhibitors (AEBSF, leupeptin, aprotinin, pepstatin 10 µg/ml each) through three cycles of freeze-thaw. The lysates were centrifuged at 30,000 x g for 1 h, and the supernatants were incubated in batch with glutathione-Sepharose beads for 2 h. Beads were washed in batch three times with buffer A plus 1% Triton X-100 and 5 mM EDTA and then with ten bead volumes in column. The columns were equilibrated in buffer A, and the proteins were eluted in buffer A plus 30 mM glutathione. Fractions containing the proteins were pooled and extensively dialyzed against buffer A. Protein concentration was quantified on a Coomassie-stained acrylamide gel using bovine serum albumin as standard. Proteins in working aliquots were flash-frozen in liquid nitrogen and stored at -80 °C. For the GST pull-down assay, 2 µg of recombinant protein were incubated for 1 h at 4°C with 10 µl of glutathione-Sepharose beads and then washed in buffer B (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3% Triton X-100, 1 mM DTT, 300 µM CaCl2). GST pull-down assays were carried out either with different combinations of purified proteins (2.7 µg of Annexin2, 0.8 µg of S100A10, 2 µg of native A2t, 5 µg of Annexin2 (1-13) as indicated in the figure legends or with cellular extracts. For GST pull-down, cell extracts from confluent MDCK cells were prepared essentially as described for cell fractionation, and the supernatant of cells lysed in Buffer L in the presence of EGTA was used. After incubation for 1 h at 4°Cin a 300-µl final volume of buffer B, the beads were spun down at 2000 x g for 2 min, washed three times with 0.5 ml of buffer B, and boiled in SDS sample buffer. Proteins were separated on 11% Tris-Tricine acrylamide gel electrophoresis, transferred to nitrocellulose, and detected by Western blot using the ECL kit.

Surface Plasmon Resonance—Real-time binding experiments were performed on a BIAcore biosensor system (Pharmacia Biosensor AB, Uppsala, Sweden). All experiments were performed at 25 °C. Anti-GST antibodies in 10 mM sodium acetate (pH 3.5), were coupled directly through their amino groups to the sensor (CM5) surface activated by N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl)carbodiimide according to the manufacturer's instructions. The remaining reactive groups were then inactivated with 1 mM ethanolamine. GST or GST-A2tBP1 binding to the antibodies, and interaction experiments with native purified A2t were done in running buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, and 0.01% Triton X-100. Sensorgrams obtained with different concentrations of A2t (297, 595, and 1190 nM) were analyzed using the Kaleidagraph software. The association and dissociation curves were fitted with an equation model of one analyte (A2t) binding two heterogenous ligands (GST-A2tBP1). In Equation 1,

Formula 1(Eq. 1)
R is the response, subscript 1 and 2 refer to each of the two ligands, t is the time (s), ks = kaCn + kd where Cn is the concentration of the analyte, and Req is the steady state response level. The correlation coefficient of the fitting, close to 1 (between 0.99485 and 0.99996) indicated the quality of the fitting. A simple explanation for this model with two ligands is that once A2t, which is a large complex (94 kDa), is bound to one of the two A2tBP1 sites on the dimer (because of GST dimerization, Ref. 36) of GST-A2tBP1, the steric constraint perturbs the binding of another A2t to the second GST-A2tBP1 site. This is in agreement with the binding stoichiometry found at steady state (up to 1.5 A2t per GST-A2tBP1 dimer at the higher concentration of the analyte tested (1.19 µM)). For the evaluation of the Kd of A2t with AtBP1, only the high affinity responses were considered.

