AMY-1, a c-Myc-binding protein, is localized in the mitochondria of sperm by association with S-AKAP84, an anchor protein of cAMP-dependent protein kinase.

We have reported that a novel c-Myc-binding protein, AMY-1 (associate of Myc-1), stimulated the transcription activity of c-Myc. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human HeLa cDNA library, and a clone encoding cAMP-dependent protein kinase anchor protein 149 (AKAP149), was obtained. AMY-1 was found to bind in vitro and in vivo to the regulatory subunit II binding region of AKAP149 and S-AKAP84, a splicing variant of AKAP149 expressed in the testis. AMY-1 was expressed postmeiotically in the testis, as S-AKAP84 was expressed. Furthermore, S-AKAP84 and regulatory subunit II, a regulatory subunit of cAMP-dependent protein kinase, made a ternary complex in cells, and AMY-1 was localized in the mitochondria of HeLa and sperm in association with AKAP149 and S-AKAP84, respectively. These results suggest that AMY-1 plays a role in spermatogenesis.

From the ‡Graduate School of Pharmaceutical Sciences, ¶College of Medical Technology, Hokkaido University, Kita-ku, Sapporo 060-0812 and §CREST, Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan We have reported that a novel c-Myc-binding protein, AMY-1 (associate of Myc-1), stimulated the transcription activity of c-Myc. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human HeLa cDNA library, and a clone encoding cAMP-dependent protein kinase anchor protein 149 (AKAP149), was obtained. AMY-1 was found to bind in vitro and in vivo to the regulatory subunit II binding region of AKAP149 and S-AKAP84, a splicing variant of AKAP149 expressed in the testis. AMY-1 was expressed postmeiotically in the testis, as S-AKAP84 was expressed. Furthermore, S-AKAP84 and regulatory subunit II, a regulatory subunit of cAMP-dependent protein kinase, made a ternary complex in cells, and AMY-1 was localized in the mitochondria of HeLa and sperm in association with AKAP149 and S-AKAP84, respectively. These results suggest that AMY-1 plays a role in spermatogenesis.
We have reported that AMY-1 ((associate of Myc-1)) bound to Myc box II in the N-proximal region of c-Myc, a transcriptional activation region of c-Myc (1), and stimulated E-box-dependent transcription activity of c-Myc (1). Two mRNAs, AMY-1S and AMY-1L, which are derived from the alternative usage of poly(A) adenylation signals, encode the same protein, AMY-1 (1). AMY-1 was found to be translocated from the cytoplasm to the nucleus only during the S phase of the cell cycle along with c-Myc, suggesting that AMY-1 is a co-activator for c-Myc (3). Moreover, AMY-1, in contrast to c-myc, was found to be a stimulating factor for the initial step in the erythrocyte differentiation of human K562 cells (2). These results suggest that AMY-1 is a trigger for K562 cells to differentiate into erythrocyte cells and that AMY-1 has a function independent of or different from c-Myc. The molecular mechanism of AMY-1 function, however, remains to be clarified. To elucidate the functions of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out, and AKAP149, an anchor protein of cAMP-dependent protein kinase (PKA) 1 (3), was identified as an AMY-1-binding protein.
AKAP is a protein that translocates PKA to the specific sites where the individual PKA works. Of the AKAPs, AKAP149 and its splicing variant, S-AKAP84 (4), have been found to anchor PKA to the mitochondria to phosphorylate the target proteins. The results showed that AMY-1 is localized in mitochondria of HeLa and sperm along with AKAP149 and S-AKAP84, respectively, suggesting that the function of AMY-1 is related to spermatogenesis.

EXPERIMENTAL PROCEDURES
Cell Culture-Human HeLa and 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum.
Plasmids-The cDNA containing the full size of AKAP149, pBS-AKAP149, was provided by C. Hanski. For pGLex-AMY-1, AMY-1 cDNA starting from the first ATG was inserted in frame into the EcoRI-XhoI sites of pGLex, a modified version of the LexA-derived bait vector for yeast two-hybrid screening (5). For pcDNA3-F-AMY-1 and pcDNA3-F-S-AKAP84, AMY-1 cDNA and S-AKAP84 cDNA starting from the first ATG were inserted into EcoRI-XhoI sites of pCMV-F, a pcDNA3 containing FLAG tag (6). For pcDNA3-RII␤-HA, RII␤ cDNA was inserted into EcoRI-NotI sites of pcDNA3-HA (6).
