Tyrosine Phosphorylation of pp185 by Insulin Receptor Kinase in a

Vol .264, No.

CHEMISTRY THEJOURNALOF BIOLOGICAL 0 1989 by The American Society for Biochemistry and Molecular Biology,lnc.

Tyrosine Phosphorylation of pp185 by Insulin Receptor Kinasein a Cell-free System* (Received for publication, September 7, 1988)

Yuko Tashiro-HashimotoS, Kazuyuki TobeQ, Osamu KoshioS, Tetsuro IzumiS, Fumimaro Takaku$, Yasuo Akanumat, and Masato KasugaStll From the $Third Departmentof Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-I Hungu, Bunkyo-ku, Tokyo 113, Japan andthe filnstitute for Diabetes Care and Research, Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, Tokyo 100, Japan

Insulin treatment of rat 33-35 henatoma cells causes tyrosine kinase activity of IR kinase, however, the targets of rapidtyrosinephosphorylation of ahighmolecular this reaction are not directly identified, except for the autoweight protein termed ppl85 besides autophosphory- phosphorylation of XR. For this reason, the molecular links in lation of the @-subunitof the insulin receptor (IR) in the signal transduction pathway from activated IR kinase to an intact cell system. To elucidate the molecular basis final cellular events remain uncertain. Several substrates were for tyrosine phosphorylationof ppl85, cell-free phos- examined in a cell-free or intact cell system, but their physphorylation of ppl85 was performed using phospho- iological significance remains unclear (13-1’7). Using antityrosine-containing proteins(PYPs) purified from de- phosphotyrosine antibody (anti-P-Tyr), tyrosine phosphoryltergent-solubilizedcell lysates by immunoprecipitation ation of approximate molecular mass 185-kDa protein (pp185) with anti-phosphotyrosineantibody.Afterinsulin is observed during the initial response of Fao hepatoma cells treatment of cells, marked increasesof tyrosine phosphorylation of ppl86 and IR were observed compared to insulin (18, 19). Tyrosine phosphorylation of pp185 is tononinsulin-treated cells. Site-specific antibodies maximum within seconds after exposure of Fao cells to insuthat specifically inactivate IR kinase inhibited tyrosine lin, and it exhibits a similar dose response as receptor autophosphorylation of ppl85 as well as the &subunit of phosphorylation. These observations put forward pp185 as IR. PYPs purified from detergent-free cell extracts one of the putative endogeneous substrates for IR kinase contained pp186 but little IR; tyrosine phosphorylation having physiological significance. This possibility was also of pp185 did not occur. Additionof IR kinase purified supported by the following observations. (i) Insulin-sensitive from human placenta to these PYPs restored insulin- tyrosine-phosphorylated proteins of similar molecular mass dependent tyrosine phosphorylation of pp185. These are observed in various types of cell lines which are sensitive results suggest that tyrosine phosphorylation of pp185 to insulin, suchas 3T3-Lladipocytes (20), human epidermoid is catalyzed directlyby IR kinase inthis cell-free sys- carcinoma cells (21), mouse neuroblastoma cells (221, and tem. isolated rat adipocytes (23). (ii) An insulin-sensitive tyrosinephosphorylated protein of similar molecular mass is observed in rat liver after insulin injection via portal vein? (iii) TyroAfter insulin binding to itsspecific receptor on target cells, a large array of biological responses is initiated. The earliest is phosphorylation of the receptor itself catalyzed by the psubunit of the receptor, which functions as a tyrosine-specific protein kinase (1-6). Recently, evidence has been accumulated supporting the notion that thefunction of this tyrosine kinase of insulin receptor (1R)l is a prerequisite for signal transduction (7-12). Even considering the importance of the

* This work has been supported by grants-in-aid for developmental scientific research and for scientific research from the Ministry of Education, Science and Culture of Japan, agrant from Sankyo Foundation of Life Science, and a grant from the Naito Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 71To whom correspondence should be addressed: The ThirdDept. of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-31 Hongo, !$unkyo-ku, Tokyo 113, Japan. The abbreviations used are: IR, insulin IeCeptOT; anti-P-Tyr, antiphosphotyrosine antibody; PYP, phosphotyrosine-containing protein; PNPP, p-nitrophenyl phosphate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TPCK, tosylphenylalanyl chloromethyl ketone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; BSA, bovine serum aibumin; anti-IRK, antibody for peptide IRK; anti-IRC, antibody for peptide IRC; NEPHGE, nonequilibrium pH gradient electrophoresis.

sine phosphorylation of pp185 is also stimulated with an insulin mimicker, such as HZ02 or anti-IR antibody (23,29). (iv) When mutant960 of IR, which has normal kinase activity, is expressed in Chinese hamster ovary cells, a defect in tyrosine phosphorylation of pp185 is paralleled by the lack of signal transduction of insulin (24). Although pp185 is expected to be an important substrate for the IR kinase, virtually no direct experimental evidence is available to support this view. Furthermore, the physiological role of pp185 has not been investigated largely because a cell-free system to examine thebiological activity of pp185 is not available. To clarify the molecular basis for tyrosine phosphorylation of pp185, we developed a cell-free phosphorylation system for pp185. We demonstrate phosphorylation of pp185 in a cell-free system, find that its phosphorylation is catalyzed via stimulation of IR kinase activity, and suggest that it is d;srect\y catalyzed by IR kinase itself. EXPERIMENTALPROCEDURES

Materials-The following materials were obtained from the sources indicated [32P]orthophosphatewas from Du Pont-New England Nuclear; [y3’P]ATP and lZ5I-proteinA were from Amersham Corp.; wheat germ agglutinin-coupled agarose was from E. Y.Laboratories; Affi-Gel 15, reagents for SDS-PAGE, and an immunobiotting appa-

* K. Tobe, 0. Koshio, Y. Tashiro-Hashimoto, F. Takaku, Y.Akanuma, and M. Kesuga, manuscript in preparation.

