The Rab GTPase-Activating Protein TBC1D4/AS160 Contains an

The Rab GTPase-Activating Protein TBC1D4/AS160 Contains anAtypical Phosphotyrosine-Binding Domain That Interacts with PlasmaMembrane Phospholipids To Facilitate GLUT4 Trafficking inAdipocytes

Shi-Xiong Tan,a Yvonne Ng,a James G. Burchfield,a Georg Ramm,c David G. Lambright,d Jacqueline Stöckli,a and David E. Jamesa,b

Diabetes and Obesity Research Program, The Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales, Australiaa; School of Biotechnology andBiomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australiab; Department of Biochemistry and Molecular Biology and Monash MicroImaging, School of Biomedical Sciences, Monash University, Melbourne, Victoria, Australiac; and Department of Biochemistry and Molecular Pharmacology, University ofMassachusetts Medical School, Worcester, Massachusetts, USAd

The Rab GTPase-activating protein TBC1D4/AS160 regulates GLUT4 trafficking in adipocytes. Nonphosphorylated AS160 bindsto GLUT4 vesicles and inhibits GLUT4 translocation, and AS160 phosphorylation overcomes this inhibitory effect. In the pres-ent study we detected several new functional features of AS160. The second phosphotyrosine-binding domain in AS160 encodesa phospholipid-binding domain that facilitates plasma membrane (PM) targeting of AS160, and this function is conserved inother related RabGAP/Tre-2/Bub2/Cdc16 (TBC) proteins and an AS160 ortholog in Drosophila. This region also contains a non-overlapping intracellular GLUT4-containing storage vesicle (GSV) cargo-binding site. The interaction of AS160 with GSVs andnot with the PM confers the inhibitory effect of AS160 on insulin-dependent GLUT4 translocation. Constitutive targeting ofAS160 to the PM increased the surface GLUT4 levels, and this was attributed to both enhanced AS160 phosphorylation and 14-3-3 binding and inhibition of AS160 GAP activity. We propose a model wherein AS160 acts as a regulatory switch in the dockingand/or fusion of GSVs with the PM.

Rab GTPases play a fundamental role in vesicle transport reac-tions in eukaryotic cells. There are more than 60 Rabs in the

human genome, suggesting that these proteins coordinate a com-plex cascade of events to ensure both fidelity and directionality tointracellular protein sorting (27, 34, 46). Rabs switch between ac-tive and inactive forms via GTP loading and GTP hydrolysis, re-spectively, and these reactions are catalyzed via guanine nucleo-tide exchange factors (GEFs) and GTPase-activating proteins(GAPs). Thus, the Rab-regulated step is often considered the ma-jor regulatory node in many transport steps. Rabs have been im-plicated in various stages of vesicle transport, including vesiclebudding, vesicle transport along microtubules, and docking oftransport vesicles with the target membrane (27, 34, 46). Consis-tent with a requirement for multiple Rabs in vesicle traffickingpathways, Rab cascades have recently been described in whichGDP/GTP loading of sequential Rabs in a pathway is coordinatelyregulated in a countercurrent manner by cognate GAPs and GEFs(10, 24, 31, 46). This elegant mechanism proposes that for twoRabs that act in series in a pathway the GEF for the downstreamRab binds in an effector-type manner to the upstream Rab, whilethe GAP for the upstream Rab binds to the GTP-bound form ofthe downstream Rab. This coupling mechanism provides a poten-tial mechanism for directional transfer in transport pathways.One of the provocative aspects of this model is that it suggests thatthe regulatory molecules involved play multiple shared roles in theprocess. For instance, the interaction between the GAP for theupstream Rab with the downstream Rab provides positive infor-mation to the pathway, which is somewhat counterintuitive to thenegative role that these particular proteins are thought to play.

Certain vesicle transport pathways are subject to exquisite reg-ulation, and so these pathways provide an ideal opportunity tointerrogate key regulatory steps. The regulation of glucose trans-

port in muscle and fat cells has become a paradigm for this modeof regulation. In this system, the facilitative glucose transporterGLUT4 is packaged into intracellular storage vesicles (GSVs) thatremain disconnected from the plasma membrane and the recy-cling pathway in the absence of insulin. Insulin causes rapid de-livery and fusion of these vesicles with the plasma membrane(PM). The canonical phosphatidylinositide 3-kinase/Akt signal-ing pathway (44) is a key determinant of this exocytic process.

The discovery of the RabGAP AS160/TBC1D4 as a major in-sulin-regulated Akt substrate in adipocytes and muscle cells was amajor advance in understanding the regulation of GLUT4 trans-location to the PM (7, 14, 19, 28, 29, 32, 35, 36, 45). AS160 pos-sesses a RabGAP/Tre-2/Bub2/Cdc16 (TBC) domain at its C ter-minus flanked by a calmodulin-binding domain and twophosphotyrosine-binding (PTB) domains at the N terminus (14,32, 45). Akt stimulates AS160 phosphorylation at Thr642, and thistriggers 14-3-3 binding (8, 28). Overexpression of an AS160 mu-tant, in which the phosphorylation sites were mutated to alanine(AS160-4P), inhibited insulin-stimulated GLUT4 translocation(7, 28, 29, 32, 35, 36, 45). Mutating a crucial arginine in the Rab-GAP domain overcame this inhibitory effect (22, 32), providing afunctional link between phosphorylation and AS160 GAP activity

Received 6 June 2012 Returned for modification 12 July 2012Accepted 28 September 2012

Published ahead of print 8 October 2012

Address correspondence to David E. James, [email protected].

S.-X.T. and Y.N. contributed equally to this article.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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(32, 45). Knockdown of AS160 in adipocytes increases basalGLUT4 translocation (2, 7, 19), suggesting that AS160 plays anegative role in insulin-stimulated GLUT4 trafficking. ThatAS160 interacts with GSVs by binding to vesicle cargo such as thelow-density lipoprotein receptor-related protein 1 (LRP1) and in-sulin-regulated aminopeptidase (IRAP) (12, 19, 26), together withthe above findings, gave rise to a model wherein AS160 binds toGSVs in the basal state to maintain its substrate Rab GTPase(s) ina GDP-loaded inactive form, retaining GLUT4 inside the cell. In-sulin-dependent phosphorylation of AS160 is thought to inhibitits GAP activity, allowing GTP loading and activation of a Rab thatregulates docking and fusion of GSVs with the PM (8, 14, 19, 28,29, 32, 36).

This model was based upon the canonical view of GAPs, whichwere thought to principally switch off their substrates by trigger-ing GTP hydrolysis. However, several observations are not en-tirely consistent with this model: reduction of AS160 in adipocytesusing shRNA results in increased GLUT4 at the PM in the basalstate but blunted insulin-stimulated translocation of GLUT4 tothe PM (2, 7); release of AS160 from GSVs is not necessary forinsulin-dependent translocation to the PM, raising the possibilitythat AS160 remains associated with GSVs until after fusion (17,35). There is a finite pool of AS160 at the PM that becomes highlyphosphorylated with insulin (23), suggesting that AS160 is possi-bly phosphorylated at the PM. Thus, we hypothesized that, inaddition to its negative regulatory role, AS160 might play an ad-ditional positive role consistent with the Rab cascade model pro-posed by Zerial and McBride (46).

We present evidence here that AS160 acts as a regulatoryswitch, playing both an inhibitory and a facilitative role in GLUT4translocation to the PM. This switch-like mechanism, which isconsistent with the Rab cascade model, is shown to be regulated byAS160 phosphorylation. Nonphosphorylated AS160 repressesGTP loading of a Rab and GLUT4 translocation, while phosphor-ylation of AS160 inactivates its GAP activity (22, 32) and allowsAS160 to fulfill an active role in facilitating GLUT4 vesicle fusionwith the PM. These two separate functions are encoded by theN-terminal PTB domain in AS160, which mediates the interactionbetween AS160 and GSV cargo proteins, thus encoding the inhib-itory function and a separate lipid-binding domain that facilitatesinteraction of AS160 with the PM.

