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Regulation of Calcyclin (S100A6) Binding by Alternative Splicing in the N-terminal Regulatory Domain of Annexin XI Isoforms*

Annexin XI is a Ca2+/phospholipid-binding protein that interacts with a member of S100 protein family, calcyclin (S100A6), in a Ca2+-dependent manner. There are two isoforms of annexin XI, annexin XI-A and -B, generated by alternative splicing in the N-terminal regulatory domain. To determine the role of the alternative splicing region in the calcyclin-binding, we identified and characterized its calcyclin binding site. Experiments with glutathioneS-transferase fusion proteins with N-terminal sites of annexin XI-A showed the calcyclin binding site to be in residues Gln49-Thr62 of rabbit annexin XI-A, which contains part of the splicing region. A synthesized peptide corresponding to Tyr43-Thr62 of annexin XI-A inhibited the interaction of annexin XI with calcyclin in liposome co-pelleting assay. The calcyclin binding site possesses a hydrophobic residue cluster conserved among S100 binding sites of annexin I and II. Recombinant annexin XI isoforms were expressed in Sf9 cells using a baculovirus expression system. In contrast to annexin XI-A, it was found that annexin XI-B protein could not bind to calcyclin by the liposome co-pelleting assay. In Sf9 cells coexpressing calcyclin with annexin XI isoforms, the calcyclin binding was observed only for annexin XI-A isoform. These results indicate that the calcyclin binding ability of annexin XI is an annexin XI-A isoform-specific character, suggesting that annexin XI isoforms might play distinct roles in cells through each alternative splicing regions.

Annexins are Ca 2ϩ /phospholipid-binding proteins that are present in a wide variety of cells and tissues. They form a structurally related family, all members consisting of a Cterminal core domain with either four or eight repeating units of about 70 amino acids and an individually unique N-terminal tail domain. Due to the Ca 2ϩ /phospholipid binding ability through the core domain, it has been shown that annexins can bind to biomembranes in a Ca 2ϩ -dependent manner and aggregate and fuse them (1)(2)(3)(4). Therefore, it has been suggested that annexins participate in a number of cellular functions including exocytosis, endocytosis, blood coagulation, regulation of phospholipase A2 activity, cell proliferation, and inflammatory responses (5). However, the exact functions of individual annexin forms remain to be determined.
To elucidate the physiological functions of annexins, their binding proteins have been investigated. It has been established, for example, that synapsin I and calspectin bind to annexin VI, whereas plasminogen and tissue plasminogen activator interact with annexin II (6). Among the various binding proteins, three members of the S100 protein family, calgizzarin (S100C), p11 (S100A10), and calcyclin (S100A6) specifically interact with annexin I, II, and XI, respectively (7)(8)(9). The S100 protein family are Ca 2ϩ -binding proteins with 2 EF-hand Ca 2ϩ binding structures. Since p11 forms heterotetramers with annexin II and affects its subcellular localization and affinity for Ca 2ϩ (10 -12), annexin-S100 protein interactions have been suggested to play important roles in intracellular functions of annexin family members. However, compared with annexin II, there is less accumulation of biochemical and biophysical information for the interaction of S100 proteins with annexin I and XI. We discovered annexin XI as a calcyclin-associated protein (CAP-50) in rabbit lung (9,13) and subsequently demonstrated that it is widely distributed in various tissues including the lung, heart, aorta, testis, and liver (14). Annexin XI is localized mainly in the nucleus and membranes of 3Y1 and COS-7 cells, whereas it is predominantly found in the cytoplasm of primary cultured hepatocytes (14 -16). The nuclear localization of annexin XI is cell type-specific and developmental-dependent (17). Since its binding protein calcyclin is overexpressed in tumor cells with the mRNA levels specifically increased in the G 1 phase of the cell cycle when stimulated by serum, plateletderived growth factor, or epidermal growth factor (18,19), it is possible that the annexin XI-calcyclin complex may play a role in cell proliferation and division.
