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J. Biol. Chem., Vol. 277, Issue 40, 36955-36961, October 4, 2002

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A-Kinase Anchoring Protein AKAP220 Binds to Glycogen Synthase Kinase-3 (GSK-3) and Mediates Protein Kinase A-dependent Inhibition of GSK-3^*

Chie Tanji^§, Hideki Yamamoto, Noriaki Yorioka^§, Nobuoki Kohno^§, Kunimi Kikuchi^¶, and Akira Kikuchi

From the Departments of  Biochemistry and ^§ Molecular and Internal Medicine, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan and the ^¶ Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nichi-7, Kita-ku, Sapporo 060-0815, Japan

Received for publication, June 21, 2002, and in revised form, July 17, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3 binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3 formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3 bound to AKAP220 decreased more markedly than the total GSK-3 activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3 bound to AKAP220 more strongly than the total GSK-3 activity. These results suggest that PKA and PP1 regulate the activity of GSK-3 efficiently by forming a complex with AKAP220.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The serine/threonine kinase GSK-3¹ was first described in a metabolic pathway for glycogen synthase regulation that is sensitive to insulin-mediated inhibition (1). GSK-3 subsequently has been shown to regulate several physiological responses including protein synthesis, gene expression, subcellular localization of proteins, and protein degradation in mammalian cells by phosphorylating many substrates (1-3). The substrates of GSK-3 include neuronal cell adhesion molecule, neurofilament, synapsin I, tau, transcription factors, adenomatous polyposis coli gene product, -catenin, and cyclin D1. The cDNAs of GSK-3 and GSK-3 in mammals have been isolated, and they encode protein kinases with molecular masses of 51 and 47 kDa, respectively (4). GSK-3 is highly conserved through evolution and plays a fundamental role in cellular responses. For example, Xenopus GSK-3 determines the cell fate and regulates axis formation during early development (5, 6). The Drosophila zeste-white3/shaggy gene product is structurally and functionally homologous to GSK-3 (7) and is required at several developmental stages during fly embryogenesis for correct embryogenic segmentation (8, 9). A Dictyostelium homolog of GSK-3 (GSKA) has been found to be important for cellular differentiation (10). In Schizosaccharomyces pombe, the skp1⁺ gene product is a homolog of GSK-3 and regulates cytokinesis (11). In Saccharomyces cerevisiae, there are four genes, MCK1, MDS1/RIM11, MRK1, and YOL128c, which encode homologs of mammalian GSK-3. Mck1 acts in the transcriptional regulation of meiosis genes (12), the chromosomal segregation processes at mitosis (13), the cell cycle delay caused by the addition of Ca²⁺ (14), and protein degradation (15). Thus, GSK-3 regulates various cellular functions.

GSK-3 is a unique protein kinase in that many GSK-3 substrates require prior phosphorylation by a priming serine/threonine kinase to form the motif S-X-X-X-S(P) (S and X indicate serine and any amino acid, respectively) before phosphorylation by GSK-3 (1, 2). The three-dimensional structure of GSK-3 has been determined (16, 17). The binding site for the priming phosphate on substrates of GSK-3 has been identified and found to contain three crucial basic residues, Arg⁹⁶, Arg¹⁸⁰, and Lys²⁰⁵. Other unique features of GSK-3 are that it is constitutively active and that various extracellular signals inhibit its activity. There are multiple regulatory mechanisms of GSK-3 in mammals (2, 3). Phosphorylation of GSK-3 is the most extensively studied mechanism of regulation. The activity of GSK-3 is decreased by phosphorylation of Ser⁹. Several kinases have been found to be capable of mediating this modification including p70 S6 kinase, p90Rsk, PKB, protein kinase C, and PKA (18-24). This inhibitory mechanism by phosphorylation of Ser⁹ is intimately connected with the unique substrate specificity requirements of GSK-3. When Ser⁹ of GSK-3 is phosphorylated, it can interact with the same residues that are involved in the binding of the priming phosphate on substrates. This transforms the amino terminus into a "pseudosubstrate" inhibitor, thereby preventing substrates from binding to the catalytic center. The inhibition of GSK-3 might allow different signals to promote the dephosphorylation of some of the many proteins that have been identified as substrates of GSK-3. However, the mechanism by which different signals inhibit GSK-3 efficiently and specifically is not known.

PKA also has broad substrate specificity. Despite this, PKA has the ability to phosphorylate individual substrates selectively in response to discrete extracellular stimuli. Evidence that anchoring of the regulatory subunit of PKA to AKAPs targets PKA in close proximity to relevant substrates and confers that specificity in the cAMP/PKA signaling pathway has been accumulated (25-27). AKAPs were first identified as proteins that co-purified with the PKA holoenzyme. Although there are more than 40 unique members in the AKAP protein family, there is no overall sequence similarity among different AKAPs. They are characterized by their interaction with type I or II regulatory subunits of the PKA holoenzyme. AKAPs also possess unique target sequences that direct the PKA-AKAP complex to cellular compartments. Furthermore, some have the ability to maintain signaling scaffolds by simultaneously associating with other kinases and phosphatases. For example, AKAP220 has been shown to bind to the catalytic subunit of PP1 in addition to PKA (28, 29). When thus bound, PP1 is inhibited, suggesting that AKAP220 functions to regulate phosphatase activity (30). However, the physiological significance of the complex formation of PKA with AKAP220 is not known.