Yeast Triple Hybrid—pLex-Ahnak-Cter and pAct2-S100A10 constructs were obtained as previously described (33). For expression of Annexin2 in yeast, a {Delta}lexA version of pEG202 vector was first obtained. For this purpose, the SphI-SphI fragment of pEG202 containing the lexA sequence was subcloned into a pGEMT vector, where the NotI-NotI fragment was previously deleted. The lexA sequence was then excised out by digesting with HindIII and EcoRI, blunting with Deep vent polymerase, and self-ligation. The {Delta}lexA SphI fragment was subcloned back into pEG202 at the SphI site. Annexin2 from pGEMTp36 (generous gift from Dr. Borsotto) was digested with and cloned into the NotI site of pEG202{Delta}lexA. Constructs in different combination as indicated in the figure legends were used to transform the AMR70 yeast strain. pAct2, pEG202{Delta}LexA, and pLex-lamin were used as control. Colonies were grown into the selective media (uracil-depleted YPD medium minus tryptophan (for pLex10), leucine (for pAct2), and histidine (for pEG202{Delta}lexA)). For beta-galactosidase assays, the activity of three independent colonies from each transformation was measured in triplicate as previously described (37).


Figure 1
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  		FIGURE 1.
Tetramerization of S100A10 and Annexin2 is required for tight interaction with the Ahnak C-terminal domain. A, in yeast, the weak Ahnak-Cter/S100A10 interaction is drastically stimulated in the presence of Annexin2. Yeasts were co-transfected with pLex-Ahnak-Cter, pAct-S100A10, or/and pEG202{Delta}LexA-Annexin2 vectors as indicated. pLex-Lamin (lanes 2, 3, and 6), pAct (lanes 1, 3, and 5), and pEG202{Delta}LexA (lanes 1, 2, and 4) were used as negative control (-). The interactions were scored by beta-galactosidase activity assays. Specific activities were obtained from three independent clones tested in triplicate (n = 9). B, Annexin2/S100A10 binding motif is involved in A2t/Ahnak C-ter interaction. Native A2t, isolated S100A10, Annexin2, and/or synthetic N-acetylated Annexin2 (1-13) peptide, were mixed as indicated with GST-Ahnak-Cter bound to glutathione-Sepharose. After washing, bound proteins were resolved by SDS-PAGE and detected by Western blot.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In a former study, we had shown that S100A10 mediates the interaction between A2t and the C-terminal domain of Ahnak (Ahnak C-ter) (33). However, the implication of Annexin2 in this interaction has not been investigated. To test whether Annexin2 is also involved in the interaction with Ahnak-Cter, we carried out yeast triple-hybrid experiments with the three components of the A2t-Ahnak complex. Because the N-terminal extremity of Annexin2 is crucial for its tetramerization with S100A10, we chose not to use it as a fusion protein. Instead, we expressed Annexin2 as a free molecule in addition to LexA-Ahnak-Cter and Gal4-S100A10 (33). By doing so, it was possible to analyze in yeast the effect of Annexin2 on Ahnak-Cter/S100A10 interaction. The interactions between Ahnak-Cter and S100A10 were then assessed in a quantitative manner through measurement of beta-galactosidase activity. Results presented in Fig. 1A show that a subtle but detectable interaction is observed between S100A10 and Ahnak-Cter when they are expressed in the absence of Annexin2. However, when Annexin2 is co-expressed with LexA-Ahnak-Cter and Gal4-S100A10, the specific activity is dramatically increased by a factor of more than 150. No effect was seen when Annexin2 was expressed alone or solely with LexA-Ahnak-Cter or Gal4-S100A10. These results indicate that in eukaryotic cells, the presence of Annexin2 and thus, most likely, the formation of the tetramer, is required for efficient S100A10 interaction with Ahnak-Cter. In this complex, the interaction between S100A10 and Ahnak-Cter would be strengthened by Annexin2 through either direct interaction of Annexin2 with Ahnak-Cter or by modulation of the S100A10 affinity for Ahnak.

