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Growth Hormone Stimulates the Tyrosine Phosphorylation of the Insulin Receptor Substrate-1 and Its Association with Phosphatidylinositol 3-Kinase in Primary Adipocytes

Growth Hormone Stimulates the Tyrosine Phosphorylation of the Insulin Receptor Substrate-1 and... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No.8, Issue of February 24, pp. 3471-3474, 1995 Communication © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. ample of a hormone presumably utilizing a wide array of in­ Growth Hormone Stimulates the tracellular proteins in its signaling chains. The initial event in Tyrosine Phosphorylation of the GH action involves binding and dimerization of its membrane receptor (1, 2). The GH receptor belongs to the cytokine recep­ Insulin Receptor Substrate-l and tor family, characterized by homologies in the extracellular Its Association with domains and lack of intrinsic tyrosine kinase activity (3). In­ stead, activation of receptor-associated cytosolic tyrosine ki­ Phosphatidylinositol3-Kinase in nases belonging to the Janus kinase family, in the case of GH Primary Adipocytes* the JAK2 tyrosine kinase (4), is an emerging theme for this family of receptors (5). In addition, a number of intracellular (Received for publication, October 5, 1994, and in revised form, key proteins have been suggested to be involved in GH signal­ December 20, 1994) ing further downstream of these or other, as yet unidentified, Martin Ridderstealet, Eva Degerman, and receptor-proximal events (6). Hans Tornqvist§ We have recently shown that the insulin-like effects ofGH in From the Departments of §Pediatrics and Medical and isolated rat adipocytes can be blocked by wortmannin, a selec­ Physiological Chemistry, University ofLund, P. O. Box tive inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase) (7). 94, S-221 00 Lund, Sweden A number of other hormones and growth factors such as insu­ Insulin receptor substrate-I (IRS-I) is tyrosine-phos­ lin, insulin-like growth factor-1 (IGF-D, platelet-derived phorylated in response to insulin resulting in associa­ growth factor, and different cytokines have been shown to tion with and activation of phosphatidylinositol 3-ki­ stimulate the activity of PI 3-kinase (8). PI 3-kinase is a dual nase (PI 3-kinase), thereby initiating some of the effects specificity lipid and serine kinase (9) consisting of a regulatory of insulin. We have recently shown that the insulin-like 85-kDa subunit (p85) containing two Src homology 2 (SH2) effects of growth hormone (GH) in adipocytes can be domains and a catalytic 110-kDa subunit (p110) (10, 11). The inhibited by the selective PI 3-kinase inhibitor wort­ role of its lipid kinase products, phosphatidylinositol 3-phos­ mannin (Ridderstr81e, M., and Tornqvist, H. (1994) Bio­ phate, phosphatidylinositol 3,4-bisphosphate, and phosphati­ chem. Biophys. Res. Commun. 203,306-310), suggesting a dylinositol 3,4,5-trisphosphate (12), as intracellular second similar role for PI 3-kinase in GH action. Here we show messengers is not known, but regulation of intracellular pro­ that mS-l is tyrosine-phosphorylated in a time- and tein trafficking (13) and/or activation ofthe ~-isoformof protein dose-dependent manner in response to GH in primary kinase C (14) have been suggested. The protein kinase activity rat adipocytes. This phosphorylation coincided with the of PI 3-kinase can phosphorylate p85 on serine resulting in a extent of interaction between IRS-I and the 85-kDa sub­ feed-back inhibition the catalytic activity of p110 (9). unit of PI 3·kinase as evidenced by coimmunoprecipita­ tion. Stimulation with 23 DM GH increased the PI 3-ki­ Insulin stimulation of glucose uptake and counteraction of nase activity associated with mS-l 4-fold. Our data lipolysis in adipocytes is believed to be mediated by activation suggest that GH·induced tyrosine phosphorylation of of PI 3-kinase (15-19). Insulin binding results in activation and IRS·I and the subsequent docking of PI 3-kinase are autophosphorylation of the insulin receptor tyrosine kinase on important postreceptor events in GO action. The mech­ specific tyrosines (20, 21). This is followed by tyrosine phospho­ anism for the phosphorylation ofIRS-I induced by GO is rylation of a major exogenous substrate for the receptor called unknown, but involvement of JAK2, the only known GO the insulin receptor substrate-1 (IRS-1) (22-24) on multiple receptor-associated tyrosine kinase, seems possible. tyrosines in YXXMIYMXM motifs (25), thus providing specific binding sites for the SH2-domains of p85 and subsequent acti­ vation of p110 (26). Furthermore, recent data suggest that PI Major advances have been made in recent years in elucidat­ 3-kinase can serine phosphorylate IRS-1 (19). In addition to ing the components of the intracellular signaling chains of insulin, IGF-I has been shown to utilize IRS-1 in intact cells different hormones, reaching from their membrane receptors to and interleukin 4 (IL-4) has been shown to utilize the function­ intranuclear events controlling transcription, or to intracellu­ ally related but immunologically distinct protein 4PS in mye­ lar target proteins controlling cellular metabolism. Growth loid cell lines (27-30). Tyrosine phosphorylation of IRS-1I4PS hormone (GH)l exerts pleiotropic actions on the growth, differ­ results in the association of several other proteins (Grb2, SH­ entiation, and metabolism of cells and constitutes a good ex- PTP2, and Nck) through their SH2 domains (31). To further explore the possible role of PI 3-kinase in GH * Financial support was given by the Swedish Medical Research Council (Project 8689), the Medical Faculty, University of Lund, action, we precipitated PI 3-kinase from GH-treated rat adipo­ Swedish Diabetic Association, and the Pahlsson, Novo Nordisk, cytes and looked for associated tyrosine-phosphorylated pro­ Crafoord and Bergwall Foundations. The costs of publication of this teins. Two major high molecular weight proteins were found. article were defrayed in part by the payment of page charges. This One of these was identified as IRS-1 by immunoblotting. IRS-1 article must therefore be hereby marked "advertisement" in accordance was tyrosine-phosphorylated and bound to the p85 subunit of with 18 U.S.C. Section 1734 solely to indicate this fact. :j:Towhomcorrespondenceshould be addressed: Dept. ofMedicaland PI 3-kinase in a dose- and time dependent manner in response Physiological Chemistry, Section of MolecularSignaling, P. O. Box94, to GH. S-221 00 Lund, Sweden. Fax: 46-46-10-40-22. 1 The abbreviations used are: GH, growth hormone; PI 3-kinase, EXPERIMENTAL PROCEDURES phosphatidylinositol 3-kinase; IRS-I, insulin receptor substrate-I; IGF-I, insulin-like growth factor-L; SH2, Src homology 2; IL-4, inter­ Adipocytes were prepared essentially according to Rodbell (32) leukin 4; PAGE,polyacrylamidegel electrophoresis. with modifications (7, 33) from 36-day-old Sprague-Dawleyrats (B&K, This is an Open Access article under the CC BY license. 