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J. Biol. Chem., Vol. 277, Issue 52, 50885-50892, December 27, 2002

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AMY-1 Interacts with S-AKAP84 and AKAP95 in the Cytoplasm and the Nucleus, Respectively, and Inhibits cAMP-dependent Protein Kinase Activity by Preventing Binding of Its Catalytic Subunit to A-kinase-anchoring Protein (AKAP) Complex^*

Makoto Furusawa^§, Takahiro Taira^§, Sanae M. M. Iguchi-Ariga^§¶, and Hiroyoshi Ariga^§

From the  Graduate School of Pharmaceutical Sciences, ^¶ College of Medical Technology, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan and ^§ CREST, Japan Science and Technology Corporation, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Received for publication, June 27, 2002, and in revised form, September 27, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have reported that a novel c-Myc-binding protein, AMY-1, binds to cAMP-dependent protein kinase-anchoring protein 149 (AKAP149) and its splicing variant, AKAP84 and is localized in the mitochondria in a complex with RII, a regulatory subunit of cAMP-dependent protein kinase (PKA) (Furusawa, M., Ohnishi, T., Taira, T., Iguchi-Ariga, S. M. M., and Ariga, H. (2001) J. Biol. Chem. 276, 36647-36651). In this study, we further found that AMY-1 competitively bound to either AKAP95 or AKAP84 in the nucleus and the cytoplasm, respectively, in a concentration-dependent manner of either AKAP. Like AKAP84, AMY-1 was found to bind to the RII-binding region of AKAP95 in vivo and in vitro and to make a ternary complex with RII. It was also found that the formation of the complex of AMY-1 with AKAP84/95 and RII prevented a catalytic subunit from binding to this AKAP complex, leading to suppression of PKA activity. These findings suggest that AMY-1 is an important modulator of PKA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have identified a novel c-Myc-binding protein, AMY-1,¹ that bound to myc box II in the N-proximal region of c-Myc, a transcriptional activation region of c-Myc, and stimulated E-box-dependent transcription activity of c-Myc (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 (1). The molecular mechanisms underlying the stimulating activity of AMY-1 toward c-Myc, however, have not been determined. Moreover, AMY-1, in contrast to c-myc, was found to be a stimulating factor for the initial step in erythrocyte differentiation of human K562 cells, suggesting 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 those of c-Myc (2). To further elucidate the functions of AMY-1, we screened AMY-1-binding proteins, and AKAP149 and S-AKAP84, anchor proteins of cAMP-dependent protein kinase (PKA), were identified as AMY-1-binding proteins (3). AKAP is known to be bound by a regulatory subunit (RII) of PKA and to play a role in translocation of PKA to specific sites where 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. We have found that AMY-1 is localized in mitochondria of HeLa cells or sperm in a ternary complex containing RII and AKAP149 or S-AKAP84, respectively (3). 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 (5-9). 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 (10-12). S-AKAP84 has, therefore, important functions in spermatogenesis, thereby suggesting that the function of AMY-1 is related to spermatogenesis.

More than 10 AKAPs have so far been identified, and they have been classified into groups according to their distributions in tissues or cells (for a recent review, see Ref. 13). All of the AKAPs comprise two domains, a targeting domain and a tethering domain, the former of which serves as a scaffold and membrane anchor and the latter of which interacts with PKA regulatory subunits. In addition to interaction with PKA, it is also thought that AKAPs bind to other signaling molecules that regulate AKAP targeting and activate other signal transduction pathways.

PKAs are known to have versatile functions in cells, and their localizations are determined by respective AKAP. After the anchoring of PKA to a specific site of the cell by binding of the RII of PKA to the RII-binding domain of AKAP, the catalytic domain of PKA (PKAc) is free to phosphorylate target proteins in specific sites. However, in addition to a combination of RII, PKAc, and AKAP, it is thought that some proteins are required to modulate the formation of this complex and activity of PKA, such molecules have not been identified.