Cloning of AMY-1-binding Proteins by a Two-hybrid System-Saccharomyces cerevisiae L40 cells containing the lacZ gene driven by the GAL1 promoter were transformed first with pGLex-AMY-1, which did not activate lacZ transcription by itself.
The transformant cells were subsequently transformed with human HeLa MATCHMAKER cDNA (CLONTECH), a cDNA library expressing the GAL4 activation domain (GALAD) fused to the cDNAs from human HeLa cells. Approximately 5.6 ϫ 10 6 colonies were screened for lacZ expression, and the results indicated the association of a GALADfused protein with LexABD-fused AMY-1. The plasmid DNAs in the lacZ-positive cells were extracted by the procedure described in the protocols from CLONTECH. Nucleotide sequences of the plasmids derived from positive colonies were determined by using an ABI377 or Li-Cor Long Reader 4200 autosequencer.
In Vitro Binding Assay-35 S-Labeled S-AKAP84 was synthesized in vitro using the reticulocyte lysate of the TnT-transcription-translation coupled system (Promega). Labeled proteins were mixed with GST or GST-AMY-1 expressed in and prepared from Escherichia coli at 4°C for 60 min in a buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris (pH 7.5), 0.05% bovine serum albumin, and 0.1% Nonidet P-40. After washing with the same buffer, the bound proteins were separated in a 10% polyacrylamide gel containing SDS and visualized by fluorography.
In Vivo Binding Assay-Ten g of pcDNA3-F-S-AKAP84 together with 10 g of pEF-AMY-1-HA was transfected into human 293T cells 60% confluent in a 10-cm dish by the calcium phosphate precipitation technique (7). Forty-eight h after transfection, the whole cell extract was prepared by the procedure described previously (1). Approximately 500 g of the 293T cell proteins was first immunoprecipitated with a * This work was supported by grants-in-aid from the Ministry of Education, Science, Culture and Sport of Japan. 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.
ʈ To whom correspondence should be addressed: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kitaku, Sapporo 060-0812, Japan. Tel.: 81-11-706-3745; Fax: 81-11-706-4988; E-mail: hiro@pharm.hokudai.ac.jp. 1 The abbreviations used are: PKA, cAMP-dependent protein kinase; mouse anti-FLAG antibody (M2; Sigma) or with nonspecific mouse IgG under the same conditions as those of the in vitro binding assay as described above. After washing with the same buffer except for 0.05% instead of 0.25% Nonidet P-40, the precipitates were separated in a 15% polyacrylamide gel containing SDS, blotted onto a nitrocellulose filter, and reacted with a rabbit anti-HA antibody (Y11; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with the mouse anti-FLAG antibody. Indirect Immunofluorescence-The human HeLa cells were fixed with a solution containing 4% paraformaldehyde and reacted with a rabbit anti-AMY-1 polyclonal antibody synthesized against the peptide corresponding to amino acid numbers 343-370, a mouse anti-AKAP149 antibody (clone 6; Transduction Laboratories), or nonimmune antiserum. The cells were then reacted with a fluorescein isothiocyanateconjugated anti-rabbit IgG or AMCA-conjugated anti-mouse IgG and observed under a fluorescent microscope. At the same time, the mitochondria in cells were stained with MitoTracker Red CM-H2Xros (Molecular Probes, Inc., Eugene, OR). The ejaculated human sperm was collected onto a cover glass by centrifugation and stained as described above.