To investigate whether Annexin2 could directly interact with Ahnak-Cter and to further characterize the A2t interaction with Ahnak, we performed in vitro binding experiments. The C-terminal domain of Ahnak was produced as a GST-tagged protein and used in a pull-down assay. S100A10 and Annexin2 subunits isolated from pig intestine (see "Experimental Procedures") were added either individually or in combination in the binding assay. Under the conditions of the assay, no interaction of Ahnak with S100A10 nor with Annexin2 was detectable (Fig. 1B, lanes 1 and 2). As expected from the triple hybrid data, when S100A10 and Annexin2 are mixed together they readily bind to Ahnak-Cter (lane 3). To investigate whether the full-length Annexin2 protein or solely the S100A10 binding site on Annexin2 influences the binding of A2t to Ahnak, an N-acetylated Annexin2 peptide (amino acids 1-13) harboring the entire S100A10 binding site was tested in the assay (19). As shown in Fig. 1B (lane 5), the peptide mimicks the action of the full-length protein in allowing complex formation between S100A10 and Ahnak-Cter. We have verified the specificity of the peptide action through integration within A2t by testing its ability to compete with full-length Annexin2. When the peptide was added in the assay in the presence of S100A10 and in excess as compared with Annexin2, Annexin2 was displaced from the complex (Fig. 1B, upper lane 4) although S100A10 is still present (Fig. 1B, bottom lane 4). Noticeably, a stronger interaction of S100A10 with Ahnak-Cter was detected in the presence of Annexin2 peptide than in the presence of full-length Annexin2 (compare lanes 3 and 4). This observation suggests that in A2t, it is the S100A10 binding domain of Annexin2 that stimulates the interaction with Ahnak. However, because Annexin2 and S100A10 used in this experiment were subjected to a denaturation/renaturation protocol (see "Experimental Procedures"), we could not exclude the possibility of an artifact caused by improper refolding. We therefore performed the experiment with the native A2t complex. Here again, the Annexin2 peptide by dissociating the complex and replacing Annexin2, induces S100A10 binding to Ahnak (Fig. 1B, lanes 6 and 7). Altogether, these data indicate that, by binding to S100A10, Annexin2 creates conditions for a strong S100A10/Ahnak interaction. We can envision that this effect is due either to a change in the S100A10 conformation or to the formation of a binding platform at the interface between S100A10 and the Annexin2 N-terminal peptide.

To map the A2t binding domain on Ahnak-Cter, we compared the interaction of A2t with a series of GST-Ahnak-Cter deletion mutants by pull-down assays (Fig. 2A). Using this strategy, we mapped the A2t binding sequence in Ahnak-Cter to a 20-amino acid peptide comprising residues 5645-5673 (Fig. 2A). When further deletions of 5 or 9 residues from the N- or C-terminal extremity, respectively, were made, the peptide lost its binding properties, indicating that these residues are crucial for the interaction. This 20-amino acid peptide that binds A2t will hereafter be referred to as A2tBP1 (for A2t Binding Peptide 1). GST pull-down assay, using cytoplasmic extracts of MDCK cells metabolically labeled with [35S]methionine/cysteine, showed that A2tBP1 is equivalent to Ahnak-Cter for its ability to bind A2t (Fig. 2B, left panel, lanes 3 and 4). Annexin2 and S100A10 whose identity was assessed by Western blot bound specifically and to a similar extent to both GST fusion constructs (Fig. 2B, right panel). Furthermore, a synthetic A2tBP1 readily antagonized the interaction between GST-Ahnak-Cter and the A2t (Fig. 2C), confirming that A2tBP1 constitutes the entire A2t binding domain of GST-Ahnak-Cter. More importantly, Fig. 2B (left panel) indicates that Annexin2 and S100A10 are the major if not the only proteins pulled-down by GST-Ahnak-Cter and GST-A2tBP1. Using GST-A2tBP1 bound to a biosensor chip via anti-GST antibodies, we next analyzed the kinetics of binding of purified native A2t complex to GST-A2tBP1 (Fig. 2D). The sensorgram shows a fast binding and a slow release kinetics indicating a high affinity between GST-A2tBP1 and A2t. Indeed, the ka and kd derived from three different curves ranged from 5 to 16 x 104 (ka) and 2.3 to 2.6 x 10-3 (kd), which gives a Kd of 3(±1.5) x 10-8 (see "Experimental Procedures"). The high affinity of A2t binding to A2tBP1 with a kd of 2.4 x 10-3 is consistent with the observation that, while the A2tBP1 synthetic peptide efficiently competed with Ahnak-Cter for binding to A2t (Fig. 2C), it only weakly displaced A2t from GST-Ahnak-Cter/A2t or GST-A2tBP1/A2t preformed complex (data not shown). To conclude, we have identified the minimal domain in the C-terminal domain of Ahnak that binds specifically and with a high affinity to the A2t complex.