In this study, we identified AKAP95, a nuclear AKAP, as an AMY-1-binding protein and found that AKAP95 and S-AKAP84 reciprocally bound to AMY-1 in the nucleus and the cytoplasm, respectively. We also found that AMY-1 inhibited PKA activity by preventing PKAc from binding to the AKAP complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Human HeLa and 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum.

Plasmids-- cDNA containing full-sized AKAP95, pBS-AKAP95, was provided by C. Hanski. To create pcDNA3-F-AKAP95, AKAP95 cDNA starting from the first ATG were inserted into EcoRI-XhoI sites of pCMV-F, a pcDNA3 containing a FLAG tag (14). Constructions of other plasmids were described previously (3).

In Vitro Binding Assay-- ³⁵S-labeled S-AKAP84 and AKAP95 were 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 µg of pcDNA3-F-S-AKAP84 or pcDNA3-AKAP95 together with 10 µg of pEF-AMY-1-HA were transfected into human 293T cells 60% confluent in a 10-cm dish by the calcium phosphate precipitation technique (15). Forty-eight h after transfection, the whole cell extract was prepared by the procedure described previously (1). Approximately 500 µg of the 293T cell proteins was first immunoprecipitated with a 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 (MBL) or with the mouse anti-FLAG antibody. The precipitated proteins were then reacted with an horseradish peroxidase-conjugated anti-rabbit IgG or an horseradish peroxidase-conjugated anti-mouse IgG and visualized by ECL system (Amersham Biosciences). To detect Rll in Fig. 5, an anti-RII antibody (Transduction Laboratories) was first conjugated with Alexa Fluor 680 using a protein labeling kit (Molecular Probes). The precipitated proteins were then reacted with this conjugated antibody and detected using the infrared imaging system (Odyssey, LI-COR).

Indirect Immunofluorescence-- The human HeLa cells were transfected with FLAG-AKAP95 and AMY-1-HA by the calcium phosphate precipitation technique. Forty-eight h after transfection, cells were fixed with a solution containing 4% paraformaldehyde and reacted with a mouse anti-anti-FLAG antibody (M2, Sigma) or with a rabbit anti-HA antibody (MBL). The cells were then reacted with an FITC-conjugated anti-rabbit IgG or rhodamine-conjugated anti-mouse IgG and observed under a confocal laser fluorescent microscope. At the same time, the nuclei in the cells were stained with 4',6-diamidino-2-phenylindole.