The N-terminal domains of the annexin family are generally considered as regulatory sites, since they contain sequences responsible for phosphorylation, proteolysis, and interaction with other proteins including S100 proteins described above. Annexin XI has the longest N-terminal domain, composed of 197 amino acids, of all known annexins (20). There are two isoforms, annexin XI-A and annexin XI-B, generated by alternative splicing in the N-terminal regulatory domain of identical primary transcripts (21). The primary isoform of annexin XI has been named annexin XI-A, and the new isoform is termed annexin XI-B (20 -23). We previously reported that the region encompassing residues Tyr 27 -Leu 52 of the N-terminal regulatory domain of rabbit annexin XI-A is critical for its calcyclin interaction based on a truncated mutant study (24). Although the exact binding site was not defined, the critical region is located in the alternative splicing region of annexin XI isoforms. In addition, the N-terminal domain of annexin XI is essential for its nuclear localization (16). Therefore, it is important to determine the exact nature of the calcyclin binding site.
In the present study, the calcyclin binding site of annexin XI-A was identified and shown to possess a hydrophobic residue cluster conserved among S100 binding sites of annexins.
We also demonstrated that the calcyclin binding of annexin XI was an annexin XI-A isoform-specific character, and annexin XI-B had no binding ability. These findings suggest that the alternative splicing-derived annexin XI isoforms play distinct roles in cell function.

EXPERIMENTAL PROCEDURES
Materials-Restriction endonucleases and DNA-modifying enzymes were from Takara Shuzo, Boehringer Mannheim Biochemica, New England Biolabs, and Fermentas MBI. Spodoptera frugiperda Sf9 cells, baculovirus transfer vector pVL1392, pBlueBacIII, baculovirus AcM-NPV DNA, and Grace's insect cell culture medium were purchased from Invitrogen. Baculovirus transfer vector pFastBac1 and DH10Bac competent cells were obtained from Life Technologies, Inc. The pGEX-3X vector, glutathione-Sepharose 4B, CNBr-activated Sepharose, and phenyl-Sepharose CL-4B, Q-Sepharose FF, and S-Sepharose FF were purchased from Pharmacia Biotech Inc. Phospholipids were from Doosan Serdary Research Laboratories, and peroxidase-conjugated anti-rabbit IgG was from MBL. The peptide corresponding Tyr 43 -Thr 62 of rabbit annexin XI-A (AXI 1 43-62 peptide) was synthesized and purchased from Peptide Institute Inc. (Osaka, Japan). All other materials and regents were of the highest quality available from commercial suppliers.
Construction of Annexin XI and Calcyclin Recombinant Baculoviruses-For subcloning of rabbit annexin XI-A cDNA into pBlueBacIII vector, the coding region of rabbit annexin XI-A cDNA in pME18S (16) was amplified by the polymerase chain reaction (PCR) with Pfu DNA polymerase and purified after cutting with BamHI and PstI. The PCR product was verified by DNA sequencing. The employed oligonucleotides corresponding to bases 20 -39 (with the initiation site) and the antisense to bases 1641-1661 (with sequence 3Ј of the termination codon) were synthesized with BamHI and PstI sites on their 5Ј ends, respectively. The sequences of the oligonucleotides used for PCR were as follows: 5Ј-AAGGATCCATGAGCTACCCTGGCTAT-3Ј and 5Ј-AACT-GCAGATTCTTGCGGCCTTTTCCTGT-3Ј. The purified PCR product was introduced into the BamHI/PstI-digested pBlueBacIII plasmid. Recombinant baculovirus of rabbit annexin XI-A was prepared with the pBlueBacIII rabbit annexin XI-A as described previously (25). For subcloning of rabbit calcyclin cDNA into the pFastBac donor plasmid, the coding region of the rabbit calcyclin in the pGEX-3X vector (26) was subcloned into the BamHI/EcoRI sites of the pFastBac donor plasmid. pFastBac rabbit calcyclin was transformed into DH10Bac competent cells for transposition into the bacmid. Bacmid DNA of calcyclin was isolated from the transformed cells was transfected into Sf9 insect cells. High titer recombinant viral stocks were obtained and used for subsequent infection of Sf9 cells. Baculoviruses of bovine annexin XI-A and XI-B were prepared as described previously (25).
