The AP-1 Complex Regulates Intracellular Localization of Insulin ...

The AP-1 Complex Regulates Intracellular Localization of Insulin Receptor Substrate 1, Which Is Required for Insulin-Like Growth Factor I-Dependent Cell Proliferation Yosuke Yoneyama, Masao Matsuo, Kazumi Take, Tomohiro Kabuta,* Kazuhiro Chida, Fumihiko Hakuno, Shin-Ichiro Takahashi Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

The activation of the insulin/insulin-like growth factor I (IGF-I) receptor and the subsequent tyrosine phosphorylation of insulin receptor substrates (IRSs) are key initial events in a variety of insulin/IGF bioactivities, including mitogenesis. It has been reported that IRS-1 associates with intracellular membrane compartments, and this localization is believed to be important for insulin/IGF signal transduction. However, the molecular mechanisms underlying IRS-1 localization remain unclear. Here we show that in L6 myoblasts, IRS-1 associates with ␮1A of the ubiquitously expressed AP-1 complex, which packages cargo proteins into clathrin-coated vesicles derived from intracellular membranes. While wild-type IRS-1 was predominantly localized to vesicular structures, IRS-1 mutants lacking three YXX⌽ motifs responsible for binding to ␮1A were mislocalized to the mannose-6-phosphate receptor-positive structures, suggesting that AP-1-dependent transport to peripheral vesicles is inhibited in these mutants. Furthermore, deletion of AP-1 binding sites in IRS-1 impaired IGF-I-induced cell proliferation, accompanied by reduced tyrosine phosphorylation of IRS-1 and its association with phosphoinositide (PI) 3-kinase. These data demonstrate the importance of AP-1-dependent localization of IRS-1 in mediating IGF-I-stimulated signaling and maximum mitogenic response.

I

t is well established that insulin and insulin-like growth factors (IGFs) display a variety of bioactivities, including induction of growth promotion, differentiation, and metabolic functions (1). Insulin and IGFs bind to specific receptors and activate their intrinsic tyrosine kinase activity. Tyrosine phosphorylation of insulin receptor substrates (IRSs) by activated receptors leads to their binding to Src homology 2 (SH2) domain-containing molecules, including the p85 phosphoinositide (PI) 3-kinase regulatory subunit and Grb2. PI 3-kinase generates phosphoinositide 3,4,5triphosphate (PIP3). PIP3 production recruits Akt kinase to the plasma membrane, resulting in its activation by Thr308/Ser473 phosphorylation. Interaction of Grb2 with tyrosine-phosphorylated IRSs leads to activation of the small GTP-binding Ras and subsequent activation of mitogen-activated protein kinase (MAPK). Since these signaling cascades are essential for various bioactivities, the IRS proteins are critical mediators of insulin/IGF signaling (2, 3). The importance of the IRS-1 isoform, one of four IRS family proteins, in insulin/IGF activity has been established by experiments using both cultured cells and knockout mice. In IRS-1 knockout mice, insulin-stimulated metabolism and somatic cell growth rate are significantly impaired (4, 5). Moreover, overexpression of IRS-1 in cultured cells enhances DNA synthesis stimulated by insulin or IGF-I (6), while RNA interference (RNAi)-mediated IRS-1 knockdown cells or fibroblasts from IRS-1 knockout mice display reduction in insulin/IGF-stimulated cell proliferation (7–10). Together, these reports demonstrate an essential role of IRS-1 in insulin/IGF-induced cell proliferation. It has been suggested that phosphorylation of IRS-1 occurs at the cell surface because insulin receptor (IR)/IGF-I receptor (IGFIR) is activated at the cell surface. Despite this claim, several studies suggest that IRS-1 is predominantly associated with intracellular membrane compartments. Subcellular fractionation studies revealed that IRS-1 is localized not only in cytosol but also in a membrane fraction called low-density microsomes (LDM) or in a high-speed pellet that is rich in vesicle compartments such as en-

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dosomes and Golgi apparatus (11–13). We have also reported that green fluorescent protein (GFP)-fused IRS-1 displays punctate localization to vesicular structures (14). Insulin-dependent activation of PI 3-kinase associated with IRS-1 is also mainly detected in the microsome and cytosol fractions and poorly detected in the plasma membrane fraction, which is correlated with the distribution of IRS-1 (15). In addition, exposure to oxidative stress induced by H2O2, which impairs insulin-stimulated glucose transport, disrupts the localization of IRS-1 and IRS-1-associated PI 3-kinase activation in LDM (16). These reports suggest that IRS-1 localization to intracellular membrane compartments is an important component of insulin/IGF action. However, IRS-1 does not contain a transmembrane domain, and there is no evidence for posttranslational modification of the protein that would enable it to associate with membrane structures. Thus, molecular mechanisms of IRS-1 localization are not well understood. It is well established that adaptor protein (AP) complexes function both in cargo selectivity and in the initial step of clathrincoated vesicle formation (17, 18). AP complexes (AP-1 to -4) participate in protein targeting of transmembrane cargos between different membrane compartments. Among them, the ubiquitously expressed AP-1 consists of two large adaptins, ␥ and ␤1, the medium adaptin ␮1A, and the small adaptin ␴1, assembled into a

Received 12 October 2012 Returned for modification 2 December 2012 Accepted 1 March 2013 Published ahead of print 11 March 2013 Address correspondence to Shin-Ichiro Takahashi, [email protected]. * Present address: Tomohiro Kabuta, Department of Degenerative Neurological Disease, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/MCB.01394-12

