Isolation and characterization of the 160,000-Da phosphotyrosyl protein, a putative participant in insulin signaling.

The 160,000-Da protein (pp 160) which is rapidly phosphorylated on tyrosine in response to insulin and thus is a putative participant in signaling from the insulin receptor has been purified to homogeneity from 3T3-L1 adipocytes. Isolation of this protein was accomplished by chromatography on an immobilized monoclonal antibody against phosphotyrosine, followed by gel electrophoresis. Sufficient protein was obtained to allow the determination of the sequences of several peptides, which in turn enabled the development of anti-peptide antibodies that specifically recognize pp 160. Immunoblotting of 3T3-L1 adipocyte lysates, together with the purified pp 160 as a standard, indicate that an insulin-treated 3T3-L1 adipocyte possesses about 230,000 copies of tyrosine-phosphorylated pp160 and that this amount is approximately 25% of the total pp160 in the cell. The number of tyrosine-phosphorylated pp160s per cell is approximately the same as that of insulin receptor beta subunits. These results provide further evidence for a role of pp160 in insulin signaling. Moreover, the availability of purified protein and knowledge of peptide sequences will allow the elucidation of the structure and function of this protein.

part of the p subunit is activated, with concomitant autophosphorylation on tyrosine (1)(2)(3). A reasonable hypothesis is that signaling from the insulin receptor then proceeds by the phosphorylation of certain target proteins upon tyrosine, since the appearance of several phosphotyrosyl polypeptides has been detected upon exposure of various cells to insulin (4)(5)(6)(7)(8). Prominent among these is a protein of 160,000-185,000 Da, which was first described by White et al. (9)' (hereafter designated pp160).
Considerable circumstantial evidence exists to suggest that pp160 is involved in signaling from the insulin receptor. First, in several cell lines and tissues this protein has been found to be the major polypeptide, in addition to the p subunit of the insulin receptor phosphorylated on tyrosine in response to insulin. These are rat (10-12) and 3T3-L1 (13)(14)(15) adipocytes, human epidermoid carcinoma cells (16), Fao (9,15,17), H4 (18), and H35 hepatoma cells (19), mouse neuroblastoma cells (20), Chinese hamster ovary cells (17,21), 3T3 fibroblasts (22,23), L6 myoblasts (24), FRTLS thyroid cells (251, and rat liver (26). Second, the phosphorylation of pp160 occurs rapidly at about the same rate and at the same concentrations of insulin as does the phosphorylation of the 8 subunit of the insulin receptor in 3T3-Ll adipocytes (14) and other cell types (9-11, 16, 20, 25). Third, an insulin receptor which is mutated from tyrosine to phenylalanine at position 960 and expressed in Chinese hamster ovary cells is not able to elicit either phosphorylation of pp160 or the typical cellular responses upon exposure to insulin, even though the receptor is still functional as an insulin-activated tyrosine kinase against itself and exogenous substrates (27). Last, partly purified pp160 is further phosphorylated on tyrosine by the purified insulin receptor (19).
In addition to its potential role in insulin signaling, pp160 may also function in signaling from the insulin-like growth factor I (IGF I)* receptor. Exposure of 3T3-Ll adipocytes (14), kidney cells (28), human epidermoid carcinoma cells (16), mouse neuroblastoma cells (ZO), and FRTL 5 thyroid cells (25) to physiological levels of IGF I leads to the rapid phosphorylation on tyrosine of a protein of this size.' In contrast, platelet-derived growth factor and epidermal growth factor do not appear to elicit the tyrosine phosphorylation of pp160 (14,16,22).
Despite this intensive investigation of pp160, the purification of this protein has not been reported. 3T3-Ll adipocytes are relatively abundant in pp160 (13,14), and we describe here the purification of the protein from this cell type. A sufficient amount was obtained to enable determination of the amino acid sequences of several peptides from the protein.
On the basis of one of these, anti-peptide antibodies which specifically recognize pp160 were developed. By immunoblotting 3T3-Ll lysates, we have found that in response to insulin the cellular content of tyrosine-phosphorylated ppl60 appears to be approximately the same as that of the insulin receptor p subunit.
' It remains to be determined whether this protein is the same in all the cell types and also whether the same protein is phosphorylated in response to both insulin and insulin-like growth factor I.
The abbreviations used are: IGF I, insulin-like growth factor I; SDS, sodium dodecyl sulfate; Tyr(P), phosphotyrosine.

12817
The 160,000-Da Protein in Insulin Action

EXPERIMENTAL PROCEDURES
Materials-Mouse monoclonal antibody 1G2 against phosphotyrosine (Tyr(P)) attached to agarose at a concentration of 15 mg/ml was obtained from Oncogene Science, Manhasset, NY. Goat IgG, attached to agarose at a concentration of 5-10 mg/ml, was obtained from Sigma. lZ51-Goat anti-rabbit IgG was the product of Du Pont-New England Nuclear.
