Major advances have been made in recent years in elucidating the components of the intracellular signaling chains of different hormones, reaching from their membrane receptors to intranuclear events controlling transcription, or to intracellular target proteins controlling cellular metabolism. Growth hormone (GH) ()exerts pleiotropic actions on the growth, differentiation, and metabolism of cells and constitutes a good example of a hormone presumably utilizing a wide array of intracellular proteins in its signaling chains. The initial event in GH action involves binding and dimerization of its membrane receptor(1, 2) . The GH receptor belongs to the cytokine receptor family, characterized by homologies in the extracellular domains and lack of intrinsic tyrosine kinase activity(3) . Instead, activation of receptor-associated cytosolic tyrosine kinases belonging to the Janus kinase family, in the case of GH the JAK2 tyrosine kinase(4) , is an emerging theme for this family of receptors(5) . In addition, a number of intracellular key proteins have been suggested to be involved in GH signaling further downstream of these or other, as yet unidentified, receptor-proximal events(6) .

We have recently shown that the insulin-like effects of GH in isolated rat adipocytes can be blocked by wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase)(7) . A number of other hormones and growth factors such as insulin, insulin-like growth factor-1 (IGF-I), platelet-derived growth factor, and different cytokines have been shown to stimulate the activity of PI 3-kinase(8) . PI 3-kinase is a dual specificity lipid and serine kinase (9) consisting of a regulatory 85-kDa subunit (p85) containing two Src homology 2 (SH2) domains and a catalytic 110-kDa subunit (p110)(10, 11) . The role of its lipid kinase products, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate(12) , as intracellular second messengers is not known, but regulation of intracellular protein trafficking (13) and/or activation of the -isoform of protein kinase C (14) have been suggested. The protein kinase activity of PI 3-kinase can phosphorylate p85 on serine resulting in a feed-back inhibition the catalytic activity of p110(9) .

Insulin stimulation of glucose uptake and counteraction of lipolysis in adipocytes is believed to be mediated by activation of PI 3-kinase (15, 16, 17, 18, 19) . Insulin binding results in activation and autophosphorylation of the insulin receptor tyrosine kinase on specific tyrosines(20, 21) . This is followed by tyrosine phosphorylation of a major exogenous substrate for the receptor called the insulin receptor substrate-1 (IRS-1) (22, 23, 24) on multiple tyrosines in YXXM/YMXM motifs(25) , thus providing specific binding sites for the SH2-domains of p85 and subsequent activation of p110(26) . Furthermore, recent data suggest that PI 3-kinase can serine phosphorylate IRS-1(19) . In addition to insulin, IGF-I has been shown to utilize IRS-1 in intact cells and interleukin 4 (IL-4) has been shown to utilize the functionally related but immunologically distinct protein 4PS in myeloid cell lines(27, 28, 29, 30) . Tyrosine phosphorylation of IRS-1/4PS results in the association of several other proteins (Grb2, SH-PTP2, and Nck) through their SH2 domains(31) .

To further explore the possible role of PI 3-kinase in GH action, we precipitated PI 3-kinase from GH-treated rat adipocytes and looked for associated tyrosine-phosphorylated proteins. Two major high molecular weight proteins were found. One of these was identified as IRS-1 by immunoblotting. IRS-1 was tyrosine-phosphorylated and bound to the p85 subunit of PI 3-kinase in a dose- and time dependent manner in response to GH.

EXPERIMENTAL PROCEDURES

Adipocytes were prepared essentially according to Rodbell (32) with modifications (7, 33) from 36-day-old Sprague-Dawley rats (B&, Stockholm, Sweden) fasted overnight prior to the experiments. After incubation with GH the cells, 4 ml of a 10% cell suspension (400 µl packed cell volume) for each condition, were homogenized in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 100 µM orthovanadate, 0.1 mM diisopropyl fluorophosphate, 1 µg/ml pepstatin A, and 10 µg/ml each of antipain and leupeptin. In some experiments cells were stimulated with GH, IGF-I, or insulin in the presence or absence of a neutralizing monoclonal anti-IGF-I antibody (recombinant human GH, human insulin, IGF-I, and anti-IGF-I (mAb 41) were generously supplied by Novo Nordisk, Bagsvaerd, Denmark). Lysates free from fat and cellular debris were supplemented with Triton X-100 to 1% (w/v) and solubilized for 1 h at 4 °C. Insoluble material was removed by centrifugation and the lysates incubated with either anti-IRS-1 antibody (0.7 µg/ml) or anti-p85 (PI 3-kinase) antisera (1:1000) from Upstate Biotechnology Inc. (Lake Placid, NY). Immunoprecipitates were collected by adding 25 µl of Protein A-Sepharose 4B (Pharmacia, Uppsala, Sweden), washed three times in 20 mM Tris-HCl, pH 7.6, containing 137 mM NaCl, dissolved in SDS sample buffer, run on 7% SDS-PAGE gels, and transferred to Immobilon-P membranes. For Western blotting a polyclonal anti-phosphotyrosine antibody (a generous gift from Dr. L. Rönnstrand, Ludwig Institute, Uppsala, Sweden) was used at 1 µg/ml, the anti-IRS-1 antibody at 0.7 µg/ml, and the anti-p85 antisera at a 1:1000 dilution. Western blotting, stripping the blots, and reblotting were performed using enhanced chemiluminescence according to the manufacturer's instructions (Amersham).