Molecular and Cellular Biology

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heterotetrameric complex. The ␮1A subunit recognizes the YXX⌽ motif (where Y is the tyrosine residue, X is any residue, and ⌽ is the bulky hydrophobic residue), which is a common sorting signal found in the cytosolic region of transmembrane cargo proteins (19). In spite of numerous studies, there is still some uncertainty about the directionality of AP-1 trafficking. For example, some studies have observed accumulation of cargo proteins in endosome or postendosome compartments in AP-1-deficient cells (20, 21), while others showed that they accumulated in the Golgi region or at the plasma membrane (22, 23). Irrespective of directionality, AP-1 is generally believed to sort cargo proteins between endosomes and the trans-Golgi network (TGN). In this study, we identified the ␮1A AP-1 subunit as an IRS-1interacting protein responsible for the association of IRS-1 with intracellular membranes. Disruption of this interaction results in altered IRS-1 localization and impaired IGF-I-stimulated IRS-1 tyrosine phosphorylation and cell proliferation. MATERIALS AND METHODS Materials. The antibodies used were obtained from the following sources. Anti-IRS-1 antibody was raised in rabbits as described previously (24). Anti-p85 antibody and anti-Myc antibody (9E10) were from Millipore (Billerica, MA). Anti-␤-actin antibody, anti-␣-tubulin antibody, and antiphosphotyrosine antibody (4G10) were from Sigma (St. Louis, MO). Anti-␥-adaptin antibody, anti-EEA1 antibody, anti-TGN38 antibody, and anti-Rab11 antibody were from BD Transduction Laboratories (Franklin Lakes, NJ). Anti-␮1A antibody and anti-Myc antibody were from Abcam (Cambridge, MA). Anti-pErk1/2 antibody, anti-Erk1/2 antibody, anti-pAkt (Ser473) antibody, and anti-Akt antibody were from Cell Signaling Technology (Danvers, MA). Anti-transferrin receptor antibody and Alexa Fluor-conjugated secondary antibodies were from Molecular Probes (Invitrogen, Carlsbad, CA). All other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Small interfering RNAs (siRNAs) were obtained from RNAi Corp. (Tokyo, Japan). The siRNAs used in this study comprised the following sequences: control, 5=-GUACCGC ACGUCAUUCGUAUC-3=; IRS-1, 5=-CAAUGAGUGUGCAUAAACUU C-3=; ␮1A(#1), 5=-CAGACGGAGAAUUCGAACUCA-3=; ␮1A(#2), 5=-C UUGUGUGUCGCUAGUAUUCU-3=; ␥-adaptin(#1), 5=-CUUCUCAU GACCAAUUGUAUC-3=; and ␥-adaptin(#2), 5=-GCAUGGUGUAUAG GUGAAUAU-3=. Cell culture. L6 myoblasts and HEK293T cells were cultured as described previously (25, 26). Plasmid construction. Full-length and the N- or C-terminal region of mouse ␮1A were cloned into pGEX-5X3. pGFP-IRS-1 was constructed as described previously (25). The plasmids expressing deletion and CAAX fusion mutants of rat IRS-1 fused with Myc-tag or GFP were cloned into pCMV-Myc or pEGFP-C1, respectively. Plasmids expressing GFP and Myc-tagged IRS-1 mutants in which tyrosines were replaced by alanines were constructed by site-directed mutagenesis, and the sequences were verified. For retroviral expression, Myc-tagged ␮1A and IRS-1 mutants were cloned into pMXs-Neo. For retroviral RNAi, short hairpin RNA (shRNA) sequences were cloned into pSIREN-RetroQ according to the manufacturer’s instructions (Clontech, Mountain View, CA). The shRNAs used in this study comprised the following sequences: luciferase, 5=-TGC GTTGCTAGTACCAAC-3=; IRS-1(#1), 5=-GATCAGGCTATCTTCCTT3=; and IRS-1(#2), 5=-CAGCAAGACCATCAGCTTT-3=. Transfection of plasmid and siRNA. The expression plasmids were transfected into L6 myoblasts using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. siRNA was transfected into L6 cells using Lipofectamine 2000 by the reverse transfection method. HEK293T cells were transfected with expression plasmids by the calcium phosphate precipitation method as described previously (25). The cells were serum starved for 16 h in Dulbecco’s modified Eagle’s medium (DMEM) sup-