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Cell-free Phosphorylation ofpp185

ratus werefrom Bio-Rad; porcine insulin (lot 615-2H2-300) was 0.1% Triton X-100, and 1 mM EDTA for at least 2 h at room kindly provided by Lilly; and TPCK-treatedtrypsin was from Cooper temperature. The sheets were incubated successively with anti-P-Tyr Biomedical. All other reagents were obtained as previously described at 3 pg/ml in rinse buffer plus 0.3% BSA for 10-16 h a t 4 "C. After (21) or were the best grade commercially available. extensive washing the rinse buffer for 90 min, the sheets were incuAntibody Production-The anti-P-Tyr was prepared in rabbits by bated for 5 h with 1 pCi of '"1-protein A (specific activity, 30 mCi/ injection of N-bromoacetyl-0-phosphotyramineconjugated to key- mg). The sheets were again washed extensively with washing buffer hole limpet hemocyanin as describedpreviously (18,21). The antibody containing 10 mM Tris-HCI (pH 7.4), 1M NaCl, 0.1% Triton X-100, was purified from serum by affinity chromatography on a phospho- and 1 mM EDTA, air-dried, and autoradiographed for 2 weeks. tyramine-Sepharose column. The final protein concentration of affinTwo-dimensional Polyacrylamide Gel Electrophoresis-Two-diity-purified antibody was approximately 1 mg/ml. The antibodies mensional polyacrylamide gel electrophoresis (NEPHGE/SDSagainst synthetic peptides were prepared by following procedure. The PAGE) was performed as previously described (21). Briefly, 32Ppeptide IRK (Thr-Arg-Asp-Ile-Tyr-Glu-Thr-Asp-Tyr-Tyr-Tyr-Arg-Lys) labeled proteins were precipitated by 15% trichloroacetic acid in the or IRC (Gly-Lys-Lys-AEn-Gly-Arg-Ile-Leu-Thr-Leu-Pro-Arg-Serpresence of 0.2 mg/ml BSA, first electrophoresed in 4% acrylamide/ Asn-Pro-Ser), corresponding to positions 1142-1153 or 1328-1343, bisacrylamide gel, 9.2 M urea, 2% Nonidet P-40, and 2% Ampholine respectively, in the @-subunitdomain of human insulin proreceptor (pH 3.5-10, LKB), and second electrophoresed in 7.5% polyacryl(25), were synthesized and coupled to the carrier protein. Antibody amide gel containing SDS. The gelswere stained with Coomassie production and characterization were conducted as previously de- Brilliant Blue, dried, and autoradiographed. scribed (12). Basically, synthetic peptide IRK or IRC conjugated to Phosphopeptide Mapping by Proteolysis in SDS-Polyacrylamide keyhole limpet hemocyanin was injected into rabbit. The antibody Gel-The bands in question were excised from dried gels, transferred (anti-IRK or anti-IRC) was purified from serum by isolation with to wells of 15% polyacrylamide gel, digested with several concentraammonium sulfate precipitation (40% saturated) and affinity chro- tions of Staphylococcus aureus V8 protease and electrophoresed acmatography on a correspondence peptide-coupling Sepharose column. cording to the method described previously (21). The gels were dried Cell Culture-H-35 rat hepatoma cells, provided by Dr. Y. Kitagawa and autoradiographed to detect the phosphopeptide fragments. (University of Nagoya), were grown in Eagle's minimum essential Phosphoamino Acid Analysis-Analysis of the phosphoamino acid medium supplemented with 6%fetal bovine serum. The cultures were composition of the proteins separatedby SDS-PAGE was carried out maintained at 37 "C in a humidified atmosphere composed of 95% air as previously described (27). The phosphoproteins contained in the and 5% CO,. dried gel fragments were incubated at 37 "C for 24 h in 0.5 ml of 50 Labeling of Cells with f2P10rthophosphate, Immunoprecipitation mM NHdHC03 containing 50 pg/ml TPCK-treated trypsin. The trypwith Anti-P-Tyr, and Analysis on SDS-PAGE-Cells were grown in tic peptides were hydrolyzed in 6 M HC1 at 110 "C for 70 min. The plastic tissue culture dishes (6-cm diameter) containing4 mlof amino acid mixture was separated by thin layer electrophoresis. medium supplemented with 6% fetal bovine serum. For labeling of Electrophoresis was performed at pH 1.9 in the first dimension and cells with [32PP]orthophosphate,15 h before the experiment the me- at pH3.5 in the second dimension. The phosphoamino acid standards dium was changed to serum-free medium. Subconfluent cells were were detected by reaction with ninhydrin, and the radioactive amino labeled for 2 h in 2.5 ml of phosphate- and serum-free RPMI 1640 acids were detected by autoradiography for several days. medium containing carrier-free [32P]orthophosphate (0.4 mCi/ml). Purification of IR Kinose from Human Placenta-IR kinase from Cells were incubated without or with insulin (100 nM), and then the human placenta was prepared by a modification of the method incubation medium was aspirated quickly, and cell monolayers were previously reported (28). Basically, human placental membranes were frozen with liquid nitrogen. The monolayers were thawed and solu- solubilized with a buffer containing 2% Triton X-100. IR kinase was bilized immediately at 4 "C with 0.4 ml of a solution containing 0.1 purified by sequential affinity chromatography on wheat germ aggluM Tris-HC1 (pH 8.0), 2 mM sodium orthovanadate, 1 mM phenyltinin-agarose and insulin-coupled Affi-Gel 15. For a cell-free phosmethylsulfonyl fluoride, 100 units/ml aprotinin, and 0.5% (v/v) Tri- phorylation, 2 pg/ml purified IR kinase was used. After incubation ton X-100. A supernatant from the Triton-solubilized extract was without or with insulin (100 nM) a t 22 "C, the phosphorylation prepared by scraping the cells from the dishes and sedimenting reaction was performed in a final volume of 50 pl as described above. insoluble materials by centrifugation at 15,000 X g for 30 min. After incubation of the extracts with anti-P-Tyr (1001(v/v) dilution) and RESULTS protein A-Sepharose (50% (v/v), 25:l dilution) at 4 "C for 2 h, the precipitates were washed three times by the same solubilization Insulin-stimulated Tyrosine-phosphorylatedProteins in Insolution. The immunoprecipitates were washed once with 50 mM tact Cells and a Cell-free System-To characterize our anti-PHepes-HCI (pH 7.5), and phosphotyrosine-containing proteins Tyr and insulin-stimulated PYPs in rat hepatoma cells (H(PYPs) were eluted from the immunoprecipitates by the addition of 35),serum-starved cells were labeled with [32P]orthoph~s10 mM PNPP in 50 mM Hepes-NaOH (pH 7.5). 3ZP-Labeledproteins were analyzed on SDS-PAGE aftersamples were boiled in Laemmli's phate, treated without or with insulin at 37 "C for 1 min, extracted with Tris-buffered 0.5% Triton X-100, and immusample buffer (26) for 5 min. Cell-free Phosphorylation of PYPs-PYPs were purified as de- noprecipitated with anti-P-Tyr. 32P-Labeled PYPs were scribed above with some modification. Cellsweregrown in 10-cm eluted from the immunoprecipitates by incubating with 10 dishes, and [32PJorthophosphatelabeling in RPMI 1640 medium was mM PNPP, then analyzed by SDS-PAGE and autoradiograomitted. In the case of detergent-free extraction, Triton X-100 was phy. As previously described, anti-P-Tyr specifically immuomitted from the solubilization buffer; however, the washing buffer for the immunoprecipitates contained 0.5% Triton X-100 as for the noprecipitated two major phosphoproteins of M, = 100,000 Triton-solubilized extraction. Reaction mixtures contained PYPs and 175,000 after insulin treatment (Fig. l A , +). This former which were eluted from anti-P-Tyr immunoprecipitates with 10 mM protein is the @-subunit of the IR, since this protein was PNPP, 50 mM Hepes-NaOH (pH 7.5), 5 mM MnCb and 5mM MgCIz. specifically immunoprecipitated with antibodies against IR The phosphorylation reaction was initiated by adding [y3'P]ATP (data not shown). The tyrosine phosphorylation of the P(20 p ~ 5 ,&i/nmol) and continued for the time intervals indicated subunit of IR may be the result of autophosphorylation catain the figure legends.Each reaction volume was 50 pl, which contained PYPs purified from approximately two dishes. In some experiments, lyzed by IR kinase itself (7-9). Next, to clarify the molecular basis of the tyrosine phosthe indicated concentration of antibody was added to the reaction 1 , attempted ~ mixture at 4 "C for 1 h before the addition of Iy3'P]ATP. The phorylation of M, = 175,000 protein ( ~ ~ 1 8 5we phosphorylation reaction was terminated by adding concentrated the phosphorylation of pp185 in a cell-free system using the Laemmli's sample buffer solution. These samples were boiled for 5 purified PYPs. Serum-starved H-35cells were treated without min. 32P-Labeledproteins were resolved on SDS-PAGE using 7.5% or with insulin at 37 "C for 1 min, extracted with Tris-buffered resolving gels and identified by autoradiography for several hours at 0.5% Triton X-100, and immunoprecipitated with anti-P-Tyr. -70 "C. Zmmunoblotting-Proteins analyzed inSDS-PAGE were trans- After washing the immunoprecipitates with anti-P-Tyr,PYPs ferred onto nitrocellulose sheets (Schleicher & Schuell) in methanol In this paper, the insulin-dependent tyrosine-phosphorylatedprobuffer containing 25 mM Tris, 192 mM glycine, 0.02% SDS, and 20% methanol at 50 V for 4 h at 4 "C. The sheets were soaked in 3% BSA tein of M,= 175,000 is designated as pp185 for consistency (18, 19, in rinse buffer containing 10 mM Tris-HC1 (pH 7.4), 150 mM NaCI, 21, 27).