PKA Assay-- Human 293T cells were transfected with FLAG-AKAP95 and AMY-1-HA by the calcium phosphate precipitation technique. Forty-eight h after transfection, the whole cell extract was prepared as described above, and the proteins were immunoprecipitated with a mouse anti-FLAG antibody (M2, Sigma). The proteins in the precipitates were subjected to a phosphorylation assay using a PKA assay kit (Invitrogen) in which [-³²P]ATP was used as a substrate, according to the manufacturer's protocol. Briefly, the preincubation was first carried out at room temperature for 15 min in a mixture containing 100 µM ATP, 10 mM MgCl₂, 250 µg/ml bovine serum albumin, 50 mM Tris, pH 7.5, 10 µM cAMP, 1 µM protein kinase inhibitor (6022) amide, and the precipitated proteins. The phosphorylation assay was then carried out at 30 °C for 5 min after adding 50 µM (Leu-Arg-Arg-Ala-Ser-Leu-Gly) Kemptide as a substrate and 100 µM [-³²P]ATP (3000 Ci/mmol) into the reaction mixture, and acid-insoluble radioactivities were measured.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of AKAP95 as an AMY-1-binding Protein and Determination of the AMY-1-binding Region-- As described previously (3), we have screened cDNAs encoding AMY-1-associating proteins by a two-hybrid system with a full-sized AMY-1 as a bait using a human HeLa cDNA library, and we obtained a cDNA encoding AKAP95 in addition to AKAP149 cDNA. In addition to an RII-binding (RIIbd) domain that is present in other AKAPs, AKAP95 possesses three other domains, a nuclear matrix targeting sequence, a nuclear localization sequence, and two zinc-finger regions (16) (Fig. 1A). We have reported that AMY-1 binds to the RIIbd of AKAP149/AKAP84 (3). To determine the direct binding of AMY-1 to AKAP95 and the importance of the RIIbd AKAP95 for its binding, an in vitro binding assay was performed by using ³⁵S-labeled wild-type AKAP95 and a deletion mutant lacking RIIbd spanning amino acid numbers 572-589 of AKAP95 synthesized in vitro. After GST-AMY-1 or GST had been expressed in and prepared from E. coli, trapped in glutathione-Sepharose 4B resin, and mixed with labeled proteins, the bound proteins were separated on-gel and visualized by fluorography (Fig. 1B). The wild-type AKAP95 bound to GST-AMY-1, whereas the fragment in which the RII-binding region had been deleted did not have binding activity toward GST-AMY-1 (Fig. 1B, lanes 3 and 6, respectively), and no binding of the two proteins to GST alone was observed (Fig. 1B, lanes 2 and 5), indicating that AMY-1 binds to the RII-binding region in AKAP95 as in the case of binding in AKAP149 and S-AKAP84 (3). To observe the complex formation of AKAP95 with AMY-1 in vivo, expression vectors for FLAG-tagged AKAP95 and its deletion mutant 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 an anti-FLAG antibody or nonspecific IgG. The precipitates were immunoblotted against an anti-HA antibody (Fig. 1C). The anti-FLAG antibody precipitated FLAG-AKAP95 (data not shown). AMY-1-HA, on the other hand, was detected in the immunoprecipitate from wild-type AKAP95-transfected cells with the anti-HA antibody but not with IgG (Fig. 1C, lanes 3 and 4, respectively). Furthermore, it was found that the deletion mutant of AKAP95, in which the RII-binding region had been deleted, did not bind to AMY-1 (Fig. 1C, lane 5). These results indicate that AMY-1 is associated with the RII-binding region of AKAP95 in ectopic expressed 293T cells. To examine the binding of AMY-1 to AKAP95 in physiological conditions, mouse AM416 cells, which are mouse NIH3T3 cells expressing exogenously added AMY-1-HA as described previously (1), were used due to the unavailability of an anti-AMY-1 antibody suitable for the immunoprecipitation experiment. The proteins in the extracts prepared from AM416 cells were first immunoprecipitated with the anti-HA antibody or nonspecific IgG and then immunoblotted against an anti-AKAP95 antibody (Fig. 1D). The anti-HA antibody was first confirmed to precipitate AMY-1-HA (Fig. 1D, lower panel). AKAP95 was detected in the immunoprecipitate with the anti-HA antibody but not with IgG (Fig. 1D, lanes 1 and 2, respectively). In both coprecipitation experiments, it was found that ~10% of AMY-1-HA and AKAP95 added to the reaction mixtures was precipitated with the anti-FLAG antibody and anti-HA antibody (Fig. 1, C and D, respectively). An affinity between AMY-1 and AKAP95 is higher than that between c-Myc and AMY-1, between which only 1-2% of c-Myc bound to AMY-1 (1). Although the affinity of AMY-1 to AKAP95 is not extremely high, these results clearly indicate that AMY-1 binds to AKAP95 in cells.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Association of AMY-1 with AKAP95. A, schematic drawings of wild-type AKAP95 and deletion mutant lacking the RII-binding region of AKAP95. NMTS, NLS, ZF, and RII indicate regions for nuclear matrix targeting sequence, nuclear localization signal, zinc finger, and RII binding, respectively. aa, amino acids. In B, GST or GST-AMY-1 was expressed in E. coli BL21(DE3) and applied to glutathione-Sepharose 4B. 35S-labeled AKAP95 or AKAP95RII 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/fifty volumes of the labeled AKAP95 or AKAP95RII used for the binding reaction were applied to the same gel (lanes 1 and 4). In C, AMY-1 and AKAP95 (wild-type and AKAP95RII) were tagged with either HA or FLAG, 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 (MBL). One/fifty volumes of the extract used for the binding reaction were applied to the same gel (input, lane 7). In D, the proteins in cell extracts prepared from AM416 cells were first immunoprecipitated with an anti-HA antibody (Y-11, Santa Cruz Biotechnology) or nonspecific IgG. The proteins in the precipitates were separated in a 10% polyacrylamide gel and blotted with an anti-AKAP95 antibody (clone 47, Transduction Laboratories). One/fifty volumes of the extract used for the binding reaction were applied to the same gel (input, lane 3). All of the experiments have been done three times, and the reproducible results were obtained. A typical example of the results was shown.