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plemented with 0.1% bovine serum albumin (BSA) 1 day after transfection and then used for pulldown assays. Retrovirus production and isolation of stable transfectant. Retrovirus production and infection were performed as described previously (26). Infected cells were selected with G418 or puromycin. Purification of GST fusion proteins. pGEX plasmids were transformed into Escherichia coli BL21. The expression of glutathione S-transferase (GST) fusion proteins was induced overnight at 26°C. Purification of GST fusion proteins with glutathione-Sepharose 4B (GE Healthcare, Pittsburgh, PA) was performed as described previously (25). The binding of GST fusion protein to glutathione-Sepharose was checked by Coomassie brilliant blue (CBB) staining and then used for pulldown assays. GST pulldown assay. Cells were harvested in cold lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 100 Kallikrein inhibitor units (KIU)/ml aprotinin, 20 ␮g/ml phenylmethylsulfonyl fluoride [PMSF], 10 ␮g/ml leupeptin, 5 ␮g/ml pepstatin, 500 ␮M Na3VO4, 10 mg/ml p-nitrophenyl phosphate [PNPP] [pH 7.4]). Cell lysates (1 mg protein) were incubated with 100 pmol of purified GST fusion protein bound to glutathione-Sepharose at 4°C for 1.5 h. Sepharose beads were washed three times with lysis buffer. Bound proteins were analyzed by immunoblotting with the indicated antibody. Immunoprecipitation of IRS-1–AP-1 complex. To assess the interaction between IRS-1 and AP-1, cells were rinsed twice with phosphatebuffered saline (PBS) and lysed in buffer A (150 mM NaCl, 10 mM HEPES, 1 mM EGTA, and 0.1 mM MgCl2, 100 KIU/ml aprotinin, 20 ␮g/ml PMSF, 10 ␮g/ml leupeptin, 5 ␮g/ml pepstatin, 500 ␮M Na3VO4, 10 mg/ml PNPP [pH 7.4]) plus 0.5% Triton X-100 by incubation for 30 min on ice. Cells were then harvested, and cell homogenates were centrifuged at 16,100 ⫻ g for 15 min. The supernatant was diluted to 1 mg/ml in buffer A plus 0.5% Triton X-100, applied to protein A-Sepharose (GE Healthcare) coated with anti-IRS-1 antibody, and incubated overnight at 4°C. The beads were then washed 5 times with buffer A plus 0.1% Triton X-100, and proteins were eluted with Laemmli’s sample buffer. Samples were analyzed by immunoblotting. Analysis of IGF-I signaling. Cells were serum starved for 16 h and then stimulated with 100 ng/ml of human recombinant IGF-I. Cell extracts were prepared with lysis buffer and subjected to immunoprecipitation and immunoblotting as described previously (25). Subcellular fractionation. Fractionation was performed as described previously with minor modifications (27). Briefly, cells were collected in a TES buffer (20 mM Tris-HCl, 1 mM EDTA, 255 mM sucrose, 500 ␮M Na3VO4, 10 ␮g/ml leupeptin, 5 ␮g/ml pepstatin, 20 ␮g/ml PMSF, 100 KIU/ml aprotinin, 10 mg/ml PNPP [pH 7.4]) and passed 20 times through a 27-gauge needle on ice. The lysates were fractionated with ultracentrifugation in an Optima TLX with a TLA-55 rotor (Beckman Coulter). The resulting pellets were resuspended in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 10 ␮g/ml leupeptin, 5 ␮g/ml pepstatin, 20 ␮g/ml PMSF, 100 KIU/ml aprotinin, 10 mg/ml PNPP [pH 8.0]). Membrane fractions and cytosolic supernatants were analyzed by immunoblotting. Immunofluorescence analysis. L6 cells grown on coverslips were serum starved for 16 h. The cells were fixed in PBS containing 4% paraformaldehyde for 20 min at room temperature or in cold methanol for 5 min at ⫺20°C. Cells were then washed with PBS and permeabilized with PBS containing 0.25% Triton X-100 for 5 min at room temperature. Cells were incubated with blocking buffer (3% BSA and 0.025% NaN3 in PBS) for 1 h at room temperature, and primary antibodies were added overnight at 4°C. The samples were again washed with PBS, incubated with a secondary antibody diluted in blocking buffer for 1 h at 37°C, and washed. Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) for visualization using a FV500 confocal microscope (Olympus, Tokyo, Japan). Images from at least five different individuals were analyzed with Fluoview version 1.4 and Photoshop CS3. Time-lapse observation. HEK293T cells were grown in glass-bottom dishes (Matsunami, Osaka, Japan) coated with collagen type I (Koken,


Regulation of IRS-1 Localization by AP-1 Complex

Tokyo, Japan). Six hours after transfection, cells were observed at 15-min intervals for 12 h with an IX81 motorized inverted microscope (Olympus, Tokyo, Japan) attached to a chamber in a humidified 5% CO2 controlled atmosphere. Image files were processed by Metamorph software (Molecular Devices). DNA synthesis assay and cell proliferation assay. DNA synthesis was measured as described previously (26). For cell proliferation assays, 4.0 ⫻ 104 cells were grown in a 35-mm dish in DMEM supplemented with 1% fetal bovine serum (FBS) with or without 100 ng/ml of IGF-I. Cell numbers were counted each day. Multiple alignment. AP-1 binding regions (APBRs) of IRS-1 from various species were aligned using ClustalX2. The output from the ClustalX2 program was shaded using Boxshade, available from the EMBnet website. The AP-1 binding regions and their accession numbers are as follows: rat (Rattus norvegicus, NP_037101.1), mouse (Mus musculus, NP_034700.2), human (Homo sapiens, NP_005535.1), chimpanzee (Pan troglodytes, XP_001134895.1), dog (Canis familiaris, XP_543274.2), cow (Bos taurus, XP_581382.2), chicken (Gallus gallus, XP_426682.2), zebrafish (Danio rerio, XP_687702.3), and frog (Xenopus laevis, NP_001084092.1). Statistical analysis. Data are expressed as means ⫾ standard errors (SE). Comparisons between two groups were performed using Student’s t test, whereas comparisons between more than two groups were analyzed by analysis of variance (ANOVA) and the Tukey post hoc test. P values of ⬍0.05 were considered statistically significant.

RESULTS

The ␮1A subunit of the AP-1 complex interacts with IRS-1. To explore the regulation of IRS-1 localization, we focused on IRS-1associated proteins. Previously, we performed yeast two-hybrid screening and succeeded in identifying many IRS-1-associated proteins (25). Among them, we identified the ␮ subunit of ubiquitously expressed AP-1 complex (␮1A) as an IRS-1-associated protein. The isolated cDNA clones encoded amino acid residues 164 to 423 of the human ␮1A C-terminal region. To confirm the results of the two-hybrid assay, the interaction between IRS-1 and ␮1A was examined in a GST pulldown assay. As shown in Fig. 1A, ␮1A can be divided into two domains—an N-terminal domain (N-␮1A) containing the ␤1-adaptin binding region that forms the AP-1 trunk domain and a C-terminal domain (C-␮1A) that recognizes YXX⌽-type cargos. GST-fused full-length ␮1A, N-␮1A, C-␮1A, and GST alone were expressed and purified from Escherichia coli. (Fig. 1A, bottom). Cell lysates from L6 myoblasts were incubated with purified GST-␮1A domains or GST only. IRS-1 specifically associated with GST-␮1A and C-␮1A but not with N-␮1A (Fig. 1B). To investigate whether endogenous IRS-1 could associate with the AP-1 complex in the cell, we performed coimmunoprecipitation analysis using L6 myoblasts. As shown in Fig. 1C, we detected that the ␥-adaptin AP-1 component coimmunoprecipitated with IRS-1, suggesting that a portion of IRS-1 associates with the AP-1 complex through ␮1A in the cell. Deletion of AP-1 suppresses IGF-I-dependent DNA synthesis. To examine the role of IRS-1 interaction with AP-1 in the biological actions of IGF-I, we performed siRNA-mediated knockdown of AP-1 subunits in L6 cells. L6 cells were transfected with siRNA against ␮1A or ␥-adaptin, resulting in the efficient knockdown of each subunit (Fig. 2A). We confirmed that in L6 cells, the majority of IRS-1 was present in the LDM fraction, which included AP-1 complex components and some endosomal markers, with a very minor portion in the plasma membrane fraction (Fig. 2B). In addition, following IGF-I stimulation, there was a