For in vitro phosphatidylinositol kinase assays, Protein A-Sepharose collected immunoprecipitates were washed twice with lysis buffer supplemented with Nonidet P-40 to 1% (w/v), three times with 10 mM Tris-HCl, pH 7.4, and 145 mM NaCl, and twice with assay buffer containing 40 mM Tris-HCl, pH 7.4, 5 mM MgCl, and 0.5 mM EGTA, and finally resuspended in 50 µl assay buffer. The assay was started by addition of 50 µl of phosphorylation mix containing 0.2 mg/ml phosphatidylinositol (a gift from Dr L. Krabisch, Lund, Sweden), 0.01 mg/ml phosphatidylserine (Sigma), and 0.2 mM [-P]ATP (1 µCi; Amersham) in assay buffer. Reactions were terminated after 10 min at 30 °C by addition of 200 µl of 1 M HCl and 400 µl of methanol:chloroform (1:1). The organic phase was recovered, and lipids were dried down and dissolved in 20 µl of chloroform:methanol (95:5), spotted onto Silica Gel 60 plates (Merck, Darmstadt, Germany), and developed in chloroform:methanol:HO:ammonium hydroxide (25%) (45:35:7.5:2.8). Phosphatidylinositol 4-phosphate, which comigrates with 3-phosphorylated phosphatidylinositol, was used as standard. P incorporated into phosphatidylinositol was visualized and quantified by Fujix Bas 2000 System.

RESULTS AND DISCUSSION

Isolated rat adipocytes were incubated in absence of hormones for 3 h in order to restore responsiveness to the insulin-like effects of GH(34) . The cells were then resuspended in fresh medium and treated with GH for varying times and at varying concentrations. Whole cell lysates were subjected to immunoprecipitation with antibodies against either p85 (p85) or IRS-1 (IRS-1). The immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine antibody (PY) or with the respective immunoprecipitating antibodies, or assayed for phosphatidylinositol kinase activity.

IRS-1 was phosphorylated on tyrosine in response to GH (Fig. 1, lane2, upperpanel) as revealed by PY immunoblotting of IRS-1 immunoprecipitates. Furthermore, when reblotting with p85, p85 was found to coimmunoprecipitate with IRS-1 in response to GH (Fig. 1, lane2, lowerpanel). Immunoprecipitation with p85 showed coimmunoprecipitation of two high molecular weight tyrosine-phosphorylated proteins (Fig. 1, lane4, upperpanel), whereas neither p85 (Fig. 1, lanes3 and 4) nor p110 (data not shown) were phosphorylated on tyrosine in response to GH. One of the p85-associated proteins migrated to the same position as IRS-1 and was, upon reblotting, recognized by IRS-1 (Fig. 1, lane4, middle panel). The other tyrosine-phosphorylated protein, tentatively called pp180, migrated above the position for IRS-1 and was not recognized by IRS-1. Apparently, p85 immunoprecipitation brought down more of the tyrosine-phosphorylated material corresponding to IRS-1 than precipitated with IRS-1 (lane 2 versuslane4). This is probably due to incomplete precipitation of IRS-1 with IRS-1 (data not shown). Yet, the presence of other tyrosine-phosphorylated proteins in the p85 precipitate cannot be excluded. To further explore the significance of our findings, the IRS-1 immunoprecipitates were assayed for association of PI 3-kinase activity. Fig. 2shows that GH stimulated a 4-fold increase (mean of 3 experiments) in phosphatidylinositol kinase activity associated with the IRS-1 immunoprecipitate. Whether this reflects activation of the kinase upon association with IRS-1 or translocation of active kinase is not known (cf. (35) ). Insulin-induced docking of PI 3-kinase to IRS-1 has been shown to result in activation of the kinase(26) .

