53BP2S, Interacting with Insulin Receptor Substrates, Modulates Insulin Signaling*

It is well known that insulin receptor substrates (IRS) act as a mediator for signal transduction of insulin, insulin-like growth factors, and several cytokines. To identify proteins that interact with IRS and modulate IRS-mediated signals, we performed yeast two-hybrid screening with IRS-1 as bait. Out of 109 cDNA-positive clones identified from a human placental cDNA library, two clones encoded 53BP2, p53-binding protein 2 (53BP2S), a short form splicing variant of the apoptosis-stimulating protein of p53 that possesses Src homology region 3 domain, and ankyrin repeats domain, and had been reported to interact with p53, Bcl-2, and NF-κB. Interaction of 53BP2S with IRS-1 was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays in COS-7 cells and 3T3-L1 adipocytes. The Src homology region 3 domain and ankyrin repeats domain of 53BP2S were responsible for its interaction with IRS-1, whereas the phosphotyrosine binding domain and a central domain (amino acid residues 750-861) of IRS-1 were required for its interaction with 53BP2S. In CHO-C400 cells, expression of 53BP2S reduced insulin-stimulated IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation. In addition, the amount of the phosphatidylinositol 3-kinase regulatory p85 subunit associated with tyrosine-phosphorylated proteins, and activation of Akt was enhanced by 53BP2S expression. Although 53BP2S also enhanced Akt activation in 3T3-L1 adipocytes, insulin-induced glucose transporter 4 translocation was markedly inhibited in accordance with reduction of insulin-induced AS160 phosphorylation. Together these data demonstrate that 53BP2S interacts and modulates the insulin signals mediated by IRSs.

It is well established that insulin and IGFs 3 display a variety of bioactivities, including induction of growth promotion, differentiation, and metabolic function. Insulin or IGFs bind to the extracellular subunits of their respective receptors and induce intramolecular conformational changes resulting in the activation of the ␤ subunit intrinsic tyrosine kinase activity (1,2). Activated receptor kinases phosphorylate several intracellular substrates, including insulin receptor substrates (IRSs), Shc Cbl, or Crk. Tyrosine phosphorylation of these substrates leads to their binding to several intermediate signaling molecules containing SH2 domains. In particular, the binding of the p85 regulatory subunit of PI 3-kinase to IRSs results in the recruitment and activation of the p110 catalytic subunit (3,4). The interaction of Grb2 with tyrosine-phosphorylated IRS and Shc leads to activation of the small GTP-binding protein Ras through the plasma membrane recruitment of the guanyl nucleotide exchange factor SOS (3,5,6). These interactions result in activation of PI 3-kinase cascade and Ras-MAPK cascade, respectively (7,8). It has become clear that activation of these two cascades play important roles in a variety of insulin or IGF actions.
Insulin is well known to induce translocation of glucose transporter 4 (Glut4), which is expressed in muscle and adipose tissue, from multiple intracellular compartments to the plasma membrane, leading to an enhancement of glucose uptake. It is well established that activation of PI 3-kinase activity, generation of the PI 3,4,5-trisphosphate, activation of the downstream effector Akt, and subsequent phosphorylation of Akt substrate, AS160, are necessary events required for the insulin stimulation of Glut4 translocation and glucose uptake (9 -17). Thus, the IRS family proteins play essential roles as intermediate mediators for this signal transduction pathway central to the biological actions of both insulin and IGF-I receptors.
Four members of IRS family proteins (IRS1-4) have been identified to date (8). These IRS family proteins share two highly homologous amino-terminal regions, the pleckstrin * This work was supported in part by Grant-in-aid for International Joint Research 08044193 (to S.-I. T.), Grant-in-aid for Scientific Research A 16208028 (to S.-I. T.) from the Ministry of Education, Science, and Culture of Japan, and by the Program for Promotion of Basic Research Activities for Innovative Biosciences (to F. H.). 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. 1 Present address; Research Institute for Health Fundamentals, Ajinomoto Co.
homology (PH) domain and the phosphotyrosine binding (PTB) domain, which play important roles in the interaction with receptor tyrosine kinase. However, the carboxyl-terminal region is not conserved except for the tyrosine residues possibly phosphorylated by the receptor tyrosine kinases (8).
In addition to tyrosine phosphorylation, there are a multiple putative serine/threonine (Ser/Thr) phosphorylation sites in the carboxyl-terminal region, several of which are reported to be involved in the modulation of insulin-induced IRS tyrosine phosphorylation and play important roles in modulation of insulin or IGF signals (18 -24). For example, in 3T3-L1 adipocytes pretreatment with tumor necrosis factor-␣ reduced insulin-induced glucose uptake through an impairment of insulinstimulated IRS-1 tyrosine phosphorylation (25)(26)(27). In contrast, we found that in rat FRTL-5 thyroid cells, chronic pretreatment with thyrotropin markedly potentiated DNA synthesis in response to IGF-I (28,29). Detailed analyses showed that thyrotropin pretreatment enhanced IGF-I-induced IRS-2 tyrosine phosphorylation, resulting in the augmentation of IGF-I signals (30,31). Nevertheless, in both experimental model systems, in vitro phosphorylation assays demonstrated that Ser/Thr phosphorylation and association of some proteins with IRS are related to alterations of IRS tyrosine phosphorylation (26). Thus the identification of IRS-associated proteins is an essential prerequisite for understanding the alteration of IRS tyrosine phosphorylation. Thus, this study was undertaken to isolate proteins that interact and modulate IRS-mediated signals. By using yeast two-hybrid screening, we have cloned a cDNA that encodes a protein known as 53BP2S, a short form splicing variant of ASPP2. Our data indicate that 53BP2S plays an important regulatory role by modulating the relative extent of IRS-1 versus IRS-2 tyrosine phosphorylation and subsequent downstream signals mediating insulin-stimulated glucose transport.