Expression and Purification of Recombinant Annexin XI-Purification of recombinant rabbit annexin XI-A and bovine annexin XI-A was performed as detailed earlier (9,25) with slight modifications. Sf9 insect cells (1.6 ϫ 10 6 cells/ml) were infected with recombinant baculovirus at a multiplicity of infection 3 in Grace's medium containing 10% fetal calf serum. Three days after infection, cells were harvested, and cell pellets were obtained by centrifugation at 1000 ϫ g for 15 min. One cell pellet from a 250-ml culture was used per purification. Frozen cell pellets were rapidly thawed in 100 ml of extraction buffer A (40 mM Tris-HCl, pH 7.5, 5 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol (DTT), 1 g/ml leupeptin, 5 g/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride), and after sonicating three times for 1 min on ice, the homogenates were centrifuged at 20,000 ϫ g for 30 min. Solid ammonium sulfate (30% saturation) was added, and the preparations were stirred for 30 min. Pellets then obtained by centrifugation at 10,000 ϫ g for 30 min were dissolved with 50 ml of buffer B (40 mM Tris-HCl, pH 7.5, 2 mM EGTA, 1 mM DTT) and dialyzed against 2 liter of buffer B overnight. After centrifugation of the dialysates at 10,000 ϫ g for 30 min, the supernatants were applied to Q-Sepharose columns that had been equilibrated with buffer B. The void fractions were pooled and precipitated by adding ammonium sulfate (30% saturation) followed by centrifugation at 10,000 ϫ g for 30 min as described above. The precipitates were dissolved in 5 ml of buffer C (50 mM MES, pH 6.0, 10 mM NaCl, 1 mM EGTA, 1 mM DTT) and dialyzed against 2 liters of buffer C. After centrifugation of the dialysates at 10,000 ϫ g for 30 min, the superna-tants were applied to S-Sepharose columns that had been equilibrated with buffer C. Elution was carried out with a 10 -300 mM NaCl linear gradient. The fractions containing annexin XI were collected and dialyzed against buffer D (40 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 0.2 mM CaCl 2 , 1 mM DTT). The dialysate was applied to a calcyclin affinity column that had been equilibrated with buffer D. After washing the column with a high salt buffer C (1 M NaCl) and subsequently buffer D, elution was carried out with buffer E (40 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 2 mM EGTA, 1 mM DTT). The fractions containing purified protein were dialyzed against buffer containing 25 mM Tris-HCl, pH 7.5, and 0.5 mM EGTA and stored at Ϫ80°C. Purification of recombinant bovine annexin XI-B was performed as follows. Frozen pellets of cells expressing recombinant annexin XI-B were prepared with baculovirus of bovine annexin XI-B as described above and rapidly thawed in extraction buffer A. After sonication, the homogenate was centrifuged at 20,000 ϫ g for 30 min. The precipitate was resuspended and incubated with buffer containing 40 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 2 mM EDTA, centrifuged at 20,000 ϫ g for 30 min, resuspended and incubated overnight with buffer containing 2% Triton X-100 and 10 mM EDTA. After extensive washing with the same buffer and centrifugation at 10,000 ϫ g for 20 min, the precipitate was dissolved in buffer containing 30 mM Tris-HCl, pH 7.5, 30 mM NaCl, 1 mM DTT, 1 mM EDTA, 8 M urea and dialyzed against 1 liter of the same buffer with and without 4 M urea. The dialysate was centrifuged at 20,000 ϫ g for 30 min, and the supernatant obtained, containing recombinant annexin XI-B protein, was dialyzed against buffer with 25 mM Tris-HCl, pH 7.5, and 0.5 mM EGTA and stored at Ϫ80°C.
Preparation of Recombinant Calcyclin and Calcyclin Affinity Sepharose-Purification of recombinant rabbit and mouse calcyclin was performed as follows. Frozen pellets of Sf9 cells expressing recombinant rabbit calcyclin were prepared as described above, rapidly thawed in extraction buffer (50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM CaCl 2 , 1 mM phenylmethylsulfonyl fluoride), and sonicated three times for 1 min. The sonicated material was centrifuged at 20,000 ϫ g for 30 min, and the supernatant-containing recombinant calcyclin was applied to a phenyl-Sepharose column. After extensive washing of the column with a high salt extraction buffer (1 M NaCl), calcyclin proteins were eluted with buffer containing 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl and 4 mM EGTA (see Fig. 3C). The pET1120 vector encoding full-length mouse calcyclin was a generous gift from Dr. W. J. Chazin (The Scripps Research Institute, San Diego, CA). This vector was transformed into Escherichia coli BL21 cells to express recombinant proteins. Overnight cultures were grown, and the recombinant proteins were induced with isopropyl-␤-D-thiogalactopyranoside. The cell cultures were spun down, resuspended in extraction buffer, and prepared as described above. Fractions containing calcyclin were collected and dialyzed against H 2 O. For preparation of the calcyclin affinity column, 35 mg of recombinant calcyclin proteins were coupled with CNBr-activated Sepharose 4B (2 g), according to the manufacturer's instructions.
were applied to glutathione-Sepharose columns. After extensive washing of the columns with a high salt resuspension buffer (1 M NaCl) and subsequently resuspension buffer, elution was carried out with resuspension buffer containing 10 mM glutathione. The fractions containing purified protein were dialyzed against resuspension buffer and stored at Ϫ80°C.