MATERIALS AND METHODSMaterials and antibodies. Polyclonal rabbit antibodies raised againstpThr308 Akt, pThr1462 TSC2, pSer21/9 glycogen synthase kinase 3�/�(GSK3�/�), and glutathione S-transferase (GST), monoclonal rabbit an-tibodies raised against total Akt (11E7) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 14C10), and monoclonal mouse antibodiesraised against pSer473 Akt (587F11) were purchased from Cell SignalingTechnologies (Beverly, MA). Polyclonal rabbit antibody raised against14-3-3� was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,CA). Polyclonal sheep antibodies raised against pThr642 AS160 were ob-tained from Peter Shepherd (Symansis, Auckland, New Zealand). Rabbitpolyclonal antibodies against human AS160 and syntaxin-4 were previ-ously described (40). The FLAG M2 and tubulin antibodies were fromSigma. Horseradish peroxidase (HRP)-conjugated secondary antibodieswere from Amersham Biosciences (Buckinghamshire, United Kingdom),and IR dye 700- or 800-conjugated secondary antibodies were from Rock-land Immunochemicals (Gilbertsville, PA). Paraformaldehyde was fromProSciTech (Thuringowa, Australia). Dulbecco modified Eagle medium(DMEM) and F-12 medium were from Invitrogen. Fetal calf serum (FCS)

was obtained from Trace Scientific (Melbourne, Australia), and the anti-biotics were from Invitrogen. Bovine serum albumin (BSA) was fromBovogen (Essendon, Australia). Bicinchoninic acid (BCA) reagent andSuperSignal West Pico chemiluminescent substrate were from Pierce(Rockford, IL). Protease inhibitor mixture tablets were from Roche Ap-plied Science (Indianapolis, IN). The Akt inhibitor, MK-2206, was gener-ously provided by Dario Alessi (University of Dundee, Dundee, UnitedKingdom). Other materials were obtained from Sigma Chemical Co. (St.Louis, MO).

Structural prediction and modeling. Protein domains were predictedusing SMART (33). The three-dimensional (3D) structure prediction isbased on the Dab1 PTB domain in complex with IP3 and the ApoER2peptide (PDB ID:1NU2). The Phyre2 server was used to identify homol-ogy model (16). Subsequent modeling and rendering was done withpyMOL.

Cell culture and transfection. 3T3-L1 fibroblasts (ATCC, Manassas,VA) were cultured and differentiated to adipocytes as described previ-ously (19). 3T3-L1 fibroblasts were infected with pWZLneo HA-GLUT4retrovirus only or together with various pBabepuro-AS160 retrovirus.After 24-h recovery period, infected cells were selected with either 2 �g ofpuromycin/ml or together with 800 �g of Geneticin/ml in DMEM sup-plemented with 10% FCS for the selection of hemagglutinin (HA)-GLUT4-infected cells or HA-GLUT4/AS160-infected cells, respectively.Surviving 3T3-L1 fibroblasts were then grown to confluence and subse-quently differentiated into adipocytes as described above. CHO IR/IRS-1cells (American Type Culture Collection, Manassas, VA) were cultured inF-12 medium containing 10% fetal calf serum (FCS), 800 �g of G418/ml,2 mM L-GlutaMAX, 100 U of penicillin/liter, and 100 �g of streptomycin/liter at 37°C in 10% CO2. HEK293E cells (American Type Culture Collec-tion) were cultured in DMEM supplemented with 10% FCS, 2 mM L-GlutaMAX, 100 U of penicillin/liter, and 100 �g/liter streptomycin at37°C in 10% CO2. CHO IR/IRS-1 cells, and HEK 293E cells were tran-siently transfected with DNA constructs using Lipofectamine LTX (Invit-rogen) or Lipofectamine 2000 (Invitrogen), respectively, according to themanufacturer’s instructions.

Western blotting analysis. Cells were washed twice with ice-coldphosphate-buffered saline (PBS) and solubilized in 2% sodium dodecylsulfate (SDS) in PBS containing phosphatase inhibitors (1 mM sodiumpyrophosphate, 2 mM sodium vanadate, 10 mM sodium fluoride) andcomplete protease inhibitor mixture. Insoluble material was removed bycentrifugation at 18,000 � g for 10 min. The protein concentration wasmeasured using the BCA method. Proteins were separated by SDS-PAGEfor immunoblot analysis. After the proteins were transferred to poly-vinylidene difluoride membranes, the membranes were incubated inblocking buffer containing 5% skim milk in Tris-buffered saline (TBS)and immunoblotted with the relevant antibodies overnight at 4°C inblocking buffer containing 5% BSA– 0.1% Tween in TBS. After incuba-tion, the membranes were washed and incubated with HRP-labeled sec-ondary antibodies and then detected by SuperSignal West Pico chemilu-minescent substrate. In some cases, IR dye 700- or 800-conjugatedsecondary antibodies were used and then scanned at the 700- and 800-nmchannels using an Odyssey IR imager. Quantification of the protein levelswas performed using the Odyssey IR imaging system software or WrightCell Imaging Facility ImageJ software.

Immunoprecipitation. After the indicated treatment, the cells werewashed with ice-cold PBS and solubilized in NP-40 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% glycerol)containing Complete protease inhibitor mixture and phosphatase inhib-itors (2 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 10mM sodium fluoride). Cell lysates were homogenized 10 times using a27-gauge needle and centrifuged at 18,000 � g for 20 min at 4°C. Onemilligram of cell lysate was incubated overnight at 4°C with 2 �g of FLAGantibody. The antibodies were then captured with protein G-Sepharosebeads for 2 h at 4°C. Immunoprecipitates were washed three times withice-cold NP-40 buffer and kept in 2� SDS sample buffer at �20°C.

Role of AS160 in Regulating GLUT4 Trafficking

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Cationic silica isolation of plasma membrane. Plasma membraneswere purified as described previously (3) with some modifications.Briefly, after treatments, the cells were washed twice with ice-cold PBS andtwice in ice-cold coating buffer (20 mM morpholineethanesulfonic acid,150 mM NaCl, 280 mM sorbitol [pH 5.0 to 5.5]). Cationic silica in a finalconcentration of 1% was added to the cells in coating buffer for 2 min onice. The cells were then washed with ice-cold coating buffer to removeexcess silica. Sodium polyacrylate (1 mg/ml, pH 6 to 6.5) was added to thecells in coating buffer, followed by incubation at 4°C for 2 min. The cellswere washed once in ice-cold coating buffer and then washed with mod-ified HES (20 mM HEPES, 250 mM sucrose, 1 mM dithiothreitol [DTT],1 mM magnesium acetate, 100 mM potassium acetate, 0.5 mM zinc chlo-ride [pH 7.4]) at 4°C and lysed as described above. Histodenz (100%;Sigma) in modified HES buffer was added to the lysate to a final concen-tration of 50%. The lysate was layered onto 0.5 ml of 70% Hisodenz inmodified HES and centrifuged in a swing-out rotor at 25,000 � g for 20 minat 4°C. The supernatant was discarded, and the pellet was resuspended in 0.5ml of modified HES buffer and centrifuged at 500 � g for 5 min at 4°C. Thepellet was resuspended in SDS-PAGE sample buffer and heated to 65°C for 10min.

Subcellular fractionation. 3T3-L1 adipocytes stably expressing theindicated constructs were washed with ice-cold PBS and harvested in ice-cold HES buffer (20 mM HEPES [pH 7.4], 1 mM EDTA, 250 mM sucrose)containing Complete protease inhibitor mixture and phosphatase inhib-itors (2 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 10mM sodium fluoride). The cells were lysed with 12 passes through a 22-gauge needle and 6 passes through a 27-gauge needle. The cell lysates werethen centrifuged at 500 � g for 10 min at 4°C to remove unbroken cells.The supernatant was centrifuged at 10,080 � g for 20 min at 4°C to yieldthe following two fractions: the pellet fraction consisting of PM and mi-tochondria/nuclei and the supernatant fraction consisting of cytosol, low-density microsomes (LDM), and high-density microsomes. The superna-tant was then centrifuged at 15,750 � g for 20 min at 4°C to obtain thepellet high-density microsome fraction. The supernatant was again cen-trifuged at 175,000 � g for 75 min at 4°C to obtain the cytosol fraction(supernatant) and the LDM fraction (pellet). To obtain the PM fraction,the pellet from the first ultracentrifuge spin was resuspended in HES buf-fer containing phosphatase and protease inhibitors, layered over high-sucrose HES buffer (20 mm HEPES [pH 7.4], 1 mm EDTA, 1.12 m su-crose), and centrifuged at 78,925 � g for 60 min at 4°C. The PM fractionwas collected above the sucrose layer, and the pellet was the mitochondrial/nuclear fraction. All of the fractions were resuspended in HES buffer con-taining phosphatase and protease inhibitors. The protein concentrationfor each fraction was determined using a BCA assay. Samples were madeup in an SDS sample buffer and then kept at �20°C.

Live-cell TIRF microscopy. 3T3-L1 adipocytes were electroporated asdescribed previously (35). Live-cell TIRF microscopy was performed us-ing a Zeiss Axiocam MRm equipped with a heated stage set at 37°C. Thecells were randomly selected by bright-field illumination prior to TIRFimaging, and images were analyzed with ImageJ software.