FIG 1 ␮1A AP-1 component interacts with IRS-1. (A) Diagrams of the re-

combinant GST-fused ␮1A deletion constructs used are shown. The number indicates the amino acid (a.a.) number of ␮1A. The N-terminal domain is shown in dark gray. The C-terminal cargo-binding domain is colored in light gray. Purified GST fusion proteins expressed in E. coli were stained with CBB. (B) Cell lysate of L6 myoblasts was incubated with purified GST-fused ␮1A deletion domains. Pulldown samples were analyzed by immunoblotting with anti-IRS-1 (␣ IRS-1) antibody. “Input” represents an aliquot corresponding to 2% of the lysates. (C) Cell lysates of L6 cells were subjected to immunoprecipitation (IP) with anti-IRS-1 antibody and immunoblotted with the indicated antibodies. “Input” represents an aliquot corresponding to 1% of the lysate.

marked decrease in the amount of LDM fraction-associated IRS-1 with a concomitant increase in the cytosolic fraction, similar to that previously reported for insulin-stimulated 3T3-L1 adipocytes (12). Subcellular fractionation analysis revealed that IRS-1 in the LDM fraction was decreased and IRS-1 in the cytosolic fraction tended to be increased by both ␮1A and ␥-adaptin depletion in spite of the fact that total IRS-1 protein was not affected (Fig. 2A, C, and D). IRS-1 was not detected in the plasma membrane fraction in either control or ␮1A/␥-adaptin-depleted cells (Fig. 2C), implying that AP-1 is not involved in IRS-1 targeting from the plasma membrane. We then examined the effect of AP-1 depletion on IGF-Iinduced DNA synthesis. L6 cells transfected with control or ␮1A/␥-adaptin siRNA were serum starved and then stimulated with IGF-I, and DNA synthesis was measured. As shown in Fig. 3A, IGF-I increased thymidine incorporation into DNA approximately 4- to 5-fold compared to the basal state in control cells. ␮1A depletion by two different siRNAs inhibited IGF-Iinduced DNA synthesis by 60 to 70%. Similar results were obtained from ␥-adaptin-depleted cells. To be sure that the reduced incorporation was not due to general toxicity of AP-1 subunit deficiency and that siRNA knockdowns were specific, we generated L6 cells stably expressing Myc-inserted mouse ␮1A (␮1A-Myc) by retroviral infection (28). ␮1A-Myc was incorporated into the endogenous AP-1 complex as assessed by coimmunoprecipitation and immunofluorescence analyses (Fig. 3B and C). ␮1A-Myc, in which three mismatched sites exist in the siRNA target sequence, was resistant to siRNAs against rat ␮1A (Fig. 3D). In addition, IGF-I-induced DNA synthesis of ␮1A-Myc-expressing cells transfected with ␮1A siRNA was restored compared with that of vector-expressing cells transfected with ␮1A siRNA (Fig. 3E). Thus, these data indicate that AP-1 complex plays a role in IGF-I-induced mitogenesis.

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FIG 2 Deletion of AP-1 suppresses localization of IRS-1 into LDM fraction. (A) L6 cells were transfected with control, ␮1A(#1) and -(#2), or ␥-adaptin(#1) and -(#2) siRNAs. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) L6 cells were treated with or without IGF-I (100 ng/ml) for 5 min and subjected to subcellular fractionation as described in Materials and Methods. Fractions (CYT, cytosol; PM, plasma membrane; HDM, high-density microsome; LDM, low-density microsome) were analyzed by immunoblotting with the indicated antibodies at the ratio of CYT to other fractions of 0.05 to 1.00 by volume. (C) Cells transfected with control, ␮1A, or ␥-adaptin siRNA were subjected to subcellular fractionation. Fractions were analyzed by immunoblotting with the indicated antibodies at the ratio of CYT to LDM or PM of 0.05 to 1.00 by volume. ␣-Adaptin, an AP-2 subunit, was evaluated as a loading control. (D) Band intensities of IRS-1 in each fraction were quantified and statistically analyzed from four independent experiments. Means ⫾ SE (n ⫽ 4) are shown. *, P ⬍ 0.05.