While analyzing the modes of activation and action of GSK-3 we found that GSK-3 binds directly to AKAP220. Here we show that GSK-3, PKA, PP1, and AKAP220 form a complex. Furthermore, we demonstrate that GSK-3 complexed with AKAP220 is regulated by PKA and PP1 more efficiently than GSK-3 free from AKAP220. These results suggest that one of the mechanisms by which PKA regulates GSK-3 is mediated by AKAP220.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Chemicals-- Plasmids that cover human AKAP220 (hAKAP220) nucleotides 175-5413 and 3866-9748 were kindly supplied by Drs. K. Taskén (University of Oslo, Oslo, Norway) (31) and N. Kusuhara (Kazusa DNA Research Institute, Chiba, Japan), respectively. Human GSK-3 cDNA was provided by Dr. J. R. Woodgett (Ontario Cancer Institute, Toronto, Canada). The anti-MBP and anti-GST antibodies were prepared in rabbits by immunization with recombinant MBP and GST, respectively. MBP and GST fusion proteins were purified from Escherichia coli according to the manufacturer's instructions. The anti-Myc antibody was prepared from 9E10 cells. COS, PC12, and 293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum, 10% fetal calf serum and 5% horse serum, and 10% fetal calf serum, respectively. The anti-AKAP220, anti-AKAP149, anti-GSK-3, anti-PP2Ac, anti-PP1c, mouse monoclonal anti-PKARII, and anti-PKAc antibodies were purchased from Transduction Laboratories (Lexington, KY). The anti-GSK-3, rabbit polyclonal anti-PKARII, anti-HA (16B12), and anti-GFP antibodies were from Upstate biotechnology (Lake Placid, NY), Santa Cruz Biotechnology (Santa Cruz, CA), Covance, and Molecular Probes, Inc. (Eugene, OR), respectively. [-³²P]ATP was obtained from Amersham Biosciences.

Plasmid Construction-- pBTM116HA/GSK-3, pCGN/GSK-3, pCGN/GSK-3^K85M, pCGN/GSK-3^Y216F, and pGEX-2T/GSK-3 were constructed as described (32). Standard recombinant DNA techniques were used to construct the following plasmids: pCGN/hAKAP220 (full length), pCGN/hAKAP220-(1-1108), pCGN/hAKAP220-(1011-1901), pEF-BOS-Myc/hAKAP220-(1011-1901), pEF-BOS-Myc/hAKAP220-(1011-1455), pEF-BOS-Myc/hAKAP220-(1017-1316), pEF-BOS-Myc/hAKAP220-(1316-1445), pEF-BOS-Myc/hAKAP220-(1456-1901), pMAL-c2/hAKAP220-(1011-1901), and pCGN/GSK-3^S9A, where Ser⁹ is mutated to Ala. In these plasmids, some plasmids were constructed by digesting the original plasmids with restriction enzymes and inserting the resultant fragments into the vectors. The other constructions were performed by inserting the fragments generated using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) into the vectors. The entire PCR products were sequenced, and the structures of all plasmids were confirmed by restriction analyses.

Complex Formation of AKAP220, GSK-3, PKA, and PP1-- To determine whether AKAP220 forms a complex with GSK-3, GSK-3, PKA, PP1, or PP2A at the endogenous level, PC12 cells (100-mm diameter dish) were lysed in 500 µl of lysis buffer (20 mM Tris/HCl, pH 7.5, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 5 mM phenylmethylsulfonyl fluoride, 25 mM -glycerophosphate, 5 mM sodium orthovanadate, and 5 mM NaF). The lysates (500 µg of protein) were immunoprecipitated with anti-AKAP220, anti-Myc, anti-GSK-3, anti-GFP, or anti-PKARII antibody, and then the immunoprecipitates were probed with the anti-AKAP220, anti-PKARII, anti-GSK-3, anti-PKAc, anti-GSK-3, anti-PP1c, and anti-PP2Ac antibodies. To demonstrate the complex formation of the deletion mutants of AKAP220 with GSK-3, PKA, and PP1 in intact cells, COS cells (60-mm diameter dish) transfected with pEF-BOS-Myc- or pCGN-derived plasmids were lysed in 200 µl of lysis buffer. The lysates (200 µg of protein) were immunoprecipitated with the anti-HA, anti-Myc, anti-GFP, or anti-PKARII antibody, and then the precipitates were probed with the anti-HA, anti-Myc, anti-GSK-3, and anti-PKARII antibodies. In these experiments, the rabbit polyclonal anti-PKARII antibody was used for immunoprecipitation and the mouse monoclonal anti-PKARII antibody was used for immunoblotting. In another experiment to show the complex formation of AKAP220, the lysates (250 µg of protein) of PC12 cells were incubated with GST-PP1c or GST (35 pmol of each) immobilized on glutathione-Sepharose 4B for 1 h at 4 °C. After GST-PP1c or GST was precipitated by centrifugation, the precipitates were probed with the anti-GSK-3 and anti-AKAP220 antibodies. To examine the direct interaction of AKAP220 with GSK-3 using the purified proteins in vitro, 0.6 µM GST-GSK-3 was incubated with MBP-AKAP220-(1011-1901) or MBP (15 pmol of each) immobilized on amylose resin in 50 µl of reaction mixture (20 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, and 1% CHAPS) for 1 h at 4 °C. After MBP-AKAP220-(1011-1901) was precipitated by centrifugation, the precipitates were probed with the anti-GST antibody.