2-Deoxyglucose uptake assay and quantitative GLUT4 transloca-tion assay. 2-Deoxyglucose uptake into 3T3-L1 adipocytes was per-formed as described previously (43).

HA-GLUT4 translocation to the PM was measured as described pre-viously (9). Briefly, 3T3-L1 adipocytes stably expressing various constructof AS160 and/or HA-GLUT4 in 96-well plates were serum-starved withKrebs-Ringer phosphate buffer (0.6 mM Na2HPO4, 0.4 mM NaH2PO4,120 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 12.5 mM HEPES[pH 7.4]) supplemented with 0.2% BSA for 2 h. Where applicable, cellswere then treated with dimethyl sulfoxide (DMSO) or MK-2206 for 30min prior to insulin stimulation for 20 min or as indicated. After stimu-lation, the cells were fixed and immunolabeled with monoclonal anti-HAantibody, followed by Alexa Fluor 488-labeled secondary antibody in theabsence or presence of saponin to analyze the amount of HA-GLUT4 atthe PM or the total HA-GLUT4 content, respectively.

GST and Flag protein purification for GST-IRAP pulldown and lipidblotting. Escherichia coli BL21(DE3) (Invitrogen) transformed with GST-AS160 fusion proteins or GST-IRAP (residues 1 to 58 of the cytosolic tail)were inoculated in 5 ml of Luria-Bertani (LB) medium and grown over-night at 37°C. Then, 500 ml of LB medium was added to the culture andgrown to an optical density at 600 nm (OD600) of 0.6. After stimulation ofprotein expression by 1 mM IPTG (isopropyl-�-D-thiogalactopyrano-side) for 4 h at 30°C, the bacteria were pelleted at 5,000 � g and resus-pended at 4°C in 25 ml of PBS (pH 7.4) containing 1 mg of lysozyme/ml,1% Triton X-100, 50 �g of DNase/ml, 50 �g of RNase/ml, 1 mM DTT, 1mM phenylmethylsulfonyl fluoride, and protease inhibitors (Roche), ul-trasonicated, and clarified by centrifugation at 15,000 � g for 30 min.GSH-Sepharose beads (Amersham Pharmacia) were incubated with GST-containing supernatant and washed extensively with PBS. Beads contain-ing GST-IRAP were used for the IRAP pulldown assay. GST-AS160 fusionproteins were eluted from beads with 20 mM glutathione and buffer ex-change with PBS using Amicon centrifuge tubes (10-kDa cutoff). Theprotein concentration was determined by a BCA assay. For Flag-AS160protein purification, HEK293E cells were transfected with Flag-AS160 for16 h. Cell lysate were harvested in NP-40 lysis buffer (50 mM Tris-HCl[pH 7.5], 1% glycerol, 1% NP-40). The lysates were clarified, and immu-noprecipitations were performed for 2 h at 4°C. After extensive washes inNP-40 lysis buffer, followed by washes in TBS, Flag-AS160 was eluted with25 �g of Flag peptide/ml at 4°C for 1 h. PIP Microstrips (Echelon) wereprocessed according to the manufacturer’s instructions with some modi-fication. Briefly, the strips were blocked with 5% nonfat milk in TBS–0.1% Tween buffer for 1 h prior to overnight incubation with 1 mg/ml ofpurified protein in TBS-0.1% Tween containing 2% BSA. After extensivewashing, the strips were incubated with anti-mouse GST antibody,washed, and probed with HRP-conjugated anti-mouse antibody. Second-ary antibody was detected using enhanced chemiluminescence as de-scribed in Western blotting.

Statistical analysis. Data are expressed as means � the standard de-viations (SD) or � the standard errors (SE), and P values were calculatedby using a two-tailed Student t test and Microsoft Excel unless statedotherwise in the figure legends.

RESULTSThe second PTB domain in AS160 binds phospholipids. Most ofthe known functions of AS160 map to the GAP domain or thephosphorylation sites. Little is known about the function of theN-terminal PTB domains. We used the SMART database to detectand align the two AS160 PTB domains with the hidden Markovmodel (HMM) for PTB domains and the Phyre2 server for sec-ondary structure prediction and identification of tertiary struc-tural homologs (Fig. 1A). Although both domains displayed theclassical PTB secondary structure aligning well with the SMARTHMM (E-values � 10�30), the second domain was somewhatatypical. The closest tertiary structural homolog identified by thePhyre2 server was the Dab1 PTB domain. Interestingly, residues190 to 239 at the N terminus of the second PTB domain in AS160were not recognized by Phyre2 but were included in the SMARTalignment, which also contains sequences corresponding to thePTB domain of Dab1, as well as both PTB domains of the Dro-sophila AS160 ortholog Pollux. Closer inspection of the SMARTHMM alignment in the context of the Dab1 tertiary structurerevealed an insertion of 107 amino acids in the �2-�2 loop, indic-ative of a “split” architecture with residues 190 to 239 correspond-ing to the �1 helix, �1 strand, and �2 helix and residues 347 to 450corresponding to the remainder of the Dab1 PTB domain (Fig.1A). Although the Dab1 PTB domain structure contains aPtdIns(4,5)P2 head group located at a site proximal to the �2-�2

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loop (Fig. 1A), the basic residues contacting the bound head groupare not conserved in AS160.

In view of our previous observation that a finite pool of AS160is located at the PM in adipocytes (23), we postulated that thismight be mediated via an interaction between the AS160 N termi-nus and phospholipids. To test this, we examined the binding ofthe AS160 N terminus to phospholipids in vitro. GST-AS160190-365, aregion that consists of the �1-�1-�2 segment, as well as the insertand �2 strand (Fig. 1A and Table 1). This region interacts withmost phosphorylated forms of phosphatidylinositol (PI) but notwith PI itself (Fig. 1B), which is not unexpected since PTB do-mains bind to various phospholipids and can localize to varioussubcellular compartments (18). To determine whether the pre-dicted insertion in the second PTB domain could mediate this

interaction, we examined the lipid binding of GST-AS160239-365.AS160239-365 did not bind any phospholipids (data not shown),suggesting that the predicted insertion alone is incapable of bind-ing to phospholipids. Positively charged lysines and arginines playa crucial role in the interaction of PTB domains with lipid (18, 20).The putative lipid-binding domain in AS160 contains severallysines and arginines (Fig. 1C) that likely form an essential part ofthis domain based on structure prediction (Fig. 1A). K209, K215,K216, K228, and R236 within the lipid binding module of AS160are highly conserved across species and are also found in theAS160 homologue TBC1D1 and the Drosophila ortholog Pollux(Fig. 1B). Mutation of two lysines at 215 and 216 to alanine abro-gated lipid binding (Fig. 1C). The phospholipid binding site in theN terminus of the second PTB domain is not located on the same

FIG 1 The second PTB domain of AS160 binds to negatively charged phospholipids. (A) Domain architecture of AS160. The amino acids corresponding to the first PTB(amino acids 31 to 191), second PTB (amino acids 197 to 449), and TBC (amino acids 915 to 1135) domains were predicted using SMART (33). In green (amino acids197 to 239) is the highly conserved region depicted in panel C. In gray is the predicted insertion, resulting in a split PTB domain and the remainder of the second PTBdomain is shown in blue. The structure shown is based on the Dab1 PTB domain in complex with Ins(1,4,5)P3 and the ApoER2 peptide (PDB ID 1NU2). A hypotheticalAS160 Ins(1,4,5)P3 is modeled in an arbitrary orientation consistent with the prediction that the K215 and/or K216 likely interact with the 4- and/or 5-phosphates. Theside chains shown correspond to the conserved basic residues in AS160 and were modeled in arbitrary rotomer conformations after mutation of the correspondingresidues in the Dab1 structure (16). The colors in the model correspond to those of the second PTB domain in the top panel. (B) Lipid binding of Flag-AS160 orGST-AS160 fusion proteins analyzed with protein-lipid overlay assays. LPA, lysophosphatidic acid; SIP, sphingosine-1-phosphate; LPC, lysophosphocholine; PI,phosphatidylinositol; PI3P, phosphatidylinositol 3-phosphate; PI4P, phosphatidylinositol 4-phosphate; PI5P, phosphatidylinositol 5-phosphate; PI(3,4)P2, phosphati-dylinositol (3,4)-bisphosphate; PI(3,5)P2, phosphatidylinositol (3,5)-bisphosphate; PI(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; PI(3,4,5)P3, phosphatidylino-sitol (3,4,5)-trisphosphate; PE, phosphatidylethanolamine; PA, phosphatidic acid; PS, phosphatidylserine; PC, phosphatidylcholine. (C) Multiple alignment of AS160residues 190 to 239 with amino acid sequence from various species of AS160, TBC1D1, and Drosophila Pollux. Red boxes highlight the conserved lysine and arginineresidues.