Colocalization of AMY-1 and AKAP95-- To determine the localization of AMY-1 and AKAP95, expression vectors for AMY-1-HA and FLAG-AKAP95 were transfected into human HeLa cells. Forty-eight h after transfection, the cells were stained with anti-HA and anti-FLAG antibodies, and they were visualized with rhodamine and FITC-conjugated secondary antibodies, respectively, under a confocal laser microscope (Fig. 2). The results showed that AMY-1-HA and FLAG-AKAP95 were located in the nucleus, and they were found to be co-localized after demonstration of the merged figure, in which the red and green colors turned yellow (Fig. 2, A-C, respectively). Co-localization of AMY-1-HA and FLAG-AKAP95 in the nucleus was confirmed by staining cell nuclei with 4',6-diamidino-2-phenylindole, by which three colors turned white (Fig. 2, D and E, respectively). Since we have reported that AMY-1 was localized in the mitochondria with S-AKAP84/AKAP149, these results suggest that AKAP95 translocates AMY-1 to the nucleus.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Colocalization of AMY-1 with AKAP95 in the nucleus. HeLa cells were cotransfected with AMY-1-HA and FLAG-AKAP95 by the calcium phosphate precipitation technique. Forty-eight h after transfection, cells were fixed, reacted with an anti-HA polyclonal antibody and an anti-FLAG monoclonal antibody, and visualized with a rhodamine-conjugated anti-rabbit antibody and an FITC-conjugated anti-mouse antibody. The same slides were also stained with 4',6-diamidino-2-phenylindole. Panels A and B and panels A, B, and D were merged (Overlay, C and E, respectively). The experiments have been done four times, and the reproducible results were obtained. A typical example of the results was shown.

Competitive Binding of AMY-1 to S-AKAP84 or AKAP95-- Since AMY-1 binds to RII-binding regions of S-AKAP84 and AKAP95 as described previously (3) and in this study, respectively, it is possible that S-AKAP84 and AKAP95 competitively bind to AMY-1. To examine this possibility, the same amounts of AMY-1-HA and FLAG-AKAP95 were cotransfected into HeLa cells along with various amounts of T7-S-AKAP84. 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-AKAP95 or with an anti-HA antibody to detect AMY-1-HA (Fig. 3). The dose-dependent expression of the introduced T7-S-AKAP84 and the constant amounts of the introduced AMY-1-HA and FLAG-AKAP95 were first detected by Western blotting with an anti-T7, the anti-HA, and the anti-FLAG antibodies, respectively (Fig. 3A). Although the anti-FLAG antibody precipitated the introduced FLAG-AKAP95 in three sets of cells at similar levels, the coprecipitated AMY-1-HA was reduced in cells cotransfected with T7-S-AKAP84 in a dose-dependent manner (Fig. 3A, lanes 1, 3, and 5). Nonspecific IgG did not precipitate either FLAG-AKAP95 or AMY-1-HA (Fig. 3A, lanes 2, 4, and 6). These results clearly showed that S-AKAP84 and AKAP95 competitively bind to AMY-1.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Competitive bindings of S-AKAP84 and AKAP95 to AMY-1 in cells. In A, expression vectors for AMY-1-HA, FLAG-AKAP95, and various doses of T7-S-AKAP84 were transfected into 293T cells. Forty-eight h after transfection, cell extracts were prepared, and the proteins in the aliquots of extracts were blotted with an anti-HA polyclonal antibody (Y-11, Santa Cruz Biotechnology), an anti-T7 monoclonal antibody (Novagen), or an anti-FLAG antibody (M2, Sigma). In B, the proteins in the extracts were immunoprecipitated (IP) with an anti-FLAG monoclonal antibody (F, M2, Sigma), and the precipitates were blotted with the anti-FLAG antibody or the anti-HA polyclonal antibody (Y-11, Santa Cruz). One/fifty volumes of the extract used for the binding reaction were applied to the same gel (input, lane 7). The experiments have been done three times, and the reproducible results were obtained. A typical example of the results was shown. G, nonspecific IgG.