To determine whether AKAP149 forms a complex with GSK-3 or PKA at the endogenous level, 293 cells (60-mm diameter dish) were lysed in 200 µl of lysis buffer. The lysates (1 mg of protein) were immunoprecipitated with the anti-Myc, anti-AKAP149, anti-GFP, or anti-PKARII antibody, and then the immunoprecipitates were probed with the anti-AKAP149, anti-PKARII, anti-GSK-3, and anti-PKAc antibodies.

Kinase Assay-- COS cells (60-mm diameter dish) expressing Myc-AKAP220-(1011-1901), Myc-AKAP220-(1011-1455), or Myc-AKAP220-(1456-1901) were lysed in 200 µl of lysis buffer. After the lysates (200 µg of protein) had been immunoprecipitated with the anti-GSK-3 or anti-Myc antibody, the immunoprecipitates were washed once with lysis buffer and three times with kinase buffer (50 mM Tris/HCl, pH 7.5, 10 mM MgCl₂, and 1 mM dithiothreitol) and then incubated with 50 µM GS peptide 1 (YRRAAVPPSPSLSRHSSPHQSEDEEE) or GS peptide 2 (YRRAAVPPSPSLSRHSSPHQS(P)EDEEE) in 30 µl of kinase buffer containing 50 µM [-³²P]ATP (400-800 cpm/pmol) for 30 min at 30 °C. The reaction mixture was then spotted onto phosphocellulose filters (Whatman P81) and washed with phosphoric acid (33, 34). The activity of GSK-3 was calculated by subtracting the activity of phosphorylation of GS peptide 1 from that of phosphorylation of GS peptide 2. When effects of Bt₂cAMP on wild-type GSK-3 and GSK-3^S9A were compared, Myc-AKAP220-(1011-1901) was expressed with HA-GSK-3 (wild type) or HA-GSK-3^S9A in COS cells.

Others-- Yeast two-hybrid screening was carried out as described (32, 35). Protein concentrations were determined using bovine serum albumin as a standard (36).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Complex Formation of GSK-3 with AKAP220 at the Endogenous Level-- To discover new functions of GSK-3, we attempted to identify a GSK-3-binding protein(s) and screened a mouse brain cDNA library by the yeast two-hybrid screen using wild-type GSK-3 as bait. When ~5 × 10⁵ transformants were screened, several clones were found to confer both His⁺ and LacZ⁺ phenotypes, and one clone was found to encode the carboxyl-terminal region of mouse AKAP220. AKAP220 was originally identified as an A-kinase anchoring protein with a molecular mass of 220 kDa (28). Among various cells tested (including COS, CHO-IR, 293, L, PC12, Rat-1, SW480, NIH3T3, F9, DLD, and C57MG), we detected significant expression of AKAP220 in PC12 cells, a line of rat pheochromocytoma cells (data not shown). Because GSK-3 was detected more than GSK-3 in PC12 cells (Fig. 1A, lane 1), we examined whether AKAP220 forms a complex with GSK-3 in PC12 cells. When the lysates of PC12 cells were immunoprecipitated with the anti-AKAP220 antibody, PKARII and PKAc were detected in the AKAP220 immune complex (Fig. 1A, lane 3). Furthermore, GSK-3 was also observed in the immune complex (Fig. 1A, lane 3). When the lysates were immunoprecipitated with the anti-Myc antibody as a control, neither PKA nor GSK-3 was co-precipitated (Fig. 1A, lane 2). When the lysates were immunoprecipitated with the anti-PKARII antibody, AKAP220 and GSK-3 were observed in addition to PKAc in the PKARII immune complex (Fig. 1A, lanes 4 and 5). Reciprocally, AKAP220 and PKARII were co-precipitated with GSK-3 (Fig. 1A, lanes 6 and 7). GSK-3 was not observed in the AKAP220 complex (Fig. 1A, lane 3), but this might have been because of the low detection of GSK-3 protein by the antibody that we used. AKAP149, another AKAP family member, formed a complex with PKA but not with GSK-3 (Fig. 1B). These results indicate that GSK-3 and PKA form a complex with AKAP220 in intact cells at the endogenous level.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Complex formation of AKAP220 with GSK-3 at the endogenous level. A, interaction of AKAP220 with PKA, GSK-3, and GSK-3. The lysates (30 µg of protein) of PC12 cells were probed with the anti-AKAP220, anti-PKARII, anti-GSK-3, anti-PKAc, and anti-GSK-3 antibodies (lane 1). The same lysates (500 µg of protein) were immunoprecipitated with the anti-AKAP220 (lane 3), anti-PKARII (lane 5), or anti-GSK-3 (lane 7) antibody, and then the immunoprecipitates were probed with the anti-AKAP220, anti-PKARII, anti-GSK-3, anti-PKAc, and anti-GSK-3 antibodies. The lysates (500 µg of protein) were also immunoprecipitated with the anti-Myc (lanes 2 and 6) or anti-GFP (lane 4) antibody for the control experiments. The bands observed in lanes 2, 4, and 6 are immunoglobulin. IP, immunoprecipitation; Ab, antibody. B, inability of AKAP149 to bind to GSK-3. The lysates (50 µg of protein) of 293 cells were probed with the anti-AKAP149, anti-PKARII, anti-GSK-3, and anti-PKAc antibodies (lane 1). The same lysates (1 mg of protein) were immunoprecipitated with the anti-Myc (lane 2), anti-AKAP149 (lane 3), anti-GFP (lane 4), or anti-PKARII (lane 5) antibody, and the immunoprecipitates were probed with the anti-AKAP149, anti-PKARII, anti-GSK-3, and anti-PKAc antibodies. The band observed in lane 2 is immunoglobulin. The results shown are representative of four independent experiments.