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surface as in Dab1 or Shc PTB domains and does not correspondto the canonical site in the structurally related Pleckstrin homol-ogy (PH) domain (data not shown). Furthermore, the conservedarginine and lysines in AS160 orthologs (Fig. 1C) are not con-served in PTB domain paralogs (data not shown). The lipid-bind-ing domain of AS160 appears to map to residues 190 to 365 sincefull-length AS160 displayed similar lipid binding profile to the190-365 fragment (Fig. 1B). Interestingly, full-length AS160 dis-played reduced binding to PI(3,4,5)3P (Fig. 1B) compared toAS160190-365. This suggests that the conformation of the N-termi-nal lipid-binding domain may be modified in the full-length pro-tein, possibly due to homodimerization (see below) or posttrans-lational modifications that may occur in the case of the full-lengthprotein made in HEK cells but not for the truncation mutantsproduced in bacteria. Regardless, we do not see any evidence forinsulin-dependent recruitment of AS160 to the membrane (datanot shown), which would be indicative of PI(3,4,5)P3 binding.These data indicate that this novel phospholipid-binding domainin AS160 involves at least two conserved lysines (KK215/216) inthe predicted �1-�2 loop of the second PTB domain. We provide

a hypothetical model of this domain in AS160 where Ins(1,4,5)P3is modeled in an arbitrary orientation consistent with the predic-tion that K215 and K216 likely interact with the 4-and/or 5-phos-phates (Fig. 1A), though presumably in a nonstereoselective man-ner, as suggested by the lipid-binding data (Fig. 1B).

AS160 is targeted to the PM via its N terminus. Phospholipidsare found in multiple cellular locations (18), and so we nextwanted to determine whether the lipid-binding domain in the Nterminus of AS160 was synonymous with its targeting to the PM.A cationic silica method was used to isolate PM from HEK cellstransfected with AS160 constructs. This method of PM isolationwas validated by blotting for a cytosolic protein (GAPDH), whichshowed a very low PM/total cell lysate (TCL) ratio, whereas a bonafide PM protein (syntaxin-4) displayed a much higher ratio con-sistent with enrichment in the PM fraction. In agreement with ourprevious data (23), we detected a pool of AS160 at the PM inHEK293 cells, and this interaction mapped to the AS160 N termi-nus (residues 1 to 924) (Fig. 2B). Further truncation analysis re-vealed that PM binding was encoded within the N-terminal half ofthe second PTB domain (Fig. 2C). Consistent with the lipid-bind-ing data (Fig. 1B), residues 190 to 239 of AS160 associated with thePM, whereas residues 239 to 365 did not (Fig. 2C and Table 1).Moreover, an AS160 mutant lacking residues 190 to 239(AS160��1�1�2) displayed reduced PM association compared tofull-length AS160 (Fig. 2C). Further evidence that PM binding wasencoded via the lipid-binding domain was provided by the obser-vation that mutation of Lys215 and 216 within AS160190-365

caused a significant reduction in PM binding (Table 1), whereasmutation of other conserved lysine and arginine residues had noeffect (Table 1). These observations were confirmed using a sepa-rate subcellular fractionation approach in 3T3-L1 adipocytes(data not shown).

Intriguingly, the C terminus of AS160, while displaying re-duced PM binding compared to full-length AS160, retained a PM/TCL ratio higher than the cytosolic protein GAPDH. This suggeststhat the C terminus may also possess PM binding activity inde-pendently of the lipid-binding domain. AS160 has been shown tohomodimerize (5, 17), and we wondered whether the ho-modimerization domain could comprise the AS160 C terminus,in which case PM binding of this mutant might be due to ho-

TABLE 1 Amino acid residues of various AS160 PTB2 fragments andthe corresponding regions of the Dab1 PTB domain, the phospholipid-binding capability, and the strength of the PM associationa

AS160 PTB2 fragment(amino acid residue)

Regioncorrespondingto the Dab1PTB domain

Phospholipidbinding

Mean PMassociation(cationicsilica)b � SD

190-239 �1-�1-�2 NDc ND239-365 Insert and �2 No 0.16 � 0.12*190-365 �1-�1-�2 insert

and �2Yes 1.00

190-365 KK215/216AA No 0.42 � 0.10*190-365 K209A ND 1.01 � 0.19190-365 K228A ND 0.89 � 0.17190-365 R236A ND 0.79 � 0.10a Domain prediction and phospholipid binding were assessed as described for Fig. 1.b PM association was determined by the cationic silica method as described for Fig. 2.Values represent the fold change compared to fragment 190-365. *, P � 0.01 (Student ttest).c ND, not determined.

FIG 2 The N terminus of AS160 that comprise the ��1�1�2 region are important for PM localization, as determined by the cationic plasma membrane isolationmethod. (A) Schematic diagrams of full-length (FL) and different fragments of human AS160 proteins used in the present study. Numbers indicate amino acids.PTB, phosphotyrosine-binding domain; RabGAP, Rab GTPase-activating protein domain. (B and C) HEK293E cells were transfected with Flag-tagged AS160full-length (FL) and mutants as indicated, and plasma membrane (PM) fractions were isolated. Total cell lysates (TCL) and PM fractions were blotted with Flag,GAPDH, or syntaxin-4 antibodies. Flag-tagged AS160 FL and mutants were quantified and normalized to GAPDH and syntaxin-4 levels. The PM/TCL ratiosrelative to FL AS160 are shown. A representative immunoblot is shown. The results are displayed as means � the standard deviations (SD; n 2 to 3) (B) ormeans � the standard errors (SE; n � 4) (C). *, P � 0.05; **, P � 0.01. GAP, AS160 GAP domain.

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modimerization with endogenous AS160. To explore this, we co-expressed various Flag-AS160 truncation mutants with Myc-tagged full-length AS160. The N-terminal fragments (i.e., the1-190, 1-440, and 1-865 fragments) interact poorly with myc-tagged full-length AS160 (Fig. 3), but fragments from this regionretained their PM interaction (Fig. 2C). This supports the conclu-sion that PM targeting of the N terminus of AS160 is not mediatedvia homodimerization. The AS160 C terminus containing theGAP domain, on the other hand, displayed a strong interactionwith full-length AS160 (Fig. 3). Hence, we surmise that the PMbinding observed with the AS160 C terminus is likely due to ho-modimerization with endogenous AS160 in HEK cells.

To confirm that AS160 binds to the PM in adipocytes, AS160

mutants were tagged with enhanced green fluorescent protein(EGFP), expressed in 3T3-L1 adipocytes, and analyzed using live-cell total internal reflection fluorescence microscopy (TIRFM)imaging. This resolves fluorescently tagged proteins at or just be-neath the PM. The fluorescence intensity in the TIRF zone wasnormalized to the total cellular fluorescence, measured via epifluo-rescence, to correct for differences in total expression. Consistentwith the biochemical data and the lipid-binding data (Fig. 1B and2C; Table 1), EGFP-AS160190-365 was localized to the TIRF zone toa greater extent than EGFP-AS160239-365 or the EGFP controls(Fig. 4). Although EGFP-AS160190-239 was also enriched in theTIRF zone (Fig. 4), this was less than was observed for EGFP-AS160190-365, indicating that the predicted insertion (i.e., 239-365)contributes to the AS160 PM binding domain (Table 1). Mutation ofKK215/216 to alanine (EGFP-AS160190-365KK215/216AA) led to a re-duction in the fluorescence detected in the TIRF zone (Fig. 4 andTable 1), a finding consistent with the lipid-binding data (Fig. 1B)and the PM isolation approach (Table 1). The observation that full-length AS160 KK215/216AA displayed reduced localization to thePM provided further confirmation that lipid binding is a majormechanism for the interaction of AS160 with the PM (Table 2).

The putative PM targeting signal in AS160190-239 is highly con-served in TBC1D1 and Pollux (Fig. 1C). We reasoned that if theseTBC proteins behaved in a similar manner to AS160, this wouldfurther support the conclusion that a lipid-binding domain inthese proteins encodes PM targeting. Hence, we next expressedTBC1D1 and Pollux and two unrelated TBC proteins, RabGAP1and TBC1D13, in HEK293 cells and determined their localization.TBC1D1 and Pollux displayed a similar enrichment at the PM toAS160, whereas this was not the case for RabGAP1, which alsocontains a PTB domain, or TBC1D13 (data not shown). Thesedata indicate that the second PTB domain of AS160 regulates PMtargeting and that this function is conserved in TBC1D1 andPollux.