EXPERIMENTAL PROCEDURES
Materials-Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), and Hanks' buffered saline solution were purchased from Nissui (Tokyo, Japan). Calf serum and fetal bovine serum were obtained from JRH Bioscience (Tokyo, Japan). Penicillin and streptomycin were obtained from Ban'yu Pharmaceutical Co. (Tokyo, Japan). Dr. Takaaki Aoyagi (Institute of Microbial Chemistry, Tokyo, Japan) generously provided leupeptin and pepstatin. The phosphotyrosine monoclonal antibodies PY20 and 4G10 were from Sigma and ICN (Irvine, CA). Polyclonal IRS-1 and IRS-2 antibodies were prepared by immunizing rabbits with synthetic peptides as reported previously (30). GFP polyclonal antibody was purchased from BD Biosciences. The FLAG M2 monoclonal antibody and HA antibody were from Sigma. Phospho-Akt-specific antibody (Ser-473) and phospho-ERK-specific antibody were from Cell Signaling (Beverly, MA). The 53BP2 antibody was purchased from BD Biosciences. The monoclonal ASPP2 antibody clone DX54.10 was from Sigma. The Myc and AS160 antibodies were purchased from Upstate (Charlottesville, VA). Phospho-AS160 (Thr-642)-specific antibody was from BioSource (Camarillo, CA). LY294002 was obtained from Sigma. Alexa Fluor 596 anti-mouse IgG and Alexa Fluor 488 anti-mouse IgG were purchased from Molecular Probes (Eugene, OR). All dishes, plates, and flasks were obtained from IWAKI (Tokyo, Japan). Other chemicals were of the reagent grade and available commercially.
Plasmid Construction-We obtained pBS-Bbp, containing a short form splicing variant of ASPP2, and 53BP2S (residues 123-1128 of ASPP2), a kind gift from Dr. Louie Naumovski (Stanford University School of Medicine, Stanford, CA) (33). IRS-1 cDNA was a kind gift from Dr. Takashi Kadowaki (Graduate School of Medicine, the University of Tokyo, Tokyo, Japan). IRS-1 cDNA containing full-length open reading frame was amplified by PCR using two primers, 5Ј-GGGGCATATG-GCGAGCCCTCCGGATA-3Ј and T7 primer. The PCR product was digested by NdeI and BamHI and was cloned into the NdeI-BamHI site of the pAS2-1 vector (BD Biosciences). The resulting plasmid was named pAS-IRS-1 and used for two-hybrid screening as bait. pGEX vectors were used for expression of fusion proteins with GST in Escherichia coli. By digesting pACT-53BP2S, the EcoRI-PstI, PstI-EcoRI, or EcoRI-EcoRI fragment, which encodes only ankyrin repeats, only the SH3 domain, or both domains, was cloned into the pGEX vector in-frame, yielding pGEX-ANK, pGEX-SH3, or pGEX-53BP2S, respectively. These plasmids were used for expression and purification of fusion proteins with GST in E. coli. pACT-ANK and pACT-SH3 were constructed in pACT2 vector (BD Biosciences) by the same way as pGEX-ANK and pGEX-SH3. pIRS-3 and pGFP-IRS-4 were constructed as described before (34). pIRS-2 was constructed as follows. Briefly, EcoRI fragment containing the full length of IRS-2 was cloned into pcDNA3. pFLAG-IRS-1, which expresses FLAG-tagged IRS-1, was constructed as follows. Full length of IRS-1 open reading frame was amplified by PCR using two primers, 5Ј-CCCCGATATCAAC-TATGGCGAGCCCTCCG-3Ј and T3 primers. The EcoRV-BamHI fragments of the PCR product was cloned into pCMV-FLAG-2 vector in-frame. Convenient restriction enzymes were used to construct the plasmids expressing some deletion mutants of IRS-1 fused with GFP. pGFP-D13 or pGFP-D14, which is a plasmid expressing IRS-1 D13 or IRS-1 D14 deletion mutant fused with GFP, respectively, was constructed as follows. IRS-1 fragment encoding the amino acid residues 660 -861 or 750 -861 was amplified by PCR using two primers, 5Ј-AGATGAAAGCTTCCAGTGG-3Ј and T3 primer or 5Ј-CCAGAAGCTTCCCAGCACAAGCC-3Ј and T3 primer. Amplified fragments were digested by HindIII and BamHI and cloned into pEGFP-C1 in-frame. BamHI fragment containing 53BP2S, a short form splicing variant of ASPP2 from the pBS-Bbp, was cloned into pEGFP-C1 or pCMV-FLAG-2 to express GFP-tagged or FLAG-tagged 53BP2S, respectively, in mammalian cells. pHA-Akt2 or pFLAG-AS160 was a kind gift from Dr. Morris Birnbaum (University of Pennsylvania).
Yeast Two-hybrid Screening-pAS-IRS-1, which expresses the full length of IRS-1 fused with Gal4-DNA binding domain in yeast, was used as bait. cDNA library expressing human placental cDNA fused with Gal4 activation domain was obtained from BD Biosciences. Yeast strain CG1945 was used for the library screening. CG1945 cells were transformed with pAS-IRS-1 and human placental cDNA library by a lithium method. Transformants that could grow in the medium lacking leucine, tryptophan, and histidine and containing 0.5 mM 3-aminotriazole were isolated. Only the colonies that turned blue in the ␤-galactosidase assay (described below) were selected. Plasmids from the candidate transformants were recovered from them into E. coli and further studied.