Calcyclin Affinity Sepharose Chromatography of Annexin XI Isoforms and GST Fusion Proteins-Binding capacities of annexin XI isoforms and GST fusion proteins for calcyclin were determined by calcyclin affinity Sepharose chromatography. Purified annexin I, XI (18 g), or GST fusion proteins (20 g) were incubated in 40 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 0.2 mM CaCl 2 with 300 l of a 50% slurry of calcyclin-Sepharose. After rotation for 1 h at 4°C, calcyclin-Sepharoses were washed three times in buffer containing 40 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.2 mM CaCl 2 . Bound annexin XI or GST fusion proteins were specifically eluted from the Sepharose beads with buffer containing 40 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM EGTA and analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) followed by Coomassie staining.
Subcellular Fractionation-Three days post-inoculation with recombinant baculovirus of annexin XI isoforms, Sf9 cells were harvested and washed three times in phosphate-buffered saline and resuspended in 40 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin A, 5 g/ml leupeptin. The cell suspensions were lysed by sonicating three times for 30 s and centrifuged at 100,000 ϫ g for 30 min, yielding the soluble cytosol and membrane pellets. Aliquots of 3 mM CaCl 2 or 3 mM EGTA were added immediately before sonication. After resuspension of the membrane pellets in the original volume of homogenate, equal volumes of soluble cytosol and membrane pellet fractions were analyzed by SDS-PAGE followed by immunoblotting.
Coexpression of Annexin XI Isoforms and Calcyclin in Sf9 Cells-Rabbit calcyclin baculovirus was infected into Sf9 cells in 25-cm 2 T flasks with and without baculovirus of bovine annexin XI isoforms. Cell monolayers were harvested by scraping from T flasks 48 h after infection. Cell pellets were obtained by centrifugation at 1000 ϫ g for 5 min, washed with phosphate-buffered saline and resuspended in 40 mM Tris-HCl, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin A, 5 g/ml leupeptin. Subcellular fractionation was performed as described above except for gentle sonication to avoid the proteolysis of calcyclin and annexin XI proteins.
Other Procedures-Liposome co-pelleting assays were performed as described previously (27), and in peptide competition study, 0.3 (3 M)-10 nmol (100 M) of the peptide was incubated with 0.06 nmol (3 g) of annexin XI protein and 0.2 nmol of calcyclin protein. Annexin I protein was purified from bovine lung as described by Glenney et al. (28). Immunoblotting was performed with an anti-bovine annexin XI antibody that can detect both annexin XI isoforms as detailed earlier (14,25).

Identification of Calcyclin Binding Site of Annexin XI-A
previous truncated mutant study indicated that the Tyr 27 -Leu 52 residues of rabbit annexin XI-A are critical for its calcyclin binding (24). To identify the calcyclin binding site of annexin XI, GST fusion proteins with N-terminal sites of rabbit annexin XI-A were constructed and expressed in E. coli. On the basis of our previous study with truncated mutants, we chose 5 sites containing 29 particular amino acid sequences in the region between Pro 29 and Tyr 81 of rabbit annexin XI-A (Fig.  1A). These fusion proteins were examined for calcyclin binding with calcyclin affinity Sepharose. These proteins were incubated with calcyclin-Sepharose in the presence of CaCl 2 . After washing three times with buffer containing 1 M NaCl to exclude nonspecific binding, the protein bound to calcyclin were eluted with buffer containing EGTA (Fig. 1B). Full-length rabbit annexin XI-A protein but not control GST protein bound to calcyclin-Sepharose in a Ca 2ϩ -dependent manner. Three GST fusion proteins, GST-AXI 34 -62, GST-AXI 40 -68, GST-AXI 49 -77, also bound to calcyclin-Sepharose, whereas the two other, GST-AXI 29 -57 and GST-AXI 53-81, did not bind. The common sequence for these demonstrating binding is Gln 49 -Thr 62 of annexin XI-A. This result shows that the calcyclin binding site is residues Gln 49 -Thr 62 of rabbit annexin XI-A.