6881

Cell-free Phosphorylation of pp185 Intact Cell

Cell-Free System

acid analysis of 180- or 103-kDa protein phosphorylated in a cell-free system was performed (Fig. IC). Each protein was phosphorylated mainly in its tyrosine residues, less phosphoInsulin insulin rylated in its serine residues, and slightly phosphorylated in (100nM) - + (100nM); Mr(kDa) Mr(kDa) its threonine residues, which differ from the phosphoamino (J.. e +PSer acid composition of pp185 or the 8-subunitof IR phosphorylcPThr (I,-180 -175 ated in an intact cell system (18, 21). These results suggested that, there were several kinases for 180- and 103-kDa protein in PYPs prepared from insulin-treated cells. These phosphoproteins were further analyzed by two-dimensional gel electrophoresis(two-dimensionalNEPHGE/ SDS-PAGE). pp185 phosphorylat,ed in intact cells migrated as a broad protein having an isoelectric pointof 5.3-6.0 (Fig. WPTyr 2.4). On the other hand, 180-kDa protein phosphorylated in a 103kDa Protein cell-free system migrated as a protein having an isoelectric 1 point of 5.2-5.5 (Fig. 2B). However, when these sampleswere FIG. 1. Insulin-stimulated tyrosine-phosphorylated pro- analyzed in the samegel (Fig. 2C), these two phosphoproteins teins in intact cells and the cell-free system.Serum-starved H- were partly overlapped. These data suggested that these two 35 cells were treated without or with insulin (100 nM) for 1 min at phosphoproteins are the same protein, and the non-overlap37 "C. These cells were scraped up into a buffer containing 0.591 ping portion may be due to the difference of the stoichiometry Triton X-100. P Y P s were purified by immunoprecipitation with anti- of phosphorylation. Although cell-free phosphorylated 103P-Tyr and eluted from the immunoprecipitates with 10 mM P N P P kDaproteinhadamoreacidicisoelectricpointthan IR as descrihed under "Experimental Procedures." Panel A , cells were phosphorylated in intact cells (Fig. 2, A and B ) ,these proteins labeled with [:'ZP]orthophosphate for 2 h before insulin treatment. "'P-Labeled PYPs were purified as describedabove and analyzed on were mostly overlapped when these sampleswere analyzed in 7.5% SDS-PAGEandautoradiography.PYPswerepurifiedfrom the same gel (Fig. 2C). These results suggested that cell-free non-insulin-treatedcells (-) or insulin-treated cells (+). Panel R, phosphorylated 103-kDa protein is the &subunit of IR. Both cell-freephosphorylationswereperformedwithPYPspurified as pp185 and IR phosphorylated in a cell-free system had more describedabovewithout[R"P]orthophosphatelabelingofthecells. acidicisoelectricpointssuggestingthesephosphoproteins The reaction mixture contained50 mM Hepes-NaOh (pH 7.5), 5 mM were more phosphorylated in a cell-free system. MnCI2, 5 mM MgCI?, and the PYPs in 10 mM PNPP. Reaction was Finally, to compare the phosphorylation sites, we performed started by the addition of [y-"P]ATP (20pM, 5pCi). After incubation for 30 min at 22 "C, "ZP-labeled proteins were analyzed by i.5% SDS- phosphopeptide mapping of pp185 and the 8-subunit of IR PAGE and autoradiography. PYPs were purified from non-insulinphosphorylated in intact cells (Fig. 3, A and C) and a cell-free A

0

C

+

treated cells (-) or insulin-treated cells (+). Panel C, phosphoamino (upper) or103-kDa (lower) acidanalysis of""P-labeled180-kDa protein shown in lane + of panel R. The migration of phosphoamino acid standards visualized by reaction with ninhydrin is indicated hy arrows. PSer, phosphoserine; PThr, phosphothreonine; P7j)r. phosphotyrosine.