RESULTS AND DISCUSSION
Identification of AKAP149/S-AKAP84 as AMY-1-binding Proteins and Determination of the AMY-1 Binding Region-To screen cDNAs encoding AMY-1-associating proteins, a full-size AMY-1 starting from amino acid number 1 was fused to the LexA-DNA binding domain and introduced into S. cerevisiae L40 cells. A human HeLa cDNA library cloned in pGADGH was then introduced into the transformant yeast cells, and the colonies resistant to a His marker followed by ␤-galactosidase expression were selected. Among a total of 5.6 ϫ 10 6 transformant cells, 79 colonies were His-and ␤-galactosidase-positive, and four of the 79 positive colonies were identified as AKAP149, a PKA anchor protein, after determination of their nucleotide sequences. Since the longest clone contained amino acids 253-903 of AKAP149, the full-size AKAP149 cDNA was kindly provided by C. Hanski. S-AKAP84 is a splicing variant of AKAP149 and contains the same amino acids spanning 1-530 as does AKAP149 (3). Both proteins are composed of at least three domains (an anchor region, a leucine zipper-like (LZ) region, and an RII-binding (RIIbd) region), and AKAP149 additionally contains a KH domain (Fig. 1A). To determine the AMY-1-binding region of AKAP149 or S-AKAP84, various deletion constructs fused to the GALAD were used for the twohybrid assay with AMY-1 as bait (Fig. 1A). The wild-types of both AKAP149 and S-AKAP84 bound to AMY-1, and the FIG. 1. Association of AMY-1 with AKAP149/S-AKAP84. A, the wild type or various deletion mutants of AKAP149 or S-AKAP84 were fused to GALAD and used for yeast two-hybrid assays in L40 cells pretransformed with LexBD-AMY-1 or a vector (PGLex). After incubation on filters, the ␤-galactosidase activity was assayed. B, GST or GST-AMY-1 was expressed in E. coli BL21(DE3) and applied to glutathione-Sepharose 4B. 35 S-Labeled S-AKAP84 (AK253-530 and AK253-530⌬RIIbd) synthesized in vitro in a coupled transcription/translation system was then applied to the column. The labeled proteins that had bound to the column were separated in a gel and visualized by fluorography. One-fiftieth volumes of the labeled S-AKAP84 used for the binding reaction were applied to the same gel (lane 5). C, AMY-1 and S-AKAP84 (AK253-530 and AK253-53⌬RIIbd) were tagged with either FLAG or HA, and their expression vectors were introduced into human 293T cells. Two days after transfection, cell extracts were prepared, and the proteins in the extracts were first immunoprecipitated (IP) with an anti-FLAG antibody (F) or nonspecific IgG (G). The proteins in the precipitates were separated in a 12.5% polyacrylamide gel and blotted with an anti-HA antibody (12CA5). One-fiftieth volumes of the extract used for the binding reaction were applied to the same gel (Input, lane 7).
C-terminal half-fragment spanning amino acids 531-903 of AKAP149 did not bind to AMY-1, suggesting that the same amino acids present in AKAP149 and S-AKAP84 contribute to the AMY-1 binding activity. The fragment spanning amino acids 253-530 was found to be sufficient for AMY-1 binding. Furthermore, deletion of the RII binding region (AK253-530⌬RIIbd) but not LZ (AK253-530⌬LZ) from this fragment abolished AMY-1 binding activity, indicating that AMY-1 binds to the RII binding region both in AKAP149 and S-AKAP84. An in vitro binding assay was then performed by using a 35 Slabeled AMY-1-binding fragment of S-AKAP84 synthesized in vitro with GST-AMY-1 expressed in and prepared from E. coli. After GST-AMY-1 or GST trapped in glutathione-Sepharose 4B resin had been mixed with labeled proteins, the bound proteins were separated on gel and visualized by fluorography (Fig. 1B). As in the case of binding in yeast, the fragment spanning amino acids 253-530 of S-AKAP84 bound to GST-AMY-1, while the fragment in which the RII binding region had been deleted did not have binding activity toward GST-AMY-1 (Fig. 1B, lanes 1  and 2), and no bindings of two proteins to GST alone were observed (Fig. 1B, lanes 3 and 4). To observe the complex formation of S-AKAP84 with AMY-1 in vivo, expression vectors for FLAG-tagged S-AKAP84 and its deletion fragments together with HA-tagged AMY-1 were transfected into human 293T cells. Forty-eight h after transfection, the cell extract was prepared, and the proteins in the extract were first immunoprecipitated with the anti-FLAG antibody or nonspecific IgG. The precipitates were immunoblotted against the anti-HA antibody (Fig. 1C). The anti-FLAG antibody did precipitate FLAG-S-AKAP84 (data not shown). AMY-1-HA, on the other hand, was detected in the immunoprecipitate from wild-type S-AKAP84-transfected cells with the anti-HA antibody but not with IgG (Fig. 1C, lanes 1 and 2, respectively). Furthermore, the fragment spanning amino acids 253-530, but not that in which the RII-binding region had been deleted, of S-AKAP84 was again found to bind to AMY-1 (Fig. 1C, lanes 3 and 5,  respectively). These results indicate that AMY-1 is associated with the RII-binding region of S-AKAP84 in ectopic expressed 293T cells.