3472 IRS-] and PI 3-Kinase in OR Action I.p. aiRS 1 apBS Sto ckholm, Swede n) fasted overnight prior to th e experime nts. After incubation with GH th e cells, 4 ml of a 10% cell sus pension (400 l.tI packed cell volume) for each condition , were homogeni zed in lysis buffer conta ining 10 rnxr Tri s-H Cl, pH 7.4, 0.25 ~I sucrose, I mxr EDTA, 100 IJ.M aPY orthova na da te, 0.1 mMdiisopropyl fluoroph osphate, I ug/ml pepst at in A, and 10 IJ.g/ml each of a nti pain and Ieup eptin. In some experiments - 205 cells wer e sti mulate d with GH, IGF-I, or insulin in th e presence or _IRS-1 abse nce of a neutralizing monoclonal anti-IGF-I antibody (recombinant - 116 hum an GH, human insul in , IGF -I, and anti-IGF-I (mAb 41) wer e gen­ erously supplied by Novo Nordi sk, Bagsvaerd , Denmark). Lysates free - 97 from fat and cellular debri s wer e supplemented with Triton X-100 to 1% (w/v) and solubilized for I h at 4 °C. Insolubl e material was removed by centrifuga tion and the lysat es incubated with eithe r anti-IRS-I anti­ body <0.7 IJ.g/mlJ or anti-p85 (PI 3-kinase) antise ra (1 : 1000) from Up­ state Biotechn ology Inc. (Lake Placid, NY). Immunoprecipitates were aIRS -1 OV- collecte d by adding 25 IJ.1of Pr otein A-Sepharose 4B (Pha rrnacia, Upp­ sa la, Sweden ), wash ed three tim es in 20 mxt Tris-HCl, pH 7.6, contain­ apBS _•• ing 137 mM NaCl, dissolved in SDS sa mple buffer, run on 7% SDS­ PAGE gels, and transferred to Imm obilon-P membranes. For Western blotting a polyclonal anti-phosphotyrosi ne antibody (a gene rous gift 1 2 3 4 from Dr. L. Riinnstrand, Ludwi g Institute, Uppsa la, Sweden ) was used FIG. 1. Tyr os ine-phosph o ry lated IR S- ! associates with PI 3-ki · at I IJ.g/ml , th e anti-IRS-I antibody at 0.7 J.Lg/ml , and th e anti-p85 n a se in r esponse to GH. Isolat ed rat adipocytes wer e prepared and antisera at a 1:1000 dilution. Western blotting, stripping th e blots, and treated as und er "Experimental Procedures." Cells were eithe r tr eat ed reblotting were performed using enha nced che miluminesce nce accord­ ( + ) or not trea ted (-) with 23 nxr GH for 10 min . Whole cell lysates wer e ing to th e manufacturer's instru ction s (Amers ham). subjec ted to immunoprecipitat ion with antibody again st th e insul in For ill vitro phosphatidylin ositol kinase assays, Protein A-Seph aro se receptor substrate- I (a IRS-I; lan es 1 a nd 2 ) or the p85 subunit of collecte d immunoprecipita tes were wash ed twice with lysis buffer sup­ phosph atidylin ositol 3-kinase (op85; lanes 3 and 4). Prot eins in th e plemented with Nonidet 1'-40 to 1% (w/v), three tim es with 10 mxt immune complexes were se parated by SDS-PAGE (7%) and trans ferred Tri s-HCI, pH 7.4, and 145 mxt NaCl, a nd twice with assay buffer to a n Imm obilon -P membrane. Th e membran e was Western blotted with an anti-phosph otyr osin e antibody (oPY; upper panel ), stri pped, conta ining 40 mxr Tri s-HCl, pH 7.4, 5 m ~1 MgCI , and 0.5 mM EGTA, and reblotte d with eithe r a IRS- I tmiddle panel ) or op85 ilotoer pan el ). and finally resusp end ed in 50 IJ.1 assay buffer. Th e assay was sta rte d by Immunoreactive bands were visua lized by enha nced che milumines­ addition of 50 IJ.1 of phosph oryl ation mix conta ining 0.2 mg/ml phos­ cence. Th e migration of molecular weight markers ar e indic ated to th e phatidylin ositol (a gift from Dr L. Krabisch, Lund, Sweden ), 0.01 mg/ml right ( x 1000). Results are representat ive of at least three individua l phosph atidylserine (Sigma), and 0.2 mxt [y_"2pIATP ( I IJ.Ci; Am ersharn) experime nts. in assay buffer. Reactions were terminated after 10 min at 30 °C by addition of 200 IJ.1 of I ~I HCI a nd 400 IJ.1 of methan ol:chloroform (1:1). The organic phase was recovered , and lipid s were dri ed down and dissolved in 20 IJ.1 of chloroform:metha nol (95:5), spotted onto Silica Gel PIP • • 60 plat es (Merck, Darmstad t, Germany), and developed in chloroform: methan ol.Hjfhammonium hydroxide (25%) (45:35:7.5:2.8). Phosph ati ­ dylin ositol 4-phosph ate, which comigra tes with 3-phosphorylated phos­ phatidylinositol , was used as sta nda rd. "21' incorp orat ed into phosphatidyl inositol was visua lized and qu an tified by Fuji x Bas 2000 Qd • Sys te m. GH RESULTS AND DISCUSSION FIG. 2. Increased PI 3-k inase a ctivity in anti-IRS- I im rnuno­ Isolated rat adipocytes were incubated in ab senc e of hor­ precipita tes fr om GH-treated cells. Isolated rat adipocytes were mon es for 3 h in order to restore responsiv eness to the insulin­ prepared and treat ed as describ ed under "Experimenta l Procedu res." Cells were eithe r treated ( +) or not treated (-) with GH (23 nxt) for 10 lik e effects ofGH (34). Th e cells were then resuspended in fresh min . Whole cell lysat es were subjected to immunoprecipitation with medium and treated with GH for varying times and at varying antibody agains t th e insulin receptor substra te- I. The precipitates were concentrations. Whole cell Iysates wer e subjected to immuno­ wash ed and imm ediately used for an ill vitro phosph atidylin ositol ki­ precipitation with antibodies against either p85 (a p85) or nase assay as described under "Experimental Procedures." The conver­ sion of phosphatidylin ositol to phosph atidylin ositol phosph ate in th e IRS-I (aIRS-I ). Th e immunoprecipitates were analyzed by presence of (y_"2PIATP was anal yzed by TLC. PIP indi cates th e migra­ Western blotting with anti-phosphotyrosine antibody (a PY) or tion of a phosphatidylinositol 4-phospha te sta nda rd. The origin, ori , with th e resp ective immunoprecipitating antibodies, or as­ contains an irrelevant residual amount of 1"2p1ATp th at was not re­ sayed for phosphatidylinositol kinase activity. moved by organic extrac tion. Results ar e representati ve of three indi­ IRS-I wa s phosphorylated on tyrosine in response to GH vidual experiments. (Fig. 1, lane 2, upper pan el) as revealed by a PY immunoblotting is probably due to incomplete precipitation of IRS-I with of a IRS-I immunoprecipitates. Furthermore, when reblotting with a p85, p85 wa s found to coimmunoprecipitate with IRS -I aIRS-I (data not shown). Yet, th e presence of other tyrosine­ in response to GH (Fig. 1, lan e 2, lower panel). Irnmunoprecipi­ phosphorylated protein s in th e a p85 precipitate cannot be ex­ tation with a p85 showed coimmunoprecipitation of two high cluded. To further explore th e significance of our findings, the aIRS-I immunoprecipitates were assayed for association of PI molecular weight tyrosine-phosphorylated protein s (Fig. 1, lan e 4, upper pan el), whereas neither p85 (Fig. 1, lan es 3 and 4) 3-kinase activity. Fig. 2 shows that GH sti mulate d a 4-fold nor pllO (dat a not shown) were phosphorylated on tyrosin e in increase (mean of 3 experi ments ) in phosphatidylinositol ki­ response to GH . One of th e p85-associated proteins migrated to nase activity associated with th e a IRS- I immunoprecipitate. Whether this reflects activation of th e kinase upon association the same position as IRS-I and wa s, upon reblotting, recog­ niz ed by a IRS-I (Fig. 1, lan e 4, middle panel). Th e other ty­ with IRS-lor translocation of active kinas e is not known (cr. sphorylated protein, tentatively called pp 180, mi­ Ref. 35). Insulin-induced docking of PI 3-kinase to IRS-I has rosin e-pho grated abov e the position for IRS-I and wa s not recognized by been shown to result in activation of th e kinase (26). a IRS-!. Apparently, crp85 immunoprecipitation brought down Having demonstrated that GH induces tyrosin e phosphoryl­ more of th e tyrosine -phosphorylated material corresponding to ation of IRS-I and as sociation of PI 3-kinase to IRS-I , we IRS-I than precipitated with a IRS-I (lan e 2 versus lane 4 ). This proce eded by inv esti gating the time course a nd dose depend- IRS-] and PI 3-Kinase in GH Action 3473 A 8 C oPY a PY aPY - l OS - l05 - 20 ~ .. ........-. IRS.' 4-- lAS·' _ ,RS·! - 116 - 116 - 116 _ 9 7 - 97 - ., ai RS·' ai RS·! ai RS· ' ------ ------ Dl>8S Dl>8S Dl>8S - - - - - _..---- Ort't,." ('I"t,." 0 GH(nM) GH( nM) 0"""""'''''0 Ti~ (IT'll") 0 ! 2 S 10 20 C?