Cell Culture"BT3-Ll fibroblasts were cultured and induced to differentiate into adipocytes as described in Ref. 29. Adipocytes were used between the 8th and the 12th day after differentiation. The amount of pp160 detected by immunoblotting SDS lysates of insulintreated cells with antibodies against Tyr(P) was unchanged during this 5-day period.
Purificatiolt of pp160-10-cm plates of 3T3-Ll adipocytes were placed in 10 ml of serum-free Dulbecco's modified Eagle's medium for 2 h before use. Cells were then treated with insulin (300 nM) for 2-4 min, a time period during which the amount of pp160 detected by immunoblotting for Tyr(P) is maximal (14). The medium was aspirated, and each dish was immediately frozen in liquid nitrogen. The frozen cells were overlaid with 2 ml/dish of homogenization buffer (20 mM Tris-HC1, 50 mM NaCl, 1 mM Na3V04, 1 mM Nethylmaleimide, 30 mM sodium pyrophosphate, 1 mM phenylmethanesulfonyl fluoride, pH 7.6) and scraped with a rubber policeman from the dish as a slush. Upon thawing, the cells were broken with 10 hand-driven strokes in a 55-ml glass homogenizer with a Teflon pestle (Arthur H. Thomas 3431D94). The suspension was then centrifuged at 45,000 rpm in a Beckman type 70Ti rotor for 1 h at 200,000 X gmaX. The infranatant solution was carefully removed with a pipette in order to leave as much of the lipid layer on the walls of the tubes as possible and then filtered through a 0.65 pm Millipore filter, type DVPP 047 00, to remove the remaining lipid. In a typical preparation 100 10-cm plates of 3T3-Ll adipocytes were processed through these steps in two 50-plate batches.
The clarified extract was then subjected to immunoaffinity chromatography at 4 "C by passing it through a 2-ml column of goat IgG immobilized on agarose in tandem with a 2-ml column of the anti-Tyr(P) antibody (1G2) immobilized on agarose. After all of the extract was applied, the goat IgG column was disconnected and the 1G2 column was washed with 200 ml of 20 mM Tris-HC1, 150 mM NaCl, 1 mM Na:%V04, pH 7.6. The Tyr(P) proteins were then eluted with 3 mM phenyl phosphate in the wash buffer and collected in 2-ml fractions. Since the protein concentration of the eluate is very low, loss of pp160 through adsorption to the collecting tubes was initially a problem. It was found that this loss did not occur with polyethylene tubes designed for low protein adsorption (4.0-ml Minisorp tubes from Nunc, catalog no. 44390), and these were then used. Flow rate was maintained throughout at 10 ml/h with a peristaltic pump. The columns were regenerated by washing with 10 ml of 1 M NaCl, 20 mM Tris-C1, 6 mM NaNn, pH 7.5. They have been used for 10 preparations over a period of 6 months without any noticeable loss in the yield of pp160.
Final separation of pp160 from other proteins was accomplished by SDS-gel electrophoresis. For isolation of pg amounts, the proteins in the fractions containing the bulk of pp160 (usually three fractions) were precipitated at 0 "C with 10% trichloroacetic acid (v/v, final concentration) in the presence of 150 pg/ml sodium deoxycholate as a carrier. The precipitate was pelleted by centrifugation at 700 X g for 30 min; the trichloroacetic acid was carefully removed by decantation followed by aspiration of remaining droplets; the pellets from the three fractions were together dissolved in 200 pl of SDS sample buffer; and the SDS sample was neutralized with Tris. Each 200-p1 sample was electrophoresed on one 6-mm lane of a large slab gel (6% acrylamide, 1.5 mm X 11 cm) in a Hoefer SE600 apparatus.
Peptide Sequencing-The polypeptides on the preparative gel were transferred to nitrocellulose at 200 mA for 16 h at 4 "C in 25 mM Tris, 192 mM glycine, 20% methanol, 0.01% SDS. Polypeptides on the nitrocellulose were stained with 0.1% Amido Black in 45% methanol, 10% acetic acid for 2 min, followed by a 5-min wash with methanol/acetic acid, and a final wash with water. pp160 appeared as a discrete band (about 4 pg of protein from a 100-plate preparation), and this was excised. The sequences of peptides were obtained according to the methods described by Aebersold et al. (30). Nitrocellulose strips containing about 12 pg of pp160 in total were digested with trypsin, and individual peptides were isolated on a narrow bore, reverse phase high pressure liquid chromatography system (Waters Peptide Analyzer; column, Vydac C4 2.1 X 150 mm, developed with an acetonitrile gradient in 0.1% trifluoroacetic acid). The collected peptides were subjected to sequence analysis in an Applied Biosystems model 477 sequenator.