Expression of AMY-1 during Spermatogenesis-Since S-AKAP84 has been reported to be expressed in testis and to be related to spermatogenesis, Northern blot analysis of AMY-1 expression was carried out using a multiple tissue bolt filter to see its expression profile ( Fig. 2A). AMY-1S and S-AKAP84 mRNAs were found to be strongly expressed in the testis, while AMY-1L, a variant of alternative usage of the poly(A) signal of AMY-1S, and AKAP149, a splicing variant of S-AKAP84, were ubiquitously expressed in tissues. Reverse transcriptasepolymerase chain reaction analysis of mRNAs was then carried out to examine the timing of expression of AMY-1S and S-AKAP84 during spermatogenesis using total RNAs extracted from the mice testis at various days after birth (Fig. 2B). Specific primers for the amplification of mRNAs of c-kit, acrosin, and protamine-1 were used to identify the times at which the expressions of spermatogonia, spermatocyte, and spermatid started (8 -10), respectively. The results showed that AMY-1S began to be weakly expressed in 2-week-old mice and was strongly expressed after the expression of spermatid, during which time S-AKAP84 was expressed. On the other hand, c-myc was found to be expressed from the time of spermatogonia. These results indicate coordinate expression between AMY-1S and S-AKAP84.
Ternary Complex among AMY-1, S-AKAP84, and RII Subunit of PKA-Since AKAP binds to RII of PKA in order to be translocated to a specific site in cells (11), it is possible that AMY-1 is in a complex with RII. Two isoforms of RII, RII␣ and RII␤, have been reported, and RII␤ was used in this experiment. To determine whether the above possibility is true, the expression vectors for FLAG-AMY-1, T7-S-AKAP84, and RII␤-HA were transfected into 293T cells in various combinations. Forty-eight h after transfection, the proteins in cell extracts were immunoprecipitated with an anti-FLAG antibody, and the precipitated proteins were blotted with the anti-FLAG antibody to detect FLAG-AMY-1 or with an anti-HA antibody to detect RII␤-HA (Fig. 3A). First, the expressions of the introduced proteins were examined by Western blotting using respective antibodies, and T7-S-AKAP84, RII␤-HA, and FLAG-AMY-1 were detected (Fig. 3A, a, lanes 1 and 3 and lanes 2 and  3, respectively). Expression of FLAG-AMY-1 was also detected (data not shown). Although the anti-FLAG antibody precipitated the introduced FLAG-AMY-1 in three sets of cells at similar levels, RII␤-HA was coprecipitated in cells cotransfected with T7-S-AKAP84 but not without T7-S-AKAP84 (Fig.  3A, b, lanes 2 and 3, respectively). These results indicate that RII␤ is associated with AMY-1 via S-AKAP84. The formation of a ternary complex of AMY-1, S-AKAP84, and RII␤ was further examined in mouse AM416 cells, which are mouse NIH3T3 cells expressing exogenously added AMY-1-HA as described previously (1). AM416 cells were transfected with a constant amount of FLAG-S-AKAP84 and with various doses of RII␤-

FIG. 2. Expressions of AMY-1 and AKAP149/S-AKAP84 mRNAs in mouse tissues and in the testis of various stages.
A, Northern blot analyses were carried out using multiple Northern blot sheets of mouse tissues (CLONTECH) with a labeled AMY-1L cDNA (upper panel), AKAP149 cDNA (middle panel), or ␤-actin (lower panel) as a probe. B, total RNAs were extracted from the mice testes at various days after birth, and the expressions of AMY-1 and S-AKAP84 were analyzed by Northern blot hybridization as described in A. Reverse transcriptase-polymerase chain reaction was also carried out with specific primers for c-myc, c-kit, acrosin, protamine-1, and ␤-actin on total RNA extracted as substrates.