~ NN~ ~ "! N N :::: F IG. 3. GH stim u lates the tyrosine phosphoryla ti on of IRS- ! a n d its associatio n with P I 3-kinase in a dose- and time-dependent manner . Isolated rat adipocytes were prepa red and treated as describ ed under "Experimenta l Procedures." Th e cells wer e stimulated eithe r with 23 ml GH for 0-20 min (A) or with 0-230 nMGI-I for 10 min (B and C ). Whole cell lysates were subjected to immunoprecipitation with an antibody aga inst the insul in receptor substrate I (a iRS-I, A and B ) or th e p85 subunit of phosph atidylinositol 3-kinase (a p85, C). Proteins in the immune complexes were separa ted by SOS-PAGE (7%) an d tran sferr ed to Immobilon-P membranes. The membranes were Western blotted with an anti-phosphoty rosine antibody (a PY; upper pan els ), st ripped, and reblotted with either a IRS-I (m iddle panels ) or a p85 tlouier pan els ). Immun o­ ation of molecular weight mark ers are ind icat ed to the right ( X 1000). reactive bands were visua lized by enha nced chemiluminesce nce. Th e migr Resu lts are representative of at least three individua l experiments. ence of these eve nts (Fig. 3). Increased ty rosine phosphoryla ­ not induce det ectabl e ty rosine phos phorylation, i.e. receptor tion ofI RS-l (Fig. 3A , upper panel) as well as the appearance of ding tyrosine kinase activation, in the 95-kDa regio n correspon p85 in the a IRS-l immunoprecipitates (Fig. 3A, lower pa nel) to the l3-sub units of these receptors, as evidenced by SDS­ were seen within 2 min of GH stimulation reachin g a maximum PAGE of a PY-immunoprecipitated solubilized membrane pro­ at 5-10 min . Since GH effects have been dem onstrated over a teins from 32P-labeled adipocytes, in comparison to the effect of wide range of concen trations (36), it was in teresting to observe ins ulin.f It has also been shown that primary rat adipocytes do the dose dependenc e of th e effects induced by GH found in the not express IGF-I rece pto rs ca pabl e of bin ding IGF-I on the cell present inves tigation. Tyrosin e ph osphoryl ation of IRS- l (Fig. surface (41). It is also possible that GH might induce local 38, upper panel) and the subse quent asso ciation of p85 in produ ction of IGF-I from the adipocytes, which by binding to response to GH appeared at 0.23 nxt, reaching a maximum at IGF-I receptors or, at higher conce nt rations, to insu lin recep­ 23 mol, and the n to decr ease at 230 mol. In addition, th e dose­ tors could resul t in th e observed phosphorylation of IRS-I. This dependen t appearance of tyrosine-phos phorylated IRS-l and possibility was investigated by stimulating adipocytes with pp180 (Fig. 3C, upper pan el) and the association of the IRS-l GH, IGF-I , or ins ulin in presence or absence of a neutralizing pro tein with p85 (Fig. 3C, m iddle panel) in the a p85 immuno­ anti-IGF-I antibody (aIGF-I). As expected, IGF-I stimulated precipitates followed the same dose dependenc e. Similar GH both lipogen esis, measured as incorporation of[ Hlglucose in to dose-response curves (ED "" 1- 2 nl.l) ha ve been observed for adipocyte tri glycerides (42), and tyrosine phosphorylation of 5 0 th e ins ulin-like effects ofGH (36) and for the ty rosine phospho­ IRS-! in a dose-dependen t manner (data not shown). Half­ rylation of th e GH receptor in response to GH in adipocyt es.f maximal stimula tion (""20 nxr) by IGF -I was 100-1000 times The observed decrease at higher concentrations is likely to higher th an th at reported for ins ulin (43), indicating that IGF-I reflect the need of receptor dim erization for sign aling (37). acts through the insulin receptor and not the IGF-I receptor. From these results we conclude that GH stimulation of adi­ Th e presenc e of a IGF-I (5.4 p.g/ml ) clearly inhibited th e effect of pocytes results in a rapid and dose-dependent tyrosine phos­ IGF-I 0 0 nxr) bu t had no effect on GH- or insulin-induced IRS-l phorylation of IRS-l and association of th e p85 subunit of PI tyr osine phosphorylation (Fig. 4). An irreleva nt monoclonal 3-kinase under conditions whe n the hormone exerts its acute mouse antibody used as a control at the sa me concentration as insulin-like effects. Previous data obtained with th e selective a IGF-I had no effect on either horm onal effects (data not PI 3-kinase inhibitor wortmannin supports th is view (7). Our shown). We conclude that GH-induced tyrosine phosphoryla­ results have th e gene ral implication that stimulation through tio n of IRS-I and subsequent association wit h PI 3-ki na se is receptors of the cytokine receptor family, like the GH receptor, not med iated through local IGF-I production and activation of ma y result in tyrosine phosphoryl ation of IRS-1 and subse ­ the IGF-I- or ins ulin rece pto rs. quent association of PI 3-ki nase in primary cells. Th is ha s also Th e kin ase responsible for the IL-4-stimulated 4PS or IRS-l been shown for IL-4 stimulation of32D myeloid progeni tor cells phosphorylation has not been identified, but it is expected to be overex pressing IRS-1 (38). On the othe r hand, Gold et al. (39) a kin ase related to the Janus kinase family proteins or the Src could not demonstrate that sti mulation of hematopoieti c cell homology protein Fyn (3 1). A role for J AK2, which is tyrosine­ lines with othe r cytokin es resulted in phosphorylation of IRS-l phosphorylated in response to GH in 3T3-F442 A preadipocytes or related proteins associating with PI 3-kinase. (4) and adipocytes.f in the phosp horylation of IRS-! therefore It is not known which tyrosine kinase is responsible for the see ms possible. GH-induced phosphorylation of IRS-I. For insulin it has been In addit ion to PI 3-kinase, several othe r SH2 domain-con­ propo sed that th e insulin receptor tyrosine 960 has a func tion taining proteins <Grb2, SH-PT P2, and Nck) have been shown to in enabling the receptor kin ase to phosphorylate IRS-l (40). associa te with IRS-l phosphorylated by the insu lin receptor Tyrosine 960 is positioned in a seque nce motif, NPXY, which is (31). GH-induced tyrosi ne phosphorylation of IRS-l might turn also found in th e IL-4 and IGF-I receptors (38). Thi s motif ha s, out to be a n important tool in th e investigation of specificity in however, not been found in the GH receptor. A role for th e th e sign als initiated by IRS-1 phosphorylation . It has for ex­ insulin- or IGF-I receptor kinases seems unlikely, since GH did ample been shown th at GH stimula tion results in activation of MAP kinase and S6 kin ase activity as well as induction of c-Fos and c-Jun (44-47). Our findings suggest that GH-induced ty­ 2 H. Eriksson, M. Ridderstrale, and H. Tornq vist , manuscript in preparation. rosine phosphorylation of IRS-I migh t be positioned upstream 3474 IR S -l and PI 3-Kinase in GH Action 6. Roup as, P., a nd Herington, A. C. (1994) Trends Endocrinol. Metab. 5, 154- 158 7. Ridderstr le., ~l.. . and Tom oy ist . H. (1994) Biochem. Bioph )'s. Res. Comm un, IGF· I Ins C GH 203, 306 - 3 10 r---1r----1r-"1r-J 8. Va rticovski, L., Ha rrison-Findik, D.. Keeler , 111. L.. and Susa , l\l . ( 994) ((IGF· 1 . . . . Biochim, Biophys. A cto 1226, 1-11 9. Dhand. R.. Hiles, 1., Panayotou , G.. Hache, S.. Fry , 111.,J.. Gout , 1., Tott y, N. F., aPY Truong, 0 ., Vicen do, 1'., Yonezawa, K.. Kasuga, M., Courtneidge, S. A., and - ZOS Waterfield, 111. D. (994 ) EMBO J . 1:1, 522- 533 ~PP 180 10. Carpenter , C. L., Duckworth, Il. C.. Auger , K R., Cohen , 13.. Scha ffha use n, Il. IRS·l S.. and Ca ntle y, I.. C. (990 ) J. Biol , Chem, 265 , 19704 -1 9711 1J. Escobedo, J . A., Nav a nkasattu sas, S., Ka va na ugh , W. 111.. Milfay, D., Fri ed, V. - II. A., an d Willia ms, I.. T. (99 1) Cell 65, 413-423 12. Whitm a n, III., Ka plan, D. H., Roberts, T. III., and Ca ntley, L. C. (1988) Nature 3:12, 644 - 646 13. Panayotou , G., and Wa terfield , M. ( HJ92) Trends Cell Bioi. 2, 358 - 360 a iRS·' .- 14. Na ka nish i, H., Brewer, K A.. a nd Exton . vl, II. ( 1993 ).J. Bioi. Chem . 268 , 13- 16 15. Kelly, K 1.., Ruderma n, N. B., a nd Che n, K S. (992 ) J. Iliol. Chern. 267, opes :1423-3428 ---_.... 