Three YXX⌽ motifs in IRS-1 are responsible for binding to ␮1A. Next, to examine the role of IRS-1 interaction with AP-1 in IGF-I-induced mitogenesis in detail, we first began to determine the AP-1 binding region of IRS-1. We constructed IRS-1 deletion mutants fused with GFP and performed pulldown assays. As shown in Fig. 4A, an IRS-1 mutant that contains a central region corresponding to amino acid residues 443 to 663 could bind to ␮1A, whereas mutants lacking this region could not. We therefore termed the amino acid residues 443 to 663 the AP-1 binding region (APBR). The rat IRS-1 APBR contains five putative YXX⌽ motifs (YICM, residues 460 to 463; YECM, residues 546 to 549; YMPM, residues 608 to 611; YMPM, residues 628 to 631; and

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YMMM, residues 658 to 661). As shown in Fig. 4B, the IRS-1 mutant in which all five tyrosine residues were replaced with alanine (IRS-1 Y5A) poorly bound to ␮1A. Although single-point mutation of any one tyrosine residue did not affect the interaction, the mutant containing Y608A, Y628A, and Y658A substitutions (IRS-1 Y3A) also displayed very weak binding to ␮1A, similar to the IRS-1 Y5A mutant. These results suggest that IRS-1 interacts with ␮1A through three tyrosine motifs (Y608, Y628, and Y658) clustered in the APBR. In addition, replacement of two tyrosine residues with alanine (Y608A and Y658A) slightly reduced the interaction with ␮1A (Fig. 4C). These data suggest that all three tyrosine motifs contribute to the interaction with ␮1A. Furthermore, multiple alignment anal-



FIG 3 Deletion of AP-1 suppresses IGF-I-dependent DNA synthesis. (A) L6 cells transfected with control, ␮1A, or ␥-adaptin siRNA were serum starved followed

by stimulation of IGF-I at a concentration of 100 ng/ml. [3H]thymidine incorporation into DNA was measured during the last 4 h. Experiments were performed in triplicate, and the means ⫾ SE are shown. *, P ⬍ 0.05. (B) Cell lysates of vector- or ␮1A-Myc-expressing cells were subjected to immunoprecipitation with anti-Myc antibody and immunoblotted with the indicated antibodies. (C) Vector- or ␮1A-Myc-expressing cells were stained with anti-Myc and ␥-adaptin antibodies. Samples were analyzed by confocal microscopy. Bar, 10 ␮m. (D) Vector- or ␮1A-Myc-expressing cells were transfected with control or ␮1A siRNAs. Cell lysates were analyzed by immunoblotting with the indicated antibodies. (E) Vector- or ␮1A-Myc-expressing cells transfected with siRNA were subjected to DNA synthesis analysis. Experiments were performed in triplicate, and the means ⫾ SE are shown. *, P ⬍ 0.05; **, P ⬍ 0.01. These are representative results of experiments independently performed three times.

ysis revealed that these motifs and the surrounding amino acid sequences were highly conserved in IRS-1 of vertebrates (Fig. 4D). The IRS-1 mutant lacking binding to ␮1A has defects in transport from the CI-MPR-positive membranes. Since IRS-1 interacted with sorting protein AP-1, we next examined how IRS-1 is transported within the cell. To address this issue, we examined the trafficking of newly synthesized GFP-IRS-1 in living cells using time-lapse microscopy. A plasmid expressing GFPIRS-1 was transfected into HEK293T cells, and fluorescent images were obtained over time shortly after its expression was observed. As shown in Fig. 5A, 6 h posttransfection the GFP-IRS-1 wild type (WT) was first observed in the perinuclear region. This structure


was gradually swollen and then started to disperse throughout the cytoplasm, resulting in punctate localization with characteristics of vesicular structures until 12 h posttransfection. In contrast, GFP-IRS-1 lacking APBR (⌬APBR) accumulated in the perinuclear region, and most of its large structure remained without dispersion for up to 12 h. We further analyzed the localization of IRS-1 mutants with reduced binding to ␮1A. In L6 myoblasts, the GFP-IRS-1 WT displayed punctate cytoplasmic localization (Fig. 5B). In contrast, the GFP-IRS-1 ⌬APBR mutant localized to the perinuclear region as seen in live imaging. This mutant was colocalized with cation-independent mannose 6-phosphate receptor (CI-MPR), which localizes mainly to the TGN and endo-

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FIG 4 Three YXX⌽ motifs in IRS-1 are responsible for binding to ␮1A. (A) Shown below the full-length IRS-1 structure (two white boxes represent the PH and PTB domains) are the deletion mutant constructs used. The lysates of HEK293T cells expressing GFP-IRS-1 deletion mutants were incubated with GST-␮1A, and pulldown samples were analyzed by immunoblotting (IB) with anti-GFP (␣ GFP) antibody. “Input” represents an aliquot corresponding to 2% of the lysates. The striped box above the IRS-1 structure indicates the AP-1 binding region (APBR). (B) APBR in IRS-1 is magnified, and five putative YXX⌽ motifs are shown. Below the WT, alanine mutant constructs are shown. The lysates of HEK293T cells expressing GFP-IRS-1 mutants were subjected to pulldown assay as shown in panel A. (C) The lysates of HEK293T cells expressing GFP-IRS-1 double mutants (Y608A Y628A, Y608A Y658A, and Y628A Y658A) were subjected to pulldown assay using GST-␮1A. (D) APBR sequences of the IRS-1 from various species were aligned using ClustalX2, and the alignment was shaded using Boxshade. Three YXX⌽ motifs are indicated by bars. Identical residues are highlighted in black, and similar residues are highlighted in gray.

somes, as did the IRS-1 Y3A mutant. In contrast, the IRS-1 Y2A mutant, which can interact with ␮1A, displayed punctate localization similar to that of the IRS-1 WT. The GFP-IRS-1 ⌬APBR and Y3A mutants were not colocalized with the TGN marker TGN38 (Fig. 5C and D). Furthermore, we analyzed the colocalization of IRS-1 with CI-MPR during its synthesis. The IRS-1 WT was targeted to a CI-MPR-positive structure at the very early stage of its synthesis and then dispersed into the cytoplasm in a time-dependent manner, no longer colocalizing with CI-MPR (Fig. 5E and F). These trafficking events were not observed in IRS-1 ⌬APBR, which was colocalized with CI-MPR during its synthesis (Fig. 5E). These data suggest that lack of three YXX⌽ motifs resulted in the mislocalization of IRS-1 to the CI-MPR-positive endosomes and blocked sorting to peripheral vesicles. Interaction with AP-1 is required for IGF-I-dependent tyrosine phosphorylation of IRS-1 and IRS-1-associated PI 3-kinase. Mislocalization of IRS-1 ⌬APBR or Y3A mutants prompted