Generation of Antibodies-Peptides were synthesized, conjugated to keyhole limpet hemocyanin, and then used to immunize rabbits, as described in Ref. 31. Antibodies were affinity-purified from serum by chromatography on the immobilized peptide, as described previously (31).
Gel Electrophoresis and Zmmunoblotting-SDS-polyacrylamide gel electrophoresis was carried out on minigels by the method of Gibbs et al. (13) (data in Fig. 1) or the larger gels described above (data in Fig. 2). The polypeptides were electrophoretically transblotted onto nitrocellulose (Schleicher and Schuell, BA83,0.2 pm) at 300 mA for 2 h (minigels) or at 200 mA for 16 h (large gels). The nitrocellulose was either stained for protein with colloidal gold (Bio-Rad) or immunoblotted with antibodies against Tyr(P) or pp160. In the case of Tyr(P) blots were first blocked for 1 h with 3% bovine serum albumin in Tris-buffered saline (150 mM NaC1, 20 mM Tris-C1, pH 7.60) and then treated with 2.0 pg/ml affinity-purified rabbit anti-Tyr(P) antibodies, prepared by the method of Pang et al. (32), in the buffer with 2 pg/ml albumin overnight. Subsequently, the blots were washed 5 times with buffer and then incubated for 2 h with '251-labeled goat anti-rabbit IgG (0.08 pCi/ml buffer containing 2 pg/ml albumin and 0.3% Tween 20). The blots were washed with buffer/Tween and autoradiographed. In the case of pp160, blots were blocked with 5% Carnation nonfat dry milk in 150 mM NaC1,lO mM sodium phosphate, pH 7.4, washed at each stage with 1% Triton X-100 in this buffer, and treated with the affinity-purified antibodies against the peptide at 50 pg/ml and the lZ51-labeled second antibody in 1% milk in this buffer. Quantitation of radiolabel on the immunoblots was done by cutting out the area of the nitrocellulose corresponding to the band on the autoradiogram and measuring the radioactivity in a y counter; the label in an appropriate blank area of the blot was used to correct for background.

RESULTS AND DISCUSSION
Purification of ppI60-pp160 was purified from the soluble proteins of insulin-treated 3T3-Ll adipocytes by a combination of immunoaffinity chromatography with a monoclonal antibody against Tyr(P) and SDS-gel electrophoresis. The details of the purification procedure are given under "Experimental Procedures." Fig. 1 presents the analysis of the fractions from the immunoaffinity chromatography for total polypeptides by protein stain (Fig. lA, fractions 11-5) and for Tyr(P) polypeptides by immunoblotting with antibodies against Tyr(P) (Fig. lB, fractions 11-5). The prominent, slightly broad band at 160 kDa detected by protein stain in fractions I2 and 3 is coincident with the Tyr(P) polypeptide in both mobility and relative intensity, and therefore is considered to be purified pp160. Further evidence for the absence of significant impurities in this band is the finding that this polypeptide was entirely absent in the fractions obtained when the purification procedure was carried out with the soluble proteins from basal 3T3-Ll adipocytes (Fig. 1, A and  B , fractions B1-5). These results, of course, do not rigorously exclude the possibility that the pp160 band includes more than one protein. The yield of pp160 was estimated by visual comparison of its protein staining intensity on a Coomasssie Blue-stained gel with several known amounts of the standards, myosin and p-galactosidase (data not shown). Approximately 4 pg were isolated from about lo9 3T3-Ll adipocytes (100 10-cm plates, 1 g of protein).
Peptides from and Antibodies against ppl60"Sequences of portions of four tryptic peptides from pp160 were obtained. None of the sequences is identical to ones in the Protein Identification Resources data bank, and so pp160 is unlikely to be a previously sequenced protein.
Antisera were raised against synthetic peptides correspond- ing to amino acid residues 4-20, 1-13, and 3-19 of the first three peptides listed above, respectively. Each antiserum reacted well with the corresponding peptide in an enzyme-linked immunoadsorbent assay, but only the antiserum against peptide (c) immunoblotted pp160. Fig. 1C shows an immunoblot of pp160 in the fractions from the purification performed with affinity-purified antibodies against this peptide. The correspondence between the pp160 band in this immunoblot and that seen by protein staining and immunoblotting for Tyr(P) (Fig. 1, A and B ) provides evidence that the antibodies react with pp160.