16. Ka nai. F.. Ito, K.. Todaka, 111., Hayash i, H., Kam oha ra , S., Ishii, K , Okada, T., Ha zeki, 0 ., Ui, M.. a nd Ebina, Y. (993) Bioc hem, Biophvs. Res. Commu n, 195, 762-768 FIG. 4. Ne u traliz in g IGF·( a n ti body does not block GH-sti m u­ 17. Okada, T.. Kawan o, Y., Sakakibara, T.. Hazeki, 0 ., an d Ui, ~1. ( 994) .]. Bioi. lated tyrosine phosphorylation of IR S-l a n d pp180. Isolated rat Chem, 269, 3568- 3573 ad ipocyt es were prepared and tr eated as descri bed un der "Experimen­ 18. Hahn, T.. Ridderstrale, M., Fredrikse n. G., Tornqvist, H., Mangia nello, V., Belfrage, 1'., a nd Deger man . E. ( 1994) FEllS Lett. :150, 3 14 - 3 18 tal Procedures." Th e cells (2 011, 10% sus pension) were stimulated wit h 19. Lam , K.. Ca r pente r, C. L., Ruderm an, N. 13.. Fri el. vl . C.. a nd Kelly, K I.. (1994) 23 nxtGH, 10 nxt IGF-I, 1 nxt insulin or vehicl e fOl'1O min in the ab sen ce J. lii ol. Chem. 269, 2064 8-20652 or presence of5.4 Jlg/ml monoclonal lGF-1antibody (a IGF-I). Whole cell 20. White, 111. F., a nd Kahn , C. R. ( 994) ,J. Bioi. Chem, 269 , 1-4 Iysat es were subjecte d to immunoprecipitation wit h an antibody to the 21. Torn qvist, II .. Gun salus, ,I. It , Nem en ofT, R. A., Fra ckelton, A. R., Pierce, M. p85 subunit of phosp hatidylinositol 3-kin ase (" p85). Proteins in th e W., a nd Avruc h, J . (1988 ) .]. Bioi. Chern, 26:1, 350- :159 immune complexes were se pa ra te d by SDS-PAGE (7%) an d tran sferred Wh ite, 1\1. F., lIIaron, H., and Kah n, H. C. (1985 ) Nature :118, 183- 186 to Immobilon-P membranes. The membranes were Western blotted 23. Tashi ro-Hash imoto, Y., Tobc, K., Kosh io, 0 ., Izumi, T., Tak aku, F., Akanuma, with an an ti-phos photyrosine antibody (uPY; upp er panel), stripped, Y., a nd Kasuga, M. (1989) .J. Bioi. Chem. 264, 6879 - 6885 and reblotted with eithe r' a n antibody against th e insu lin recepto r 24. Sun, X. J , Rothen berg, P., Kah n, H. C.. Backer, ,J. 111. Araki, K , Wilden, P. A., substra te 1 (,d RS-I) (middle pan el ) or " p85 (lower pan el). Immunore­ Cahill, D. A., Golds tein, B. J ., an d White, 1\1. r. (99 1) Nature :152, 73-77 25. Shoelson, S. Eo, Chatterj ee, 5 ., Cha udhuri , M.. a nd White, 1\1. F. ( 1992) Proc. active bands wer e visua lized by enha nced che milumines cence. The Notl. Acad. Sci . U. S . A. 89, 2027-20:11 migration of molecular weight markers are indicated to the right 26. Backer, ,I. 1\1., Myers, 111. G, J r., Sho elson, S. E., Chin, D. J ., Sun , X. J ., ( X 1000). Resul ts are represent ati ve of at least thr ee individual Mira lpeix, 111.. Hu, 1'.. Margolis, 13.. Skolnik , E. Y., Schless inger , J .. and experiments. Wh ite, 111 . F. ( 1992 ) EMIl O ,I. 9, :1469 -:1479 27. Izu mi, T., White, 111. F., Kad owak i, T., Tak aku, F., Akan uma, Y., an d Kasuga, 1\1. (987 ) J. Bioi. Chem, 262, 1282- 1287 of such events. Identification of the sites on IRS-! phosphoryl­ 28. Wang, 1..-111., Keegan, A. D., Pa ul , W. Eo, Heida ra n. 111. A.. Gutkind, J . S.. a nd ated in response to GH as well as the identi ty of the kinase Pierce, ,J. H. (1992) EMlJO ,J. 11, 4899- 4908 responsibl e for these actions will be of significant imp ortance. 29. Myers, 111. G., .Ir., Sun, X. ,J, Chea tha m, B., J ach na, B. R., Glash een , ~; . 111., Backer, J . Ill., an d White, M. F. (993) Endocrin ology 132 , 142 1-1 430 In additio n to the tyrosine- phosphoryla ted IRS-! , tyrosine­ 30. Wan g, L.-1II.. Keegan , A. D., Lienh a rd, G. E.. Pacini, S., Gu tki nd , ,J. S.. lIIyers, phosphorylated pp!80 was coimmunoprecipitated by a p85 in M. G.. Sun , X.-J .. White, 111. F.. Aaronson , S. A., Pa ul, W. E., a nd Pier ce, J . response to GH (Fig. 1) as well as IGF-I and insulin (Fig. 4). H . ( 993 ) Proc. Natl. Acad. S ci. lJ. S . A. 90, 4032- 4036 3 1. Myers, 111. G., -Jr., Sun, X. ,J., a nd White, 111. F. (994 ) Trends nt«. S ci. 19, Th e iden tity of pp!80 is not kn own, but it might be functionally 289 -293 related to IRS-! since it is tyrosine -phosphoryla ted and asso­ :12. Hodhell, 111. (1964) d . Bioi. Chern. 2:19, 375- 380 33. Gliernan , ,I. (1967) Dinbctolog ica :I, :182-388 cia tes wit h p85 in response to these horm ones. Interestingly, 34. Goodman, II . 1\1. ( 98 1) Endocrinology 109, 120 - 129 insuli n, IGF-I , a nd IL-4 stimulation of myeloid cell lines results 35. Kelly, K L.. and Huderm an , N. B. (993 ) J. Bioi. Chem . 268, 439 1- 4398 36. Gric ht ing, G., Levy, L. K , and Goodma n, H. l\l . (983 ) Endocrinol ogy 113, in tyrosine phosphorylation of a protein wit h relative molecular 1111- 1120 weight and fun ction similar to th at of IRS-! call ed 4PS (30). :17. Ilondo, 111. 111., Darnh olt , A. B., Cunningha m, B. A., Wells, ,I. A., De Meyts, 1'., Two recen t reports on transge nic mice lacking IRS-! expres­ a nd Sh ymko , H. 1\1. ( 994) Endocrin ology 1:14, 2397- 2403 38 . Keegan , A. D., Nelms, K , Whit e, 111., Wan g, L.-M., Pierce, J . II., a nd Paul. W. sion indica tes that a protein migra ting above th e position for E. (994) Cell 76, 8 11-820 IRS-! and designated IRS-2 exists in adi pocytes, liver, and 39. Gold, 1\1. R., Dur onio, V., Sax en a, S. 1'.. Schrade r, ,I. W., a nd Aebersold, R. muscle cells with functions similar to those of IRS-! (48, 49). (994 ) J. Bioi. Chern, 269, 5403 -54 12 40. White, 111. F., Livingston, J . N.. Backer , J . M., La uris, V., Dull , T. •J, Ullrich, A.. The tissue distribution of th ese proteins has not yet been es­ a nd Ka hn , R. (988 ) Cell 54, 641- 649 ta blished, but the pp!80 pr otein foun d here migh t be identical 41. Massague, ,J., a nd Czech, 111. P. (1982) J. Bioi. Chem . 257, 5038- 5045 42. Moody, A. J ., Stan , 111. A., Stan, 1\1., an d Gliema nn , J . ( 1974) Harm . Metab. Re». or related to 4PS or IRS-2. 6, 12- 1 43. Avruch , ,J., Tornqvist , II., Gun salus, J . R., Yurkow, E. ,I., Kyri akis. vl, 111.. a nd Acknowledgm ent- We gratefully acknowledge the excellent techni­ Pr ice, D. J . (990 ) Handbook of Experimental Pharmacology: l neulin , Vol. cal assista nce of Ann-Kri st in Holmen -Palbrink. 92, pp. 313-366, Spri nge r-Verlag, Berli n 44. Ander son, N. G. (1992) Bioc hem . J . 284, 649 - 652 REFERENCES 45 . Ca mpbell, G. S., Pan g, L.. lIliyasak a, T., Sa ltiel, A. H., and Curte r-Su, C. (1992) .]. Bioi. Chem . 267, 6074- 6080 J. Leung. D. W., Spencer . S. A., Cachianes, G.. Hammond s, R. G., Collins, C., Henzel, W. J ., Barn a rd , R., Wat er s, 111. ,J., a nd Wood, W. 1. (1987) Nature 46 . Schlootwe g, 111. C., de Groot, R. P., Herrma nn-Erlee, 111. P. 111., Koornneef, I., Kruijer, W.. a nd Kra mer, Y. 111. (991) ,I. Mal. Endocrinol. 6, 179 - 188 33 0, 537- 543 2. Cunningha m, B. C., Ultsch, 111., De Vos, A., Mulkerrin, M. G., Cla user , K R.. 47. Gurla nd, G., Ashcom, G.. Cochran, B. H., a nd Sch wartz, J . (990 ) En docrinology 127, 3 187- 3 196 and Wells, ,J. A. (199 1) S cience 254 , 821-825 3. Cosman, D.. Lym an , S. D., Idzerda, R. L., Beckm a nn , 111. 1'., Pa rk, I.. S., 48. Tarnemoto, H.. Kadowaki , T., Tobe, K , Yagi , T., Sakura, II. , Hayakawa, T., Ter a uch i, Y., Ueki, K , Kaburagi , Y., Sato h, S., Seki hara, H., Yosh ioka, S., Goodwin, R. G., a nd Ma rch, C. (990 ) Trends Bioi. Sci . 15, 265- 270 4. Argetsinger, L. S.. Cam pbell, G. S., Ya ng, X., Witthuhn, 8. A.. Silven noine n, Hori koshi , H., Furuta , Y., Ikawa, Y., Kasuga,lII ., Yazaki, Y., a nd Aiza wa, S. ( 1994) Na ture 372, 182-186 0., Ihle, ,J. N., a nd Carter-Su , C. (1993 ) Cell 74, 237-244 5. Ihle,.J. N., Witthuhn, B. A., Quelle, F. W., Yam am oto, K , Thierfeld er , W. E., 49. Araki, Eo, Lipes, 111. A., Patti, M. E., Bru nin g, ,J. C.. Haag, B. J ohnson , R. S.. a nd Kaha, C. H. (1994 ) Nature 372, 186-1 90 Kreider, 8. , an d Silven noinen, O. (994 ) Trends Biochem . S ci. 19, 222- 227 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Growth Hormone Stimulates the Tyrosine Phosphorylation of the Insulin Receptor Substrate-1 and Its Association with Phosphatidylinositol 3-Kinase in Primary Adipocytes