␤-Galactosidase Assay-Yeast transformants were grown for 2 days on the nylon filter in the medium lacking leucine and tryptophan. The colonies on the filter were frozen by liquid nitrogen and incubated for 30 min at 30°C in Z buffer (38.6 mM ␤-mercaptoethanol, 1 mg/ml 5-bromo-4-chloro-3indolyl-␤-D-galactopyranoside (X-gal), 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 ). When colonies turned blue, these gene products were scored as having a positive interaction.
Transient Transfection of COS-7, CHO-C400, and 3T3-L1 Adipocytes-The expression plasmids were transfected into COS-7 cells using the DEAE-dextran procedure. Briefly, cells were grown to be subconfluent on 100-mm dishes. The medium was then changed to 4 ml of transfection buffer (DMEM containing 10 g of DNA, 0.3 mg/ml DEAE-dextran, 50 mM Tris-HCl, pH 7.4). After 4 h of incubation, the medium was aspirated, and the cells were treated for 2 min with 4 ml of glycerol buffer (DMEM supplemented with 10% glycerol, 50 mM Tris-HCl, pH 7.4). Subsequently, the cells were washed three times with Hanks' buffered salt solution and cultured in 6 ml of growing medium for further 44 h. Then the cells were used for pulldown assay.
CHO-C400 cells were transfected with expression plasmids by the calcium phosphate precipitation method. Briefly, cells were grown to be subconfluent on 100-mm dishes. One ml of DNA solution (10 g/ml plasmid DNA, 50 mM Hepes, pH 7.05, 0.7 mM Na 2 PO 4 , 125 mM CaCl 2 ) was incubated at room temperature for 30 min and added to the cultured dishes. Four hours later, medium was changed by fresh medium, and cells were cultured for additional 2 days and then used for some experiments.
Transient transfection of 3T3-L1 adipocytes was described previously (35). Briefly, fully differentiated 3T3-L1 adipocytes were detached from the tissue culture plates by trypsin buffer (0.25% trypsin, 0.02% EDTA in PBS), and the cells were collected by centrifugation and then washed twice with PBS. The cells were resuspended in 0.5 ml of PBS and electroporated at 1.5 mV and 0.95 mA (GenePulser II, Bio-Rad). DMEM containing 10% fetal bovine serum was added to the electroporated cells; the cells were then allowed to adhere to tissue culture dishes for 24 h, and the adipocytes were then serum-starved for 2 h before experiments. In some experiments, the electroporated adipocytes were seeded on coverslips.
Purification of GST Fusion Proteins-pGEX plasmid was transformed into E. coli BL21 (DE3) pLysS. Isopropyl ␤-D-thiogalactopyranoside was added to 1 mM in the final concentration, and the expression of GST fusion protein was induced overnight at 26°C. Cells were harvested and resuspended in PBS with 1% Triton X-100 and lysed by sonication three times for 30 s on ice. The lysates were centrifuged, and supernatant was added to the glutathione-Sepharose column pre-equilibrated in the PBS buffer. The column was washed with PBS three times, and the GST fusion proteins were eluted by elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione). Eluted solution was fractionated by 1 ml. Each fraction was subjected to protein assay using protein assay kit (Bio-Rad). The most concentrated fraction was used for experiments.
GST Pulldown Assay-COS-7 cells were transfected with expression plasmids as described above. Two days after transfection, cells were harvested by cold lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 100 kallikrein-inactivating units/ml aprotinin, 20 mg/ml phenylmethanesulfonyl fluoride, 10 mg/ml leupeptin, 5 mg/ml pepstatin). The lysates were centrifuged at 14,000 ϫ g for 20 min at 4°C. The supernatant was subjected to protein assay using the protein assay kit. Cell lysates (1 mg of protein) were incubated with 100 pmol of purified GST, GST-53BP2S, GST-ANK, or GST-SH3 fusion protein at 4°C for 2.5 h. Forty l of glutathione-Sepharose beads (50% (v/v)) was then added, and incubation was continued for additional 2.5 h. Sepharose beads were collected by centrifuge and washed three times with washing buffer containing 50 mM Tris-HCl, 1 mM EDTA, and 0.1% Triton X-100. Bound proteins were subjected to SDS-PAGE, transferred to nylon membrane, and immunoblotted with the indicated antibody.
Analyses of Insulin Signaling in CHO-C400-CHO-C400 cells transfected with pEGFP-53BP2S were grown to confluency, and the quiescent cells were stimulated with insulin (100 nM) for indicated times. Cell extracts were prepared in lysis buffer, and 1 mg of total lysate protein was used for immunoprecipitation with IRS-1, IRS-2, or 4G10 antibody. Precipitants were separated by 8% SDS-PAGE and immunoblotted with 4G10 or p85 antibody. One hundred g of total cell lysates were separated by 12% SDS-PAGE and immunoblotted with phos-pho-Akt-specific antibody (Ser-473) or with phospho-ERK antibodies.
Immunofluorescence Analysis-Electroporated 3T3-L1 adipocytes were washed once with PBS and fixed/permeabilized with a solution containing 3.7% formaldehyde and 0.2% Triton X-100 in PBS for 10 min at room temperature. Cells were then washed with PBS and incubated with blocking buffer (1% bovine serum albumin and 5% donkey serum in PBS) for 1 h at room temperature, and primary antibodies (1:200 for anti-Myc, 1:100 for phospho-Akt-specific antibody (Ser-473), or 1:100 for FLAG antibody) were added for 1 h at room temperature. The samples were again washed with PBS, incubated with a secondary antibody conjugated to Texas Red (1:100 dilution), Alexa Fluor 488 (1:1000 dilution), or Alexa Fluor 596 (1:1000 dilution) for 40 min and washed, and the coverslips were mounted on Vectashield for visualization using a Zeiss LSM510 confocal microscope.