Determination of the GSK-3-binding Site on AKAP220-- Various constructs of AKAP220 used in this study are shown in Fig. 2. To examine which region of AKAP220 is necessary for the complex formation with GSK-3, HA-AKAP220 (full length), HA-AKAP220-(1-1108), or HA-AKAP220-(1011-1901) was expressed in COS cells (Fig. 3A, lanes 1-4). Endogenous GSK-3 was complexed with HA-AKAP220 (full length) and HA-AKAP220-(1011-1901) but not with HA-AKAP220-(1-1108) (Fig. 3A, lanes 5-8). These results indicate that GSK-3 forms a complex with the carboxyl-terminal region of AKAP220. To further analyze the binding region, various deletion mutants of Myc-AKAP220 were expressed in COS cells. GSK-3 was co-precipitated with Myc-AKAP220-(1011-1901), Myc-AKAP220-(1011-1455), and Myc-AKAP220-(1017-1316) but not with Myc-AKAP220-(1316-1445) and Myc-AKAP220-(1456-1901) (Fig. 3B). These results demonstrate that GSK-3 forms a complex with a region within amino acids 1017-1316 of AKAP220. Axin is another GSK-3-binding protein and plays an essential role in the Wnt signaling pathway (32). Previously we showed that two kinase-inactive mutants of GSK-3, GSK-3^K85M and GSK-3^Y216F, do not associate with Axin (32). In contrast to Axin, AKAP220 associated with both kinase-inactive mutants as well as wild-type GSK-3 (Fig. 3C), suggesting that AKAP220 and Axin associate with GSK-3 in different manners. To examine whether GSK-3 binds to AKAP220 directly, MBP-AKAP220-(1011-1901) and GST-GSK-3 were purified (Fig. 3D, lanes 1 and 3). GST-GSK-3 but not GST precipitated with MBP-AKAP220-(1011-1901) immobilized on amylose resin, and GST-GSK-3 did not precipitate with MBP (Fig. 3D, lanes 5-7). These results indicate that the binding of GSK-3 and AKAP220 is direct. To examine whether GSK-3 complexed with AKAP220 is active, the kinase assay was performed. In this assay GS peptides 1 and 2 were used as substrates. It is known that GSK-3 specifically phosphorylates GS peptide 2 but not GS peptide 1 (33). When the lysates expressing Myc-AKAP220-(1011-1901) were immunoprecipitated with the anti-Myc antibody, the activity to phosphorylate GS peptide 2 was observed specifically in the Myc-AKAP220-(1011-1901) immune complex (Fig. 3E), indicating that active GSK-3 forms a complex with AKAP220. Consistent with the results shown in Fig. 3B, GSK-3 activity was observed in the Myc-AKAP220-(1011-1455) immune complex but not in the Myc-AKAP220-(1456-1901) immune complex (Fig. 3E).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Schematic representation of human AKAP220 constructs used in this study. The white boxes indicate the PKARII-binding site or PP1-binding motif, respectively.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Determination of the GSK-3-binding site on AKAP220. A, interaction of the AKAP220 deletion mutants with GSK-3 in intact cells. The lysates (20 µg of protein) of COS cells expressing HA-AKAP220 deletion mutants were probed with the anti-HA and anti-GSK-3 antibodies (lanes 2-4). The same lysates (200 µg of protein) were immunoprecipitated with the anti-HA antibody, and the immunoprecipitates were probed with the anti-HA and anti-GSK-3 antibodies (lanes 6-8). The lysates of COS cells transfected with empty vectors were used as a control (lanes 1 and 5). Arrowheads indicate expression of HA-AKAP220 (full length) or its mutants. Full, full length; IP, immunoprecipitation; Ab, antibody. B, complex formation of the deletion mutants of AKAP220 with GSK-3. The lysates (20 µg of protein) of COS cells expressing various deletion mutants of Myc-AKAP220 were probed with the anti-Myc and anti-GSK-3 antibodies (lanes 2-6). The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc and anti-GSK-3 antibodies (lanes 7-11). The lysates of COS cells transfected with empty vectors were used as a control (lane 1). Ig, immunoglobulin. C, interaction of AKAP220 with kinase-inactive mutants of GSK-3. The lysates (20 µg of protein) of COS cells expressing the indicated proteins were probed with the anti-Myc and anti-HA antibodies (lanes 2-5). The same lysates were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc and anti-HA antibodies (lanes 6-9). The lysates of COS cells transfected with empty vectors were used as a control (lane 1). WT, wild type. D, direct interaction of AKAP220 with GSK-3. Purified proteins (0.5 µg of each protein) used in this experiment were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining (lanes 1-4). GST-GSK-3 or GST (0.6 µM) was incubated with MBP-AKAP220-(1011-1901) (lanes 5 and 6) or MBP (lane 7) (15 pmol of each) immobilized on amylose resin, and then MBP-AKAP220-(1011-1901) and MBP were precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. Results shown are representative of three independent experiments. E, activity of GSK-3 in the AKAP220 complex. After the lysates (200 µg of protein) of COS cells expressing Myc-AKAP220-(1011-1901) (lanes 1 and 2), Myc-AKAP220-(1011-1455) (lanes 3 and 4), or Myc-AKAP220-(1456-1901) (lanes 5 and 6) had been immunoprecipitated with the anti-Myc antibody, the immunoprecipitates were incubated with GS peptide 1 (lanes 1, 3, and 5) or GS peptide 2 (lanes 2, 4, and 6). The activities of phosphorylation of GS peptides 1 and 2 were measured. Results shown are expressed as means ± S.E. from three independent experiments.