Competitive bindings of S-AKAP84 and AKAP95 to AMY-1 were further investigated in terms of the localization of AMY-1 in cells (Fig. 4). Expression vectors for AMY-1-HA and S-AKAP84 were transfected into 293T cells with or without an expression vector for FLAG-AKAP95. Forty-eight h after transfection, the cells were stained with anti-HA and anti-FLAG antibodies and then visualized with rhodamine and FITC-conjugated secondary antibodies, respectively, under a confocal laser microscope (Fig. 4). In cells that had not been transfected with FLAG-AKAP95, AMY-1-HA was found to be localized in the mitochondria as described previously (3) (Fig. 4A, a-c). In cells transfected with FLAG-AKAP95 along with AMY-1-HA and S-AKAP84, on the other hand, AMY-1-HA was found to be colocalized with FLAG-AKAP95 in the nuclei, in which green and red colors turned to yellow color in a merged figure (Fig. 4A, d-f). Since the size of HA-tagged AMY-1 was small enough for it to diffuse to the nucleus, GFP-tagged AMY-1 was cotransfected with FLAG-AKAP95 into 293T cells, and their distributions in cells were examined. Since the molecular mass of GFP is 27 KDa, GFP-AMY-1 is not able to diffuse to the nucleus by itself. Although GFP-AMY-1 was found to be located in the cytoplasm of cells that had been transfected with GFP-AMY-1 alone, it was found to be colocalized with FLAG-AKAP95 in the nuclei of cells that had been transfected with GFP-AMY-1 and FLAG-AKAP95 (Fig. 4B, a-c). Since the expression levels of FLAG-AKAP95 and S-AKAP84 in cells were similar, as shown in Fig. 3, these results suggest that AKAP95 has a much stronger binding affinity to AMY-1 than does S-AKAP84, thereby leading to the translocation of AMY-1 from the mitochondria to the nucleus.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Relocalization of AMY-1 from the cytoplasm to the nucleus by AKAP95 in cells. In A, HeLa cells were cotransfected with AMY-1-HA and S-AKAP84 with or without FLAG-AKAP95 by the calcium phosphate precipitation technique (a-c and d-f, respectively). Forty-eight h after transfection, cells were fixed, reacted with an anti-HA polyclonal antibody and an anti-FLAG monoclonal antibody, and visualized with an FITC-conjugated anti-rabbit antibody and a rhodamine-conjugated anti-mouse antibody. Panels a and b and panels d and e were merged (Overlay, c and e, respectively). In B, HeLa cells were cotransfected with AMY-1-GFP and FLAG-AKAP95 and treated as described above. Cells were stained with an anti-FLAG monoclonal antibody and visualized with a rhodamine-conjugated anti-mouse antibody (B, panel b). AMY-1-GFP was visualized by own light emission (B, panel a), and panels a and b were merged (Overlay, B, panel c). All of the experiments have been done three times, and the reproducible results were obtained. A typical example of the results was shown.