To identify other A2t-specific binding partners, we searched in data banks for proteins containing motifs homologous to A2tBP1. Surprisingly, in addition to Ahnak A2tBP1 from other mammalian species, only two peptides both belonging to Ahnak molecules showed significant homology with A2tBP1. The A2tBP1 sequence is strictly conserved in the C-terminal domain of all mammalian Ahnak for which sequences are available, indicating that this sequence is probably necessary for the function of the molecule. The two other sequences homologous to A2tBP1 are located at two different sites within the N-terminal domain of Ahnak. In human Ahnak, the first peptide corresponding to residues 302-321 shows 65% identity/75% homology with the A2tBP1 peptide (Fig. 3A, upper lanes). Its conservation among mammalian species ranges from 65% identity (Rattus versus Canis) to 90% identity (human versus Taurus). The second peptide localized at position 426-447 of human Ahnak harbors 50% identity/65% homology with A2tBP1 (Fig. 3A, bottom lanes). Among mammalian Ahnak proteins, this domain is also well conserved when compared with the human sequence with 68% identity/78% homology to 81% identity/95% homology for the Rattus and the Canis sequences, respectively. These homologies with A2tBP1 and conservation during evolution prompted us to investigate by GST pull-down assays using MDCK cell extracts, whether these two peptide motifs could interact with A2t. As shown in Fig. 3B, the C-terminal domain and A2tBP1 interacted significantly with A2t in these assays (lanes 5 and 6; see also Fig. 1). The N-terminal domain containing the two putative binding sequences also interacted with A2t (lane 3) even though its affinity for A2t seemed lower than that of the C-terminal domain. The peptide corresponding to residues 302-321 of human Ahnak bound to A2t to a similar extent as the N-terminal domain from which it is derived (lane 7). In contrast, the peptide 426-447 of human Ahnak did not interact detectably with A2t (lane 8). GST alone and the median domain (M) of Ahnak containing 4 Ahnak internal repeats (38), used as controls, did not show any interaction with A2t. Taken together, these data indicate the existence, in Ahnak, of a second sequence closely related to A2tBP1 and capable of interacting with A2t, albeit with a slightly less avidity than A2tBP1. This A2t binding domain was called A2tBP2.


Figure 2
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  		FIGURE 2.
Identification of a 20-amino acid motif on Ahnak C-ter specific for S100A10/Annexin2 complex. A, mapping of a 20-amino acid motif from Ahnak C-ter that interacts with the A2t complex. GST-Ahnak C-terminal domain or deletion mutants (as indicated) were bound to glutathione-Sepharose and incubated with MDCK cell extract. After pull-down, and washing, bound Annexin2 and S100A10 proteins were resolved by SDS-PAGE and detected by Western blot. The presence of bound Annexin2 and S100A10 detected by Western blot is indicated on the right and by a gray box on the schematic representation of the Ahnak. Amino acid positions of the fragments within the Ahnak full-length sequence is indicated on the left. The sequence of the smallest fragment competent for Annexin2/S100A10 binding corresponding to Ahnak-(5645-5673) is presented on the bottom. Bold letters indicate residues whose deletion impaired Annexin2/S100A10 binding. B, Annexin2 and S100A10 are specific Ahnak C-ter and A2tBP1 targets. [35S]methionine/cysteine-labeled MDCK whole cell extracts (lane 1) were incubated with GST (lane 2) and GST-A2tBP1 (lane 3) or GST-Ahnak C-ter (lane 4) bound to glutathione-Sepharose. Interacting proteins were resolved on SDS-PAGE and detected by autoradiography (left panel) or Western blot using anti-Annexin2 and anti-S100A10 monoclonal antibodies (right panel). C, A2t binding strength of A2tBP1 is comparable to that of Ahnak C-ter. MDCK cell extracts were incubated with GST fusion Ahnak C-ter in the absence or in the presence of increasing A2tBP1concentrations. Bound proteins were analyzed by Western blotting using anti-Annexin 2 and anti-S100A10 antibodies. The molar A2tBP1/Ahnak C-ter ratio is indicated. D, real-time surface plasmon resonance recording. The association curve (light box) of the sensorgrams were recorded after injection (light arrowhead) of A2t (595 nM) onto GST-A2tBP1 (upper curve) or GST (lower curve) immobilized on the sensor chip. After 10 min, A2t injection was stopped (dark arrowhead), and the dissociation curve was recorded (dark box).