Assay of the Glut4 Translocation to the Plasma Membrane-Fully differentiated 3T3-L1 adipocytes were transfected with pEGFP vector or pEGFP-53BP2S along with pGlut4-myc by Proteins precipitated by glutathione-Sepharose beads were subjected to immunoblotting (IB) with indicated antibodies. Input represents an aliquot corresponding to 1% of the lysates used in each binding reaction. These are representative immunoblots independently performed three times. D, interaction of 53BP2S mutants with IRS-1 was analyzed in pulldown assay. Cell lysate of COS-7 expressing FLAG-tagged IRS-1 was incubated with purified GST, GST-53BP2S, GST-ANK, or GST-SH3. Proteins precipitated by glutathione-Sepharose beads were subjected to immunoblotting with anti-FLAG antibody. Input represents an aliquot corresponding to 3% of the lysates used in each binding reaction. These are representative immunoblots independently performed three times. electroporation. Twenty four hours later, cells were serumstarved for 2 h followed by stimulation with or without insulin (100 nM) for 20 min. Cells were fixed, permeabilized, and followed by incubation with a Myc antibody. The ratio of the cells displaying Glut4-myc plasma membrane fluorescence was determined by counting the 50 cells that were co-expressing both the GFP constructs and Glut4-myc in three independent experiments. Quantification of Glut4 translocation was determined as follows. Fully differentiated 3T3-L1 adipocytes were transfected with 200 g of pFLAG vector or 200 g of pFLAG-53BP2S along with 50 g of pGlut4-myc-eGFP by electroporation. Cells were serum-starved for 2 h followed by stimulation with or without insulin (100 nM) for 20 min. Cells were fixed without permeabilization and incubated with a Myc antibody. The ratio of Glut4 translocation was determined by comparison of the total Myc fluorescence intensity with the total GFP intensity. Statistical Analysis-Results are expressed as means Ϯ S.E. For comparison, the data were analyzed by analysis of variance followed by Student's t test, and the difference was considered significant at p Ͻ 0.05.

Identification of Proteins That Interact with Insulin Receptor
Substrate-1-To identify proteins that interact with IRS-1, the fulllength rat IRS-1 cDNA was fused with the Gal4 DNA binding domain in pAS2-1 vector and used as bait for a two-hybrid screening. We screened a human placental cDNA library fused with Gal4 activation domain in pACT2, and from 1 ϫ 10 6 clones, 109 IRS-1 interacting candidates were identified. Among them, 33 clones included cDNAs encoding 14-3-3 isoforms (␤, ⑀, and ), which were previously shown to interact with IRS-1 or IRS-2 (36). In addition to the various clones identified, two contained the same insert, a region of ASPP2 cDNA sequence corresponding to amino acid residues 881-1128. ASPP2 is an 1128-amino acid protein that consists of four ankyrin repeats domain and an Src homology (SH) 3 domain in the carboxyl-terminal region (Fig. 1A). ASPP2 was originally isolated as 53BP2 or the Bcl-2 binding protein Bbp (33,37). Recently, it was reported that 53BP2 is a 1005amino acid protein and that ASPP2 contained an additional 123 amino acids to the amino terminus of 53BP2. In addition, 53BP2 is an alternative splicing variant of ASPP2, and they proposed that we call this splicing variant 53BP2S (38). In this study, we used 53BP2S as an ASPP2 splicing variant.
53BP2S Interacts with IRS Family Proteins in Vitro-The clones isolated in the two-hybrid screen contained the carboxyl-terminal region that includes the SH3 domain and ankyrin FIGURE 2. Interaction of 53BP2S with IRS-1 deletion mutants in the GST pulldown assay. Schematic structure of IRS-1 protein is shown. Two boxes represent PH or PTB domain, respectively. Boxes above the IRS-1 structure indicate the 53BP2S binding domains. Below the IRS-1 structure, constructs of some deletion mutants (D1-D14) are shown. Each deletion mutant was fused with GFP at the amino-terminal end. Plasmids expressing each deletion mutant of IRS-1 (D1-D14) were transfected into COS-7. Cell lysate of COS-7 expressing each deletion mutant was incubated with purified GST or GST-53BP2S. Proteins precipitated by glutathione-Sepharose beads were subjected to immunoblotting with anti-GFP antibody. The results of each pulldown assay are shown on the right side of the deletion constructs. Input represents an aliquot corresponding to 1% of the lysates used in each binding reaction. DECEMBER 28, 2007 • VOLUME 282 • NUMBER 52
To confirm the interaction of 53BP2S with IRS-1 by the two-hybrid assay, 53BP2S was tested in a GST pulldown assay. Isolated 53BP2S cDNA (amino acid residues 758 -1005) fused with GST (GST-53BP2S) was constructed in a pGEX vector. GST-53BP2S protein or GST alone was then expressed and purified from E. coli. (Fig. 1B). Cell lysates isolated from COS-7 cells expressing full-length FLAG tagged IRS-1 (FLAG-IRS-1) were incubated with purified GST-53BP2S or GST only. Protein complexes precipitated with glutathione-Sepharose beads were separated by SDS-PAGE, and FLAG-IRS-1 bound to GST-53BP2S was detected by immunoblotting with the FLAG antibody. FLAG-IRS-1 associated with GST-53BP2S but not with GST alone (Fig.  1C), indicating that 53BP2S specifically associates with IRS-1 in vitro as well as in the yeast two-hybrid assay.