Inhibition of PKA Activity by AMY-1-- Since we have reported that AMY-1 makes a ternary complex with S-AKAP84/AKAP149 and a regulatory subunit of PKA (RII), it is possible that AMY-1 also makes a ternary complex with AKAP95 and RII. To explore this possibility, expression vectors for FLAG-RII were cotransfected with AMY-1-HA and AKAP95-HA 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 an anti-HA antibody to detect AMY-1-HA and AKAP95-HA. It has been shown that AMY-1 does not directly bind to RII (3). As in the case of AMY-1 and S-AKAP84, it was found that AMY-1 made a ternary complex with AKAP95 and RII (data not shown). Since it is known that PKA is comprised of RII and a catalytic subunit (PKAc) is anchored by AKAP to the respective site in cells and that PKAc exerts its phosphorylation activity at this site, it is possible that AMY-1 affects the phosphorylation activity of PKA. To explore this possibility, expression vectors for FLAG-S-AKAP84 or FLAG-AKAP95 were cotransfected with AMY-1-HA into 293T cells, and the proteins in cell extracts were immunoprecipitated with the anti-FLAG antibody at 48 h after transfection (Fig. 5, A and B, respectively). The dose-dependent expression of the introduced AMY-1-HA and the constant amounts of the introduced FLAG-S-AKAP84 or FLAG-AKAP95 were first detected by Western blotting with the anti-HA and the anti-FLAG antibodies, respectively (Fig. 5, A, panel a, and B, panel a, respectively). Expressions of constant amounts of the endogenous PKAc and endogenous RII were also confirmed by Western blotting with an anti-PKAc antibody and an the anti-RII antibody, respectively (Fig. 5, A, panel a, and B, panel a, lower two panels). When the proteins immunoprecipitated with the anti-FLAG antibody were blotted with the anti-PKAc antibody, it was found that although the amounts of precipitated RII were constant, the amounts of precipitated PKAc were reduced by AMY-1-HA in a dose-dependent manner in cells that had been transfected with both FLAG-S-AKAP84 and FLAG-AKAP95 (Fig. 5, A, panel b, and B, panel b, respectively), suggesting that AMY-1 negatively affects PKA activity. To directly examine the effect of AMY-1 on PKA activity, PKA activities in the same precipitates as those used for detection of PKAc by Western blotting were measured using a mixture containing [-³²P]ATP and a synthetic peptide Kemptide as a substrate (Fig. 5, A, panel c, and B, panel c). As suggested by the results described above, it was found that PKA activities in the precipitates from cells that had been transfected with both FLAG-S-AKAP84 and FLAG-AKAP95 were partially inhibited by introduced AMY-1 in a dose-dependent manner and that the inhibited level of kinase activity in the extract from cells that had been transfected with FLAG-AKAP95 was higher than that in the extract from cells that had been transfected with FLAG-S-AKAP84. These results indicate that AMY-1 negatively regulates PKA activity.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   Effect of AMY-1 on kinase activity of PKA. In A, panel a, 293T cells were transfected with expression vectors for FLAG-S-AKAP84 alone or with AMY-1-HA. Forty-eight h after transfection, cell extracts were prepared, and the proteins in an aliquot were blotted with an anti-FLAG antibody, an anti-HA antibody, an anti-PKAc antibody (clone 5B, Transduction Laboratories), or an anti-RII antibody. In A, panel b, the cell extracts prepared in A, panel a, were immunoprecipitated (IP) with an anti-FLAG monoclonal antibody (F) or nonspecific IgG (G), and the precipitates were blotted with an anti-PKAc antibody or an Alexa Fluor 680-conjugated anti-RII antibody and detected by ECL system (Amersham Biosciences) or the infrared imaging system (Odyssey, LI-COR), respectively. In A, panel c, the kinase activity was measured in a mixture containing 200 µg of proteins in the precipitates prepared in A, panel b, [32P]ATP, and Kemptide as a substrate as described under "Experimental Procedures," and the trichloroacetic acid-insoluble radioactivity was measured. B, 293T cells were transfected with expression vectors for FLAG-AKAP95 alone or with AMY-1-HA and subjected to analyses for the expression of each protein (B, panel a), for the immunoprecipitated proteins with an anti-FLAG antibody (B, panel b), and for the PKA activity (B, panel c) as described in A. All of the experiments have been done five times, and the reproducible results were obtained. A typical example of the results was shown.