Effect of Synthesized Peptide Corresponding to Tyr 43 -Thr 62 of Annexin XI on the Annexin XI-Calcyclin Interaction-To confirm the calcyclin binding site of annexin XI obtained with GST fusion proteins with N-terminal sites of annexin XI, the synthesized peptide corresponding to residues Tyr 43 -Thr 62 of annexin XI (AXI 43-62 peptide) was examined to see if it inhibited the annexin XI-calcyclin interaction. Calcyclin was incubated with annexin XI and Ca 2ϩ /phosphatidylserine liposome in the presence or absence of the AXI 43-62 peptide. Almost all calcyclin protein bound to annexin XI and was coprecipitated with annexin XI-phosphatidylserine liposome complex in a Ca 2ϩ -dependent manner in the absence of the peptide (Fig. 2). AXI 43-62 peptide inhibited the binding of calcyclin to annexin XI in a dose-dependent manner. This result supports that the residues Gln 49 -Thr 62 of rabbit annexin XI-A functions as the calcyclin binding site.
Expression and Purification of Annexin XI Isoforms in Bacu-

FIG. 1. Ca 2؉ -dependent calcyclin binding of GST fusion proteins with N-terminal sites of annexin XI-A.
A, schematic representation of GST fusion proteins with N-terminal sites of rabbit annexin XI-A. The numbers above each construct refer to the amino acid positions for rabbit annexin XI-A. B, Ca 2ϩ -dependent interaction of GST-AXI fusion proteins with calcyclin. Each protein was incubated with calcyclin-Sepharose in the presence of 0.2 mM CaCl 2 , respectively. After incubation, the supernatant (unbound fraction, S) was collected. The Sepharose was washed three times and eluted with 5 mM EGTA (bound fraction, P), as described under "Experimental Procedures." Supernatants and EGTA-eluted fractions were subjected to SDS-PAGE (12.5% gel) followed by Coomassie staining. Molecular masses are indicated on the left (kDa).

lovirus/Sf9 Cells-We expressed and purified recombinant annexin XI isoforms in Sf9 cells with recombinant baculoviruses.
Recombinant bovine annexin XI-A was highly expressed as a 54-kDa protein and a 60-kDa form due to a post-translational modification as previously reported (Fig. 3A) (25). Recombinant annexin XI-A proteins were solubilized from Sf9 cells with EGTA-containing buffer and purified by 30% ammonium sulfate precipitation and Q-and S-Sepharose column chromatography. After purification, a major 54-kDa protein and a minor 48-kDa protein appeared on SDS-PAGE. The 54-kDa protein could bind to calcyclin affinity Sepharose in a Ca 2ϩ -dependent manner, but 48-kDa protein could not. Both proteins were detected on Western blots with anti-annexin XI antibody (data not shown). Therefore, the 54-kDa protein was concluded to be the full-length form of annexin XI and the 48-kDa protein is a result of proteolysis during purification, possibly around amino acid position 60 in the N-terminal domain. Recombinant annexin XI-B protein was extracted in 8 M urea-containing buffer from Sf9 cell pellets after extensive washing with buffer containing EGTA and Triton X-100, because it existed in membrane fraction in buffer containing EGTA or detergents such as Triton X-100 (Fig. 3B). After dialysis with EGTA buffer to exclude urea, the annexin XI-B protein was soluble in the buffer and 90% pure.
Calcyclin Binding Ability of Annexin XI Isoforms-Part of the calcyclin binding site of rabbit annexin XI-A shown in Fig.  1 is located within the alternative splicing region of bovine annexin XI-A, a homologue of rabbit annexin XI-A (Fig. 4A). The amino acid sequences in the alternative splicing region of annexin XI isoforms differ considerably. Therefore, it is speculated that calcyclin is not able to bind to annexin XI-B. To examine it, the calcyclin binding abilities of annexin XI isoforms were examined by the liposome co-pelleting assay with phosphatidylserine-containing vesicles. Annexin XI-A, XI-B, and annexin I (S100C-binding protein) proteins were incubated with phosphatidylserine liposome and calcyclin protein in the presence of CaCl 2 . Calcyclin was co-precipitated with annexin XI-A, whereas it was not co-precipitated with annexin XI-B and annexin I and remained in the supernatant (Fig. 4B), indicating that calcyclin formed a complex only with annexin XI-A, not with annexin XI-B and annexin I. The GST fusion protein with Val 52 -Pro 80 of bovine annexin XI-B (GST-AXIB 52-80), which contains the region corresponding to the calcyclin binding site of annexin XI-A, was also examined. GST-AXIB 52-80 did not bind to calcyclin-Sepharose (Fig. 4C). These results demonstrate that annexin XI-B has no calcyclin binding ability and suggest that the alternative splicing of annexin XI isoforms might regulate the function of annexin XI through the calcyclin binding.