Figure 1: Tyrosine-phosphorylated IRS-1 associates with PI 3-kinase in response to GH. Isolated rat adipocytes were prepared and treated as under ``Experimental Procedures.'' Cells were either treated (+) or not treated(-) with 23 nM GH for 10 min. Whole cell lysates were subjected to immunoprecipitation with antibody against the insulin receptor substrate-1 (IRS-1; lanes1 and 2) or the p85 subunit of phosphatidylinositol 3-kinase (p85; lanes3 and 4). Proteins in the immune complexes were separated by SDS-PAGE (7%) and transferred to an Immobilon-P membrane. The membrane was Western blotted with an anti-phosphotyrosine antibody (PY; upperpanel), stripped, and reblotted with either IRS-1 (middlepanel) or p85 (lowerpanel). Immunoreactive bands were visualized by enhanced chemiluminescence. The migration of molecular weight markers are indicated to the right ( 1000). Results are representative of at least three individual experiments.

Figure 2: Increased PI 3-kinase activity in anti-IRS-1 immunoprecipitates from GH-treated cells. Isolated rat adipocytes were prepared and treated as described under ``Experimental Procedures.'' Cells were either treated (+) or not treated(-) with GH (23 nM) for 10 min. Whole cell lysates were subjected to immunoprecipitation with antibody against the insulin receptor substrate-1. The precipitates were washed and immediately used for an in vitro phosphatidylinositol kinase assay as described under ``Experimental Procedures.'' The conversion of phosphatidylinositol to phosphatidylinositol phosphate in the presence of [-P]ATP was analyzed by TLC. PIP indicates the migration of a phosphatidylinositol 4-phosphate standard. The origin, ori, contains an irrelevant residual amount of [P]ATP that was not removed by organic extraction. Results are representative of three individual experiments.

Having demonstrated that GH induces tyrosine phosphorylation of IRS-1 and association of PI 3-kinase to IRS-1, we proceeded by investigating the time course and dose dependence of these events (Fig. 3). Increased tyrosine phosphorylation of IRS-1 (Fig. 3A, upperpanel) as well as the appearance of p85 in the IRS-1 immunoprecipitates (Fig. 3A, lowerpanel) were seen within 2 min of GH stimulation reaching a maximum at 5-10 min. Since GH effects have been demonstrated over a wide range of concentrations(36) , it was interesting to observe the dose dependence of the effects induced by GH found in the present investigation. Tyrosine phosphorylation of IRS-1 (Fig. 3B, upperpanel) and the subsequent association of p85 in response to GH appeared at 0.23 nM, reaching a maximum at 23 nM, and then to decrease at 230 nM. In addition, the dose-dependent appearance of tyrosine-phosphorylated IRS-1 and pp180 (Fig. 3C, upperpanel) and the association of the IRS-1 protein with p85 (Fig. 3C, middlepanel) in the p85 immunoprecipitates followed the same dose dependence. Similar GH dose-response curves (ED 1-2 nM) have been observed for the insulin-like effects of GH (36) and for the tyrosine phosphorylation of the GH receptor in response to GH in adipocytes. ()The observed decrease at higher concentrations is likely to reflect the need of receptor dimerization for signaling(37) .

Figure 3: GH stimulates the tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase in a dose- and time-dependent manner. Isolated rat adipocytes were prepared and treated as described under ``Experimental Procedures.'' The cells were stimulated either with 23 nM GH for 0-20 min (A) or with 0-230 nM GH for 10 min (B and C). Whole cell lysates were subjected to immunoprecipitation with an antibody against the insulin receptor substrate 1 (IRS-1, A and B) or the p85 subunit of phosphatidylinositol 3-kinase (p85, C). Proteins in the immune complexes were separated by SDS-PAGE (7%) and transferred to Immobilon-P membranes. The membranes were Western blotted with an anti-phosphotyrosine antibody (PY; upperpanels), stripped, and reblotted with either IRS-1 (middlepanels) or p85 (lowerpanels). Immunoreactive bands were visualized by enhanced chemiluminescence. The migration of molecular weight markers are indicated to the right ( 1000). Results are representative of at least three individual experiments.

From these results we conclude that GH stimulation of adipocytes results in a rapid and dose-dependent tyrosine phosphorylation of IRS-1 and association of the p85 subunit of PI 3-kinase under conditions when the hormone exerts its acute insulin-like effects. Previous data obtained with the selective PI 3-kinase inhibitor wortmannin supports this view(7) . Our results have the general implication that stimulation through receptors of the cytokine receptor family, like the GH receptor, may result in tyrosine phosphorylation of IRS-1 and subsequent association of PI 3-kinase in primary cells. This has also been shown for IL-4 stimulation of 32D myeloid progenitor cells overexpressing IRS-1(38) . On the other hand, Gold et al.(39) could not demonstrate that stimulation of hematopoietic cell lines with other cytokines resulted in phosphorylation of IRS-1 or related proteins associating with PI 3-kinase.