Mapping the GLUT4 storage vesicle binding domain ofAS160. Since the GSV cargo protein IRAP also binds to the secondPTB domain in AS160 (26, 28), we next sought to determinewhether there is an overlap between the IRAP and PM bindingdomains. GST-IRAP interacted with AS1601-365 but not withAS160365-1299 (Fig. 5A) and only weakly to AS160��1�1�2 (Fig.5B), suggesting that, analogous to the PM binding domain, the

FIG 4 TIRF microscopy analysis demonstrate the requirement of Lys215 and Lys216 of the N terminus of AS160 for PM localization. (A) 3T3-L1 adipocytes wereelectroporated with different EGFP-tagged AS160 truncation mutants and imaged using live-cell TIRFM and epifluorescence. Scale bar, 10 �m. (B) Quantifi-cation of images in panel A. The fluorescence intensity in the TIRF zone of each cell was normalized to the total cellular fluorescence, measured via epifluores-cence. The ratio of each construct was normalized to that of EGFP control. The results are displayed as means � the SD. *, P � 0.05; **, P � 0.01. A total of 40to 60 cells were analyzed for each condition.

FIG 3 Full-length AS160 interacts with the C terminus region of AS160.HEK293 cells were transfected with myc-tagged AS160 and with either Flagvector, Flag-tagged AS160, or mutants as indicated. Lysates were immunopre-cipitated with Flag antibody and immunoblotted with antibodies against Flag(top panel) or myc (bottom panel). Quantification is displayed as the means �the SD (n 2).




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AS160 �1�1�2 domain is sufficient to mediate IRAP binding.Interestingly, AS160KK215/215AA, which bound weakly to the PM(Table 2), retained a significant interaction with IRAP (Fig. 5B andTable 2). This suggests that the critical lysine residues (KK215/

216) required for PM binding are dispensable for the IRAP inter-action. Intriguingly, the region within the second PTB domainthat contains the lysines was highly conserved in Pollux, consis-tent with its strong interaction with the PM, whereas the remain-

TABLE 2 Interaction of AS160 mutants with the PM, IRAP, or LRP1 and effect of the corresponding phospho-mutants on HA-GLUT4 translocation

AS160 mutantMean PM association(cationic silica)a � SD Mean IRAP bindingb � SD LRP1 bindingc

Corresponding phospho-mutant effect onGLUT4 translocation (mean % inhibitionof GLUT4 translocationd � SD)

AS160 FL 1.00 1.00 WT 62.12 � 8.70*AS160 KK215/216AA 0.52 � 0.10* 0.65 � 0.16* ND 60.05 � 5.02*AS160��1�1�2 0.56 � 0.13* 0.47 � 0.08* 22 16.55 � 3.04*365-1299 0.64 � 0.08* 0.13 � 0.08* 222 4.65 � 7.25Pollux-AS160 1.80 � 0.33 0.03 � 0.03* ND 11.73 � 0.78*a The PM association was determined by the cationic silica method as described for Fig. 2. Values represent the fold change compared to AS160 FL. *, P � 0.01 versus AS160 FL.b The IRAP binding was determined using a GST-IRAP pulldown assay as described for Fig. 5. Values represent the fold change compared to AS160 FL. *, P � 0.01 versus AS160 FL.c The number of arrows indicates the relative strength of the effect. ND, not determined.d The percent inhibition of insulin-stimulated HA-GLUT4 translocation was determined as described for Fig. 6. Values represent the percent inhibition compared to thecorresponding wild type (WT). *, P � 0.01 versus WT AS160.

FIG 5 AS160 interacts with PM and IRAP via a distinct mechanism. (A and B) GST-IRAP pulldown (PD) of Flag-tagged constructs as indicated that were expressed inHEK293 cells. Total cell lysate (TCL) and PD were immunoblotted with antibodies against Flag and GST. The amount of Flag-tagged protein bound to GST-IRAP wasexpressed as a ratio of PD/TCL relative to FL AS160. The results are displayed as means � the SD (n 3). **, P � 0.01. (C) HEK293E cells were transfected withFlag-tagged constructs as indicated. TCL and PM fractions were blotted with Flag, GAPDH, or syntaxin-4 antibodies. The amount of Flag-tagged AS160 at the PM wasexpressed as the ratio of PM to TCL relative to FL AS160. The results are displayed as means � the SD (n 3). *, P � 0.05. (D) HEK 293E cells were transfected with thelight chain of LRP1 and/or Flag vector, Flag-tagged AS160, AS160��1�1�2, or 365-1299. The cell lysate was harvested and immunoprecipitated with anti-Flag antibody.Total cell lysate and the immunoprecipitates were immunoblotted with anti-Flag or anti-LRP1 antibody. (E) Sedimentation analysis of AS160 mutants with GLUT4vesicles. Low-density microsomes were isolated from 3T3-L1 adipocytes expressing various Flag-tagged AS160 constructs. Sucrose gradient centrifugation was per-formed as described in Materials and Methods. Gradient fractions were subjected to SDS-PAGE, followed by Western blotting with anti-Flag or GLUT4 antibodies.

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der of the N terminus was less conserved (data not shown). Hence,we postulated that the IRAP binding domain might not be con-served in the Drosophila paralog. Consistent with this, Pollux dis-played considerably less IRAP binding than AS160 (Fig. 5B).Moreover, a Pollux-AS160 chimera comprised of Pollux1-334 andAS160365-1299 retained PM but not IRAP binding (Fig. 5B and Cand Table 2). These data indicate that distinct structural determi-nants within the second PTB domain define phospholipid/PMand GSV cargo interactions.

It was shown that AS160 binds to another GSV cargo proteinLRP1 and the cytosolic tail of LRP1 shares sequence similaritywith IRAP (12). Consistent with a shared mode of binding, weobserved that LRP1 coimmunoprecipitated with full-lengthAS160 but not with AS160 lacking the �1�1�2 domain (Fig. 5D).These data indicate that AS160 targeting to GSVs is likely medi-ated via its interaction with multiple proteins in GSVs.

The inhibitory function of nonphosphorylated AS160 isthought to involve its interaction with GSVs via binding to cargoproteins such as IRAP and LRP1 (12, 26, 28). To confirm that thein vitro binding data recapitulate AS160 localization to GSVs, weexamined AS160 binding to GSVs isolated by density sedimenta-tion. Consistent with the IRAP pulldown data, a significant pool ofwild-type AS160 and AS160KK215/215AA cofractionated withGLUT4 vesicles, whereas this was not the case for AS160365-1299,Pollux-AS160, and AS160-��1�1�2 (Fig. 5E). These data indi-cate that the second PTB domain in AS160 interacts with the PMand with GSVs via discrete binding modes.

The inhibitory role of AS160-4P depends on GSV cargo bind-ing. Since AS160 possesses distinct binding sites for GSV cargoand phospholipids, we surmised that this likely demarcates sepa-rate functions. The best-described function of AS160 is its inhib-itory role in GLUT4 translocation. This was established by over-expressing an AS160 mutant (AS160 4P) in adipocytes that wasunable to be phosphorylated and so presumably locked into aGAP active state (32). We reasoned that if this negative role ismediated by binding to GSVs via the N-terminal cargo bindingdomain in AS160, then abolition of the cargo binding domain butnot the PM binding domain in AS160 should overcome its inhib-itory effect on GLUT4 trafficking. We therefore generated 4P mu-tants (S318A, S588A, T642A, and S751A) of a series of AS160constructs and 3P mutants of those lacking Ser318. Consistentwith this hypothesis, neither Pollux-AS160-3P nor AS160 365-1299-3P, both of which fail to bind to IRAP, exerted any inhibitoryeffect on insulin-stimulated GLUT4 translocation (Fig. 6A andTable 2). Strikingly, both AS160-��1�1�2-4P and AS160 KK215/216AA-4P, which retain IRAP but not PM binding, inhibited in-sulin-stimulated GLUT4 translocation (Fig. 6A and Table 2). Fur-thermore, there was a strong correlation (R2 0.87) between thedegree of cargo binding and the inhibition of insulin-stimulatedGLUT4 translocation across these mutants (Fig. 6B). These dataindicate that the inhibitory function of AS160 on GLUT4 traffick-ing is encoded via its interaction with GSVs and not with the PM.

