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Biochemical and Biophysical Research Communications 276, 1302–1305 (2000)

doi:10.1006/bbrc.2000.3612, available online at http://www.idealibrary.com on

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nsulin-Dependent Phosphorylation of a 70-kDa Proteinn Light Microsomes from Rat Adipocytes

armen Martinez,1 Gino Vallega, and Paul F. Pilch2

epartment of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118

eceived August 28, 2000

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In order to discover possibly novel insulin receptorubstrates and/or downstream targets in the insulinignaling pathway, we established a cell-free systemor this purpose using purified insulin receptor andubcellular fractions from rat adipocytes as a sourse ofellular substrates. Under these conditions, we haveound a 70-kDa protein (pp70) in fat cells that isyrosine-phosphorylated by the activated insulin re-eptor. Using sucrose velocity gradient sedimentatione also show that pp70 cofractionate a particulate

raction containing IRS-1 but not with GLUT-4 vesicle-nriched fractions. Our results suggest that pp70 maye an endogenous substrate for the insulin receptoryrosine kinase. © 2000 Academic Press

Key Words: insulin receptor; endogenous substrates;at adipocytes; tyrosine phosphorylation.

Insulin signaling is initiated by insulin binding to itseceptor and activation of the receptor’s intrinsic ty-osine kinase activity, which in turn, leads to phos-horylation of intracellular substrate proteins on ty-osine residues. There are 4 such insulin receptorubstrates (IRSs 1–4) that have been identified, whichollowing their tyrosyl phosphorylation, act as dockingroteins for several downstream SH2 domain-ontaining proteins such as phosphatidylinositol-3-inase (PI3-K), whose activation is necessary for mostr all of insulin’s metabolic responses (reviewed in (1,)). In rat adipocytes, substrate proteins that becomehosphorylated on tyrosine residues in response to in-ulin, include pp160–180 (IRS-1/2) (3–6) and pp60IRS-3) (7–9). The phosphorylated substrates are foundfter subcellular fractionation in both cytosolic andarticulate fractions from rat and 3T3-L1 adipocytes,ut it is only in the latter fraction that association withRS1 occurs (4, 10–12). These results have suggested

1 Current address: Area de Bioquimica, Facultad de Quimicas,niversidad de Castilla-La Mancha, Ciudad Real, Spain.2 To whom correspondence should be addressed. Fax: 617-638-

208. E-mail: pilch@biochem.bumc.bu.edu.

1302006-291X/00 $35.00opyright © 2000 by Academic Pressll rights of reproduction in any form reserved.

e a key feature of signal transduction. Previous stud-es from our lab have shown that the activated insulineceptor undergoes rapid endocytosis in rat adipocytesnd remains highly active with respect to both auto-hosphorylation and exogenous kinase activity uponndocytosis (13). It can be hypothesized that insulineceptor kinase would encounter specific substrates inhe internal membrane fraction. In an effort to test thisossibility we have used a cell-free system to reconsti-ute insulin action. Light microsomes isolated from ratdipocytes were incubated with purified insulin recep-ors that had been previously activated. The goal of theresent work was to identify insulin-regulated phos-hoproteins in internal membranes of rat adipocytes, aajor target cell for insulin action.

ATERIALS AND METHODS

Preparation of a cell-free system for insulin action: Insulin receptorurification from cells overexpressing its cDNA. Plasma mem-ranes were obtained from NIH-3T3 cells transfected with humannsulin receptor cDNA (1502 cells, kindly provided by Dr. Simeonaylor, NIH, Bethesda, MD) as previously reported (14). Solubiliza-ion of membranes was carried out in 1% Triton X-100 in the pres-nce of 30 mM HEPES, pH 7.4, 50 mM sodium fluoride, 10 mModium pyrophosphate, 1 mM PMSF and 1 mM each of aprotinin,eupeptin and pepstatin, for 1 h at 4°C. Following centrifugation120,000g for 1 h) to remove insoluble material, the membrane ex-racts were passed over WGA-agarose. Unbound material wasashed from column with WGA buffer (30 mM HEPES, pH 7.4,.05% Triton X-100 and protease inhibitors). Lectin-bound proteinsere eluted with 0.3 M N-acetylglucosamine and 10% glycerol inGA buffer. Insulin binding assays were performed on the fractions

ollected as described (15). Fractions corresponding to the highestmount of insulin binding activity were used for the study.WGA-purified insulin receptors were incubated overnight at 4°C in

he absence or presence of 100 nM insulin and then phosphorylatedith 50 mM unlabeled ATP, 12 mM MgCl2, 4 mM MnCl2 in 30 mMEPES, pH 7.4, for 10 min at room temperature, in order to activate

he receptor kinase. The same insulin receptor preparation used inhe kinase assay was tested for activity by autophosphorylation. Tohis end, insulin receptors unstimulated (2) and stimulated with 100M insulin (1) were incubated at room temperature for 10 min with0 mCi [g-32P]ATP in a solution containing 12 mM MgCl2 and 4 mMnCl2. The reaction was stopped by addition of 60 mM EDTA.