HA, and cell extracts were prepared 48 h after transfection. Expression levels of introduced FLAG-S-AKAP84, RII␤-HA, and AMY-1-HA were determined by Western blot analysis (Fig.  3B, a). The proteins in the extract were then immunoprecipitated with the anti-FLAG antibody, and the precipitates were blotted with the anti-HA antibody (Fig. 3B, b). Two or three bands of RII␤-HA were observed in trasfected cells. Faster migrating bands may be degradation products of RII␤-HA, and the similar patterns of RII␤ have been reported in human testis (12). The result showed that RII␤-HA was coprecipitated with the S-AKAP84-AMY-1 complex in a dose-dependent manner, suggesting that RII␤ and AMY-1 mutually, but not competitively, bound to the RII-binding region of S-AKAP84. Colocalization of AMY-1 with AKAP149/S-AKAP84 in the Mitochondria in HeLa and Sperm-Previous studies have shown that AMY-1 was usually located in the cytoplasm and translocated from the cytoplasm to nuclei only during the S phase of the HeLa cell upon the expression of c-Myc (1) and that S-AKAP84 was localized in the mitochondria of HeLa, elongating spermatid (4), and mature sperm (12). To determine the cellular localization of AKAP149/S-AKAP84 and AMY-1 more precisely, human HeLa cells and the ejaculated human sperm were stained with anti-AMY-1 and anti-AKAP149/S-AKAP84 antibodies, and the proteins were detected by fluorescein isothiocyanate-and AMCA-conjugated second antibodies, respectively, and then visualized under a confocal laser microscopy (Fig. 4). Mitochondria were also stained with MitoTracker, which gives a red color. In HeLa cells, AMY-1 (green), AKAP149 (blue), and mitochondria (red) were co-localized as shown by the white color (Fig. 4A, Overlay). In sperm, both AMY-1 and S-AKAP84/AKAP149 were localized in the neck of the sperm, which was identified as mitochondria by Mito-Tracker staining, and these three were colocalized as shown by the white color (Fig. 4B, Overlay). To distinguish which AKAP, S-AKAP84 or AKAP149, is located in the ejaculated human sperm and HeLa cells, proteins extracted were blotted with the anti-AKAP149/S-AKAP84 antibody and with an anti-actin antibody as an internal control (Fig. 5). Results clearly showed that S-AKAP84 was strongly expressed and a faint amount of AKAP149 was expressed in sperm (Fig. 5, lane 2). In HeLa cells, on the other hand, only AKAP149 was expressed (Fig. 5,  lane 1). These results clearly show that AMY-1 is colocalized with AKAP149 or S-AKAP84 in the mitochondria of HeLa cells or sperm, respectively.
It has been reported that tyrosine phosphorylation of proteins plays a key role in the acquisition of fertilization activity and that tyrosine phosphorylation is stimulated by dibutylic cAMP, 8-bromo-cAMP, or inhibitors of phosphodiesterase during fertilization (13)(14)(15)(16)(17). It has also been reported that an inhibitor of PKA inhibits and that an inhibitor of serine/threonine-protein phosphatase stimulates both tyrosine phosphorylation and fertilization, suggesting that PKA plays a crucial role in tyrosine-phosphorylation-mediated fertilization (18 -20). It has been shown that AKAP determines the place where PKA works in cells and that S-AKAP84 and AKAP149 anchor PKA to the mitochondria in spermatid, sperm, and various somatic cells, respectively (3,4). In this study, we found that AMY-1 was located in the mitochondria in sperm and HeLa cells in a complex with S-AKAP84/AKAP149 and RII␤ and that both AMY-1 and S-AKAP84 were expressed after the appearance of spermatocytes in the testis, while the expression profile of c-myc during spermatogenesis was not parallel with that of AMY-1. These results suggest that AMY-1 plays a role independent of that of c-Myc in spermatogenesis. In addition to the functions of S-AKAP84 in spermatogenesis, it has been reported that S-AKAP84/AKAP149 may be involved in apoptosis regulation by anchoring PKA to mitochondria in which the unphosphorylated active form of BAD, a protein for proapoptosis, is bound to Bcl-XL, an antiapoptosis protein, to be inactivated. After BAD is phosphorylated by PKA in mitochondria, the phosphorylated inactive form of BAD is translocated to the cytoplasm and binds to 14-3-3 protein, thereby leading to the free active form of Bcl-XL that functions as an antiapoptosis protein (21). Transgenic mice carrying overexpressing AMY-1 are apt to become infertile (data not shown). Although the precise mechanisms of the AMY-1 function in testis are not clear at present, it is possible that AMY-1 affects the apoptosis of spermatogenesis-related proteins by modification of PKA anchored by S-AKAP84. Furthermore, since AMY-1 and RII␤ were found to bind mutually to the RII-binding region of S-AKAP84/AKAP149, which spans 22 amino acids, it is possible that both proteins bind to the same surface of the RIIbinding region side by side or that each protein binds to the opposite surface of the RII-binding region of S-AKAP84/ AKAP149. To examine these possibilities, structural analysis of the complex containing AMY-1 is now being carried out.