Four members of IRS family proteins, IRS-1, IRS-2, IRS-3, and IRS-4, have been identified to date. Among them, PH and PTB domains are highly conserved, but other regions are divergent except for the tyrosine residues possibly phosphorylated by receptor tyrosine kinases (2). Fig.  1C shows that IRS-2, IRS-3, and GFP-tagged IRS-4 could also interact with 53BP2S (Fig. 1C).
Interaction of IRS-1 with 53BP2S Requires Both Ankyrin Repeats and SH3 Domain of 53BP2S-To investigate which region of 53BP2S is required for the interaction with IRS-1, two-hybrid ␤-galactosidase and GST pulldown assays were carried out. pACT-ANK or pACT-SH3 was constructed to contain only ankyrin repeats or only the SH3 domain in pACT2 vector, respectively (Fig. 1A). In the two-hybrid system, neither interaction between IRS-1 and the ankyrin repeats nor between IRS-1 and the SH3 domain was detectable by the ␤-galactosidase assay (Table 1). Similarly, we examined the precipitation of IRS-1 using an ankyrin repeat (GST-ANK) and an SH3 domain fusion protein (GST-SH3), respectively (Fig. 1B). Neither GST-ANK nor GST-SH3 could pull down FLAG-tagged IRS-1 (Fig. 1D), indicating that both the ankyrin repeats and the SH3 domain are required for the interaction with IRS-1. Consistent with these data, the interaction of 53BP2S with p53 and Bcl-2 is also required for both the ankyrin repeats and SH3 domain (33,37,39).

53BP2S Interacts with PTB Domain and a Central Region (Amino Acid Residues 750 -861) of IRS-1-To identify regions
of IRS-1 that are required for the interaction with 53BP2S, we examined IRS-1 deletion mutants fused with GFP (D1-D14), and pulldown assays were performed for each mutant (Fig. 2). An IRS-1 mutant that contains only the PH domain (D8) could not, whereas a mutant that contains the PTB domain (D9) could interact with GST-53BP2S (amino acid residues 758 -1005). These data indicate that the IRS-1 PTB domain, but not the PH domain, is sufficient for the interaction with 53BP2S. Surprisingly, a mutant in which PH and PTB domains were both deleted (D5) still interacted with 53BP2S. More detailed analyses identified another 53BP2S binding domain, a central region containing 112 amino acid residues (750 -861), that was sufficient for the interaction with IRS-1 (Fig. 2). The cross-reactivity of the four IRS proteins (Fig. 1C) probably results from the interaction of 53BP2S with the PTB domains, as these domains are highly homologous between the isoforms.
Endogenously Expressed 53BP2S Interacts with IRS-1 in 3T3-L1 Adipocytes-We next investigated the interaction of endogenously expressed 53BP2S with IRS-1. To confirm the expression of 53BP2S in 3T3-L1 cells, RT-PCR was carried out using total cellular RNA from 3T3-L1 preadipocytes. 53BP2S cDNA fragment was not amplified from first strand cDNA without SuperScript2 (RTϪ), whereas 53BP2S cDNA fragment  -1 and 53BP2S. A, left panel, total RNA was extracted from 3T3-L1 preadipocytes. Using this total RNA, first strand cDNA was synthesized with (RTϩ) or without (RTϪ) SuperScript2. PCR was carried out using these first strand cDNA as templates. PCR condition was as follows: 30 cycles of sequential incubations at 94°C for 30 s, 50 or 55°C for 60 s, and 72°C for 60 s followed by final extension at 72°C for 5 min. Right panel, total RNA was extracted from 3T3-L1 cells at differentiation stage 0, 2, 4, 6, and 8 day. PCR condition was 25 cycles of sequential incubation at 94°C for 30 s, 55°C for 60 s, and 72°C for 60 s for both 53BP2S and 36B4. These are representative data independently performed five times. B, cell lysates of fully differentiated 3T3-L1 adipocyte cells were immunoprecipitated (IP) by IRS-1 antibody or preimmune serum. Total cell lysates of HEK293 cells or 3T3-L1 adipocytes and immunoprecipitants by IRS-1 antibody were immunoblotted (IB) with anti-ASPP2 antibody (Sigma) or anti-IRS-1 antibody. Input represents an aliquot corresponding to 1% of the lysates used in immunoprecipitation assay. C, cell lysate of quiescent 3T3-L1 adipocyte cells or stimulated with insulin (100 nM) for 2 min was incubated with purified GST or GST-53BP2S. Proteins precipitated by glutathione-Sepharose beads were detected by anti-IRS-1 antibody. Input represents an aliquot corresponding to 10% of the lysates used in each binding reaction. These are representative immunoblots independently performed three times. D, pEGFP or pEGFP-53BP2S was transiently transfected into 3T3-L1 adipocytes by electroporation. Cells were serum-starved for 2 h followed by stimulation with insulin or without insulin for 2 min. Cell lysates were immunoprecipitated by IRS-1 antibody or preimmune serum and immunoblotted with anti-53BP2 antibody.
was amplified when PCR was carried out using first strand cDNA with SuperScript2 (RTϩ), indicating that 53BP2S mRNA was expressed in 3T3-L1 preadipocytes (Fig. 3A). RT-PCR confirmed the expression of 53BP2S in differentiated 3T3-L1 adipocytes. The expression of 53BP2S was increased by differentiation of 3T3-L1 adipocytes, suggesting that 53BP2S plays an important role in differentiated 3T3-L1 cells (Fig. 3A).