Ridderstråle, Martin; Degerman, Eva; Tornqvist, Hans
Journal of Biological Chemistry – Feb 1, 1995
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0021-9258
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10.1074/jbc.270.8.3471
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No.8, Issue of February 24, pp. 3471-3474, 1995 Communication © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. ample of a hormone presumably utilizing a wide array of in­ Growth Hormone Stimulates the tracellular proteins in its signaling chains. The initial event in Tyrosine Phosphorylation of the GH action involves binding and dimerization of its membrane receptor (1, 2). The GH receptor belongs to the cytokine recep­ Insulin Receptor Substrate-l and tor family, characterized by homologies in the extracellular Its Association with domains and lack of intrinsic tyrosine kinase activity (3). In­ stead, activation of receptor-associated cytosolic tyrosine ki­ Phosphatidylinositol3-Kinase in nases belonging to the Janus kinase family, in the case of GH Primary Adipocytes* the JAK2 tyrosine kinase (4), is an emerging theme for this family of receptors (5). In addition, a number of intracellular (Received for publication, October 5, 1994, and in revised form, key proteins have been suggested to be involved in GH signal­ December 20, 1994) ing further downstream of these or other, as yet unidentified, Martin Ridderstealet, Eva Degerman, and receptor-proximal events (6). Hans Tornqvist§ We have recently shown that the insulin-like effects ofGH in From the Departments of §Pediatrics and Medical and isolated rat adipocytes can be blocked by wortmannin, a selec­ Physiological Chemistry, University ofLund, P. O. Box tive inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase) (7). 94, S-221 00 Lund, Sweden A number of other hormones and growth factors such as insu­ Insulin receptor substrate-I (IRS-I) is tyrosine-phos­ lin, insulin-like growth factor-1 (IGF-D, platelet-derived phorylated in response to insulin resulting in associa­ growth factor, and different cytokines have been shown to tion with and activation of phosphatidylinositol 3-ki­ stimulate the activity of PI 3-kinase (8). PI 3-kinase is a dual nase (PI 3-kinase), thereby initiating some of the effects specificity lipid and serine kinase (9) consisting of a regulatory of insulin. We have recently shown that the insulin-like 85-kDa subunit (p85) containing two Src homology 2 (SH2) effects of growth hormone (GH) in adipocytes can be domains and a catalytic 110-kDa subunit (p110) (10, 11). The inhibited by the selective PI 3-kinase inhibitor wort­ role of its lipid kinase products, phosphatidylinositol 3-phos­ mannin (Ridderstr81e, M., and Tornqvist, H. (1994) Bio­ phate, phosphatidylinositol 3,4-bisphosphate, and phosphati­ chem. Biophys. Res. Commun. 203,306-310), suggesting a dylinositol 3,4,5-trisphosphate (12), as intracellular second similar role for PI 3-kinase in GH action. Here we show messengers is not known, but regulation of intracellular pro­ that mS-l is tyrosine-phosphorylated in a time- and tein trafficking (13) and/or activation ofthe ~-isoformof protein dose-dependent manner in response to GH in primary kinase C (14) have been suggested. The protein kinase activity rat adipocytes. This phosphorylation coincided with the of PI 3-kinase can phosphorylate p85 on serine resulting in a extent of interaction between IRS-I and the 85-kDa sub­ feed-back inhibition the catalytic activity of p110 (9). unit of PI 3·kinase as evidenced by coimmunoprecipita­ tion. Stimulation with 23 DM GH increased the PI 3-ki­ Insulin stimulation of glucose uptake and counteraction of nase activity associated with mS-l 4-fold. Our data lipolysis in adipocytes is believed to be mediated by activation suggest that GH·induced tyrosine phosphorylation of of PI 3-kinase (15-19). Insulin binding results in activation and IRS·I and the subsequent docking of PI 3-kinase are autophosphorylation of the insulin receptor tyrosine kinase on important postreceptor events in GO action. The mech­ specific tyrosines (20, 21). This is followed by tyrosine phospho­ anism for the phosphorylation ofIRS-I induced by GO is rylation of a major exogenous substrate for the receptor called unknown, but involvement of JAK2, the only known GO the insulin receptor substrate-1 (IRS-1) (22-24) on multiple receptor-associated tyrosine kinase, seems possible. tyrosines in YXXMIYMXM motifs (25), thus providing specific binding sites for the SH2-domains of p85 and subsequent acti­ vation of p110 (26). Furthermore, recent data suggest that PI Major advances have been made in recent years in elucidat­ 3-kinase can serine phosphorylate IRS-1 (19). In addition to ing the components of the intracellular signaling chains of insulin, IGF-I has been shown to utilize IRS-1 in intact cells different hormones, reaching from their membrane receptors to and interleukin 4 (IL-4) has been shown to utilize the function­ intranuclear events controlling transcription, or to intracellu­ ally related but immunologically distinct protein 4PS in mye­ lar target proteins controlling cellular metabolism. Growth loid cell lines (27-30). Tyrosine phosphorylation of IRS-1I4PS hormone (GH)l exerts pleiotropic actions on the growth, differ­ results in the association of several other proteins (Grb2, SH­ entiation, and metabolism of cells and constitutes a good ex- PTP2, and Nck) through their SH2 domains (31). To further explore the possible role of PI 3-kinase in GH * Financial support was given by the Swedish Medical Research Council (Project 8689), the Medical Faculty, University of Lund, action, we precipitated PI 3-kinase from GH-treated rat adipo­ Swedish Diabetic Association, and the Pahlsson, Novo Nordisk, cytes and looked for associated tyrosine-phosphorylated pro­ Crafoord and Bergwall Foundations. The costs of publication of this teins. Two major high molecular weight proteins were found. article were defrayed in part by the payment of page charges. This One of these was identified as IRS-1 by immunoblotting. IRS-1 article must therefore be hereby marked "advertisement" in accordance was tyrosine-phosphorylated and bound to the p85 subunit of with 18 U.S.C. Section 1734 solely to indicate this fact. :j:Towhomcorrespondenceshould be addressed: Dept. ofMedicaland PI 3-kinase in a dose- and time dependent manner in response Physiological Chemistry, Section of MolecularSignaling, P. O. Box94, to GH. S-221 00 Lund, Sweden. Fax: 46-46-10-40-22. 1 The abbreviations used are: GH, growth hormone; PI 3-kinase, EXPERIMENTAL PROCEDURES phosphatidylinositol 3-kinase; IRS-I, insulin receptor substrate-I; IGF-I, insulin-like growth factor-L; SH2, Src homology 2; IL-4, inter­ Adipocytes were prepared essentially according to Rodbell (32) leukin 4; PAGE,polyacrylamidegel electrophoresis. with modifications (7, 33) from 36-day-old Sprague-Dawleyrats (B&K, This is an Open Access article under the CC BY license. 