Complex Formation of GSK-3, AKAP220, PKA, and PP1-- It has been shown that AKAP220 binds to PP1 in addition to PKA (29). We therefore examined whether GSK-3, PKA, and PP1 bind simultaneously to AKAP220. The site of AKAP220 that binds to PKA is the region within amino acid residues 1650-1663 (31). When various deletion mutants of Myc-AKAP220 were expressed in COS cells and the lysates were immunoprecipitated with the anti-PKARII antibody, AKAP220-(1011-1901) and AKAP220-(1456-1901) but not AKAP220-(1011-1455) formed a complex with PKARII (Fig. 4A), consistent with the previous report (31). Taken together with the observation that GSK-3 interacts with AKAP220-(1017-1316), these results clearly demonstrate that GSK-3 and PKA bind to the different sites of AKAP220. It has been reported that PP1 binds to residues 1195-1198 of AKAP220 (29, 31). Indeed, PP1 was observed with GSK-3 and PKA in the AKAP220 immune complex when the lysates of PC12 cells were immunoprecipitated with the anti-AKAP220 antibody (Fig. 4B). Under the same conditions, PP2A was only slightly immunoprecipitated with AKAP220 (Fig. 4B). When the lysates of PC12 cells were incubated with GST-PP1c, endogenous GSK-3 and AKAP220 were detected in the GST-PP1c complex (Fig. 4C). Furthermore, overexpression of HA-PP1c did not affect the complex formation of GSK-3 and Myc-AKAP-(1011-1901) (Fig. 4D). Although we did not definitively conclude that the sites of AKAP220 that bind to PP1 and GSK-3 are different, it is likely that GSK-3, PKA, and PP1 can bind to AKAP220 simultaneously and that they form a quaternary complex.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Complex formation of AKAP220, GSK-3, PKA, and PP1. A, interaction of PKARII with AKAP220 in intact cells. The lysates (20 µg of protein) of COS cells expressing Myc-AKAP220-(1011-1901) (lane 2), Myc-AKAP220-(1011-1455) (lane 3), or Myc-AKAP220-(1456-1901) (lane 4) were probed with the anti-Myc and anti-PKARII antibodies (lanes 1-4). The same lysates (200 µg of protein) were immunoprecipitated with the anti-PKARII (lanes 7-9) or anti-GFP antibody (lane 5), and the immunoprecipitates were probed with the anti-Myc and anti-PKARII antibodies (lanes 5-9). The lysates of COS cells transfected with empty vectors were used as a control (lanes 1 and 6). Ig, immunoglobulin; IP, immunoprecipitation; Ab, antibody. B, interaction of GSK-3, PKA, and PP1 with AKAP220 at the endogenous level. The lysates (30 µg of protein) of PC12 cells were probed with the anti-AKAP220, anti-PKARII, anti-GSK-3, anti-PP1c, anti-PP2Ac, and anti-PKAc antibodies (lane 1). The lysates (500 µg of protein) were immunoprecipitated with the anti-Myc (lane 2) or the anti-AKAP220 (lane 3) antibody, and the immunoprecipitates were probed with the anti-AKAP220, anti-PKARII, anti-GSK-3, anti-PP1c, anti-PP2Ac, and anti-PKAc antibodies. The band observed in lane 2 is immunoglobulin. C, interaction of GST-PP1c with the AKAP220 and GSK-3 complex. GST-PP1c (lane 1) and GST (lane 2) (0.5 µg of each protein) used in this experiment were subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining. The lysates (30 µg of protein) of PC12 cells were probed with the anti-AKAP220 and anti-GSK-3 antibodies (lane 3). The lysates (250 µg of protein) of PC12 cells were incubated with GST-PP1c (lane 4) or GST (lane 5) (35 pmol of each) immobilized on glutathione-Sepharose 4B, and then GST-PP1c and GST were precipitated by centrifugation. The precipitates were probed with the anti-AKAP220 and anti-GSK-3 antibodies. D, complex formation of AKAP220 with GSK-3 and PP1c. The lysates (20 µg of protein) of COS cells expressing the indicated proteins were probed with the anti-Myc, anti-GSK-3, and anti-HA antibodies (lanes 2-4). The same lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc, anti-GSK-3, and anti-HA antibodies (lanes 5-7). The lysates of COS cells transfected with empty vectors were used as a control (lane 1). The results shown are representative of three independent experiments.