Coexpression of Annexin XI Isoforms with Calcyclin in Sf9 Cells-To determine whether annexin XI isoforms form complexes with calcyclin in cells, lysates from normal Sf9 cells, cells expressing calcyclin, and cells coexpressing calcyclin and each annexin XI isoform were subjected to coprecipitation assays with membrane components of Sf9 cells followed by SDS-PAGE to detect calcyclin or immunoblotting to detect annexin XI. In Sf9 cells expressing only recombinant calcyclin, about 50% of the calcyclin existed in the cytosolic fraction under the experimental conditions applied (Fig. 5). When calcyclin was coexpressed with annexin XI-A, almost all was translocated to the precipitated membrane fraction by binding to annexin XI-A in a Ca 2ϩ -dependent manner. However, when coexpressed with annexin XI-B, calcyclin was not translocated, remaining in the cytosolic fraction in both the presence and absence of Ca 2ϩ . This result confirms that the annexin XI-B isoform is not a calcyclin-binding protein. In this subcellular fractionation study, a difference for subcellular distributions of annexin XI isoforms was observed. Annexin XI-A translocated from the cytosolic fraction to the membrane fraction in a Ca 2ϩ -dependent manner (Fig. 5, lower panel). In contrast, annexin XI-B was localized completely in the membrane fraction both in the absence and presence of Ca 2ϩ . This localization was also observed in Sf9 cells expressing only recombinant annexin XI-B (data not shown).

DISCUSSION
In the present study, use of multiple GST fusion proteins with N-terminal sites of rabbit annexin XI-A identified its calcyclin binding site, located within residues Gln 49 -Thr 62 of the N-terminal regulatory domain. Previously, we reported that a truncated form lacking the N-terminal 26 amino acids of annexin XI-A retained calcyclin binding ability, whereas loss of the N-terminal 52 amino acids abolished the interaction (24), suggesting that the region of Tyr 27 -Leu 52 is critical for the calcyclin binding or might be the calcyclin binding site. Between the residues Tyr 27 -Leu 52 of rabbit annexin XI-A and the residues Gln 49 -Thr 62 of the calcyclin binding site presented in this study, the common sequence is Gln 49 -Leu 52 . As shown in Fig. 1B, GST-AXI 49 -77, but not GST-AXI 53-81, could bind to calcyclin. Therefore, it is strongly suggested that the region between Gln 49 -Leu 52 of annexin XI-A is essential for the bind-ing, and the region of Tyr 27 -Asn 48 is not necessarily essential. In addition, it is also suggested that the region of Asn 58 -Thr 62 of annexin XI-A is essential for the binding, since GST-AXI 34 -62, but not GST-AXI 29 -57, could bind to calcyclin. Considering that the binding of annexin XI with calcyclin is mediated by a hydrophobic interaction (26), it is probable that only hydrophobic residues, Leu 52 and Met 59 , within Gln 49 -Leu 52 and Asn 58 -Thr 62 , respectively, may play an important role.