Immunoblotting of 3T3L1 adipocyte extracts demonstrated the presence of a specific protein band that migrated identical human ASPP2 expressed in human embryonic kidney cell HEK293 cells (Fig. 3B). More importantly, immunoprecipitation of endogenous IRS-1 resulted in the co-immunoprecipitation of the endogenous 53BP2S in 3T3-L1 adipocytes (Fig. 3B). These data demonstrate that 53BP2S and IRS-1 directly interact in vivo.
53BP2S Interacts with IRS-1 Not through Recognition of Phosphotyrosine Residues-Because yeast has few tyrosine kinases, the expressed IRS-1 is not tyrosine-phosphorylated. To investigate whether the interaction of IRS-1 with 53BP2S could be modulated by IRS1 tyrosine phosphorylation, precipitation assays using phosphorylated IRS-1 or nonphosphorylated IRS-1 was carried out. Briefly, cell lysates of quiescent or insulin-stimulated 3T3-L1 adipocyte cells were incubated with GST-53BP2S (amino acid residues 758 -1005) or GST alone. Endogenous IRS-1 protein bound to GST-53BP2S was visualized by immunoblotting with anti-IRS-1 antibody. As expected, IRS-1 proteins derived from quiescent 3T3-L1 adipocyte cells were precipitated by GST-53BP2S (Fig. 3C). IRS-1 proteins precipitated in lysates isolated from either quiescent or insulinstimulated cell did not display any immunoreactivity against a phosphotyrosine-specific antibody. In addition, the amount of IRS-1 co-precipitated by 53BP2S was markedly decreased following insulin stimulation by immunoblotting with an IRS-1 antibody (Fig. 3C). Taken together, these data indicate that formation of a 53BP2S-IRS-1 complex is inhibited by insulin stimulation, most likely because of IRS-1 tyrosine phosphorylation.
To evaluate the mechanism of interaction between IRS-1 and exogenously expressed 53BP2S in adipocytes, fully differentiated 3T3-L1 adipocytes were electroporated with pEGFP or the pEGFP-53BP2S expression vectors. Electroporated cells were starved for 2 h, and cell lysates were immunoprecipitated with an IRS-1 antibody. As observed in the GST pulldown assays, 53BP2S was co-immunoprecipitated with IRS-1 (Fig. 3D). In addition, insulin stimulation resulted in a decreased amount of 53BP2S that co-immunoprecipitated with IRS-1 (Fig. 3D). These data are consistent with the GST pulldown results and further document that post-translational modification of IRS-1, possibly tyrosine phosphorylation, inhibited the interaction between IRS-1 and 53BP2S.
Effect of 53BP2S Expression on Insulin Signals in CHO-C400 Cells-To examine biological function of 53BP2S in insulin/ IGF-I signaling mediated by IRS proteins, we assessed the effect of 53BP2S expression on insulin signaling. GFP or GFP-53BP2S expression vectors were introduced into CHO-C400 cells by calcium phosphate transfection, and the activation of signaling targets in response to insulin was assessed. Expression of 53BP2S resulted in an increase in insulin-stimulated IRS-2 tyrosine phosphorylation, whereas IRS-1 tyrosine phosphorylation was de-creased (Fig. 4B). In addition, the amount of IRS-1-associated p85 PI 3-kinase was decreased in 53BP2S-transfected cells, whereas the amount of p85 PI 3-kinase in IRS-2-immunocomplex was enhanced compared with GFP-transfected cells (Fig. 4A). To measure the total amount of p85 PI 3-kinase associated with total tyrosine-phosphorylated proteins, whole cell lysates were immunoprecipitated with phosphotyrosine antibody (4G10), and the amount of p85 PI 3-kinase in the immunocomplex was measured. Tyrosine phosphorylation of proteins at 180 kDa was enhanced by 53BP2S expression. Similarly, the amount of p85 PI 3-kinase immunoprecipitated by 4G10 was enhanced by 53BP2S overexpression (Fig. 5A). These data suggested that PI 3-kinase activation was elevated in 53BP2S-expressing cells. Because Akt activation was induced by PI 3-kinase, we next determined Akt activation in 53BP2S-expressing cells. It is well known that full activation of Akt kinase activity requires PDK1-dependent phosphorylation of threonine 308 followed by PDK2 phosphorylation on serine 473 (40). Consistent with an increase in the amount of p85 PI 3-kinase associated with tyrosine-phosphorylated proteins, insulin-stimulated Akt serine 473 phosphorylation was enhanced by GFP-53BP2S expression without any change in ERK activation (Fig. 5B). These results indicate

. Effects of 53BP2S expression on insulin-induced IRS tyrosine phosphorylation.
A, CHO-C400 cells were transfected with pEGFP or pEGFP-53BP2S by the calcium phosphate method. Forty eight hours later, cells were starved in serum-free medium and stimulated with or without insulin for the indicated time. Cell lysates were prepared from each cell and immunoprecipitated (IP) by anti-IRS-1 antibody or anti-IRS-2 antibody. Immunoprecipitants were separated by SDS-PAGE and immunoblotted (IB) with anti-phosphotyrosine antibody (4G10) or anti-p85 antibody. B, CHO-C400 cells transfected with pEGFP or pEGFP-53BP2S were starved in serum-free medium and stimulated with or without insulin for 2 min. Cell lysates were prepared from each cell and immunoprecipitated by anti-IRS-1 antibody or anti-IRS-2 antibody. Immunoprecipitants were separated by SDS-PAGE and immunoblotted with anti-phosphotyrosine antibody, anti-IRS-1 antibody, or anti-IRS-2 antibody. Quantified data from each blot are shown in the graphs below the immunoblot images. Values are the means Ϯ S.E. of three different experiments and expressed as relative to insulin-stimulated GFP-transfected control. *, difference between GFP-expressing cells and GFP-53BP2S-expressing cells with insulin stimulation is significant with p Ͻ 0.05. DECEMBER 28, 2007 • VOLUME 282 • NUMBER 52 that the enhancement of IRS-2 tyrosine phosphorylation and association of p85 PI 3-kinase with IRS-2 compensated for the reduction of IRS-1 tyrosine phosphorylation and p85 PI 3-kinase association with IRS-1 in these cells. The net effect results in a 53BP2S enhancement of Akt activation.