3472 IRS-] and PI 3-Kinase in OR Action I.p. aiRS 1 apBS Sto ckholm, Swede n) fasted overnight prior to th e experime nts. After incubation with GH th e cells, 4 ml of a 10% cell sus pension (400 l.tI packed cell volume) for each condition , were homogeni zed in lysis buffer conta ining 10 rnxr Tri s-H Cl, pH 7.4, 0.25 ~I sucrose, I mxr EDTA, 100 IJ.M aPY orthova na da te, 0.1 mMdiisopropyl fluoroph osphate, I ug/ml pepst at in A, and 10 IJ.g/ml each of a nti pain and Ieup eptin. In some experiments - 205 cells wer e sti mulate d with GH, IGF-I, or insulin in th e presence or _IRS-1 abse nce of a neutralizing monoclonal anti-IGF-I antibody (recombinant - 116 hum an GH, human insul in , IGF -I, and anti-IGF-I (mAb 41) wer e gen­ erously supplied by Novo Nordi sk, Bagsvaerd , Denmark). Lysates free - 97 from fat and cellular debri s wer e supplemented with Triton X-100 to 1% (w/v) and solubilized for I h at 4 °C. Insolubl e material was removed by centrifuga tion and the lysat es incubated with eithe r anti-IRS-I anti­ body <0.7 IJ.g/mlJ or anti-p85 (PI 3-kinase) antise ra (1 : 1000) from Up­ state Biotechn ology Inc. (Lake Placid, NY). Immunoprecipitates were aIRS -1 OV- collecte d by adding 25 IJ.1of Pr otein A-Sepharose 4B (Pha rrnacia, Upp­ sa la, Sweden ), wash ed three tim es in 20 mxt Tris-HCl, pH 7.6, contain­ apBS _•• ing 137 mM NaCl, dissolved in SDS sa mple buffer, run on 7% SDS­ PAGE gels, and transferred to Imm obilon-P membranes. For Western blotting a polyclonal anti-phosphotyrosi ne antibody (a gene rous gift 1 2 3 4 from Dr. L. Riinnstrand, Ludwi g Institute, Uppsa la, Sweden ) was used FIG. 1. Tyr os ine-phosph o ry lated IR S- ! associates with PI 3-ki · at I IJ.g/ml , th e anti-IRS-I antibody at 0.7 J.Lg/ml , and th e anti-p85 n a se in r esponse to GH. Isolat ed rat adipocytes wer e prepared and antisera at a 1:1000 dilution. Western blotting, stripping th e blots, and treated as und er "Experimental Procedures." Cells were eithe r tr eat ed reblotting were performed using enha nced che miluminesce nce accord­ ( + ) or not trea ted (-) with 23 nxr GH for 10 min . Whole cell lysates wer e ing to th e manufacturer's instru ction s (Amers ham). subjec ted to immunoprecipitat ion with antibody again st th e insul in For ill vitro phosphatidylin ositol kinase assays, Protein A-Seph aro se receptor substrate- I (a IRS-I; lan es 1 a nd 2 ) or the p85 subunit of collecte d immunoprecipita tes were wash ed twice with lysis buffer sup­ phosph atidylin ositol 3-kinase (op85; lanes 3 and 4). Prot eins in th e plemented with Nonidet 1'-40 to 1% (w/v), three tim es with 10 mxt immune complexes were se parated by SDS-PAGE (7%) and trans ferred Tri s-HCI, pH 7.4, and 145 mxt NaCl, a nd twice with assay buffer to a n Imm obilon -P membrane. Th e membran e was Western blotted with an anti-phosph otyr osin e antibody (oPY; upper panel ), stri pped, conta ining 40 mxr Tri s-HCl, pH 7.4, 5 m ~1 MgCI , and 0.5 mM EGTA, and reblotte d with eithe r a IRS- I tmiddle panel ) or op85 ilotoer pan el ). and finally resusp end ed in 50 IJ.1 assay buffer. Th e assay was sta rte d by Immunoreactive bands were visua lized by enha nced che milumines­ addition of 50 IJ.1 of phosph oryl ation mix conta ining 0.2 mg/ml phos­ cence. Th e migration of molecular weight markers ar e indic ated to th e phatidylin ositol (a gift from Dr L. Krabisch, Lund, Sweden ), 0.01 mg/ml right ( x 1000). Results are representat ive of at least three individua l phosph atidylserine (Sigma), and 0.2 mxt [y_"2pIATP ( I IJ.Ci; Am ersharn) experime nts. in assay buffer. Reactions were terminated after 10 min at 30 °C by addition of 200 IJ.1 of I ~I HCI a nd 400 IJ.1 of methan ol:chloroform (1:1). The organic phase was recovered , and lipid s were dri ed down and dissolved in 20 IJ.1 of chloroform:metha nol (95:5), spotted onto Silica Gel PIP • • 60 plat es (Merck, Darmstad t, Germany), and developed in chloroform: methan ol.Hjfhammonium hydroxide (25%) (45:35:7.5:2.8). Phosph ati ­ dylin ositol 4-phosph ate, which comigra tes with 3-phosphorylated phos­ phatidylinositol , was used as sta nda rd. "21' incorp orat ed into phosphatidyl inositol was visua lized and qu an tified by Fuji x Bas 2000 Qd • Sys te m. GH RESULTS AND DISCUSSION FIG. 2. Increased PI 3-k inase a ctivity in anti-IRS- I im rnuno­ Isolated rat adipocytes were incubated in ab senc e of hor­ precipita tes fr om GH-treated cells. Isolated rat adipocytes were mon es for 3 h in order to restore responsiv eness to the insulin­ prepared and treat ed as describ ed under "Experimenta l Procedu res." Cells were eithe r treated ( +) or not treated (-) with GH (23 nxt) for 10 lik e effects ofGH (34). Th e cells were then resuspended in fresh min . Whole cell lysat es were subjected to immunoprecipitation with medium and treated with GH for varying times and at varying antibody agains t th e insulin receptor substra te- I. The precipitates were concentrations. Whole cell Iysates wer e subjected to immuno­ wash ed and imm ediately used for an ill vitro phosph atidylin ositol ki­ precipitation with antibodies against either p85 (a p85) or nase assay as described under "Experimental Procedures." The conver­ sion of phosphatidylin ositol to phosph atidylin ositol phosph ate in th e IRS-I (aIRS-I ). Th e immunoprecipitates were analyzed by presence of (y_"2PIATP was anal yzed by TLC. PIP indi cates th e migra­ Western blotting with anti-phosphotyrosine antibody (a PY) or tion of a phosphatidylinositol 4-phospha te sta nda rd. The origin, ori , with th e resp ective immunoprecipitating antibodies, or as­ contains an irrelevant residual amount of 1"2p1ATp th at was not re­ sayed for phosphatidylinositol kinase activity. moved by organic extrac tion. Results ar e representati ve of three indi­ IRS-I wa s phosphorylated on tyrosine in response to GH vidual experiments. (Fig. 1, lane 2, upper pan el) as revealed by a PY immunoblotting is probably due to incomplete precipitation of IRS-I with of a IRS-I immunoprecipitates. Furthermore, when reblotting with a p85, p85 wa s found to coimmunoprecipitate with IRS -I aIRS-I (data not shown). Yet, th e presence of other tyrosine­ in response to GH (Fig. 1, lan e 2, lower panel). Irnmunoprecipi­ phosphorylated protein s in th e a p85 precipitate cannot be ex­ tation with a p85 showed coimmunoprecipitation of two high cluded. To further explore th e significance of our findings, the aIRS-I immunoprecipitates were assayed for association of PI molecular weight tyrosine-phosphorylated protein s (Fig. 1, lan e 4, upper pan el), whereas neither p85 (Fig. 1, lan es 3 and 4) 3-kinase activity. Fig. 2 shows that GH sti mulate d a 4-fold nor pllO (dat a not shown) were phosphorylated on tyrosin e in increase (mean of 3 experi ments ) in phosphatidylinositol ki­ response to GH . One of th e p85-associated proteins migrated to nase activity associated with th e a IRS- I immunoprecipitate. Whether this reflects activation of th e kinase upon association the same position as IRS-I and wa s, upon reblotting, recog­ niz ed by a IRS-I (Fig. 1, lan e 4, middle panel). Th e other ty­ with IRS-lor translocation of active kinas e is not known (cr. sphorylated protein, tentatively called pp 180, mi­ Ref. 35). Insulin-induced docking of PI 3-kinase to IRS-I has rosin e-pho grated abov e the position for IRS-I and wa s not recognized by been shown to result in activation of th e kinase (26). a IRS-!. Apparently, crp85 immunoprecipitation brought down Having demonstrated that GH induces tyrosin e phosphoryl­ more of th e tyrosine -phosphorylated material corresponding to ation of IRS-I and as sociation of PI 3-kinase to IRS-I , we IRS-I than precipitated with a IRS-I (lan e 2 versus lane 4 ). This proce eded by inv esti gating the time course a nd dose depend- IRS-] and PI 3-Kinase in GH Action 3473 A 8 C oPY a PY aPY - l OS - l05 - 20 ~ .. ........-. IRS.' 4-- lAS·' _ ,RS·! - 116 - 116 - 116 _ 9 7 - 97 - ., ai RS·' ai RS·! ai RS· ' ------ ------ Dl>8S Dl>8S Dl>8S - - - - - _..---- Ort't,." ('I"t,." 0 GH(nM) GH( nM) 0"""""'''''0 Ti~ (IT'll") 0 ! 2 S 10 20 C?