Preparation of a cell-free system for insulin action: Subcellularfeca1lb

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Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ractionation of rat adipocytes. Adipocytes were isolated from thepididymal fat pads of male Sprague-Dawley rats (150–175 g) byollagenase digestion (16). Adipose cells were equilibrated for 30 mint 37°C and then incubated either in the absence (C) or presence of0 nM insulin (INS) for the times indicated as described in (17). Theight microsomal (LM) fraction was prepared essentially as describedy Simpson et al. (18).For velocity sedimentation analysis of intracellular microsomes,

he LM fraction (1 mg of protein) obtained from basal (C) or insulin-reated (10 nM for 15 min) rat adipocytes (INS) was resuspended inES buffer (20 mM HEPES, pH 7.4, 250 mM sucrose, 1 mM EDTA,mM benzamidine, 5 mM sodium orthovanadate, 10 mM sodium

yrophosphate, 50 mM sodium fluoride, 1 mM PMSF and 1 mM eachf aprotinin, pepstatin and leupeptin) and overlaid onto a 4.6 ml0–35% continuous sucrose gradient. Gradients were centrifuged at75,000g for 55 min, and fractions (200 ml) collected from the bottomf each gradient. Aliquots of odd-numbered fractions (15 ml) wereubjected to 10% SDS-PAGE. The gel was then transferred to nitro-ellulose membrane and immunoblotted with anti-GLUT4 monoclo-al antibody 1F8 (19) and anti-rat carboxy-terminal IRS-1 antibodyUpstate Biotechnology). GLUT4 containing fractions and IRS-1 con-aining fractions were independently pooled, diluted with PBS inresence of protease inhibitors and centrifuged at 230,000g for 90in. Pellets were resuspended in PBS.

In vitro reconstitution experiments. Activated (1IR) and nonacti-ated (2IR) insulin receptors, in a final concentration of 100–200 pMf insulin-binding capacity, were incubated with 50 mg of LM fractionor 20 mg of pooled fractions of sucrose gradient) and 10 mCi ofg-32P]ATP (42 mM ATP) for 10 min at room temperature. The reac-ion was stopped by adding 60 mM EDTA and tyrosine phosphory-ated proteins were immunoprecipitated with 25 mg of anti-hosphotyrosine antibody PY20 (Transduction Laboratories),vernight at 4°C in presence of 1% Triton X-100. Insulin receptorsutophosphorylated as described above were immunoprecipitatedith a-P-Tyr antibodies at the same time. 20 ml of protein–Trisacryl beads (Pierce) were then added and pellets were washedith 30 mM HEPES, pH 7.4, 1% Triton X-100 and 150 mM NaCl.roteins were eluted with Laemmli sample buffer (20), boiled for 5in and separated using 10% SDS-PAGE. Radioactive proteins were

dentified by autoradiography of the stained and dried gel.

ESULTS AND DISCUSSION

Insulin receptors were first activated and then addedo a preparation of light microsomes isolated from ratdipocytes. Insulin-dependent activation of the insulineceptor was assessed by increased tyrosine phosphor-lation of the IR b-subunit in an autophosphorylationssay (Fig. 1, lanes 5 and 6). Incubation of insulineceptors and microsomes with [g-32P]ATP results inhe tyrosine phosphorylation of several proteins ashown in the autoradiograms of immunoprecipitated-Tyr-proteins. Most of these proteins can be seen even

n the absence of insulin receptor in the assay (data nothown). The phosphorylation of a protein of 70-kDaas demonstrated to be dependent on the presence ofctivated receptors (Fig. 1, lanes 2 and 4 vs lanes 1nd 3).The band of insulin receptor b-subunit was also ob-

erved in the autoradiography in spite of having beenreviously phosphorylated with unlabeled ATP, mostikely because there is a turnover of the tyrosine-bound

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hosphate due to the presence of active phosphoty-osine phosphatases in the internal membranes (21).The identity of pp70 is not yet known. It is unlikely

hat pp70 was a degradation product of the insulineceptor since previous immunoprecipitation with a-IRntibodies does not result in the removing of the phos-hoprotein when using a two-step immunoprecipita-

FIG. 1. In vitro tyrosine phosphorylation of a 70-kDa proteinrom rat adipocytes LM by the activated insulin receptor. WGA-urified insulin receptor (10–20 fmol) was first activated in vitro inhe presence of insulin (100 nM) and ATP (50 mM). The microsomalraction (LM) was isolated from adipocytes unstimulated (C) andtimulated with 10 nM insulin (INS) for either 15 min (A) or 3.5 minB). This microsomal fraction was then incubated with activatedIR1) or nonactivated (IR2) insulin receptors and with labeled ATPor 10 min. Immunoprecipitation with anti-phosphotyrosine antibod-es was then conducted as described under Materials and Methods.utophosphorylation of the insulin receptor with labeled ATP waserformed as a control for receptor activation (lanes 5 and 6). Auto-hosphorylated insulin receptors were immunoprecipitated withnti-P-Tyr under the same conditions as before. A representativexperiment is shown.