The AS160-PM interaction facilitates GLUT4 translocation.Since GSV binding seems to mediate the negative regulatory effectof AS160 on GLUT4 translocation, we next wondered whether theinteraction of AS160 with the PM could facilitate a downstreamstep in the movement of GLUT4 vesicles to the PM. Consistentwith a positive role for AS160 in this process, some studies haveshown that knockdown of AS160 in adipocytes inhibits insulin-dependent GLUT4 translocation (2, 7). Hence, in addition to its

inhibitory role under basal conditions, AS160 may have a positiverole on insulin-regulated GLUT4 trafficking that may be attrib-uted to its interaction with the PM. To explore this, we targetedAS160 to the PM by tagging it with the myristoylation/palmitoyla-tion signal from Lyn, which is referred to as Lyn-AS160 from thispoint on. Lyn-AS160 was enriched at the PM in adipocytes (Fig.7A). PM levels of HA-GLUT4 were increased by 2-fold in non-stimulated adipocytes expressing Lyn-AS160, and insulin-depen-dent GLUT4 translocation was potentiated at submaximal insulinlevels (Fig. 7B). Moreover, GLUT4 translocation to the PM wassignificantly higher over the whole time course in cells expressingLyn-AS160 (Fig. 7C). Glucose transport in adipocytes expressingLyn-AS160 was also increased (data not shown). This was not dueto increased GLUT4 expression (data not shown) or to reducedGLUT4 endocytosis, since the latter would have led to a potenti-ation of cell surface GLUT4 levels at maximal insulin stimulation(Fig. 7B). One possibility is that Lyn-AS160 dimerizes with endog-enous AS160, sequestering it at the PM away from GSVs and thusoverriding its inhibitory effect. However, we did not observe asignificant change in the subcellular distribution of endogenous

FIG 6 The degree of AS160-IRAP interaction correlates with the inhibitoryeffect of the AS160-phospho-mutants on GLUT4 translocation. (A) 3T3-L1adipocytes expressing Flag-tagged AS160 wild-type (WT) or mutants as indi-cated were serum starved for 2 h and not stimulated (white bar) or stimulatedwith 100 nM insulin (black bar) for 20 min. The amount of HA-GLUT4 at thePM was determined by anti-HA fluorescence immunolabeling of nonperme-abilized cells as described in Materials and Methods. The results are displayedas means � the SD (n 3). *, P � 0.05; **, P � 0.01. (B) Correlation betweenthe degree of AS160 binding to IRAP and inhibition of HA-GLUT4 transloca-tion to the PM by various AS160 phospho-mutants. The results are displayedas means � the SD (n 3).




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AS160 in cells overexpressing Lyn-AS160 (Fig. 7A). This supportsa positive regulatory role for AS160 at the PM in regulatingGLUT4 translocation.

To confirm that targeting of AS160 to the PM alone was suffi-cient to mediate a physiologically relevant increase in glucosetransport, we constructed an assay that coupled cellular glucosetransport to sensitivity in response to cellular stress. The uptake ofthe nonmetabolizable glucose analogue 2-deoxyglucose into cellsinhibits glycolysis, reduces ATP levels, activates the stress kinaseAMP-activated kinase (AMPK), and ultimately triggers apoptosis(42). Wild-type cells or cells overexpressing Lyn-AS160 were in-cubated with 2-deoxyglucose, and AMPK phosphorylation wasmeasured as a marker of cell stress. As shown (Fig. 7D), Lyn-AS160-overexpressing cells exhibited a significant increase in theirstress response, as indicated by elevated levels of phosphorylatedAMPK and the AMPK substrate ACC. This clearly indicates thattargeting AS160 to the PM causes a physiological increase in cel-lular glucose uptake.

PM-targeted AS160 is constitutively phosphorylated. The in-crease in GLUT4 at the PM in Lyn-AS160 overexpressing cells maybe attributed to enhanced insulin signaling. However, we did notobserve any significant change in the phosphorylation of Akt,GSK3, or TSC2 (Fig. 8A). Intriguingly, however, the phosphory-lation of Lyn-AS160, as well as endogenous AS160, at Thr642 wasincreased under basal conditions, whereas this was not observedin cells expressing Flag-AS160 (Fig. 8A). The Akt inhibitor MK-2206 (38) partially blocked phosphorylation of Lyn-AS160 andtotally inhibited phosphorylation of endogenous AS160 (Fig. 8A).The partial inhibition of Lyn-AS160 phosphorylation under basalconditions may denote slow turnover of phospho-AS160 at thePM or the presence of a small pool of active Akt at the PM that isrefractory to MK-2206. Alternatively, another kinase at the PMmay phosphorylate AS160. These data indicate that constitutivetargeting of AS160 to the PM is sufficient to facilitate its phosphor-ylation (Fig. 8A) and enhance GLUT4 translocation in the absenceof insulin (Fig. 7B). In addition, Lyn-AS160 may interact withendogenous AS160 at the PM (5, 17) to facilitate phosphorylationof both proteins. Consistent with this model, endogenous AS160was coimmunoprecipitated with Lyn-AS160 and with wild-typeFlag-AS160 in 3T3-L1 adipocytes (Fig. 8B). This likely represents alow proportion of endogenous AS160 that was recruited to the PM(Fig. 7A and 8B).

One interpretation of these data is that AS160 is phosphory-lated principally at the PM and not on GSVs (19, 32). To test this,we determined the phosphorylation status of the mutant AS160365-1299 that does not localize to the PM (Fig. 8C). Since AS160 isphosphorylated rapidly (39), we stimulated cells with insulin (1nM) at 28°C. The mutant was phosphorylated at a slower ratecompared to endogenous AS160 (Fig. 8C). To determine whetherthese effects were specific to AS160 at the PM rather than someother membrane, we next targeted AS160 to GLUT4 vesicles byfusing it to GLUT4 (35). This construct exhibited very low levels ofThr642 phosphorylation in the absence of insulin compared toLyn-AS160 (data not shown). These data suggest that AS160 isprimarily phosphorylated at the PM.

To determine whether the positive effect of Lyn-AS160 couldbe due to increased phosphorylation of endogenous AS160, wemade use of the knowledge that phosphorylation of endogenousAS160 is inhibited by the Akt inhibitor, whereas PM-targetedAS160 is less affected under both basal and insulin-stimulated

FIG 7 AS160 targeted to the PM increases GLUT4 translocation. (A) 3T3-L1adipocytes expressing Flag-tagged AS160 or Lyn-AS160 were fractionated to ob-tain different subcellular fractions, TCL, cytosol (Cyt), high-density microsomes(HDM), low-density microsomes (LDM), plasma membranes (PM), and mito-chondria/nuclei (M/N). Fractions were blotted with Flag antibody (first and sec-ond panels). Fractions isolated from cells expressing Lyn-AS160 were immuno-blotted with antibody against AS160 (bottom panel). Endo., endogenous. (B)Dose response of HA-GLUT4 translocation in 3T3-L1 adipocytes expressing Flag-tagged AS160 and Flag-tagged Lyn-AS160. 3T3-L1 adipocytes were serum starvedfor 2 h and stimulated with the indicated doses of insulin for 10 min. The amountof HA-GLUT4 at the PM was determined by anti-HA fluorescence immunolabel-ing of nonpermeabilized cells as described in Materials and Methods. The resultsare displayed as means � the SE (n 4). *, P � 0.05; **, P � 0.01. (C) Time courseof HA-GLUT4 translocation in 3T3-L1 adipocytes expressing Flag-tagged AS160and Flag-tagged Lyn-AS160. 3T3-L1 adipocytes were serum starved for 2 h andstimulated with 1 nM insulin as indicated. The results are means � the SE (n 3).*, P � 0.05; **, P � 0.01. (D) 3T3-L1 adipocytes expressing Flag-tagged AS160 orLyn-AS160 were incubated with 5 mM 2-deoxyglucose for the indicated periods oftime. Total cell lysates were harvested and immunoblotted with pSer79 ACC, totalACC, pThr172 AMPK, or total AMPK antibodies.

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conditions (Fig. 8A). In cells expressing Flag-AS160, both endog-enous AS160 and Flag-AS160 were highly phosphorylated withinsulin, and both were robustly inhibited by MK-2206 (Fig. 9A),as was insulin-stimulated GLUT4 translocation (Fig. 9B). In Lyn-AS160 expressing cells, MK-2206 inhibited phosphorylation ofendogenous AS160 in the basal state with minimal effect on Lyn-AS160 phosphorylation (Fig. 9A). Inhibition of endogenousAS160 phosphorylation did not affect the stimulatory effect ofLyn-AS160 on GLUT4 translocation (Fig. 9B). Consistent withthis, the stimulatory effect of Lyn-AS160 on GLUT4 translocationwas retained in insulin-stimulated cells treated with MK-2206(Fig. 9B). A similar trend was observed by measuring glucose up-

take in wild-type 3T3-L1 adipocytes expressing Lyn-AS160, indi-cating that this effect was independent of HA-GLUT4 expression(data not shown). This indicates that the positive effect of Lyn-AS160 on GLUT4 translocation is not due to increased phosphor-ylation of endogenous AS160 and is likely associated with Lyn-AS160 phosphorylation at the PM.