-

MW(kDa)

MW(kDa)

200-

A

t 175

-

were eluted from the immunoprecipitates by incubating with 116P N P P . S i n c e P N P P h a sa similar structure to the phospho96- e 100 tyrosine, we checked the effectof the concentrationof P N P P I 1 I onthetyrosinekinaseactivity.Inthecase of IRkinase, autophosphorylation of IR kinase was inhibited completely with 100 mM P N P P ; however, more than 90% of the auto200t 180 phosphorylation activity was maintained in the presence of B 10 mM PNPP (data not shown). Thus, we used 10 mM P N P P toelutePYPsfromimmunoprecipitateswithanti-P-Tyr. 116Cell-freephosphorylationwasconductedbyadding5 mM 96- . c 103 MnCIP, 5 mM MgClr, and 20 p~ [Y-~*P]ATP to the PYPs. I I 1 After the incubationat 22 "C for 30 min,"'P-labeled proteins were analyzed by SDS-PAGE and autoradiography. As shown in Fig. 1, panel B , when phosphorylation reaction was per200formed with PYPs prepared from non-insulin-treated cells, C "2P-labeled protein bands did not appear under our conditions (Fig. l B , -). However, when the same reaction was performed 116with the PYPs from insulin-treated cells at 37 "C for 1 min, :12Plabeling of 180- and 103-kDa protein bands was observed 96(Fig. l B , +). Exceptforthesetwomajorphosph6rylated I I PI 4.'5 510 5.5 6.0 bands, several less "P-labeled bands were also observed including broad bandsof approximately 130 kDa. However, the FIG. 2. Two-dimensional electrophoresis of proteins phosphosphorylation of these bands including the 130-kDa protein phorylated in intact cells anda cell-free system.Serum-starved H-S5 cells were stimulated with insulin (100 nM) for 1 min. PYPs in was not reproducible, and sometimes faintly stained bands withCoomassieBluewerecomigratedwithsome of these intact cells were prepared as described in Fig. 1A (panel A ) ; PYPs in a cell-free system were prepared as described in Fig. 1H (panel R ) , bands. Furthermore, 130-kDa protein bands were also someand these samples were combined (panel C). Each sample was anatimes observed when cell-free phosphorylation was done with lyzedwithtwo-dimensional gel electrophoresis(NEPHGE/SDSPAGE) as descrihed under "Experimental Procedures." PYPs prepared from non-insulin-treated cells. Phosphoamino

Cell-free Phosphorylation of pp185

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system (Fig. 3, R and D ) by partial proteolysis using several Reaction Time ( m i d concentrations of S. aureus V8 protease. The phosphopeptide 0 10 30 6 0 mapping of pp185 phosphorylated in intact cells and thecelle Mr(kDa) free system seemed very similar (Fig. 3, A and B ) ; however, 2oo0 Ilr) -pp185 these were quite different from phosphopeptide mapping of the /%subunit of IR (Fig. 3, C and D ) . Although some phos* 6 , ; : 116phopeptides of pp185 phosphorylated in the cell-free system migrateddifferentlyfrom pp185 phosphorylatedinintact 97o @b&? P-abuntt cells, there were many phosphopeptides migrated in the same 66position. The resultsof two-dimensional gel analysis and this phosphopeptide mapping analysissuggested that pp185 phosphorylated in the cell-free system and intact cells were not 45only the same protein but also phosphorylated in the common sites. The slight differences of phosphopeptide mapping between the two can be due to the differencesof phosphoamino acid compositions and the stoichiometryof the phosphorylaFIG. 4. Time course of cell-freephosphorylation. Serumtion. (100 nM) for 1 min at starved H-35 cells were treated with insulin Characterization of Cell-free Phosphorylation of 180- and 37 “C. PYPs were purified from these cells, and cell-free phosphoryl103-kDa Proteins-Tyrosine phosphorylation of pp185 was ation was performed as describedin Fig. 1,panel R. Incubation times reproduced in our cell-free system; we examined correlation with[-y-:”P]ATP were changed as describedin the figure. These of phosphorylation in anintact cell and cell-free system. samples were analyzed by 7.5% SDS-PAGE and autoradiography. pp185 or t.he 8-subunit of IR phosphorylated in an intact cell incubation Time (mid syst.em was M , = 175,000 or 100,000, respectively, under our 0 0.5 1 10 30 6 0 electrophoresisconditions (Fig. lA, +). However,cell-free Mr(kDa) phosphorylation of theseproteinsresultedinthetyrosine 200phosphorylation of M , = 180,000 or 103,000 proteins (Fig. lB, Cpp185 +). Fig. 4 shows the time courseof cell-free phosphorylation. The degree of phosphorylation of both proteins increased up 116to 30 min, and phosphorylation increased gradually after 30 97*p-Subuntt min. Since their mobilities on SDS-PAGE were broader and slower asphosphatecontentincreased,the differences in 66molecular weight of each proteinbetween intact cell and cellfree systems may be due to differences in their phosphorylation level. 45Next, we examined whether the phosphorylationprofile of pp185 in an intact cell system was reflected in the cell-free phosphorylation of pp185. In an intactcell system, phosphorylation of the 0-subunit of IR and pp185was detectable FIG. 5. Effects of insulin preincubation time on the cell-free within 10 s and reached a maximum within30 s after insulin phosphorylation. Serum-starved H-35 cells were treated with in(100 nM) treatment.Thephosphorylation level of the 0- sulin (100 nM) for various time periods(as described in t,he figure) at subunit of IR decreased slowly a t least up to 1 h whereas that 37 “C. PYPs from each were purified, and cell-free phosphorylation of each fraction was performedas described in Fig. 1,panel R. These samples were analyzed by 7.5% SDS-PAGE and autoradiography.