Novel Function of 53BP2S in the Insulin Signaling
Effect of 53BP2S Expression on PI 3,4,5-P 3 Production and Akt Activation in 3T3-L1 Adipocytes-To determine the effect of 53BP2S expression on insulin signals in 3T3-L1 adipocytes, we measured the insulin stimulation of PI 3-kinase pathway activation in 53BP2S-expressing cells. We took advantage of the pleckstrin homology (PH) domain of Grp1 fused to GFP that specifically interacted with PI 3,4,5-P 3 . Fully differentiated 3T3-L1 adipocytes were transfected with pFLAG and pGrp-PH-GFP or pFLAG-53BP2S and pGrp-PH-GFP. Twenty four hours later, cells were serum-starved for 2 h, followed by stimulation with insulin for 5 min. Cells were fixed, permeabilized, and stained with the FLAG antibody. Under basal conditions, Grp-PH-GFP probe was primarily localized to nuclei (Fig. 6). However, insulin stimulation induced translocation of the probe to the plasma membrane.
In FLAG-tagged 53BP2S-expressing cells, the plasma membrane staining of Grp-PH-GFP could be observed, indicating that PI 3,4,5-P 3 was normally produced in response to insulin even in 53BP2S-expressing cells (Fig. 6). Taken together, these data suggested that PI 3-kinase was activated in 53BP2S-expressing cells.
To investigate the effect of 53BP2S expression on Akt activation in vivo, we established the single cell assay of Akt activation. At first, adipocyte cells were stained with phospho-Akt-specific antibody (Ser-473) with or without insulin stimulation. We found the nuclei staining, which is independent of insulin stimulation, but we also observed staining on the plasma membrane, which is dependent of insulin stimulation (Fig. 7A). The plasma membrane labeling was specific for Akt activation, as it was prevented by the PI 3-kinase-specific inhibitor LY294002 (Fig. 7A). Although the number of cells displaying the Akt staining on the plasma membrane under basal conditions was essentially 0%, insulin stimulation resulted in greater than 90% of the cells phospho-Akt-positive. Also in 53BP2S-expressing cells, insulin-induced phospho-Akt staining was normally observed (Fig. 7B). In this assay, we could not detect the enhancement of insulin-induced Akt phosphorylation by 53BP2S expression. To compare the relative effect of 53BP2S expression on the extent of Akt, we co-transfected 3T3-L1 adipocytes with 50 g of pHA-Akt2 and 200 g of pGFP or 50 g of pHA-Akt2 and 200 g of pGFP-53BP2S. In this way, most cells expressing HA-Akt2 were expected to also express GFP or GFP-53BP2S. Cell lysates were prepared from these transfectants and immunoprecipitated with the HA antibody, followed by immunoblotting with phospho-Akt-specific antibody (Ser-473). As shown in Fig. 7C, HA-Akt2 was activated following insulin stimulation that was enhanced by 53BP2S expression.
Expression of 53BP2S Inhibits the Glut4 Translocation Induced by Insulin in 3T3-L1 Adipocytes-Because insulinstimulated Glut4 translocation is an important readout for insulin signaling, we next examined the effect of 53BP2S in the 3T3-L1 adipocytes. Fully differentiated 3T3-L1 adipocytes were transfected with plasmid expressing GFP and Glut4-myc or GFP-53BP2S and Glut4-myc. Twenty four hours later, cells were serum-starved for 2 h, followed by stimulation with insu- lin for 20 min. Cells were fixed, permeabilized, and stained with the Myc antibody (Fig. 8A). Fifty cells expressing both Glut4myc and GFP or Glut4-myc and GFP-53BP2S were counted, and the percentage of cells in which Glut4-myc was translocated to plasma membrane is shown in Fig. 8B. Insulin stimulation resulted in an ϳ4-fold increase in the number of transfected cells displaying Glut4 translocation to the plasma membrane (16.7 Ϯ 1.8 to 58.2 Ϯ 4.3%) in GFP-transfected cells. In contrast, overexpression of GFP-53BP2S significantly inhibited insulin-stimulated plasma membrane Glut4 translocation (14.5 Ϯ 1.6 to 23.9 Ϯ 3.3%). Under these conditions, there was no effect of 53BP2S on the basal level of Glut4 translocation demonstrating that 53BP2S specifically inhibited insulin signal events necessary for Glut4 translocation.
To confirm this apparent inhibition of insulin-stimulated Glut4 translocation by 53BP2S, we next quantified the extent of Glut4 translocation using a double-labeled Glut4 reporter (Glut4-myc-eGFP). Briefly, 3T3-L1 adipocytes were electroporated with 200 g of pFLAG vector or 200 g of pFLAG-53BP2S along with 50 g of pGlut4-myc-eGFP by electroporation. Cells were fixed without permeabilization and incubated with Myc antibody. We assessed the relative Myc fluorescent in comparison with the total cell GFP fluorescence. The results further demonstrate that 53BP2S-expressing cells have a marked attenuation of insulin-stimulated Glut4 translocation (Fig. 8C).