 


Figure 3
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  		FIGURE 3.
Identification of a second A2t binding motif in the Ahnak protein. A, comparison of A2tBP1 with Ahnak residues 302-321 (upper lanes) and 426-447 (bottom lanes). Dark gray and light gray indicate identical and homologue residues, respectively. B, GST-Ahnak N-terminal domain and GST-Ahnak residues 302-321 bind to S100A10 and Annexin2. GST alone (lane 2) or fused to the N terminus (lane 3), part of the median domain (M, lane 4), the C-terminal domain (lane 5), A2tBP1 (lane 6), Ahnak-(302-321) (lane 7), and Ahnak-(426-447) (lane 8) were used in the pull-down assay with MDCK cell extracts. Bound S100A10 and Annexin 2 were detected by Western blot.

 
To confirm that in eukaryotic cells, A2tBPs interact with A2t, the peptide-coding sequences were cloned in-fusion with the EGFP gene, and stable transformants of MDCK cells were obtained. Because A2t has been shown to interact with the membranous fraction in a calcium-dependent manner, the cells were lysed either in the presence of calcium or in the presence of EGTA. Cellular debris including membranes were pelleted, and partitioning of A2t subunits and A2tBPs into the pellet and in the supernatant was analyzed by Western blot. Results presented in Fig. 4A show that as expected, Annexin2 and S100A10 are essentially present within the pelletable fraction in the presence of calcium (compare upper lanes 1 to 2 and 3 to 4). In contrast, in the presence of EGTA, both were found in the supernatant (lanes 2 and 4) and not in the pellet (lanes 1 and 3). When we analyzed the fractionation of EGFP fusion peptides, we observed that A2tBP1 and to a lesser extent A2tBP2 promote a calcium-dependent association of EGFP with the pelletable fraction. Whereas EGFP alone distributed into the supernatant fraction independently of the presence of calcium (lanes 5 and 6), A2tBP1 and 2 fused to EGFP were found in the pelletable fraction in the presence of calcium (compare bottom lanes 7 to 10). A2tBP2 showed a less prominent recruitment compared with EGFP-A2tBP1. These data indicate that calcium promotes a recruitment of Annexin2, S100A10, A2tBP1, and to a lesser extent A2tBP2 to insoluble cellular components. Because this effect has already been shown for A2t (39) and given the binding specificity and affinity of A2tBP1 for A2t, we can assume that in these experiments, A2tBPs fractionation is dependent on their interaction with A2t. We then carried out co-immunoprecipitation experiments to confirm the in vivo interaction of A2t with A2tBPs in MDCK cells (Fig. 4B). For this purpose, we used protein fractions obtained under the same (Ca2+/EGTA) conditions as described above. Fig. 4B shows that Annexin2 and S100A10 present in the calcium pellet (Fig. 4A, lanes 1 and 3) or in the EGTA supernatant (Fig. 4A, lanes 2 and 4) co-immunoprecipitated with EGFP-A2tBP1 (Fig. 4B, lanes 7 and 8) and to a lesser extent with EGFP-A2tBP2 (Fig. 4B, lanes 9 and 10). As expected, Annexin2 and S100A10 were not found associated to immunoprecipitated EGFP (Fig. 4A, lane 6) present in the EGTA supernatant (Fig. 4B, lane 2). Together, these data indicate that in MDCK cells, overexpressed A2tBP1 is associated in a tight complex with A2t.