pp185

-A-

P -subon& of

-B-

IR

of pp185 rapidly decreased by 10 min a t almost 20% of the maximum level, even in the presence of insulin (21). In an 96 96intact cell system, the mobility of pp185 on SDS-PAGE was 66 66slower as the incubation time of the cells with insulin was 43 prolonged up to 1 h (18, 19). Cell-free phosphorylation was 43performed with the PYPs purified from insulin-treated cells a t various time periods (Fig. 5). By changing the time of 31 31insulin preincubation of the cells, maximal cell-free phosphorylation of pp185 was found tobe obtained a t 30 s and rapidly decreased by 10 min. Mobility of pp185 was slower as the 22 22preincubation time of insulin with thecells was prolonged up to 1h (Fig. 5). These observations were nearly consistent with 14 14the data for tyrosine phosphorylation of pp185 in an intact cell system. Since cell-free phosphorylation was almost maxFIG. 3. Phosphopeptide mapping of pp185 or the 8-subunit imal after 30 min of incubation with [y-”PIATP (Fig. 4), the of IR phosphorylated in intact cells or in a cell-free system. phosphate content of pp185 in a cell-free system might be pp185 (A and H ) or the $subunit of IR (C and D ) was phosphorylated directly proportional to the protein content of pp185. Thus in intact cells(A and C) or in a cell-free system ( R and D )as described the similar time courses of susceptibility to maximal phosin Fig. 1 and electrophoresed on 7.5% SDS-PAGE. Phosphopeptide phorylation suggested t h a t a t 30 s after insulin treatment of mappingwasperformed as described under “Experimental Procethe cells, the number of tyrosine-phosphorylated pp185 moldures.” Phosphoproteins were digested with either 0 (lane I), 0.018 ecules was a t a maximum level in an intactcell system. (lane 21, 0.16 (lane 3), or 1.5 (lane 4 ) pg/lane S. aureus VR protease For further characterizationof cell-free phosphorylation of and electrophoresed on15% SDS-PAGE. Autoradiographsof gels are shown. pp185 and the 8-subunit of IR, we examined the effects of w

1

(km’

2

3

4 1

2

3

4

Phosphorylation Cell-free

of pp185

6883

ambientfactors in thereactionmixturecontainingPYPs purified from insulin-treated cells. In an intact cell system, tyrosine phosphorylation of pp185 and the @-subunit of IR was very sensitive to insulin treatmentof the cells (Fig. 1A). However, addition of insulin (100 nM) directly to the reaction mixture did not change the phosphorylation of pp185 and the @-subunitof IR in thecell-free system (Table I). These results suggested that in our cell-free system, insulin-sensitive tyro- @-subunitsine kinase(s) for pp185 and IR was already activated and anti-P-Tyr specifically immunoprecipitated the activated kinase. The kinase(s) for pp185 and @-subunitof IR was more *@ 0 ' 0.03 ' dependent on Mn2+ than M$+, and without these divalent 0.3 [Antibody] rnglrnl cations, phosphorylationof pp185 or the @-subunit of IR was 7 not observed (Table I). Moreover 0.1 mg/ml BSA was not phosphorylated in ourcell-free system (data not shown), and FIG.6. Effects of antibodies on the cell-free phosphorylaphosphorylation of pp185 and the @-subunit of IR was not tion. Panel A, serum-starved H-35 cells were treated with insulin changed in the presence of 0.1 mg/ml BSA (Table I). Thus, (100 nM) for 1 min a t 37 " c . Purification of PYPs, preparation of phosphorylation of pp185 and the @-subunitof IR was cata- reaction mixtures containing antibody, andcell-free phosphorylation were conducted as described in Fig. 1, panel B, and under "Experilyzed by the kinase and not phosphate exchange reactions. mental Procedures." Concentrationsof anti-IRK, anti-IRC, or control Inhibition of Cell-free Phosphorylation of pp185 and the @- antibody added to the reaction mixtures are indicated in the figure. subunit of IR Kinase by Anti-IR Antibody (Anti-IRK)-We After the cell-free phosphorylation reactionwas terminated, ?'Phad previously reported that site-specific antibodies to the labeled proteins were analyzed by 7.5% SDS-PAGE and autoradiogkinase domain of IR (anti-IRK) inhibited kinase activity of raphy. Panel B, quantitative representation of the data shown in IR kinase for autophosphorylation andexogenous substrates panel A. After autoradiography, corresponding bands were cut out, the radioactivity in each bandwas counted. Radioactivity of "P(12). This antibody was added to the reaction mixture con- and labeled pp185or the P-subunit of IR phosphorylated without antibody taining the PYPspurified from insulin-treated cells. After 1 is represented as 100%. 0 , A, W, ppl85; 0, A, 0,/3-subunit of IR. h of incubation with anti-IRK at 4 "C, the phosphorylation Cell-free phosphorylation was performed in thepresence of anti-IRK reaction was started by adding [y3'P]ATP to this reaction (0,O), anti-IRC (A,A), or control antibody (W, 0). mixture and incubated for 30 min a t 22 "C. Anti-IRK (0.03 mg/ml) inhibited phosphorylation of the @-subunitof IR to tion of IR(anti-IRC) was more potent than anti-IRK in 50% of the control level, and 0.3 mg/ml of the same antibody immunoprecipitation of IR, anti-IRC did not inhibit the auinhibited phosphorylation of the @-subunit of IR to 30% of tophosphorylation of theIR kinase(12). Inour cell-free the controllevel (Fig. 6 ) . The decreases in phosphorylation of system, even in thepresence of 0.3 mg/ml anti-IRC, phosphopp185 and the 130-kDa protein were observed concomitantly. rylation of the @-subunitof IR was not inhibited a t all (Fig. That is, 0.03 mg/ml anti-IRK inhibited phosphorylation of 6, A and B). Phosphorylation of pp185 and the 130-kDa pp185 to 50% of the control level, and 0.3 mg/ml anti-IRK protein was slightly inhibited in the presence of 0.3 mg/ml inhibited phosphorylationof pp185 to 10%(Fig. 6 ) .Thus 90% anti-IRC (Fig. 6). Interestingly, the heavy chain of IgG of of phosphorylation of pp185 was abolished bythe inactivation anti-IRC was phosphorylated in our cell-freesystem (Fig. 6A). of IR kinase. Anti-IRK specifically immunoprecipitated IR There might be several reasons for the decrease of phosphofrom ["S]methionine-labeled cells (12) anddid not immuno- rylation of pp185 in the presence of anti-IRC. One possible precipitate pp185 or other phosphorylated proteins besides IR reason is that anti-IRC andpp185 competed with each other in the reaction mixture (data not shown). These results sug- as a substrate for IR kinase. Another possible reason is that gested that the tyrosine phosphorylation of pp185 originated the interaction between IR kinase and pp185 might be disfrom IR kinase activity. turbed sterically by the covering of the C-terminal portionof Although the site-specific antibody to the C-terminal porIR with anti-IRC. Separation of pp185 from the Tyrosine Kinasefor pp185 by TABLE I Extraction ofCells with Detergent-free Solution"ppl85 is a Effects of ambient factors on the cell-free phosphorylation soluble proteinthatcanbeextracted with detergent-free Serum-starved H-35 cells were treated with insulin (100 nM) for 1 buffer (19). After cells were treated with insulin (100 nM) for min a t 37 "C. PYPs were prepared as described in Fig. 1, panel B, 1min a t 37 "C and disruptedby freezing with liquid nitrogen, and the phosphorylation reaction was performed. Each reaction mixture contained 50 mM Hepes (pH 7.5), 5 mMMnC12, 5 mMMgC12, PYPs were purified from detergent-free or detergent-solubiand PYPs in 10 mM P N P P (control), with added insulin (100 nM), lized extracts of these cells. Each set of PYPs was analyzed with added BSA (0.1 mg/ml), or without Mn2+ and/or Mg2'. Reactions by SDS-PAGE and immunoblotting with anti-P-Tyr. The were started by the addition of [-y-"PIATP. After incubation for 30 min a t 22 "C, "'P-labeled proteins were analyzed by 7.5% SDS-PAGE. amount of pp185 was not so much different in these PYPs Corresponding bands were cut out, and '*P present in the band was when examined by immunoblotting with anti-P-Tyr (Fig. 7 B , measured by liquid scintillation spectroscopy. Results were expressed lanes 1 and 2). In contrast, since IR kinase is a membraneas a percentage of control values. spanning protein and not a soluble protein, it could be exAmbient factors % of control tracted only from Triton-solubilizedcell lysates (Fig. 7B, lanes in the 1 and 2). We examined whether the tyrosine kinase for pp185 reaction PP 185 &subunit was extractedwithdetergent-free buffer or not. Cell-free mixture phosphorylation wasperformed withPYPs purified from Control 100 100 detergent-freeor detergent-solubilized extracts of insulin101 99 +Insulin (100 nM) treated cells. When PYPs were purified from detergent-free +0.1 mg/ml RSA 101 104 "$+ 94 92 extracts, cell-free phosphorylation of pp185 was not observed -Mn2+ 49 38 under our conditions (Fig. 7A, lane 2). Long exposure of the " F , -Mn2+2 2 same gels revealed only slightly phosphorylated pp185. Phos"