Regulation of GSK-3 by PKA and PP1 in the AKAP220 Complex-- Finally we examined the physiological significance of the binding of GSK-3 to AKAP220. Because PKA and PP1 regulate the GSK-3 activity (23, 24, 37), we examined the regulation of GSK-3 in the AKAP220 complex. Myc-AKAP220-(1011-1901) was expressed in COS cells, and the cells were treated with Bt₂cAMP to activate PKA. When the lysates were immunoprecipitated with the anti-GSK-3 antibody, the total GSK-3 activity decreased in a manner dependent on the doses of Bt₂cAMP (Fig. 5A). When cells were treated with 1 mM Bt₂cAMP, the decrease in the activity of total GSK-3 was about 20%. When the same lysates were immunoprecipitated with the anti-Myc antibody, the activity of GSK-3 in the AKAP220 complex decreased more markedly than that of total GSK-3 (Fig. 5A). COS cells expressing Myc-AKAP220-(1011-1901) were treated with calyculin A, an inhibitor of both PP1 and PP2A, and the lysates were immunoprecipitated with the anti-GSK-3 antibody. Calyculin A inhibited GSK-3 in a dose-dependent manner and produced about 50% inhibition of GSK-3 (Fig. 5B). Moreover, calyculin A inhibited the activity of GSK-3 complexed with AKAP220 strongly (Fig. 5B). To analyze the modes of the inhibitory action of Bt₂cAMP and calyculin A, cells were treated with both reagents. Calyculin A did not enhance Bt₂cAMP-dependent inhibition of GSK-3 in the AKAP220 complex (Fig. 5C). Similarly, Bt₂cAMP did not stimulate calyculin A-dependent inhibition of GSK-3 (Fig. 5D). These results suggest that PKA and protein phosphatase regulate GSK-3 in the AKAP220 complex efficiently and that their modes of action toward GSK-3 are the same.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Regulation of GSK-3 activity by PKA and PP1 in the AKAP220 complex. A and B, effect of Bt2 cAMP or calyculin A on the GSK-3 activity. After COS cells expressing Myc-AKAP220-(1011-1901) were treated with Bt2cAMP (A) or calyculin A (B) for 30 min, the activities of total GSK-3 () and GSK-3 complexed with AKAP220 () were measured. In the complex immunoprecipitated with the anti-GSK-3 antibody from the cells without treatment, the activities of phosphorylation of GS peptides 1 and 2 were 1850 ± 390 and 18980 ± 1100 cpm, respectively. In the complex immunoprecipitated with the anti-Myc antibody from the cells without treatment, the activities of phosphorylation of GS peptides 1 and 2 were 1370 ± 270 and 3370 ± 560 cpm, respectively. The activity of GSK-3 was determined by subtracting the activity of phosphorylation of GS peptide 1 from the activity of phosphorylation of GS peptide 2. The results shown are expressed as a percentage of the activity of GSK-3 without Bt2cAMP or calyculin A treatment, and they are means ± S.E. from three independent experiments. C, effect of calyculin A on Bt2cAMP-dependent inhibition of GSK-3. COS cells expressing Myc-AKAP220-(1011-1901) were treated with the indicated concentrations of Bt2cAMP in the presence () or absence () of 1 nM calyculin A for 30 min. GSK-3 activity in the AKAP220 complex was measured, and the results shown are means ± S.E. from three independent experiments. D, effect of Bt2cAMP on calyculin A-dependent inhibition of GSK-3. COS cells expressing Myc-AKAP220-(1011-1901) were treated with the indicated concentrations of calyculin A in the presence () or absence () of 0.125 mM Bt2cAMP for 30 min. GSK-3 activity in the AKAP220 complex was measured, and the results shown are means ± S.E. from three independent experiments.