14-3-3 binding and GAP inactivation is required for the fa-cilitative role of AS160. We next sought to determine whetherincreased AS160 phosphorylation at the PM was required for itsfacilitative effect on GLUT4 translocation. To test this, GLUT4trafficking was examined in cells expressing the AS160 mutantsAS160-4P or Lyn-AS160-4P. Although neither of these mutantsare phosphorylated in response to insulin, the latter mutant isenriched at the PM. AS160-4P inhibited GLUT4 translocation un-der basal and insulin-stimulated conditions (Fig. 10A), an obser-vation consistent with previous findings (32). Whereas Lyn-AS160 increased basal HA-GLUT4 levels (Fig. 10A), this was notobserved in cells expressing Lyn-AS160-4P. Lyn-AS160-4P de-creased HA-GLUT4 translocation in insulin-stimulated cells, al-beit to a lesser extent than AS160-4P (Fig. 10A). This suggests thatphosphorylation of AS160 is required for the positive regulatoryrole of AS160 at the PM. To determine whether this phosphory-lation-dependent positive effect is due to changes in AS160 GAPactivity, similar studies were performed using AS160 mutants inwhich an arginine residue known to be crucial for GAP activitywas mutated (22, 25) (Lyn-4P-R/A). Intriguingly, cells expressingLyn-4P-R/A retained the stimulatory effect on HA-GLUT4 trans-

FIG 8 Overexpression of AS160 at the PM increases Thr642 phosphorylationand does not affect Akt signaling. (A) Flag-tagged AS160 expressing 3T3-L1adipocytes were serum starved for 2 h. The cells were treated with either 0.1%DMSO (lanes D) or 10 �M MK-2206 (lanes M) for 30 min before 100 nMinsulin stimulation. Total cell lysates were subjected to Western blot analysiswith pThr642 AS160, total AS160, pThr308 Akt, pSer473 Akt, total Akt,pSer21/9 GSK3�/�, pThr1462 TSC2, or 14-3-3� antibodies. (B) Untrans-fected 3T3-L1 adipocytes or 3T3-L1 adipocytes expressing Flag-tagged AS160were serum starved for 2 h (lanes B) and treated with 100 nM insulin for 20 min(lanes I). The total cell lysates (TCL) were harvested and immunoprecipitatedusing Flag antibodies. The TCL and Flag immunoprecipitates (IP) were sub-jected to Western blot analysis using total AS160 antibody. Endo., endoge-nous. (C) 3T3-L1 adipocytes expressing 365-1299 AS160 were serum starvedfor 2 h at 37°C. The cells were incubated at 28°C for an additional 10 min andtreated with 1 nM insulin for the indicated times. The total cell lysates wereimmunoblotted with pThr642 AS160, total AS160, pThr308 Akt, pSer473 Akt,or total Akt antibodies.

FIG 9 The increase in Thr642 AS160 phosphorylation and GLUT4 transloca-tion of cells expressing Flag-tagged Lyn-AS160 cannot be completely inhibitedby the Akt inhibitor MK-2206. (A) Flag-tagged AS160 expressing 3T3-L1 adi-pocytes were serum starved for 2 h. The cells were treated with either 0.1%DMSO, 1 �M MK-2206 (MK), or 10 �M MK-2206 for 30 min before 1 or 100nM insulin stimulation. Total cell lysates were subjected to Western blot anal-ysis with pThr642 AS160, total AS160, pThr308 Akt, pSer473 Akt, or total Aktantibodies. (B) HA-GLUT4 translocation in 3T3-L1 adipocytes expressingFlag-tagged AS160 or Flag-tagged Lyn-AS160. The cells were serum starved for2 h and treated with either 0.1% DMSO, 1 �M MK-2206 (MK), or 10 �MMK-2206 for 30 min before 1 nM insulin stimulation. The results are displayedas means � the SD (n 3). *, P � 0.05; **, P � 0.01.




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location under basal conditions and, if anything, this mutationpotentiated the stimulatory effect on GLUT4 translocation ob-served in cells expressing Lyn-AS160 (Fig. 10A and Table 3). Thissuggests that inactivation of the GAP, which is normally consid-ered to be encoded by AS160 phosphorylation, facilitates the pos-itive function of AS160 at the PM. One interpretation of these datais that targeting of an inactive GAP per se is sufficient to achievethis positive effect. We tested this by targeting the GAP domain ofAS160 (Lyn-GAP) or its inactive counterpart (Lyn-GAP R/A) tothe PM. Neither of these mutants had a significant effect onGLUT4 translocation (data not shown). These data indicate thatin addition to requiring an inactive GAP, the positive effect ofAS160 requires other domains in AS160.

We have previously shown that insulin-dependent AS160phosphorylation triggers binding of 14-3-3 (19, 28). We nextwanted to test whether the positive effects of AS160 at the PM aredue to phosphorylation per se or to the concomitant binding of14-3-3 proteins to AS160. To test this, we introduced a constitu-

tive 14-3-3 binding site into the AS160 4P mutant (4P-R18). Wehave previously shown that this mutation increases 14-3-3 bind-ing in AS160 and that this mutation overcomes the inhibitoryeffects of the AS160-4P mutant on insulin-stimulated GLUT4translocation (28). Strikingly, cells expressing Lyn AS160-4P-R18displayed higher levels of cell surface GLUT4 in the absence ofinsulin than was observed in cells expressing Lyn-AS160 (Fig.10B). This effect was reversed by mutating two lysines in the R1814-3-3 binding site that disrupt 14-3-3 binding (Lyn-AS160 4P-R18KK) (Fig. 10B and Table 3). These data indicate that there is afunctional link between AS160 phosphorylation, 14-3-3 binding,and inhibition of AS160 GAP activity at the PM that facilitatesGLUT4 translocation.

DISCUSSION

We provide evidence that the RabGAP AS160 plays both negativeand positive regulatory roles in vesicle transport. This supportsthe existence of Rab regulatory networks whereby individual com-ponents play active roles both in promoting and repressing fluxthrough the pathway. This is consistent with the notion of Rabcascades where the function of multiple Rabs that act in series in apathway can be coupled by a countercurrent mechanism encodedboth by GEFs and GAPs (10, 24, 31, 46). The current studies ex-tend this model by showing that phosphorylation and/or 14-3-3binding switches AS160 from a negative to a positive regulator ofvesicle fusion. In view of the vast number of different RabGAPsfound in the human genome, these studies have broad implica-tions for the role of this family of proteins in eukaryotic vesicletransport.

In addition to the TBC domain, RabGAPs possess a range ofmodular domains, the function of which in many cases has notbeen ascertained. A key observation in the present study was theidentification of a lipid-binding domain encoded within the sec-ond PTB domain in the N terminus of AS160 that conferred itslocalization to the PM. This was a striking observation becauseprevious studies had suggested that AS160 acted principally as anegative regulator of GLUT4 trafficking by binding to intracellu-lar GLUT4 vesicles and inhibiting GTP loading of a Rab that wasrequired to facilitate docking of the vesicles at the PM. The iden-

FIG 10 The facilitative effect of AS160 on GLUT4 translocation requires 14-3-3 binding and inactivation of AS160 GAP. (A) 3T3-L1 adipocytes expressingHA-GLUT4 and WT AS160 or mutants as indicated were serum starved for 2 h (basal) and stimulated with 100 nM insulin for 20 min. The cell surfaceHA-GLUT4 levels were determined and are expressed as the percentage of HA-GLUT4 at the cell surface of insulin-stimulated cells expressing WT AS160. Theresults are displayed as means � the SD (n 6). **, P � 0.01 (WT AS160 versus AS160 mutants or as indicated). (B) 3T3-L1 adipocytes expressing HA-GLUT4and Flag-tagged WT AS160 or mutants were serum starved for 2 h. The cell surface HA-GLUT4 levels were determined and are expressed as the percentage ofHA-GLUT4 at the cell surface of insulin-stimulated (100 nM insulin, 20 min) cells expressing WT AS160. The results are displayed as means � the SE (n � 4).**, P � 0.01 (WT AS160 versus AS160 mutants or as indicated).