Figure 4
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  		FIGURE 4.
EGFP-A2tBP1 interacts with S100A10 and Annexin2 in MDCK cells. A, EGFP-A2tBP1 expressed in MDCK cells co-fractionate in a calcium-dependent manner with Annexin2 and S100A10. MDCK cells stably transformed with EGFP (lanes 1-6), EGFP-A2tBP1 (lanes 7 and 8), or EGFP-A2tBP2 (lanes 9 and 10) were lysed in the presence of 0,3 mM Ca2+ (upper panel) or 1 mM EGTA (lower panel). Lysates were spun down, and pellets (P) and supernatants (S) were analyzed by Western blot using anti-Annexin2 (lanes 1 and 2), anti-S100A10 (lanes 3 and 4), or anti-EGFP (lanes 5-10). B, EGFP-A2tBPI co-immunoprecipitates with Annexin2 and S100A10. Calcium concentration of the pellets (lanes 1, 3, 5, and 7) and supernatants (lanes 2, 4, 6, and 8) from A were adjusted to 0.3 mM. EGFP (lanes 1 and 2), EGFP-A2tPB1 (lanes 3 and 4), and EGFP-A2tBP2 (lanes 5 and 6) were immunoprecipitated using anti-EGFP antibodies, and the presence of co-immunoprecipitated Annexin2 (two upper panels) and S100A10 (two bottom panels) was analyzed by Western blot.

 


Figure 5
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  		FIGURE 5.
Annexin2 and A2tBP1 re-localize from the cytosol to the plasma membrane in response to cellular stress. MDCK cells stably transfected with EGFP, Annexin2-EGFP, EGFP-A2tBP1 and 2 as indicated were either treated with 1 mM H2O2 or scraped (Scrap) with a fine tip pastette (dotted line). EGFP fluorescence of living cells was recorded after 5 min with a confocal microscope. Scale bars indicated in the bottom panels are of 100 micrometers in control and H2O2 treatments and 50 micrometers for the scraping experiments.