To determine the reason for the different inhibitory activities of AMY-1 toward PKA, binding affinities of S-AKAP84 and AKAP95 to RII or AMY-1 were then examined. To examine affinities of two AKAPs to RII, expression vectors for FLAG-S-AKAP84 and FLAG-AKAP95 were cotransfected with RII-HA into 293T cells in various combinations. Since RII binds to the RII-binding regions of AKAPs, deletion mutants of FLAG-S-AKAP84 and FLAG-AKAP95 lacking RII-binding regions were also cotransfected as negative controls. Forty-eight h after transfection, the proteins in cell extracts were immunoprecipitated with an anti-FLAG antibody or nonspecific IgG, and the precipitated proteins were blotted with an anti-HA antibody to detect RII-HA (Fig. 6A). First, expressions of the introduced proteins were examined by Western blotting using respective antibodies, and similar levels of FLAG-S-AKAP84, FLAG-AKAP95, their deletion mutants, and RII-HA were detected (Fig. 6A, panel a). It was found that RII-HA was not precipitated with the anti-FLAG antibody in extracts from cells that had been transfected with deletion mutants of both FLAG-S-AKAP84 and FLAG-AKAP95 (Fig. 6A, panel b, lanes 3 and 7, respectively) or with nonspecific IgG in cell extracts (Fig. 6A, panel b, lanes 2, 4, 6, and 8). It was also found that a smaller amount of RII-HA was precipitated in extracts from cells that had been transfected with FLAG-AKAP95 than in extracts from cells that had been transfected with FLAG-S-AKAP84 (Fig. 6A, panel b, lanes 1 and 5, respectively), suggesting that S-AKAP84 possesses much higher binding affinity to RII than AKAP95 does.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Different binding affinities of S-AKAP84 and AKAP95 to RII or AMY-1. In A, panel a, various combinations of expression vectors for FLAG-S-AKAP84EX spanning amino acid numbers 253-530, FLAG-AKAP95, and RII-HA were transfected into 293T cells. Forty-eight h after transfection, cell extracts were prepared, and the proteins in an aliquot were blotted with an anti-FLAG antibody and an anti-HA antibody. In A, panel b, the cell extracts prepared in A, panel a were immunoprecipitated (IP) with an anti-FLAG monoclonal antibody (F) or nonspecific IgG (G), and the precipitates were blotted with an anti-HA antibody. B, 293T cells were transfected with various combinations of expression vectors for FLAG-S-AKAP84, FLAG-AKAP95, and AMY-1-HA and subjected to analyses for the expression of each protein (B, panel a) and for the immunoprecipitated proteins with an anti-FLAG antibody (B, panel b) as described in A. All of the experiments have been done three times, and the reproducible results were obtained. A typical example of the results was shown.

The affinities of two AKAPs to AMY-1 were then examined by the same experimental strategies as those used for RII-HA by using transfection of AMY-1-HA instead of that of RII-HA (Fig. 6B). It was first confirmed that the levels of expression of the introduced proteins were similar (Fig. 6B, panel a). Contrary to the results regarding the amount of precipitated RII-HA in cells that had been transfected with either AKAP, it was found that a larger amount of AMY-1-HA was precipitated in extracts from cells that had been transfected with FLAG-AKAP95 than in extracts from cells that had been transfected with FLAG-S-AKAP84 (Fig. 6B, panel b, lanes 1 and 5, respectively), suggesting that AKAP95 possesses much higher binding affinity to AMY-1 than S-AKAP84 does. From these results, it is thought that a ternary complex of AMY-1, S-AKAP84/AKAP95, and RII prevents PKAc from binding to the RII-AKAP complex and that different affinity of AMY-1 to either AKAP determines its inhibitory activity toward PKA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we identified AKAP95, a nuclear AKAP, as an AMY-1-binding protein as well as S-AKAP84/AKAP149 and found that AMY-1 was colocalized with AKAP95 in the nucleus and that AMY-1 partially inhibited PKA activity by preventing PKAc from associating RII. We have reported that AMY-1 was translocated from the cytoplasm to the nucleus during the S-phase of the cell cycle along with c-Myc and stimulated transcription activity of c-Myc (1). We have also reported that during periods other than the S-phase, AMY-1 was located in the cytoplasm, exclusively in the mitochondria, in which AMY-1 was associated with S-AKAP84/AKAP149, suggesting that AMY-1 has at least two roles: one in the nucleus and the other in the cytoplasm (1, 3). It has been reported, on the other hand, that AKAP95 was located in the nucleus during G₁-S phases and that it was excluded from the condensed chromatin and localized outside the metaphase plate during mitosis (17-19). In the present study, we found that a larger amount of AKAP95 than that of S-AKAP84 translocated AMY-1 from the cytoplasm to the nucleus, in which AMY-1 and AKAP95 were colocalized. These results together with other reported results suggest that AMY-1 binds to AKAP95 in the nucleus during the S-phase of the cell cycle and to S-AKAP84/AKAP149 in the cytoplasm during phases of the cell cycle other than the S-phase, thereby facilitating the exertion of different activities of AMY-1 in the cytoplasm and the nucleus.