TABLE 3 Effect of expressing AS160 mutants on basal PM HA-GLUT4levels in this studya

AS160 mutant GAP activity Phosphorylation 14-3-3 bindingMean PM GLUT4level � SEb

AS160 Active No No 21.29 � 0.96AS160 4P Constitutive Absent No 13.45 � 0.58*Lyn-AS160 Low Increased Yes 30.24 � 1.14†Lyn-4P Constitutive Absent No 22.60 � 1.22Lyn-4P R/A Inactive Absent No 37.62 � 16.70†Lyn-4P R18 Inactive Absent Constitutive 37.09 � 3.06†Lyn-4P R18 KK Constitutive Absent No 28.62 � 3.44

a The presumed GAP activity and ability to phosphorylate or bind to 14-3-3 wereincluded in the analysis. The GAP activity and 14-3-3 binding results are based on theprediction that phosphorylation on Thr642 leads to 14-3-3 binding (28) and that thisphosphorylation presumably inhibits GAP activity based on the 4P mutation (32). Theabsence of GAP activity is based on the mutation of the critical arginine residue toalanine in the GAP domain (22, 25). Constitutive 14-3-3 binding is based on the R18mutation as reported elsewhere (28).b That is, the level relative to the insulin-stimulated level. The PM GLUT4 level wasdetermined as described for Fig. 10. *, P � 0.01 for cells expressing AS160 4P with asignificantly lower PM GLUT4 level compared to those expressing AS160; †, P � 0.01for cells expressing AS160 mutants with a higher PM GLUT4 level compared to thoseexpressing AS160.

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tification of a PM binding domain was also intriguing in light ofprevious observations identifying a pool of highly phosphorylatedAS160 at the PM in adipocytes (23).

PTB domains bind to phosphopeptides and phospholipids, al-though the locations of the phospholipid binding sites differ dras-tically between PTB domains (6). For example, the PI(4,5)P2

binding site in Dab1 is not likely to be a canonical binding site forphospholipids, as indicated by the lack of conservation of the rel-evant basic residues in paralogs. Indeed, the phospholipid bindingsite in the Shc PTB domain is located on a completely differentsurface compared to Dab1 (30). Using the Dab1 PTB domain as ahomology model, it is predicted that the phospholipid binding sitein the second PTB domain of AS160 is located on a surface prox-imal to the peptide binding groove and not where the PI(4,5)P2

site is located in the Dab1 (Fig. 1A) or Shc PTB domains. Intrigu-ingly, the lysine and arginine residues conserved in AS160 or-thologs are not broadly conserved in other PTB domain paralogs.Further, the second PTB domain of AS160 carries an insertion of107 amino acids not found in other PTB domains. Therefore, it islikely that the second PTB domain of AS160 is unique. Also note-worthy is that the second PTB domain in AS160 regulates bindingto GSV cargo proteins, although this function is distinct fromphospholipid binding (Fig. 6). Nevertheless, the fact that both ofthese functions are encoded within the same domain suggests thatthe inhibitory and facilitative roles that are encoded by these dis-tinct interactions coevolved. By examining a series of AS160 mu-tants with various degrees of IRAP binding, we mapped the neg-ative regulatory function of AS160 to the IRAP binding domain(Fig. 5 and 6). However, given that this domain also interacts withother proteins in GSVs, such as LRP1 (Fig. 5), this suggests that theinteraction with GSVs is mediated via interactions with multiplecargo components. This is consistent with the observation that theintracellular distribution of AS160 is unaltered in adipocytes from

IRAP�/� mice and 3T3-L1 adipocytes that have reduced IRAPexpression (13, 15).

Thus, the interaction of AS160 with GSVs likely confers aninhibitory effect on GLUT4 translocation by inhibiting a Rab as-sociated with GSVs. Evidence indicates that the inhibition ofAS160 RabGAP activity is mediated by AS160 phosphorylationand 14-3-3 binding (28, 32), although this has not been formallyproven. The present study extends this model. We propose thatAS160 associated with GSVs might become phosphorylated at thePM when it encounters active Akt at this location. Consistent withthis, it has been shown that Akt functions principally at the PM (1)and that insulin stimulates GSV trafficking to the PM in an Akt-independent manner (43). Moreover, AS160 is highly phosphor-ylated at the PM and not at other locations (23). Notably, consti-tutive targeting of AS160 to the PM enhanced its phosphorylationin the absence of insulin, and this was accompanied by increasedGLUT4 translocation. This surprising result suggests that underbasal conditions there must be a small amount of active Akt at thePM that under normal circumstances is insufficient to phosphor-ylate endogenous AS160. By targeting AS160 to the PM, we havelikely shifted the equilibrium in favor of AS160 phosphorylation.These findings indicate that phosphorylation of AS160 at the PMpositively regulates GLUT4 trafficking. AS160 phosphorylation atthe Thr642 site encodes 14-3-3 binding (28), and here we showthat constitutive binding of 14-3-3 to AS160 was sufficient to rep-licate its facilitative role in GLUT4 trafficking. Collectively, thesefindings suggest that phosphorylation and 14-3-3 binding notonly suppress the GAP activity of AS160 but that this also confersan additional facilitative regulatory function. We have yet to re-solve the nature of this role, but we speculate that phosphorylated/14-3-3-bound AS160 at the PM plays an active role in the dockingof GSVs at the PM. It will be intriguing to determine whetherother RabGAPs display a similar dual role. Hence, we conclude

FIG 11 Model for the positive role of AS160 at the PM. In the absence of insulin, the AS160 PTB domain interacts with the PM, depicted by red lines. Thisinteraction is transient and with no further GSV-PM interaction the vesicle dissociates from the PM. With insulin, the generation of PI(3,4,5)P3 (orange) leadsto the activation of Akt at the PM, phosphorylation of AS160 (depicted by stars), 14-3-3 binding, and inhibition of AS160 GAP activity and the concomitant GTPloading of a cognate Rab on GSVs. This creates an additional binding site for GSVs at the PM via the active Rab and a putative PM effector. Hence, in this modelthe PM binding domain in AS160 encodes two positive regulatory features: a spatial effect to colocalize AS160 with upstream regulators such as Akt anddownstream regulators that are required for vesicle docking/fusion and a structural function to physically tether the vesicles at the PM.




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that AS160 possesses two separate and mutually exclusive func-tions that can be interchangeably regulated by Akt-dependentphosphorylation and 14-3-3 binding. An elegant feature of the useof its PTB domain for targeting AS160 to the PM is that by strate-gically localizing a very small pool of AS160 to the precise site ofvesicle docking at the PM, this makes it relatively easy for Akt todisarm the inhibitory function of AS160 only at this location topromote the facilitative role of AS160 on the docking and fusionreaction (Fig. 11).

The fact that we detected a stimulatory effect of PM targetedAS160 on GLUT4 trafficking in the absence of any other pertur-bation was noteworthy. First, insulin-dependent GLUT4 translo-cation requires many steps other than phosphorylation of AS160,including trafficking of GLUT4 vesicles to the adipocyte cortex,actin rearrangement (21), recruitment of the exocyst complex tothe PM (11), and posttranslational modification of SNARE pro-teins (4) or their regulators such as Munc18c (37, 40, 41). Second,in addition to disarming the GAP, one imagines that in the ab-sence of an active effect on the Rab GEF this might have a relativelyminor effect. Hence, the impact of constitutive targeting of AS160to the PM, in the absence of any of these other changes, on GLUT4trafficking probably points to the highly significant dual role ofAS160 in this process.

In summary, we have mapped out the critical residues for theassociation of AS160 with phospholipids at the PM and unravel anadditional role for AS160 in GLUT4 translocation. These dataimplicate AS160 as a major fork in the pathway that determinesthe probability that GSVs will either fuse with the PM or recycleback to the cell interior. This is a highly efficient way of couplingthe activity of the Rab on the vesicle to the correct location in thecell, to which the vesicle is destined to fuse, and the nutrient statusof the cell, which is encoded by the activity state of Akt also foundat the same location.

ACKNOWLEDGMENTS

We thank Morris Birnbaum (University of Pennsylvania) for providingthe Pollux cDNA. The Akt inhibitor, MK-2206, was generously providedby Dario Alessi (University of Dundee). The LRP1 plasmid and antibodywere kindly provided by Joachim Herz (University of Texas, Southwest-ern Medical Center). We thank Roger Daly and Antony Cooper (GarvanInstitute of Medical Research) for providing invaluable feedback on themanuscript.

This study was supported by grants from the NHMRC of Australia andDiabetes Australia Research Trust (to D.E.J.) and the National Institutesof Health (DK060564 to D.G.L.). D.E.J. is an NHMRC Senior PrincipalResearch Fellow.

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The Rab GTPase-Activating Protein TBC1D4/AS160 Contains an