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us to examine the phenotype of the IRS-1 mutants in IGF-I-stimulated signaling and mitogenic activity. To this end, we set up rescue experiments in which IRS-1 mutants were reintroduced into IRS-1-depleted L6 cells. We first generated L6 cells stably expressing shRNA against IRS-1 by retroviral infection. In these cells, IGF-I-dependent phosphorylation of Akt, but not MAPK Erk1/2, was inhibited compared to that in control cells expressing shRNA against luciferase (Fig. 6A). IGF-I-induced DNA synthesis and cell proliferation were also inhibited in shIRS-1-expressing cells (Fig. 6B and C). Next, we reexpressed Myc-tagged IRS-1 mutants, which were resistant to shIRS-1(#2) targeting the IRS-1 3= untranslated region (UTR), by retroviral infection (Fig. 7A). IGFI-dependent tyrosine phosphorylation and its association of the p85 subunit of the PI-3 kinase with IRS-1 ⌬APBR or Y3A were inhibited compared to the case in the IRS-1 WT (Fig. 7C and E). This occurred without any effect on the tyrosine autophosphorylation of IGF-IR itself (Fig. 7D). In addition, Akt phosphorylation,



FIG 5 The IRS-1 mutant lacking the binding to ␮1A has defects in transport from the CI-MPR-positive membranes. (A) Time-lapse microscopy of HEK293T

cells expressing the GFP-IRS-1 WT or ⌬APBR mutant. Montage images composed of observed points are shown with a time scale (hours:minutes). Bar, 10 ␮m. (B) L6 myoblasts expressing the GFP-IRS-1 WT or ⌬APBR, Y2A, or Y3A mutant (green) were stained with anti-CI-MPR antibody (red) and analyzed by confocal microscopy. Bars, 10 ␮m. (C) L6 cells expressing the GFP-IRS-1 WT or ⌬APBR or Y3A mutant (green) were stained with anti-TGN38 antibody (red). Samples were analyzed by confocal microscopy. Bars, 10 ␮m. (D) The bar graphs represent Pearson’s coefficient calculated for GFP-IRS-1 and CI-MPR or TGN38 colocalization. Means ⫾ SE are shown. *, P ⬍ 0.05 versus IRS-1 WT; n ⬎ 40 cells for all conditions. (E) HEK293T cells expressing the GFP-IRS-1 WT or ⌬APBR mutant (green) were fixed at the indicated times and stained with anti-CI-MPR antibody (red). (F) The percentages of the cells expressing GFP-IRS-1 WT that colocalized with CI-MPR at the indicated times are shown in the graph. Means ⫾ SE (n ⬎ 40) are shown.

but not Erk1/2 phosphorylation of rescued IRS-1 WT cells, was upregulated compared to that of rescued vector-containing cells, while that of rescued ⌬APBR or Y3A mutant cells was not (Fig. 7E), indicating that mislocalization of IRS-1 caused by lack of interaction with AP-1 reduced tyrosine phosphorylation of IRS-1 and subsequent Akt phosphorylation.


However, it is also possible to ascribe this inhibition to the deletion of tyrosine residues in APBR because Y608, Y628, and Y658 are known to be phosphorylated by IR/IGF-IR and function as binding sites of PI 3-kinase (29, 30). To examine whether mislocalization or the deletion of tyrosine residues inhibited the phosphorylation of IRS-1 and subsequent association of PI 3-kinase,

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FIG 6 Depletion of IRS-1 suppresses IGF-I-dependent Akt phosphorylation and cell proliferation. (A) L6 cells stably expressing shRNA against luciferase (shLuc) or IRS-1 were treated with or without IGF-I (100 ng/ml) for 5 min. Cell lysates were subjected to immunoblotting with the indicated antibodies. Band intensities of pAkt (Ser473) and pErk1/2 were quantified and are shown as means ⫾ SE (n ⫽ 4). *, P ⬍ 0.05. (B) L6 cells stably expressing shLuc or shIRS-1(#1) and -(#2) were subjected to DNA synthesis analysis. Experiments were performed in triplicate, and means ⫾ SE are shown. *, P ⬍ 0.05. (C) Cells were grown in DMEM containing 1% FBS with or without IGF-I (100 ng/ml), and the cell number was counted each day. Experiments were performed in triplicate, and means ⫾ SE are shown. *, P ⬍ 0.05.

we constructed an IRS-1 mutant in which the C terminus is fused with the partial CAAX sequence of H-Ras (IRS-1 CAAX). The C-terminal sequence (C181-MS-C184-K-C186-VLS) of H-Ras contains the CAAX motif (CVLS) (31). Palmitoylation of C181 and C184 in addition to farnesylation of C186 anchors H-Ras on the plasma membrane through the endoplasmic reticulum and Golgi apparatus, and the H-Ras mutant lacking C181 mislocalizes to the perinuclear region containing the Golgi apparatus (32). The IRS-1 CAAX mutant that contained the partial sequence of H-Ras (CKCVLS) lacking palmitoylated C181 was not targeted to PM and mainly was localized to the perinuclear region (Fig. 7B). Immunofluorescence analysis revealed that a large portion of IRS-1

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CAAX was colocalized to CI-MPR, mimicking the localization of the ⌬APBR or Y3A mutant. Tyrosine phosphorylation of the IRS-1 CAAX mutant and its association with p85 were inhibited similar to the case in the ⌬APBR or Y3A mutant (Fig. 7C and E). Akt phosphorylation of rescued IRS-1 CAAX cells was comparable to that of rescued vector-containing cells, consistent with the mislocalization of IRS-1 being responsible for reduced IRS-1 tyrosine phosphorylation and PI 3-kinase association (Fig. 7E). Interaction with AP-1 is required for IGF-I-induced cell proliferation. Next, we examined DNA synthesis and cell proliferation in rescued IRS-1 mutant cells. Consistent with tyrosine phosphorylation of IRS-1 and Akt phosphorylation, IGF-I-induced