Cell-free Phosphorylation of pp18.5 B

A

lmmunoblotting

1 2 3 4 5 6

1 1' 2 2'

pp185-

B

CPSer

Y

cor.

0 @subunit-

C PTtr -PTyr

L . "

pyps + Purified IR-kinase lnsuiin(100nM)

+

+ +

-- ; 1

-2

FIG. 8. Phosphorylation of pp185 with purified IR kinase. Panel A, serum-starved H-35 cells were treated with insulin (100 nM) FIG.7. Localization of tyrosine kinase for pp185. Serum- for 1 min a t 37 "C. Cells were scraped into detergent-free solution, starved H-35 cells were treated with insulin (100 nM) for 1 min a t and PYPswere purified as described in Fig. 5 and under"Experimen37 "C. The cells were disrupted by freezing with liquid nitrogen, tal Procedures." Reaction mixtures containing PYPs ([anes 1, 2, 3, scraped intoa buffer containing 0.5% Triton X-I00 or detergent-free and 4 ) and/or 2 pg/ml purified IR kinase (lanes 3 , 4 , 5 , and 6) were incubated without(lanes 1,3, and 5) or with (lanes 2,4, and 6) insulin solution, and PYPs were purified as described under "Experimental Procedures." Panel A, cell-free phosphorylation was performed with (100 nM) for 45 min a t 22 "C. Cell-freephosphorylation was performed PYPs purified from detergent-solubilized extracts (lane I ) or deter- as described under "Experimental Procedures." "P-Labeled proteins gent-free extracts (lane 2). The phosphorylation reaction was started were analyzed by 7.5% SDS-PAGE and autoradiography. Panel B, by adding [r-"'P]ATP, and "P-labeled proteins were analyzed by phosphoamino acid analysis of "P-labeled pp185 shown in [ane 4 of 7.5% SDS-PAGE and autoradiography.Panel B, purified PYPs from pane/ A . The migration of phosphoamino acid standards visualized detergent-solubilized extract (lane I ) or detergent-free extract (lane by reaction with ninhydrin is indicatedby arrows. PSer, phosphoserine; PThr, phosphothreonine; PTyr, phosphotyrosine. 2) were directly analyzed by SDS-PAGE, and cell-free phosphorylation of each PYP from detergent-solubilized extracts (lane 1 ' ) or detergent-freeextracts (lane 2') wasperformed with non-labeled ATP. After the SDS-PAGE, tyrosine phosphorylationwas analyzed chromatography using wheat germagglutinin-agarose and with an immunoblottingtechnique with anti-P-Tyr as described insulin-coupled Affi-Gel 15. When the phosphorylationreacunder "Experimental Procedures." tion was performed withpurified IR kinase in the presence of