Because 53BP2S expression enhanced Akt activation and the downstream readout of Glut4 translocation was inhibited, 53BP2S must affect another target in the Glut4 translocation pathway. Recently it was reported that insulin induced phosphorylation of AS160 (Rab-GAP), one of Akt substrate, followed by activation of Rab10 was required for Glut4 translocation in 3T3-L1 adipocytes (16,17). To evaluate the effect of 53BP2S expression on the AS160 phosphorylation, we co-transfected 3T3-L1 adipocytes with 50 g of pFLAG-AS160 and 200 g of pGFP or 50 g of pFLAG-AS160 and 200 g of pGFP-53BP2S. Cell lysates were prepared from and immunoprecipitated with the FLAG antibody, followed by immunoblotting with phospho-AS160-specific antibody (Thr-642). As shown in Fig. 8D, FLAG-AS160 was phosphorylated following insulin stimulation, and this phosphorylation was repressed by 53BP2S expression.

DISCUSSION
This study was undertaken to identify proteins, which interact with IRS and modulate insulin signaling. Using yeast twohybrid screens, we isolated 53BP2S as one of IRS-interacting proteins. The interaction between 53BP2S and IRS proteins was confirmed by using both pulldown and co-immunoprecipitation binding assays. Expression of 53BP2S reduced insulininduced IRS-1 tyrosine phosphorylation with a concomitant enhancement of IRS-2 tyrosine phosphorylation in CHO-C400 cells. In addition, Akt activation was enhanced by 53BP2S expression. Consistent with these data, Akt activation was enhanced by 53BP2S expression also in 3T3-L1 adipocytes; however, Glut4 translocation to plasma membrane in response to insulin was significantly inhibited by 53BP2S expression.
Previous studies demonstrated that chronic tumor necrosis factor-␣ pretreatment inhibited insulin-induced IRS-1 tyrosine phosphorylation, leading to a decrease in insulin sensitivity (26). Previously, we reported that chronic thyrotropin pretreatment enhances the IGF-I-induced IRS-2 tyrosine phosphorylation, leading to augmentation of IGF-I-induced DNA synthesis (28 -31). Although multiple studies have demonstrated that post-translational modification, such as Ser/Thr phosphorylation, plays important roles in modulating IRS tyrosine phosphorylation (18 -24), we have also observed that various IRS-associated proteins can have dramatic effects on IRS phosphorylation. These data indicated the possibility that IRS-associated proteins play important roles in modulation of insulin/IGF bioactivities.
In this study, we identified the interaction regions of IRS-1, PTB domain, and the central region with 53BP2S. Although PTB domain is highly homologous among IRS family proteins, the central region is a unique sequence of IRS-1 compared with other IRS members, suggesting that the binding mechanism of 53BP2S with IRS-1 or IRS-2 could be different. This might account for the opposite effect of 53BP2S on tyrosine phosphorylation of IRS-1 and IRS-2. On the other hand, we also identified the interaction region of 53BP2S. Both SH3 and ankyrin repeat domains were required for association, indicating that SH3 and ankyrin repeats domains cooperatively form the conformational structure that is important for this interaction.
The interaction domains of IRS-1 with 53BP2S did not contain the putative tyrosine phosphorylation sites, and this interaction was clearly detected in the basal state. These data suggest that 53BP2S interacts with IRS-1 in the absence of tyrosine phosphorylation and that 53BP2S interaction modulates IRS tyrosine phosphorylation mediated by the insulin receptor tyrosine kinase. Thus, 53BP2S is a likely candidate for a protein  p85 PI 3-kinase regulatory subunit result in the preferential association of the catalytic subunit with p50 and enhanced insulin sensitivity (51). Thus, adequate cellular distribution or isoform-selective activation of IRS, PI 3-kinase, and Akt could be disturbed by overexpression of 53BP2S. This disturbance could account for the inability of activated Akt to phosphorylate AS160. In addition, several studies have suggested the presence of additional signaling pathways that may function in concert with IRS-1/PI 3-kinase signaling through caveolin-enriched lipid raft microdomains (52,53). Although we have not assessed the effect of 53BP2S on lipid raft-dependent signaling, there is no evidence that the IRS/PI 3-kinase functions through these microdomains (52). However, it is more likely that 53BP2S inhibition could occur by blocking additional IRS-1mediated signals. In addition, we could not rule out the possibility that 53BP2S inhibited the Akt downstream pathway required for Glut4 translocation independent of modulation of IRS-mediated insulin signals.
53BP2S and ASPP2 protein has been also isolated as a binding partner of many proteins, including p53, Bcl2, NF-B, PP1, and APCL (33,37,39,54,55), suggesting that 53BP2S/ASPP2 is involved in apoptotic pathways. IGF-I and IRS-1 are also well known to have anti-apoptotic activity. Ueno et al. (56) showed that IRS-1 interacts with Bcl-2 and has an anti-apoptotic effect. It is possible that Bcl-2 and 53BP2S/ASPP2 compete each other for interacting with IRS and regulate anti-apoptotic role IRS proteins. Whether the interaction between IRS-1 and 53BP2S/ ASPP2 is involved in apoptosis awaits further study.
In summary, we have found that 53BP2S protein directly interacts with IRS family proteins both in pulldown and coimmunoprecipitation assays independent of IRS tyrosine phosphorylation, and we have identified the specific binding regions responsible. Importantly, 53BP2S functions to regulate IRS isoform tyrosine phosphorylation induced by insulin, resulting in modulation of insulin signals. These lead to attenuation of Glut4 translocation to the plasma membrane by as of yet unknown mechanisms.