 
To test whether A2tBP1 could be used to trace A2t within cells, we carried out live fluorescence analysis of EGFP-A2tBPs and Annexin2-EGFP overexpressed in MDCK (Fig. 5). As previously described, EGFP alone was localized throughout the cells with an accumulation within the nucleus (panel A). When EGFP was fused to Annexin2, its localization became essentially cytoplasmic (panel D). A2tBPs also targeted EGFP to the cytoplasm (panels G and J) as expected if they were to interact in the cytoplasm with A2t. Annexin2 was shown to be relocalized in response to oxidative stress (40). We therefore tested whether Annexin2-EGFP and EGFP-A2tBPs localization under our cellular conditions would change in response to H2O2 treatment. As shown in panels E, H, and K, H2O2 treatment led to a fast re-localization to the plasma membrane of Annexin2-EGFP and EGFP-A2tBP1 and 2, respectively. Localization changes were observable after 2 min of treatment (data not shown) and completed after 5 min. These modifications of intracellular localization were entirely reversible when transferring the cells back to fresh culture medium (data not shown). We notice that in agreement with its weaker interaction with A2t, A2tBP2 was less efficient than A2tBP1 in targeting EGFP to the cytoplasm in resting cells (compare panels G and J) and to the plasma membrane of H2O2-treated cells (compare panels H and K). The contrast in EGFP fluorescence between the nucleus and the cytoplasm under both conditions and between the cytoplasm and the plasma membrane in treated cells is stronger with EGFP-A2tBP1 than with EGFP-A2tBP2. We also tested the response of the EGFP fusion proteins to a mechanical stress induced by wounding of a cell monolayer. Here again, Annexin2-EGFP, EGFP-A2tBP1, and EGFP-A2tBP2 within the cells close to the wound re-localized to the plasma membrane during the few minutes required for the observation (panels F, I, and L, respectively). Neither H2O2 nor wounding had any effect on GFP cellular distribution. It is interesting to note that these relocalization events are detectable only in living cells since after fixation and immunodetection, EGFP-A2tBP1, Annexin2-EGFP, and endogenous Annexin2 are essentially detected at the plasma membrane in resting cells (data not shown). Together, these data indicate that EGFP-A2tBP1 expressed in MDCK follows the Annexin2-EGFP localization. Given our previous data indicating that A2tBP1 binding is specific for A2t, we can assume that EGFP-A2tBP1 localization is dependent on and reflects the endogenous A2t localization.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this article, we have shown that efficient S100A10 interaction with Ahnak requires its tetramerization with Annexin2. The fact that the Annexin2 contribution localizes to the first 13 amino acids of Annexin2 corresponding to its S100A10 binding site suggests that it might act by changing the conformation of S100A10 and thereby increasing S100A10 affinity for Ahnak. The crystal structure of the S100A10 dimer alone or in complex with the Annexin2 binding site has been solved (20). S100A10, as the other members of the S100 proteins, is composed of two EF-hand motifs linked by a flexible loop. In the S100A10 dimer, the flexible loop (L2) of each subunit adopts a different conformation leading to an asymmetric structure. In the complex, the two S100A10 molecules adopt the same conformation in which the flexible loops form short stretch of {alpha}-helices implicated in part in binding to the Annexin2 peptide. Such a conformational change could explain the increase in the interaction of the Annexin2 peptide-S100A10 complex with Ahnak. However, when bound to S100A10, the Annexin2 peptide is exposed to the surface of the complex. We thus cannot exclude that it may form together with S100A10 a platform in which each subunit would participate to Ahnak binding. A2t formed with full-length Annexin2 is less efficient in binding Ahnak than the Annexin2 peptide-S100A10 complex. One can speculate that this difference is caused by steric constraint for the formation of the A2t/Ahnak-Cter complex providing a way for A2t to modulate its binding with its targets through a change in Annexin2 conformation. Crystallographic studies would be useful to reveal the real nature of A2t binding to its targets. Co-crystallization of S100A10 with full-length Annexin2 has not been achieved to date. This might be because of the flexibility between the N-terminal S100A10 binding region and the core domain. In this regard, A2tBP1 might be a powerful tool for a co-crystallization assay with A2t.

A2tBP1 was identified in this report as a twenty amino acid peptide in AhnakC-ter that mediates the interaction with A2t. A2tBP1 is the first binding motif specific for the tetramer characterized to date. This peptide is 100% conserved among different mammalian Ahnak species, indicating that it is likely to be involved in key functions. Because the only cellular binding partners detected for A2tBP1 are the A2t subunits, we propose that Ahnak binding to A2t is required for crucial cellular functions probably including the targeting of Ahnak to cholesterol-rich membrane domains and the establishment of cell polarity (33). Surprisingly, the only two motifs found in data banks as homologs of A2tBP1 are in the Ahnak molecule itself. No homology was found when comparing A2tBP1 to known S100A10 or Annexin2 targets (data not shown) suggesting that they probably do not share the same mode of interaction with A2t. Among the two A2tBP1 homolgous peptides that are localized in the N-terminal domain of Ahnak, only one, named A2tBP2 interacts in vitro with A2t, albeit with a lower apparent binding affinity for A2t than A2tBP1. Unfortunately, sequence comparison could not clearly point out specific residues of A2tBP1 conserved in A2tBP2 and absent from the third non-A2t-interacting homologue (data not shown). Identification of A2tBP1 residues involved in the interaction with A2t will require mutagenesis or structural investigations.