Several studies on the properties and functions of AKAP95 have recently been performed. These studies have shown that AKAP95 binds to chromatin and functions in chromosome condensation by targeting the condensin complex in the M-phase of the cell cycle (17-19). It has also been reported that PKA binding to AKAP95 is dispensable for the chromosome condensation activity of AKAP95 but that maintenance of the chromosome in a condensed form requires PKA activity and PKA binding to AKAP95 (17). In addition to these M-phase phenomena of AKAP95, it is known, as described above, that AKAP95 is localized exclusively in the nuclear matrix and associated with p68 RNA helicase, which has been suggested to play a role in the chromatin remodeling reaction, indicating the possibility that AKAP95 plays a role in assembly of hormonally responsive transcriptional complexes (20). Furthermore, it has been reported that HA95/HAP95/NAKAP95, which is a nuclear protein homologous to AKAP95 but lacking RII binding activity, affected transcriptional coactivator functions of Epstein-Barr virus-related herpes virus in Epstein-Barr virus-transformed cells, suggesting again a role of HA95 or AKAP95 as a scaffold for transcriptional regulation (21). Although the precise mechanisms underlying the transcriptional-stimulating activity of AMY-1 toward c-Myc have not been determined, it is possible that AKAP95 participates in this transcriptional apparatus to play roles in the modification of chromatin structures that facilitate or inhibit tethering of coactivators or corepressors to c-Myc-target genes. Our preliminary results suggest that AAT-1, a novel AMY-1-binding protein, also binds to AKAPs with AMY-1 and is a member of the c-Myc-AMY-1 transcriptional complex. We are now investigating this possibility.

We also found in this study that AMY-1 partially inhibited PKA phosphorylation activities that had been tethered by S-AKAP84 or AKAP95 and that the inhibitory activity tethered by AKAP95 was stronger than that tethered by S-AKAP84. The mechanism underlying this inhibitory activity of AMY-1 toward PKA is likely to be the abrogation of the binding activity of PKAc to RII, and the different inhibitory activities tethered by the two AKAPs are likely to be due to different affinities of AKAP to either RII or AMY-1; the affinity of AKAP95 to AMY-1 is stronger than that of S-AKAP84, and the affinity of AKAP84 to RII is weaker than that of S-AKAP84. Although it has been reported that AKAP activities leading to anchoring of PKA are regulated by kinases and phosphatases (13), this is the first report of AKAP activities being regulated by AMY-1, an AKAP-associated protein. Taken together, the results suggest that AMY-1 is a modulator of PKA.

	ACKNOWLEDGEMENTS

We are grateful to C. Hanski for the cDNA of AKAP95. We also thank Yoko Misawa and Kiyomi Takaya for technical assistance.

	FOOTNOTES

^* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan. Tel.: 81-11-706-3745; Fax: 81-11-706-4988; E-mail: hiro@pharm.hokudai.ac.jp.

Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M206387200

	ABBREVIATIONS

The abbreviations used are: AMY-1, associate of Myc-1; GST, glutathione S-transferase; AKAP, A-kinase-anchoring protein; RII, regulatory subunit II of A-kinase; PKA, cAMP-dependent protein kinase; PKAc, catalytic domain of PKA; RIIbd, RII-binding domain; HA, hemagglutinin; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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