FIG 7 Interaction with AP-1 is required for IGF-I-dependent tyrosine phosphorylation of IRS-1 and IRS-1 association with PI 3-kinase. (A) L6 cells stably expressing shIRS-1(#2) were infected with retrovirus for reexpression of Myc-tagged IRS-1 mutants. Cell lysates were subjected to immunoblotting with the indicated antibodies. C-terminal fusion of the CAAX sequence inhibited the binding of an anti-IRS-1 antibody that recognizes the C terminus of IRS-1. p85 PI 3-kinase was evaluated as a loading control. (B) L6 cells expressing the Myc-IRS-1 WT or CAAX were stained with anti-Myc (red) and CI-MPR (green) antibodies and analyzed by confocal microscopy. Bars, 10 ␮m. (C) L6 cells stably expressing both shIRS-1(#2) and Myc-IRS-1 mutants were treated with or without IGF-I (100 ng/ml) for 5 min and subjected to immunoprecipitation with anti-Myc antibody and immunoblotting with the indicated antibodies. (D) L6 cells stably expressing both shIRS-1 and Myc-IRS-1 mutants were treated as in panel C and subjected to immunoprecipitation with anti-IGF-IR␤ and immunoblotting with the indicated antibodies. (E) Protein amounts of the Myc-IRS-1 mutant, phosphorylated IRS-1, p85 associated with IRS-1, phosphorylated Akt (Ser473), and phosphorylated Erk1/2 were quantified, and pY IRS-1/Myc-IRS-1 and p85/Myc-IRS-1 were calculated. Experiments were performed in triplicate, and means ⫾ SE are shown. *, P ⬍ 0.05. Shown are representative results from experiments independently performed three times.


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FIG 8 Interaction with AP-1 is required for IGF-I-induced cell proliferation. (A) L6 cells stably expressing both shIRS-1(#2) and Myc-IRS-1 mutants were serum starved followed by IGF-I stimulation. [3H]thymidine incorporation into DNA was measured during the last 4 h. Experiments were performed in triplicate, and means ⫾ SE are shown. *, P ⬍ 0.05. (B) L6 cells stably expressing both shIRS-1(#2) and Myc-IRS-1 mutants were grown in DMEM containing 1% FBS with or without IGF-I (100 ng/ml), and the cell number was counted each day. Experiments were performed in triplicate, and means ⫾ SE are shown. *, P ⬍ 0.05. Shown are representative results from experiments independently performed three times.

DNA synthesis of rescued IRS-1 WT cells was upregulated compared to rescued vector-containing or other IRS-1 mutant cells (Fig. 8A). In addition, proliferation of rescued IRS-1 WT cells was enhanced by IGF-I addition, while that of rescued vector-containing or other IRS-1 mutant cells was not (Fig. 8B). These data demonstrate the importance of IRS-1–AP-1 interaction in maximum mitogenic response to IGF-I. DISCUSSION

IRS-1 is known to be a pivotal mediator of insulin/IGF signaling cascades. Previous studies using subcellular fractionation proposed that the localization of IRS-1 to the intracellular membrane is important for efficient insulin/IGF signaling and biological responsiveness (12, 13, 16, 33). Earlier biochemical investigation indicated a protein-protein interaction is required for IRS-1 targeting to vesicles and membrane-anchored cytoskeleton. Despite

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these reports, there remains a lack of compelling evidence supporting a mechanism responsible for intracellular IRS-1 localization. In this study, we have identified the AP-1 complex as an important determinant of IRS-1 localization to the intracellular membrane compartment. Furthermore, deletion of the interaction with the AP-1 complex impairs IGF-I-dependent IRS-1 tyrosine phosphorylation and cell proliferation, suggesting AP-1 as a critical regulator of IRS-1 localization in IGF signaling and its mitogenic activation. The AP-1 complex is extensively characterized as a clathrincoated vesicle adaptor and is often reported to transport transmembrane proteins. In this study, we have shown that AP-1 controls the localization of IRS-1, which is cytosolic protein, into vesicular structures. IRS-1 interacted with the cargo recognition domain of ␮1A through YXX⌽-type motifs. As ␮1A directly recognizes YXX⌽ motifs in AP-1 cargos (19), it is likely that IRS-1 is