XXVTGPGEFL*MQVDDXVVAQNM, (c) XIPGANLGTS-
Amount of Tyrosine-phosphorylated pp160 in 3T3-Ll Adipocytes-The purification procedure yielded pp160 in its Tyr(P) form and thus provided a standard with which to estimate the amount of the Tyr(P) form of the protein in insulin-treated 3T3-Ll adipocytes. SDS lysates of basal and insulin-treated 3T3-Ll adipocytes, side by side with known amounts of purified pp160, were immunoblotted with antibodies against Tyr(P) (Fig. 2 A ) . As we expected, pp160 was only detected in the lysate of insulin-treated adipocytes ( Fig.   2 A , lanes 3 and 4 versus lanes I and 2). Quantitation of the signals in this and a duplicate experiment revealed that IO7 cells (a 10-cm plate) contained 600 ng (average of values of 680 and 510 ng from the two experiments) of Tyr(P) pp160, which corresponds to 230,000 copies/cell. A 3T3-Ll cell possesses about 125,000 insulin receptors (33) and only 6,000 IGF I receptors (34). Since each insulin receptor consists of two 6 subunits, this amount of the Tyr(P) form of pp160 is approximately equal to the amount of the 6 subunit.
Total Amount of pp160 in 3T3-Ll Adipocytes-In order to estimate the total amount of pp160 (both tyrosine nonphosphorylated and phosphorylated), we used the anti-peptide antibodies to immunoblot the SDS lysates of basal and insulin-treated cells together with known amounts of the purified Tyr(P) form of pp160 as the standard (Fig. 2B). The interpretation of the results was complicated by the finding that the signal from pp160 in the SDS lysate of insulin-treated cells was about 1.3 times stronger than that from pp160 in basal cells (Fig. 2B, compare lanes 3 and 4 with lunes 1 and  2). Since the cells were treated with insulin for only 3 min, the difference is unlikely to be due to the synthesis of more pp160; rather we ascribe the stronger signal to more efficient immunoblotting of the Tyr(P) form, which is present only in the insulin-treated cells.
Even with this complication it was still possible to estimate the total amount of pp160 in the cells in the following way (illustrated with one set of data). With the SDS lysate of insulin-treated cells, the pp160 signal (cpm) (Fig. 2B, lane 3, 3030 cpm) on the immunoblot consists of contributions from both the Tyr(P) and non-Tyr(P) forms, whereas the pp160 signal from the SDS lysate of basal cells (lune 1, 2410 cpm) is given by the same total amount of entirely non-Tyr(P) form. The contribution of the Tyr(P) form to the total signal in the insulin lysate (1340 cpm) can be calculated from the amount of this form (9.5 ng), given by the data in Fig. 2 A , and the specific activity of this form (cpm/ng), given by the standards (Fig. 2B, lanes 5-7, 141 cpm/ng), which are entirely the Tyr(P) form. The remaining portion of the total pp160 signal in the insulin lysate (3030 -1340 = 1690 cpm) is due to the non-Tyr(P) form, and this cpm value divided by that of the pp160 band in the basal lysate (lune 1, 2410 cpm) gives the percentage of non-Tyr(P) form present after insulin treatment (70%). The difference between this percentage and 100% is in turn the percentage of Tyr(P) form after insulin treatment (30%), from which value and the ng of this form (given by Fig. 2A ) the total amount of pp160 is calculated. Two separate experiments of the type presented in Fig. 2 were performed and analyzed in this way. The percentage of pp160 phosphorylated on tyrosine in response to insulin was 26 f 4% (values f S.D., from four determinations); the total amount of pp160 per lo7 cells averaged 2.4 pg.
The determination of the amount of the Tyr(P) form of pp160 and therefore also of the total amount of pp160 assumes that the Tyr(P) form of pp160 in the cell lysate and in the purified preparation is immunoblotted with equal efficiency by the antibodies against Tyr(P). There is suggestive evidence that pp160 may be phosphorylated on more than one tyrosine residue (19). Consequently, it remains possible that the Tyr(P) content of the purified pp160 used as the standard differs from that of pp160 in the lysate of insulin-treated cells, even though the purified pp160 was isolated from identically stimulated cells. Thus, until this assumption can be examined, the values given above should be considered as estimates.
Conclusions-The purification of microgram amounts of pp160 has allowed the determination of the sequences of several peptides from the protein and the development of anti-peptide antibodies that recognize pp160. These should enable the cloning of the cDNA encoding this protein, an effort now under way in our laboratory. The findings that the amount of the Tyr(P) form of insulin-treated pp160 in 3T3-L1 adipocytes appears to be approximately the same as the amount of the insulin receptor p subunit and that probably a substantial percentage of pp160 is phosphorylated on tyrosine in response to insulin provide further evidence for a role of pp160 in insulin signaling.