In the members of the annexin family, annexin I and II have been shown to bind to other S100 proteins, S100C (calgizzarin) and p11 (S100A10), by interaction of the 13 and 12 N-terminal amino acids, respectively (29 -32). These are again mediated by hydrophobic-hydrophobic bonding (30,(32)(33)(34). Comparison of the primary structures of the S100 binding sites reveals a conserved amino acid arrangement consisting of four hydrophobic amino acids, residues Leu 52 , Met 55 (Val in bovine), FIG. 4. Ca 2؉ -dependent calcyclin binding of purified recombinant annexin XI isoforms. A, structure of annexin XI and amino acid sequences of the alternative splicing region of annexin XI isoforms (bold letters); residues 19 -55 of rabbit annexin XI-A (20), residues 20 -56 of bovine annexin XI-A, and residues 20 -58 of bovine annexin XI-B (21). B, liposome co-pelleting assay. Purified annexin XI isoforms and annexin I proteins were incubated with calcyclin and liposome in the presence of 0.2 mM CaCl 2 , respectively. After incubation and pelleting by ultracentrifugation, the supernatant (unbound fraction, S) was collected. The pellet (bound fraction, P) was resuspended in sample buffer for SDS-PAGE. Equivalent amounts of each fractions, relative to total volumes, were subjected to SDS-PAGE (10% gel) followed by Coomassie staining. Molecular masses are indicated on the left (kDa). C, calcyclin affinity Sepharose assay. GST-AXIB52-80 fusion protein was incubated with calcyclin-Sepharose in the presence of 0.2 mM CaCl 2 . After incubation, the supernatant (unbound fraction, S) was collected. The Sepharose was washed three times and eluted with 5 mM EGTA (bound fraction, P), as described under "Experimental Procedures." SDS-PAGE was performed as described above. Molecular masses are indicated on the left (kDa). Ala 56 , Met 59 of annexin XI-A, residues Val 4 , Phe 7 , Leu 8 , Ala 11 of annexin I, and residues Val 4 , Ile 7 , Leu 8 , Leu 11 of annexin II (Fig. 6A). CD spectroscopy indicates that the p11 binding site of annexin II forms an amphipathic ␣-helix (29), and Chou-Fasman secondary analysis has predicted that both the S100C binding site of annexin I and the calcyclin binding site of annexin XI presented here form similar amphipathic ␣-helices. In these ␣-helices, the hydrophobic residues described above are aligned on one side, whereas hydrophilic residues are aligned on the opposite side (Fig. 6B). Thus the secondary structures of S100 binding sites appear to be conserved among annexin I, II, and XI. Despite this similarity, annexin XI protein only binds to calcyclin among the S100 protein family including S100C and p11 (27). In addition, calcyclin does not interact with other annexin family proteins (annexin II, V, VI, VII) as described previously (9). In this study, annexin I protein could not bind to calcyclin (Fig. 4B). These suggest that differences of amino acids other than hydrophobic residues in sequences of S100 binding sites or three-dimensional structures of each annexins and S100 proteins might be involved in determining the binding specificity.
Among protein-protein interactions through hydrophobic faces within amphipathic helices, perhaps the best characterized are those of calmodulin with calmodulin-dependent enzymes such as myosin light chain kinase (35,36). Calmodulin, a Ca 2ϩ -binding protein with four EF-hand structures similar to the two EF-hands of S100 proteins, binds to specific peptide regions of many target proteins to regulate their activity in a Ca 2ϩ -dependent manner. Formation of amphipathic ␣-helices has been shown by CD spectroscopy and NMR analysis (36). By analogy it would be expected that the functions of annexins might be affected by binding with S100 proteins. Indeed, the formation of heterotetramers of annexin II and p11 alters the subcellular localization and the affinity for Ca 2ϩ in phospholipid binding and chromaffin granule aggregation of annexin II, which is suggested to play an essential role in catecholamine secretion in chromaffin cells (2, 10 -12, 37). Both annexin I and S100C have been identified as components of the cornified envelope in human epidermal keratinocytes (38). Sites of crosslinkage have been identified at residues within the S100C binding site of annexin I and the Lys 3 and C-terminal residues near the annexin I binding site of S100C. Thus the annexin I-S100C interaction might be required for anchorage to the envelope. Annexin XI was discovered as calcyclin-associated protein (CAP-50) in our laboratory (9,13) and has been shown to localize in the nucleus of continuous cell lines such as 3Y1 and COS-7 cells (16). It has been suggested that calcyclin is involved in cell proliferation and division, since it is overexpressed in tumor cells, and transcription of its mRNA can be induced by serum and growth factors (18,19). Although a nuclear localization signal of annexin XI has still been unidentified, it has been indicated that the nuclear localization of annexin XI is mediated through its N-terminal regulatory domain (16). Since the calcyclin binding site of rabbit annexin XI-A is located at residues Gln 49 -Thr 62 in this domain, it is possible that the nuclear localization of annexin XI might thereby be regulated by its binding of calcyclin.