recognized by ␮1A in the same manner as conventional cargo proteins. In addition, depletion of AP-1 subunits decreased the amount of LDM-associated IRS-1, indicating association of IRS-1 with the intracellular microsomal fraction is required for AP-1. Analyses using IRS-1 ⌬APBR or Y3A mutants provides further evidence of direct involvement of AP-1 in sorting IRS-1. First, time-lapse observations revealed that newly synthesized IRS-1 is dramatically transported from the perinucleus to peripheral vesicles, and ⌬APBR mutants are defective in this transport. Second, the IRS-1 WT overlapped with CI-MPR at the very early stage of its synthesis, but at the later stage dispersed into peripheral vesicles that did not contain CI-MPR. In contrast, the IRS-1 mutant lacking the AP-1 binding was localized to CI-MPR-positive structure even after the later stage of the synthesis. CI-MPR is known to localize mainly to the TGN and late endosomes (34), and the IRS-1 WT or ⌬APBR or Y3A mutants did not overlap the TGN marker, TGN38. These results suggested that IRS-1 is sorted as one of the AP-1 cargos from CI-MPR-positive post-TGN compartment, such as late endosomes. Kural et al. recently revealed that AP-1 complex temporarily associates with cargo-containing membranes (35). By biochemical analyses, it was found that IRS-1 interacted with the AP-1 complex in a relatively stable fashion, which may help the packaging of cytosolic IRS-1 into AP-1-associated vesicles. We have not yet been able to identify the destination of the IRS-1 sorted by AP-1. The fact that we detected little or no overlap between endogenous IRS-1 and conventional endosome markers (e.g., EEA1, Rab11, and transferrin receptor) (data not shown) indicates that IRS-1 is localized to a unique vesicular compartment. The sorting directionality of AP-1 is reported to be both anterograde (TGN/post-TGN compartment to peripheral endosome) and retrograde (endosome to TGN) and seems to depend on the type of cargo protein (20–23, 36). Thus, defining the intracellular localization of IRS-1 will help us understand the more detailed AP-1 sorting pathway of IRS-1. It is also possible that IRS-1 is localized to intracellular vesicles from plasma membrane through interaction with IR/IGF-IR or AP-1-mediated internalization. The N-terminal pleckstrin homology (PH)-phosphotyrosine binding (PTB) domain of IRS-1 interacts with activated IR/IGF-IR and possesses the ability to bind phosphoinositides (37–39). IRS-1 mutants lacking the PH or PH-PTB domain were localized to peripheral vesicles, as seen in the IRS-1 WT (data not shown), suggesting that interaction with IR/IGF-IR is not necessary for IRS-1 localization. Furthermore, we did not observe accumulation of IRS-1 in plasma membrane in cells with AP-1 depletion or in AP-1 binding site IRS-1 mutants. These results demonstrate that internalization by interaction with IR/IGF-IR or AP-1 does not contribute to IRS-1 localization. In addition, IRS-1 associates with other proteins related to vesicle trafficking, such as the ␴3 AP-3 subunit (40, 41). These proteins might control IRS-1 localization in cooperation with AP-1. Among them, AP-3 was undetectable in L6 cells and its role in IRS-1 localization could not be experimentally examined in this system (data not shown). We could not rule out the possibility that disruption of interaction with AP-1 leads IRS-1 to traffic in other sorting pathways, resulting in mislocalization into the CIMPR-positive compartment. In any case, several studies using subcellular fractionation suggested that intracellular membrane compartments, such as the LDM, harbor the population of IRS-1 that actively participates in signal transduction (11, 33, 42, 43). However, as IRS-1 exists both


in cytosol and in intracellular membranes, it is difficult to define precisely the importance of intramembrane-associated IRS-1 in insulin/IGF signaling. In this study, using IRS-1 mutants lacking AP-1 binding we have shown that AP-1-dependent localization of IRS-1 into vesicular structures is required for IGF-I-dependent tyrosine phosphorylation and its association with PI 3-kinase. Mislocalization of IRS-1 reduced IGF-I-dependent tyrosine phosphorylation of IRS-1 and the association with PI 3-kinase without affecting phosphorylation of IGF-IR. Several reports suggest that internalization of IR/IGF-IR is required for proper signal transduction (44–47). These observations raise the possibility that the IRS-1–IGF-IR association should be spatially restricted in the intracellular membrane compartment, and therefore a defect in AP-1 transport to such compartment could result in reduced tyrosine phosphorylation of IRS-1 by IGF-IR. It has been structurally demonstrated that the ␮ subunit of the AP complex could not bind to the phosphorylated YXX⌽ motif (19). In addition, AP-1 binding sites in IRS-1 overlap PI 3-kinase binding sites. We speculate that a mechanism exists that allows for the AP-1-dependent sorting and subsequent dissociation, thereby targeting IRS-1 for IGF-I-mediated tyrosine phosphorylation and PI 3-kinase activation in a spatially compartmentalized manner. We also found that IGF-I-dependent phosphorylation of Akt, a downstream molecule of PI 3-kinase, was impaired in IRS-1 ⌬APBR- or Y3A mutant-rescued cells, accompanied by the decrease in DNA synthesis. It is well established that activated Akt stimulates cell proliferation through phosphorylation of multiple downstream substrates (48). Thus, coordination of internalized activated receptor and IRS-1 in specific vesicular structures might induce compartmentalized activation of Akt, which in turn stimulates downstream signaling and mitogenesis. In addition, the amount of intracellular membrane-associated IRS-1 is well correlated with insulin-induced glucose uptake (15, 16, 33), indicating that AP-1-dependent IRS-1 localization is also important for insulin/IGF bioactivities other than cell proliferation. Based on our results, we conclude that the AP-1 complex is a key regulator of IRS-1 localization to the intracellular membrane compartment required for IGF-I-stimulated signaling and maximum mitogenic response. ACKNOWLEDGMENTS We thank Hiroshi Ohno (RIKEN, Kanagawa, Japan) for the kind gift of mouse ␮1A cDNA. Human recombinant IGF-I was a kind gift from Astellas Pharma, Inc., Tokyo, Japan. We extremely appreciate helpful discussions with Jeffry E. Pessin (Department of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, NY), Stuart Newfeld (School of Life Sciences, Arizona State University, AZ), and Takashi Umehara (RIKEN, Kanagawa, Japan). In addition, we thank Susan Hall (University of North Carolina at Chapel Hill, NC) for help with writing the manuscript. This work was supported in part by a Grants-in-Aid for Scientific Research [Scientific Research (A) (2)#16208028 and Scientific Research (A) (2)#22248030] to S.I.T., the Core-to-Core Program from the Japan Society for the Promotion of Science Research (JSPS) to S.-I.T., and the Program for Promotion of Basic Research Activities for Innovative Biosciences to F.H., as well as the Program for Basic and Applied Research for Innovations in Bio-Oriented Industry to S.-I.T. Y.Y. obtained Fellowships of JSPS for Young Scientists.

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