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Vol. 276, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ion protocol (data not shown). Insulin stimulation ofdipocytes for either 3.5 or 15 min does not markedlyffect the ability of the activated IR to phosphorylatehe 70-kDa protein in light microsomes.

To determine if p70 is associated with GLUT4-ontaining vesicles or with other subcellular fractions,e performed a velocity sedimentation analysis of lighticrosomes. The distribution of GLUT4-containing

esicles and IRS-1 was determined by Western blottingith specific antibodies (Fig. 2A). Fractions enriched inLUT4 (fractions 3–12) and IRS-1 (fractions 14–22)ere independently pooled and used as substrates for

he activated insulin receptor in the in vitro kinasessay. As shown in Fig. 2B the insulin-dependent ty-osine phosphorylation of p70 is only observed whenractions at the top of the gradient are used. These

FIG. 2. The 70-kDa protein colocalizes with IRS-1 in the sameractions of a sucrose velocity gradient. (A) Light microsomes fromnsulin-treated (10 nM, 15 min) and untreated rat adipocytes wereentrifuged in a 10–35% continuous sucrose gradient as describednder Materials and Methods. Aliquots of odd-numbered fractions15 ml) were resolved by SDS-PAGE and subjected to immunoblot-ing with antibodies specific for GLUT4 and IRS-1. (B) Fractionsnriched in GLUT4 (fractions 3–12) and fractions enriched in IRS-1fractions 14–22) were independently pooled and used as substratesor the activated insulin receptor in the in vitro kinase assay asescribed in the legend to Fig. 1.

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ontain cytoskeletal elements (10). Paxillin is a cy-oskeletal protein of 70 kDa that is phosphorylated onyrosine (22). However, we were not able to detectyrosine phosphorylation of the 70-kDa protein in pax-llin immunoprecipitates (results not shown).

The exogenous insulin receptor becomes more phos-horylated when using fractions enriched in GLUT4nd devoid of IRS-1. It can be speculated that there arective phosphotyrosine phosphatases at IRS-1 contain-ng fractions. In fact, we have recently shown that theistribution of PTP-1B in light microsomes from adi-ocytes is similar to that of IRS-1 as determined byucrose velocity gradient fractionation (21). PTP-1B isphosphotyrosine phosphatase that has been impli-

ated in the negative regulation of insulin signaling23). Interestingly, in our assay the phosphatases seemo be more active when cells have been previouslyncubated with insulin as demonstrated comparing thehosphorylation of p70 in lanes 6 and 8 from Fig. 2B.Several proteins with M r range of 70,000 Da have

een reported to be tyrosine phosphorylated in re-ponse to insulin, for example, a 70-kDa protein inR-overexpressing NIH3T3 cells (A14 fibroblasts)reated with PAO (phenylarsine oxide, a potent ty-osine phosphatase inhibitor) prior insulin stimulation24). This protein, detected in the supernatant fractionfter centrifugation at 100,000g, associates with theH2 domains of both Grb2 and p120GAP and behavess a RNA-binding protein.Most recently, the tyrosine phosphorylation of a 68-

Da protein that binds RNA has also been reported inT3-L1 adipocytes in response to insulin and osmotichock (25). This protein was found in the detergent-nsoluble fraction of LM from 3T3-L1 cells, a fractionhat would correspond to what we see in the presenttudy, and thus, these pp70 species may be the same.his protein is not paxillin or IRS3 (data not shown)nd, as noted by Hresko and Mueckler (25), it does noteem to correspond to other known tyrosine phosphor-lated proteins in the 70-kDa size range.In summary, a 70-kDa protein from rat adipocytesicrosomes is tyrosine-phosphorylated by activated in-

ulin receptors in a cell-free reaction. Our results showhat pp70 colocalize with IRS-1 in the same fractions ofsucrose velocity gradient, and it may correspond to aovel protein recently described, but not identified in aimilar fraction from cultured murine adipocytes. Ef-orts are underway to determine the identity and func-ion of this protein.

CKNOWLEDGMENTS

This work was supported by Grant DK 30425 from the Nationalnstitutes of Health (to P.F.P.). C. Martinez is the recipient of aostdoctoral fellowship from M.E.C. (Spain).

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