Mn2+, M$+, and [y-"P]ATP,only autophosphorylation of phoamino acid analysis of pp185 in this fraction revealed the @-subunitof IR was observed after insulin bindingfor 45 mainly phosphoserine, withlesser extents of phosphotyrosine min a t 22 "C (Fig. 8A, lanes 5 and 6).On the other hand, after (data not shown). However, when PYPs were purified from cells were treated with insulin (100 nM) for 1 min at 37 "c, Triton-solubilized cell lysates, cell-free phosphorylation of PYPs were prepared from detergent-free extracts of these of pp185 was pp185 was evident (Fig. 7A, lane I ) . Since these two fractions cells. In this fraction, tyrosine phosphorylation contained the pp185 (Fig. 7B, lanes 1 and 2 ) , more than 95% almost abolished even in the presence of insulin (100 nM) in of the tyrosine kinase activity we observed for pp185 wasnot the reaction mixture (Fig. 7A, lane 2 and Fig. 8A, lanes 1 and extracted with detergent-free buffer (Fig. 7A, lanes 1 and 2). 2). After purified IR kinase was added to reaction mixtures or insulin Thus tyrosine kinasefor pp185 could be extracted only from containing these PYPs and incubated withoutwith Triton-solubilized cell lysates. Furthermore, cell-free phos- (100 nM) a t 22 "C for 45 min, cell-free phosphorylation was in an phorylation of PYPs was analyzed with immunoblotting. In performed. Phosphorylation of pp185wasobserved this case, cell-free phosphorylation was done withnon-labeled insulin-dependent manner (Fig. 8A, lanes 3 and 4), and phosATP. When the PYPswere prepared from Triton-solubilized phoamino acid analysis of pp185 phosphorylated in thisfraccell lysates, pp185 was more tyrosine-phosphorylated after tion revealed primarily tyrosine phosphorylation (Fig. 8B). the addition of ATP, Mn2+, and Mg2' (Fig. 7B, lanes 1 and Sinceourpreparation of IRkinase purifiedfrom human placenta was not very active, the relative intensity of phos1 ' ) . However, when the PYP fraction was preparedfrom detergent-free buffer, no significant increase of tyrosine phos- phorylation of pp185 and the @-subunitof IR was low comphorylation of pp185 was observed even after cell-free phos- pared with the phosphorylationof the 130-kDa band in Fig. of IR andpp185 were phorylation (Fig. 7B, lanes 2 and 2 ' ) . In the case of IR, 8A. In this assay system, the @-subunit tyrosine-phosphorylated @-subunitwas observed only in the the only insulin-dependent tyrosine-phosphorylated proteins PYPs purified from Triton-solubilized cell lysates (Fig. 7B, even after the longerexposure of the film. Theseresults lanes 1 and 2), and more tyrosine-phosphorylated @-subunit suggested that pp185 was directly phosphorylated by the purified and insulin-stimulated IRkinase. was observed after cell-free phosphorylation was performed Mg+ to (Fig. 7B, lanes 1 and 1 By adding ATP, Mn2+, and DISCUSSION the PYPs preparedfrom detergent-free extracts, the tyrosine phosphorylation of these proteins was not changed (Fig. 7B, Tyrosine kinase activity is shared by growth factor receplanes 2 and 2 ' ) . These results are consistent with the dataof tors and viral transforming proteins, suggesting that such cell-free phosphorylation with [-p3*P]ATP (Fig. 7 , A and B ) . activity may be important in the regulation of cellular metabBoth analyses, "P labeling and immunoblotting, suggested olism and growth. In the case of growth factor receptors, that tyrosine kinase for pp185 could be extracted only with binding of a ligand activates the tyrosine kinase activity of its solubilizing buffer containing 0.5% Triton X-100. specific receptor. Aftertheactivation of receptor onthe Phosphorylation of pp185 with Purified IR Kinase-IR ki- plasma membrane, the receptor must transduce its signal to nase was purified from human placenta by two-step affinity another molecule to initiate subsequentphysiological events. I ) .

Cell-free Phosphorylation of pp185 TOdetermine the signal transduction mechanism of insulin in target cells, it is necessary to identify and characterize the direct substrates of IR kinase in intact cells. In intact cells, afterinsulintreatment, insulin-sensitive tyrosine phosphorylation of pp185 and the @-subunitof IR was observed in immunoprecipitates of anti-P-Tyr. In a cellfree system, incubation of the anti-P-Tyrimmunoprecipitate with [T-~'P]ATP,Mn", and Mg2+ resulted in a marked increase of tyrosine phosphorylation of pp185 and the @-subunit of IR. As shown in the results of two-dimensional electrophoresis and phosphopeptide mapping, cell-free phosphorylation of PYPs reproduced the tyrosine phosphorylation of pp185 and the@-subunitof IR observed in intact cells. Wehave previously reported that the stoichiometry of phosphorylation of pp185 phosphorylated in intactcells after insulin treatment was high compared with that of receptor phosphorylation by comparison of 32P-labelingexperiments with 35S-labelingexperiments (21). It is difficult to estimate the stoichiometry of phosphorylation of pp185 phosphorylated in our cell-free experiments. However, the stoichiometry may not be low since any bands at the pp185 position were not detected by the silver staining of the gel in our cell-free experiments. In the cell-free system, the tyrosine kinase responsible for phosphorylation of the @-subunitof IR is IR kinase itself, because anti-IRK which inhibits IR kinase activity decreased phosphorylation of IR substantially in our cell-free system. The tyrosine kinase for pp185 was also inhibited by anti-IRK, even though this antibody was specifically immunoprecipitated only IR (12). The tyrosine phosphorylation of pp185 was not due to autophosphorylation, because in PYPs purified from detergent-free extraction of insulin-treated cells, the tyrosine phosphorylation of pp185 was not observed even in the presence of pp185. These results suggest that IR kinase activity regulates or directly mediates tyrosine phosphorylation of pp185 in a cell-free system. In an intact cell system, White et al. (19) andwe4 observed that increasing the concentration of the human IR in Chinese hamster ovary cells by transfection with a plasmid containinghuman IR cDNA caused a higher level of tyrosine phosphorylation of both psubunit and pp185. Thus tyrosine phosphorylation of pp185 is regulated by IR kinase activity in both intact cells and a cell-free system. Furthermore, we suggest that IR kinase itself directly catalyzes the phosphorylation of pp185 in a cell-free system for the following reasons. Since little if any tyrosine phosphorylation of pp185 occurred in PYPs obtained from detergentfree extracts of insulin-treated cells in our assay conditions, the tyrosine kinase activity for pp185 is mainly absent from this fraction. When purified IR kinase was added to this fraction, pp185 was tyrosine-phosphorylated in an insulindependent manner. Thus, it is highly likely that purified IR kinase directly tyrosine phosphorylates pp185. It is, of course, conceivable that IR kinase activates some other tyrosine kinase(s) which in turn phosphorylates pp185. However, this possibility is less likely, since the p-subunit of IR and pp185 R. Yamamoto, Y. Shibasaki, K. Momomura, 0. Koshio, and M. Kasuga, unpublished data.