Annexin XI isoforms are generated by alternative splicing of identical primary transcripts (21). Annexin XI-A has a 37amino acid insert, whereas annexin XI-B has a totally different 39-amino acid insert in the splicing region. As it was speculated that their calcyclin binding ability would differ in view of the results of our previous truncated mutant study (24), it was found that annexin XI-B could not bind to calcyclin by a liposome co-pelleting assay (Fig. 4B). By contrast between the amino acid sequences of annexin XI isoforms, two arrange-FIG. 6. Alignment of amino acid sequences of S100 binding sites of the annexin family. A, the amino acid residues of 49 -62 for rabbit annexin XI-A, 2-14 for human annexin I (31,32), and 2-14 for human annexin II (29) are aligned. Conserved hydrophobic residues among the S100 binding sites of these proteins are enclosed in boxes. B, helical wheel of S100 binding sites of annexin XI, I, and II. Covered areas represent the hydrophobic faces of each amphipathic helices. Conserved hydrophobic residues among the S100 binding sites of these proteins are indicated by open circles. ments consisting of four hydrophobic amino acids shown in the calcyclin binding sites Pro 30 -Asp 43 and Asp 43 -Arg 56 are also found in the alternative splicing region of annexin XI-B (Fig.  4A). However, since annexin XI-B has no calcyclin binding ability, these sites does not function as calcyclin binding sites. In Val 52 -Thr 65 of annexin XI-B, corresponding to the calcyclin binding site of annexin XI-A, the four hydrophobic amino acid arrangement is not conserved (Fig. 4A). Indeed, this region of annexin XI-B could not function as a calcyclin binding site (Fig. 4C, GST-AXIB 52-80).
It was observed that the subcellular distributions of recombinant annexin XI isoforms were distinct in Sf9 cells (Fig. 5). Annexin XI-B existed in the membrane fraction, both in the absence and presence of Ca 2ϩ , in contrast to the Ca 2ϩ -dependent membrane association of annexin XI-A. In hydrophobicity plot analysis with the method by Kyte and Doolittle (39), it was revealed that annexin XI-B possesses a long hydrophobic amino acid region between Leu 33 and Met 62 containing part of the splicing region (Fig. 7). The hydrophobic region consisting of 30 residues is of sufficient length to span the membrane lipid bilayer and to possess a high probability of ␣-helix formation. Such a long hydrophobic site was not found in the splicing region of annexin XI-A. It is possible that annexin XI-B might be an integral membrane protein generated through a hydrophobic interaction and that the alternative splicing region of annexin XI-B might function as the Ca 2ϩ -independent membrane-associating domain, not as a calcyclin binding site. Previously, we have examined the cellular distribution of transfected annexin XI isoforms in COS-7 cells with immunocytofluoresence (25). Endogenous annexin XI is localized in the nucleus of COS-7 cells, some in cytoplasm. Transfected annexin XI-A proteins are mainly localized in nucleus and in cytoplasm with aggregation pattern. Transfected annexin XI-B proteins are mainly distributed in cytoplasm, particularly in intracellular structures likely resembling the endoplasmic reticulum and plasma membrane, suggesting the association of annexin XI-B proteins with endoplasmic reticulum membrane and plasma membrane. Based on their primary structures, this is probably unique within annexin family members. Although several laboratories have proposed the existence of EGTA-resistant membrane-associated annexins, for example, through an N-terminal myristoylation (40 -43), so far there has been no example presented for penetration into the membrane lipid bilayer through hydrophobic insertion in annexin family. Little is known about variation in the biochemical and biophysical characteristics of annexin XI isoforms, although a number of studies have been made of annexin XI. It is difficult to distinguish between the isoforms by biochemical methods such as immunoblotting and immunohistochemistry, because they differ in length by only six nucleotides or two amino acids, and our antibody prepared with bovine annexin XI proteins cross-reacts with both (25). On the basis of a reverse transcription-PCR study of MDBK cells and chondrocytes, it appears likely that annexin XI-A is the major and annexin XI-B is the minor form (21). Further study for the alternative splicing region of annexin XI-B might propose the novel regulation mechanism of annexin function by the N-terminal reguratory domain.
In summary, the present investigation provided evidence that the calcyclin binding site is located within residues Gln 49 -Thr 62 , containing part of the alternative splicing region of rabbit annexin XI-A. This binding site demonstrates a similar structure to S100 protein binding sites of annexin I and II, potentially forming amphipathic ␣-helix. Calcyclin binding ability is a character specific to annexin XI-A isoform. The results thus indicate that alternative splicing gives rise to annexin XI isoforms with distinct cellular roles.