PKA phosphorylates Ser⁹ of GSK-3 directly and reduces its activity (23, 24). We examined whether inhibition of the GSK-3 activity in the AKAP220 complex is through the phosphorylation of Ser⁹. To this end, we expressed Myc-AKAP220-(1011-1091) with either HA-GSK-3 (wild type) or HA-GSK-3^S9A in COS cells and measured the GSK-3 activities (Fig. 6). GSK-3^S9A is a GSK-3 mutant in which Ser⁹ is changed to Ala. The inhibition of the GSK-3 activity in the AKAP220 immune complex from the lysates expressing HA-GSK-3^S9A by Bt₂cAMP was attenuated as compared with that from the lysates expressing HA-GSK-3 (wild type). Because the endogenous GSK-3 was also included in the AKAP220 complex (Fig. 6), the decrease of the GSK-3 activity in the lysates expressing Myc-AKAP220-(1011-1901) and HA-GSK-3^S9A may reflect the inhibition of endogenous GSK-3 by Bt₂cAMP. These results suggest that the phosphorylation of Ser⁹ of GSK-3 is also important for the regulation of GSK-3 complexed with AKAP220.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of Bt2cAMP on GSK-3S9A complexed with AKAP220. COS cells expressing Myc-AKAP220-(1011-1901) with either HA-GSK-3 (wild type, lanes 1 and 2) or HA-GSK-3S9A (lanes 3 and 4) were treated with (lanes 2 and 4) or without (lanes 1 and 3) 1 mM Bt2cAMP for 30 min, and the activities of GSK-3 complexed with AKAP220 were measured. The results shown are expressed as a percentage of the activity of GSK-3 without Bt2cAMP treatment, and they are means ± S.E. from three independent experiments. The lower panel shows the complex formation of HA-GSK-3 (wild type) or HA-GSK-3S9A with Myc-AKAP220-(1011-1901). The lysates (200 µg of proteins) described above were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-GSK-3 antibody. WT, wild type.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we demonstrated that GSK-3 forms a complex with AKAP220, as shown by the following results. (i) GSK-3 associated with AKAP220 as well as PKA and PP1 in intact cells at the endogenous level. (ii) Purified GSK-3 bound directly to purified AKAP220. (iii) PKA and GSK-3 interacted with different sites of AKAP220. (iv) PP1 did not inhibit the binding of GSK-3 to AKAP220. These results suggest that GSK-3, PKA, and PP1 bind to AKAP220 simultaneously and that they form a quaternary complex. Because it has been shown that PKA forms a complex with GSK-3 in intact cells (23), our results may explain the mechanisms of this interaction. There are more than 40 members of the AKAP protein family (26, 27). Although we have not examined all of the possibilities, at least GSK-3 did not form a complex with AKAP149, suggesting that GSK-3 binds to AKAP220 specifically.

What is the physiological significance of the complex formation of GSK-3 and AKAP220? We showed that Bt₂cAMP inhibits the activity of GSK-3 complexed with AKAP220 more efficiently than the total GSK-3 activity. It has already been reported that PKA inhibits GSK-3 by a similar mechanism with PKB and p90Rsk. These protein kinases phosphorylate Ser⁹ of GSK-3 directly, and this phosphorylation prevents the catalytic site of GSK-3 from interacting with substrates (16, 17). However, Bt₂cAMP does not inhibit the total GSK-3 activity completely. For instance, treatment of Rat-1, NIH3T3, and 293 cells with PKA-stimulating reagents such as 8-bromo-cAMP, forskolin, and isoproterenol leads to about a 40% decrease in GSK-3 activity (23, 24). Consistent with these observations, our results demonstrated that treatment with Bt₂cAMP produces only a 20% inhibition of the total GSK-3 activity in COS cells. Interestingly, about 80% of the activity of GSK-3 complexed with AKAP220 was inhibited after treatment of the cells with Bt₂cAMP. We also demonstrated that the inhibition of the GSK-3 activity in the AKAP220 immune complex from the lysates expressing HA-GSK-3^S9A by Bt₂cAMP is attenuated in comparison with that from the lysates expressing HA-GSK-3 (wild type). These results suggest that the inhibition of GSK-3 by PKA in the AKAP220 complex is caused by the phosphorylation of Ser⁹. Therefore, PKA would phosphorylate and inhibit GSK-3 efficiently in the AKAP220 complex.

Because insulin and EGF, which activate PKB and p90Rsk, respectively, inhibit the GSK-3 activity by about 50% (20-22), PKB and p90Rsk may also form a complex with GSK-3 and regulate its activity effectively. Indeed, PKB associates with GSK-3 under the overexpression conditions (38), but whether the interaction is direct or indirect is not known. Dvl, a component of the Wnt signaling pathway, inhibits GSK-3-dependent phosphorylation of -catenin by an unknown mechanism (39, 40). However, PKB and Dvl did not form a complex with AKAP220 (data not shown). Therefore, AKAP220 may associate with PKA selectively among the GSK-3 upstream molecules and enhance the signaling between PKA and GSK-3. It is intriguing to speculate that GSK-3 forms a complex with various upstream molecules through the third proteins, resulting in efficient and specific regulation of the kinase activity.