~ NN~ ~ "! N N :::: F IG. 3. GH stim u lates the tyrosine phosphoryla ti on of IRS- ! a n d its associatio n with P I 3-kinase in a dose- and time-dependent manner . Isolated rat adipocytes were prepa red and treated as describ ed under "Experimenta l Procedures." Th e cells wer e stimulated eithe r with 23 ml GH for 0-20 min (A) or with 0-230 nMGI-I for 10 min (B and C ). Whole cell lysates were subjected to immunoprecipitation with an antibody aga inst the insul in receptor substrate I (a iRS-I, A and B ) or th e p85 subunit of phosph atidylinositol 3-kinase (a p85, C). Proteins in the immune complexes were separa ted by SOS-PAGE (7%) an d tran sferr ed to Immobilon-P membranes. The membranes were Western blotted with an anti-phosphoty rosine antibody (a PY; upper pan els ), st ripped, and reblotted with either a IRS-I (m iddle panels ) or a p85 tlouier pan els ). Immun o­ ation of molecular weight mark ers are ind icat ed to the right ( X 1000). reactive bands were visua lized by enha nced chemiluminesce nce. Th e migr Resu lts are representative of at least three individua l experiments. ence of these eve nts (Fig. 3). Increased ty rosine phosphoryla ­ not induce det ectabl e ty rosine phos phorylation, i.e. receptor tion ofI RS-l (Fig. 3A , upper panel) as well as the appearance of ding tyrosine kinase activation, in the 95-kDa regio n correspon p85 in the a IRS-l immunoprecipitates (Fig. 3A, lower pa nel) to the l3-sub units of these receptors, as evidenced by SDS­ were seen within 2 min of GH stimulation reachin g a maximum PAGE of a PY-immunoprecipitated solubilized membrane pro­ at 5-10 min . Since GH effects have been dem onstrated over a teins from 32P-labeled adipocytes, in comparison to the effect of wide range of concen trations (36), it was in teresting to observe ins ulin.f It has also been shown that primary rat adipocytes do the dose dependenc e of th e effects induced by GH found in the not express IGF-I rece pto rs ca pabl e of bin ding IGF-I on the cell present inves tigation. Tyrosin e ph osphoryl ation of IRS- l (Fig. surface (41). It is also possible that GH might induce local 38, upper panel) and the subse quent asso ciation of p85 in produ ction of IGF-I from the adipocytes, which by binding to response to GH appeared at 0.23 nxt, reaching a maximum at IGF-I receptors or, at higher conce nt rations, to insu lin recep­ 23 mol, and the n to decr ease at 230 mol. In addition, th e dose­ tors could resul t in th e observed phosphorylation of IRS-I. This dependen t appearance of tyrosine-phos phorylated IRS-l and possibility was investigated by stimulating adipocytes with pp180 (Fig. 3C, upper pan el) and the association of the IRS-l GH, IGF-I , or ins ulin in presence or absence of a neutralizing pro tein with p85 (Fig. 3C, m iddle panel) in the a p85 immuno­ anti-IGF-I antibody (aIGF-I). As expected, IGF-I stimulated precipitates followed the same dose dependenc e. Similar GH both lipogen esis, measured as incorporation of[ Hlglucose in to dose-response curves (ED "" 1- 2 nl.l) ha ve been observed for adipocyte tri glycerides (42), and tyrosine phosphorylation of 5 0 th e ins ulin-like effects ofGH (36) and for the ty rosine phospho­ IRS-! in a dose-dependen t manner (data not shown). Half­ rylation of th e GH receptor in response to GH in adipocyt es.f maximal stimula tion (""20 nxr) by IGF -I was 100-1000 times The observed decrease at higher concentrations is likely to higher th an th at reported for ins ulin (43), indicating that IGF-I reflect the need of receptor dim erization for sign aling (37). acts through the insulin receptor and not the IGF-I receptor. From these results we conclude that GH stimulation of adi­ Th e presenc e of a IGF-I (5.4 p.g/ml ) clearly inhibited th e effect of pocytes results in a rapid and dose-dependent tyrosine phos­ IGF-I 0 0 nxr) bu t had no effect on GH- or insulin-induced IRS-l phorylation of IRS-l and association of th e p85 subunit of PI tyr osine phosphorylation (Fig. 4). An irreleva nt monoclonal 3-kinase under conditions whe n the hormone exerts its acute mouse antibody used as a control at the sa me concentration as insulin-like effects. Previous data obtained with th e selective a IGF-I had no effect on either horm onal effects (data not PI 3-kinase inhibitor wortmannin supports th is view (7). Our shown). We conclude that GH-induced tyrosine phosphoryla­ results have th e gene ral implication that stimulation through tio n of IRS-I and subsequent association wit h PI 3-ki na se is receptors of the cytokine receptor family, like the GH receptor, not med iated through local IGF-I production and activation of ma y result in tyrosine phosphoryl ation of IRS-1 and subse ­ the IGF-I- or ins ulin rece pto rs. quent association of PI 3-ki nase in primary cells. Th is ha s also Th e kin ase responsible for the IL-4-stimulated 4PS or IRS-l been shown for IL-4 stimulation of32D myeloid progeni tor cells phosphorylation has not been identified, but it is expected to be overex pressing IRS-1 (38). On the othe r hand, Gold et al. (39) a kin ase related to the Janus kinase family proteins or the Src could not demonstrate that sti mulation of hematopoieti c cell homology protein Fyn (3 1). A role for J AK2, which is tyrosine­ lines with othe r cytokin es resulted in phosphorylation of IRS-l phosphorylated in response to GH in 3T3-F442 A preadipocytes or related proteins associating with PI 3-kinase. (4) and adipocytes.f in the phosp horylation of IRS-! therefore It is not known which tyrosine kinase is responsible for the see ms possible. GH-induced phosphorylation of IRS-I. For insulin it has been In addit ion to PI 3-kinase, several othe r SH2 domain-con­ propo sed that th e insulin receptor tyrosine 960 has a func tion taining proteins <Grb2, SH-PT P2, and Nck) have been shown to in enabling the receptor kin ase to phosphorylate IRS-l (40). associa te with IRS-l phosphorylated by the insu lin receptor Tyrosine 960 is positioned in a seque nce motif, NPXY, which is (31). GH-induced tyrosi ne phosphorylation of IRS-l might turn also found in th e IL-4 and IGF-I receptors (38). Thi s motif ha s, out to be a n important tool in th e investigation of specificity in however, not been found in the GH receptor. A role for th e th e sign als initiated by IRS-1 phosphorylation . It has for ex­ insulin- or IGF-I receptor kinases seems unlikely, since GH did ample been shown th at GH stimula tion results in activation of MAP kinase and S6 kin ase activity as well as induction of c-Fos and c-Jun (44-47). Our findings suggest that GH-induced ty­ 2 H. Eriksson, M. Ridderstrale, and H. Tornq vist , manuscript in preparation. rosine phosphorylation of IRS-I migh t be positioned upstream 3474 IR S -l and PI 3-Kinase in GH Action 6. Roup as, P., a nd Herington, A. C. (1994) Trends Endocrinol. Metab. 5, 154- 158 7. Ridderstr le., ~l.. . and Tom oy ist . H. (1994) Biochem. Bioph )'s. Res. Comm un, IGF· I Ins C GH 203, 306 - 3 10 r---1r----1r-"1r-J 8. Va rticovski, L., Ha rrison-Findik, D.. Keeler , 111. L.. and Susa , l\l . ( 994) ((IGF· 1 . . . . Biochim, Biophys. A cto 1226, 1-11 9. Dhand. R.. Hiles, 1., Panayotou , G.. Hache, S.. Fry , 111.,J.. Gout , 1., Tott y, N. F., aPY Truong, 0 ., Vicen do, 1'., Yonezawa, K.. Kasuga, M., Courtneidge, S. A., and - ZOS Waterfield, 111. D. (994 ) EMBO J . 1:1, 522- 533 ~PP 180 10. Carpenter , C. L., Duckworth, Il. C.. Auger , K R., Cohen , 13.. Scha ffha use n, Il. IRS·l S.. and Ca ntle y, I.. C. (990 ) J. Biol , Chem, 265 , 19704 -1 9711 1J. Escobedo, J . A., Nav a nkasattu sas, S., Ka va na ugh , W. 111.. Milfay, D., Fri ed, V. - II. A., an d Willia ms, I.. T. (99 1) Cell 65, 413-423 12. Whitm a n, III., Ka plan, D. H., Roberts, T. III., and Ca ntley, L. C. (1988) Nature 3:12, 644 - 646 13. Panayotou , G., and Wa terfield , M. ( HJ92) Trends Cell Bioi. 2, 358 - 360 a iRS·' .- 14. Na ka nish i, H., Brewer, K A.. a nd Exton . vl, II. ( 1993 ).J. Bioi. Chem . 268 , 13- 16 15. Kelly, K 1.., Ruderma n, N. B., a nd Che n, K S. (992 ) J. Iliol. Chern. 267, opes :1423-3428 ---_.... 