6885

were the only insulin-dependent tyrosine-phosphorylated proteins detected in our cell-free phosphorylation system. Since phosphopeptide maps of pp185 phosphorylated in between intact cells and the cell-free system were very similar, it may be suggested that IR kinase directly phosphorylates pp185 also in intact cells. A molecular link of tyrosine phosphorylation from IR kinase to pp185 was directly elucidated in our study. This observation provides further support for an important role for pp185 in the insulin signaling pathway. The next obvious question is the function of pp185 and its alterations by tyrosine phosphorylation. The in vitro system described here should be useful in answering this question. Acknowledgments-Wewish to thank Dr. Kenneth M. Yamada (National Cancer Institute, National Institutes of Health, Bethesda, MD) forhis critical readingof the manuscript. We also wishto thank Drs. K. Momomura and T. Shiba for generous support and useful discussions. REFERENCES 1. Kasuga, M., Karlsson, F. A,, and Kahn, C. R. (1982)Science 215,185-187 2. Roth, R. A,, and Cassell, D. J. (1983)Science 219, 299-301 3. Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D. L., and Kahn, C. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2137-2141 4. Shia, M. A., and Pilch, P. F. (1983)Biochemistry 22, 717-721 5. Rosen, 0.M., Herrera, R., Olowe, Y., Petruzzelli, L. M., and Cohh, M. H. (1983)Proc. Natl. Acad. S a . U. S. A . 80,3237-3240 6. Yu, K. T., and Czech, M. P. (1964)J. Biol. Chem. 259,5277-5286 7. Ellis, L., Clauser, E., Morgan, D. 0.. Edery, M., Roth, R. A,, and Rutter, W. J. (1986)Cell 45,721-732 8. Chou, C. K., Dull, T. J., Russe1,D. S., Gherzi, R., Lehwohl, D., Ullrich, A., and Rosen, 0.M. (1987)J. Bml. Chem. 262,1642-1647 9. Ehina, Y., Araki, E., Taira, M., Shimada, F., Mori, M., Craik, C. S., Siddle, K.. Pierce. S. B.. Roth. R. A,. and Rutter. W. J. (1987) . . Proc. Natl. Acad. Sei. U. S. A . 84;704-708 ' 10. Morgan, D. O.,Ho, L., Korn, L. J., and Roth,R. A. (1986)Proc. Natl. Acad. Sci. U. S. A. 83,328-332 11. Morgan, D. O.,and Roth, R. A. (1987)Proc. Natl. Acad. Sci. U. S. A . 84, 41-45 12. Izumi, T., Saeki, Y., Akanuma, Y., Takaku, F., and Kasuga, M. (1988)J. Biol. Chem. 263,10386-10393 13. Kadowaki, T., Fujita-Yamaguchi, Y., Nishida, E., Takaku, F., Akiyama, T., Kathuria, S., Akanuma, Y., and Kasuga, M. (1985)J. Biol. Chem. 260, 4016-4020 14. Zick, Y., Sagi-Eisenberg, R., Pines, M., Gierschik, P., and Spiegel, A. M. (1986)Proc. Natl. Acad. Sci. U. S. A . 83,9294-9297 15. Yu. K. T.. Khalaf.. N.., and Czech. M. P. (1987) . . J. Biol. Chem. 262.78657873 ' Laird, D.M., and Lane, M. D. (1987)Proc. Natl. Acad. Sci. 16. Bernier, M., Laird: U. S. A. 84,1844-1848 84.184, Accili, D., Marcus-Samueles, B., Rees-Jones, R., and Taylor, 17. Perrotti, N., Accili S. I. (1987)Proc. Natl. Acad. Sei. U. S. A . 84,3137-3140 S. 18. White, M. F., Maron, R., and Kahn, C. R. (1985)Nature 318, 183-186 19. White, M. F.,.Stegmann, E. W., Dull, T. J., Ullrich, A., and Kahn, C. R. 262,9769-9777 47~-4777 (1987)J. E d . Chem. 262, 20. Gibhs, E. M., Allard, W. J., aand Lienhard, G. E. (1986)J. Biol. Chem. 261, 16597-16603 21. Kadowaki, T., Koyasu, S., Nishida, E., Tobe, K., Izumi, T., Takaku, F., Sakai, H., Yahara, I., and Kasuga, M. (1987)J. Biol. Chem. 262, 73427350 22. Shemer, J., Adamo, M., Wilson, G. L., Heffez, D., Zick, Y., and LeRoith, D. (1988)J . Biol. Chem. 262,15476-15482 23. Momomura, K., Tobe, K., Seyama, Y., Takaku, F., and Kasuga, M. (1988) Biochem. Biophys. Res. Commun. 155, 1181-1186 24. White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R. (1988)Cell 54,641-649 25. Ullrich, A,, Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A,, Coussens, L., Liao, Y.C., Tsubokawa, M., Mason, A., Seehurg, P. H., Grunfeld, C., Rosen, 0. M., and Ramachandran,J. (1985) Nature 313, 756-761 26. Laemmli, U. K. (1970)Nature 227,680-685 27. Izumi, T., White, M.F., Kadowaki, T., Takaku, F., Akanuma, Y., and Kasuga, M. (1987)J. Biol. Chem. 262,1282-1287 28. Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y., and Itakura, K. (1983)J . Biol. Chem. 258,5045-5049 29. Takayama, S., Tobe, K., Momomura, K., Koshio, O., Tashiro-Hashimoto, Y., Akanuma, Y., Hirata, Y., Takaku, F., and Kasuga, M. (1989)J. Clin. Endocrinol. Metab., in press