Furthermore, our results demonstrated that calyculin A efficiently inhibits the activity of GSK-3 complexed with AKAP220. Calyculin A is an inhibitor of protein phosphatases, especially PP1 and PP2A (41). Therefore, protein phosphatases may activate GSK-3 by dephosphorylating it. However, it is unlikely that PP2A affects GSK-3 in the AKAP220 complex, because PP2A associated with AKAP220 very weakly in comparison with PP1 (Fig. 4B), and okadaic acid, a potent inhibitor of PP2A, did not inhibit GSK-3 in COS cells (data not shown), consistent with the previous observations using SY5Y cells (42). Furthermore, using rat brain slices it has been shown that calyculin A suppresses GSK-3-dependent phosphorylation of tau by inhibiting PP1 (37). Thus, we conclude that PP1 but not PP2A is involved in the regulation of GSK-3 in the AKAP220 complex. We also demonstrated that the effects of Bt₂cAMP and calyculin A are not additive, suggesting that PKA and PP1 regulate GSK-3 bound to AKAP220 in a similar manner. It has been reported that AKAP220 binds to and inhibits PP1 and that the binding of PKARII to AKAP220 enhances the phosphatase inhibition (30). Taken together, these facts suggest that AKAP220 may regulate GSK-3 in two ways. One is that PKA directly phosphorylates and inhibits GSK-3 efficiently in the AKAP220 complex; the other is that AKAP220 and PKA inhibit PP1 co-operatively, thereby enhancing the phosphorylation of GSK-3 and inhibiting the GSK-3 activity.

GSK-3 is not only regulated by various upstream regulators but also has multiple substrates (1-3). How does GSK-3 find its appropriate substrates? So far, we have found that GSK-3 binds to Axin, which functions as a scaffold protein in the Wnt signaling pathway by interacting with -catenin and adenomatous polyposis coli gene product (43, 44). Axin enhances the phosphorylation of -catenin by GSK-3 by positioning GSK-3 close to -catenin, resulting in the inhibition of the Wnt signaling pathway (32). Thus, anchoring proteins such as AKAP and Axin enhance the specificity of GSK-3 relative to both upstream regulators and downstream substrates. Another unique characteristic of GSK-3 is that the phosphorylation of some substrates by GSK-3 requires prior phosphorylation by distinct kinases (1, 2). For instance, in the cases of the G subunit of PP1 and adenomatous polyposis coli gene product GSK-3 phosphorylates these substrates after PKA phosphorylates them. Therefore, it is intriguing to speculate that substrates of PKA and GSK-3 are also present in the AKAP220 complex. We are currently trying to identify the AKAP220-binding proteins.

Because PKA is involved in many parallel signaling pathways, it is important to understand the mechanisms by which this kinase is activated and recognizes substrates. It is generally thought that the AKAP family proteins may function to coordinate multiple components of the signal transduction pathway (25-27). This concept is in accord with our results, which indicate that AKAP220 binds to PKA, GSK-3, and PP1, resulting in regulation of GSK-3 by PKA. AKAPs are also known to direct PKA to discrete intracellular locations (25-27). For example, AKAP15/18 associates with plasma membranes through lipids (45). mAKAP is targeted to the perinuclear membranes of cardiomyocytes (25). AKAP350/450 is present in centrosomes (46). AKAP220 is expressed abundantly in rat testis and exhibits punctual staining patterns in rat testis cell lines (28). Furthermore, AKAP220 is present in human male germ cells and mature sperm, suggesting that AKAP220 is involved in spermatogenesis (31). Although we found that AKAP220 is present in PC12 cells, its functions are not clear. GSK-3 induces apoptosis in cerebellar granule neurons, and PKA prevents it by inhibiting GSK-3 (24). Therefore, AKAP220 may be involved in cellular differentiation and death. Further studies will be necessary to understand the whole picture of the physiological roles of the AKAP220 complex.

	ACKNOWLEDGEMENTS

We thank Drs. S. Nakashima and T. Hinoi for technical assistance and Drs. K. Taskén, N. Kusuhara, and J. R. Woodgett for donation of plasmids.

	FOOTNOTES

^* This work was supported by grants-in-aid for scientific research (B) and for scientific research on priority areas (C) from the Ministry of Education, Science, and Culture, Japan and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders.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: Dept. of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Tel.: 81-82-257-5130; Fax: 81-82-257-5134; E-mail: akikuchi@hiroshima-u.ac.jp.

Published, JBC Papers in Press, July 29, 2002, DOI 10.1074/jbc.M206210200

	ABBREVIATIONS

The abbreviations used are: GSK-3, glycogen synthase kinase-3; PKB, protein kinase B; PKA, protein kinase A; AKAP, A-kinase anchoring protein; PP1, type 1 protein phosphatase; hAKAP220, human AKAP220; MBP, maltose-binding protein; GST, glutathione S-transferase; PP2Ac, catalytic subunit of protein phosphatase 2A; PP1c, catalytic subunit of PP1; PKAR, regulatory subunit of PKA; PKAc, catalytic subunit of PKA; HA, hemagglutinin; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GS, glycogen synthase; Bt₂cAMP, dibutyryl cyclic AMP..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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