16. Ka nai. F.. Ito, K.. Todaka, 111., Hayash i, H., Kam oha ra , S., Ishii, K , Okada, T., Ha zeki, 0 ., Ui, M.. a nd Ebina, Y. (993) Bioc hem, Biophvs. Res. Commu n, 195, 762-768 FIG. 4. Ne u traliz in g IGF·( a n ti body does not block GH-sti m u­ 17. Okada, T.. Kawan o, Y., Sakakibara, T.. Hazeki, 0 ., an d Ui, ~1. ( 994) .]. Bioi. lated tyrosine phosphorylation of IR S-l a n d pp180. Isolated rat Chem, 269, 3568- 3573 ad ipocyt es were prepared and tr eated as descri bed un der "Experimen­ 18. Hahn, T.. Ridderstrale, M., Fredrikse n. G., Tornqvist, H., Mangia nello, V., Belfrage, 1'., a nd Deger man . E. ( 1994) FEllS Lett. :150, 3 14 - 3 18 tal Procedures." Th e cells (2 011, 10% sus pension) were stimulated wit h 19. Lam , K.. Ca r pente r, C. L., Ruderm an, N. 13.. Fri el. vl . C.. a nd Kelly, K I.. (1994) 23 nxtGH, 10 nxt IGF-I, 1 nxt insulin or vehicl e fOl'1O min in the ab sen ce J. lii ol. Chem. 269, 2064 8-20652 or presence of5.4 Jlg/ml monoclonal lGF-1antibody (a IGF-I). Whole cell 20. White, 111. F., a nd Kahn , C. R. ( 994) ,J. Bioi. Chem, 269 , 1-4 Iysat es were subjecte d to immunoprecipitation wit h an antibody to the 21. Torn qvist, II .. Gun salus, ,I. It , Nem en ofT, R. A., Fra ckelton, A. R., Pierce, M. p85 subunit of phosp hatidylinositol 3-kin ase (" p85). Proteins in th e W., a nd Avruc h, J . (1988 ) .]. Bioi. Chern, 26:1, 350- :159 immune complexes were se pa ra te d by SDS-PAGE (7%) an d tran sferred Wh ite, 1\1. F., lIIaron, H., and Kah n, H. C. (1985 ) Nature :118, 183- 186 to Immobilon-P membranes. The membranes were Western blotted 23. Tashi ro-Hash imoto, Y., Tobc, K., Kosh io, 0 ., Izumi, T., Tak aku, F., Akanuma, with an an ti-phos photyrosine antibody (uPY; upp er panel), stripped, Y., a nd Kasuga, M. (1989) .J. Bioi. Chem. 264, 6879 - 6885 and reblotted with eithe r' a n antibody against th e insu lin recepto r 24. Sun, X. J , Rothen berg, P., Kah n, H. C.. Backer, ,J. 111. Araki, K , Wilden, P. A., substra te 1 (,d RS-I) (middle pan el ) or " p85 (lower pan el). Immunore­ Cahill, D. A., Golds tein, B. J ., an d White, 1\1. r. (99 1) Nature :152, 73-77 25. Shoelson, S. Eo, Chatterj ee, 5 ., Cha udhuri , M.. a nd White, 1\1. F. ( 1992) Proc. active bands wer e visua lized by enha nced che milumines cence. The Notl. Acad. Sci . U. S . A. 89, 2027-20:11 migration of molecular weight markers are indicated to the right 26. Backer, ,I. 1\1., Myers, 111. G, J r., Sho elson, S. E., Chin, D. J ., Sun , X. J ., ( X 1000). Resul ts are represent ati ve of at least thr ee individual Mira lpeix, 111.. Hu, 1'.. Margolis, 13.. Skolnik , E. Y., Schless inger , J .. and experiments. Wh ite, 111 . F. ( 1992 ) EMIl O ,I. 9, :1469 -:1479 27. Izu mi, T., White, 111. F., Kad owak i, T., Tak aku, F., Akan uma, Y., an d Kasuga, 1\1. (987 ) J. Bioi. Chem, 262, 1282- 1287 of such events. Identification of the sites on IRS-! phosphoryl­ 28. Wang, 1..-111., Keegan, A. D., Pa ul , W. Eo, Heida ra n. 111. A.. Gutkind, J . S.. a nd ated in response to GH as well as the identi ty of the kinase Pierce, ,J. H. (1992) EMlJO ,J. 11, 4899- 4908 responsibl e for these actions will be of significant imp ortance. 29. Myers, 111. G., .Ir., Sun, X. ,J, Chea tha m, B., J ach na, B. R., Glash een , ~; . 111., Backer, J . Ill., an d White, M. F. (993) Endocrin ology 132 , 142 1-1 430 In additio n to the tyrosine- phosphoryla ted IRS-! , tyrosine­ 30. Wan g, L.-1II.. Keegan , A. D., Lienh a rd, G. E.. Pacini, S., Gu tki nd , ,J. S.. lIIyers, phosphorylated pp!80 was coimmunoprecipitated by a p85 in M. G.. Sun , X.-J .. White, 111. F.. Aaronson , S. A., Pa ul, W. E., a nd Pier ce, J . response to GH (Fig. 1) as well as IGF-I and insulin (Fig. 4). H . ( 993 ) Proc. Natl. Acad. S ci. lJ. S . A. 90, 4032- 4036 3 1. Myers, 111. G., -Jr., Sun, X. ,J., a nd White, 111. F. (994 ) Trends nt«. S ci. 19, Th e iden tity of pp!80 is not kn own, but it might be functionally 289 -293 related to IRS-! since it is tyrosine -phosphoryla ted and asso­ :12. Hodhell, 111. (1964) d . Bioi. Chern. 2:19, 375- 380 33. Gliernan , ,I. (1967) Dinbctolog ica :I, :182-388 cia tes wit h p85 in response to these horm ones. Interestingly, 34. Goodman, II . 1\1. ( 98 1) Endocrinology 109, 120 - 129 insuli n, IGF-I , a nd IL-4 stimulation of myeloid cell lines results 35. Kelly, K L.. and Huderm an , N. B. (993 ) J. Bioi. Chem . 268, 439 1- 4398 36. Gric ht ing, G., Levy, L. K , and Goodma n, H. l\l . (983 ) Endocrinol ogy 113, in tyrosine phosphorylation of a protein wit h relative molecular 1111- 1120 weight and fun ction similar to th at of IRS-! call ed 4PS (30). :17. Ilondo, 111. 111., Darnh olt , A. B., Cunningha m, B. A., Wells, ,I. A., De Meyts, 1'., Two recen t reports on transge nic mice lacking IRS-! expres­ a nd Sh ymko , H. 1\1. ( 994) Endocrin ology 1:14, 2397- 2403 38 . Keegan , A. D., Nelms, K , Whit e, 111., Wan g, L.-M., Pierce, J . II., a nd Paul. W. sion indica tes that a protein migra ting above th e position for E. (994) Cell 76, 8 11-820 IRS-! and designated IRS-2 exists in adi pocytes, liver, and 39. Gold, 1\1. R., Dur onio, V., Sax en a, S. 1'.. Schrade r, ,I. W., a nd Aebersold, R. muscle cells with functions similar to those of IRS-! (48, 49). (994 ) J. Bioi. Chern, 269, 5403 -54 12 40. White, 111. F., Livingston, J . N.. Backer , J . M., La uris, V., Dull , T. •J, Ullrich, A.. The tissue distribution of th ese proteins has not yet been es­ a nd Ka hn , R. (988 ) Cell 54, 641- 649 ta blished, but the pp!80 pr otein foun d here migh t be identical 41. Massague, ,J., a nd Czech, 111. P. (1982) J. Bioi. Chem . 257, 5038- 5045 42. Moody, A. J ., Stan , 111. A., Stan, 1\1., an d Gliema nn , J . ( 1974) Harm . Metab. Re». or related to 4PS or IRS-2. 6, 12- 1 43. Avruch , ,J., Tornqvist , II., Gun salus, J . R., Yurkow, E. ,I., Kyri akis. vl, 111.. a nd Acknowledgm ent- We gratefully acknowledge the excellent techni­ Pr ice, D. J . (990 ) Handbook of Experimental Pharmacology: l neulin , Vol. cal assista nce of Ann-Kri st in Holmen -Palbrink. 92, pp. 313-366, Spri nge r-Verlag, Berli n 44. Ander son, N. G. (1992) Bioc hem . J . 284, 649 - 652 REFERENCES 45 . Ca mpbell, G. S., Pan g, L.. lIliyasak a, T., Sa ltiel, A. H., and Curte r-Su, C. (1992) .]. Bioi. Chem . 267, 6074- 6080 J. Leung. D. W., Spencer . S. A., Cachianes, G.. Hammond s, R. G., Collins, C., Henzel, W. J ., Barn a rd , R., Wat er s, 111. ,J., a nd Wood, W. 1. (1987) Nature 46 . Schlootwe g, 111. C., de Groot, R. P., Herrma nn-Erlee, 111. P. 111., Koornneef, I., Kruijer, W.. a nd Kra mer, Y. 111. (991) ,I. Mal. Endocrinol. 6, 179 - 188 33 0, 537- 543 2. Cunningha m, B. C., Ultsch, 111., De Vos, A., Mulkerrin, M. G., Cla user , K R.. 47. Gurla nd, G., Ashcom, G.. Cochran, B. H., a nd Sch wartz, J . (990 ) En docrinology 127, 3 187- 3 196 and Wells, ,J. A. (199 1) S cience 254 , 821-825 3. Cosman, D.. Lym an , S. D., Idzerda, R. L., Beckm a nn , 111. 1'., Pa rk, I.. S., 48. Tarnemoto, H.. Kadowaki , T., Tobe, K , Yagi , T., Sakura, II. , Hayakawa, T., Ter a uch i, Y., Ueki, K , Kaburagi , Y., Sato h, S., Seki hara, H., Yosh ioka, S., Goodwin, R. G., a nd Ma rch, C. (990 ) Trends Bioi. Sci . 15, 265- 270 4. Argetsinger, L. S.. Cam pbell, G. S., Ya ng, X., Witthuhn, 8. A.. Silven noine n, Hori koshi , H., Furuta , Y., Ikawa, Y., Kasuga,lII ., Yazaki, Y., a nd Aiza wa, S. ( 1994) Na ture 372, 182-186 0., Ihle, ,J. N., a nd Carter-Su , C. (1993 ) Cell 74, 237-244 5. Ihle,.J. N., Witthuhn, B. A., Quelle, F. W., Yam am oto, K , Thierfeld er , W. E., 49. Araki, Eo, Lipes, 111. A., Patti, M. E., Bru nin g, ,J. C.. Haag, B. J ohnson , R. S.. a nd Kaha, C. H. (1994 ) Nature 372, 186-1 90 Kreider, 8. , an d Silven noinen, O. (994 ) Trends Biochem . S ci. 19, 222- 227

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