We have shown that A2tBP1 binds specifically and with high affinity to the A2t complex but not to the individual S100A10 or Annexin2 subunits. A2t was the only detectable A2tBP1 cellular binding partner in GST pull-down assays using metabolically labeled cell extracts. In addition, EGFP-A2tBP1 co-fractionated in a calcium-dependent manner and co-immunoprecipitated with S100A10 and Annexin2. These data suggest that EGFP-A2tBP1 could be used to follow A2t localization in living cells. Indeed, EGFP-A2tBP1 localization is identical to that of Annexin2-EGFP in resting cells and changes with the same dynamics as Annexin2-EGFP upon cellular stress. Given our previous data, we can reasonably assume that it is the interaction of A2tBP1 with endogenous A2t that is responsible for EGFP-A2tBP1 localization in cells. This statement is strengthened by the observation that A2tBP2, which has a reduced binding affinity to A2t, is also less efficient in targeting EGFP to sites of Annexin2 localization.

Because, A2t is thought to be involved in membrane trafficking/remodeling and is likely to associate with various cellular components including small organelles (5-9,41), the identification of its cellular location is of major importance to study its mechanisms of action. Carrying out live imaging experiments will be required to study dynamic events in which endogenous A2t takes part and to avoid artifacts caused by immunodetection techniques. We believe that EGFP-A2tBP1 might constitute a critical tool to dissect functions strictly involving the complex and not the individual subunits. In this respect, our live experiments strongly suggest that A2t complex exists in the cytoplasm of resting cells and that its plasma membrane localization relies on cellular signaling.

The subunit of A2t, which is implicated in membrane binding, may depend on the cellular context. It has been shown in chromaffin cells that p11 is constitutively expressed at the subplasmalemmal region of the cell and that Annexin2 plasma membrane localization relies on nicotinic activation and calcium rise (42). In huvec, S100A10 is required for cell surface translocation of Annexin2 upon temperature stress (43). In contrast, in living HepG2 cells, Annexin2 is the subunit that mediates A2t complex binding to the plasma membrane (44).

Several mechanisms can be envisioned to participate in the stress-induced A2t targeting to the plasma membrane. On the onehand,becauseA2tinteractionwiththemembraneiscalcium-dependent and because both H2O2 treatment (45) and cellular wounding (46) can induce a rapid increase of intracellular calcium, one can speculate that the observed membrane localization of A2t is caused by transient calcium increase. On the other hand, oxidization treatment as well as wounding (47) may induce alteration of some membrane lipids such as peroxidation (48) that would promote binding of A2t. Alternatively, stress-induced A2t membrane localization could involve direct modification of Annexin2 such as tyrosine phosphorylation (43).


   FOOTNOTES
 
* This study has been supported by Grant 5643 from the Association pour la Recherche sur le Cancer (ARC) (to C. D.) and Grant 11597 from the Association Française contre les Myopathies (AFM). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: INSERM EMI 0104/TS-DRDC, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. Tel.: 33-4-38-78-47-27; Fax: 33-4-38-78-50-58; E-mail: jbaudier{at}cea.fr. 2 To whom correspondence may be addressed: INSERM EMI 0104/TS-DRDC, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. Tel.: 33-4-38-78-47-27; Fax: 33-4-38-78-50-58; E-mail: cdelphin{at}cea.fr.

3 The abbreviations used are: A2t, Annexin2 tetramer; A2tBP, A2t-binding peptide; GST, glutathione S-transferase; MDCK, Madin-Darby canine kidney; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DTT, dithiothreitol; mAb, monoclonal antibody; EGFP, enhanced green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Zhuxiang Nie and Takashi Hashimoto for the pC-DY plasmid, Dr. Marc Borsotto for the pGEMTp36 plasmid, and Dr. Stephen Moss for the Annexin2-EGFP plasmid. We are indebted to Dr. Gerard Klein for the analysis of real-time plasmon resonance data. We gratefully acknowledge Dr. Jean-Christophe Deloulme and Dr. Laurence Aubry for critical analysis of the manuscript.



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 RESULTS
 DISCUSSION
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