It is not known which tyrosine kinase is responsible for the GH-induced phosphorylation of IRS-1. For insulin it has been proposed that the insulin receptor tyrosine 960 has a function in enabling the receptor kinase to phosphorylate IRS-1(40) . Tyrosine 960 is positioned in a sequence motif, NPXY, which is also found in the IL-4 and IGF-I receptors(38) . This motif has, however, not been found in the GH receptor. A role for the insulin- or IGF-I receptor kinases seems unlikely, since GH did not induce detectable tyrosine phosphorylation, i.e. receptor tyrosine kinase activation, in the 95-kDa region corresponding to the -subunits of these receptors, as evidenced by SDS-PAGE of PY-immunoprecipitated solubilized membrane proteins from P-labeled adipocytes, in comparison to the effect of insulin. It has also been shown that primary rat adipocytes do not express IGF-I receptors capable of binding IGF-I on the cell surface(41) . It is also possible that GH might induce local production of IGF-I from the adipocytes, which by binding to IGF-I receptors or, at higher concentrations, to insulin receptors could result in the observed phosphorylation of IRS-1. This possibility was investigated by stimulating adipocytes with GH, IGF-I, or insulin in presence or absence of a neutralizing anti-IGF-I antibody (IGF-I). As expected, IGF-I stimulated both lipogenesis, measured as incorporation of [H]glucose into adipocyte triglycerides(42) , and tyrosine phosphorylation of IRS-1 in a dose-dependent manner (data not shown). Half-maximal stimulation (20 nM) by IGF-I was 100-1000 times higher than that reported for insulin(43) , indicating that IGF-I acts through the insulin receptor and not the IGF-I receptor. The presence of IGF-I (5.4 µg/ml) clearly inhibited the effect of IGF-I (10 nM) but had no effect on GH- or insulin-induced IRS-1 tyrosine phosphorylation (Fig. 4). An irrelevant monoclonal mouse antibody used as a control at the same concentration as IGF-I had no effect on either hormonal effects (data not shown). We conclude that GH-induced tyrosine phosphorylation of IRS-1 and subsequent association with PI 3-kinase is not mediated through local IGF-I production and activation of the IGF-I- or insulin receptors.

Figure 4: Neutralizing IGF-I antibody does not block GH-stimulated tyrosine phosphorylation of IRS-1 and pp180. Isolated rat adipocytes were prepared and treated as described under ``Experimental Procedures.'' The cells (2 ml, 10% suspension) were stimulated with 23 nM GH, 10 nM IGF-I, 1 nM insulin or vehicle for 10 min in the absence or presence of 5.4 µg/ml monoclonal IGF-I antibody (IGF-I). Whole cell lysates were subjected to immunoprecipitation with an antibody to the p85 subunit of phosphatidylinositol 3-kinase (p85). Proteins in the immune complexes were separated by SDS-PAGE (7%) and transferred to Immobilon-P membranes. The membranes were Western blotted with an anti-phosphotyrosine antibody (PY; upperpanel), stripped, and reblotted with either an antibody against the insulin receptor substrate 1 (IRS-1) (middlepanel) or p85 (lowerpanel). Immunoreactive bands were visualized by enhanced chemiluminescence. The migration of molecular weight markers are indicated to the right ( 1000). Results are representative of at least three individual experiments.

The kinase responsible for the IL-4-stimulated 4PS or IRS-1 phosphorylation has not been identified, but it is expected to be a kinase related to the Janus kinase family proteins or the Src homology protein Fyn(31) . A role for JAK2, which is tyrosine-phosphorylated in response to GH in 3T3-F442A preadipocytes (4) and adipocytes, in the phosphorylation of IRS-1 therefore seems possible.

In addition to PI 3-kinase, several other SH2 domain-containing proteins (Grb2, SH-PTP2, and Nck) have been shown to associate with IRS-1 phosphorylated by the insulin receptor(31) . GH-induced tyrosine phosphorylation of IRS-1 might turn out to be an important tool in the investigation of specificity in the signals initiated by IRS-1 phosphorylation. It has for example been shown that GH stimulation results in activation of MAP kinase and S6 kinase activity as well as induction of c-Fos and c-Jun(44, 45, 46, 47) . Our findings suggest that GH-induced tyrosine phosphorylation of IRS-1 might be positioned upstream of such events. Identification of the sites on IRS-1 phosphorylated in response to GH as well as the identity of the kinase responsible for these actions will be of significant importance.

In addition to the tyrosine-phosphorylated IRS-1, tyrosine-phosphorylated pp180 was coimmunoprecipitated by p85 in response to GH (Fig. 1) as well as IGF-I and insulin (Fig. 4). The identity of pp180 is not known, but it might be functionally related to IRS-1 since it is tyrosine-phosphorylated and associates with p85 in response to these hormones. Interestingly, insulin, IGF-I, and IL-4 stimulation of myeloid cell lines results in tyrosine phosphorylation of a protein with relative molecular weight and function similar to that of IRS-1 called 4PS(30) . Two recent reports on transgenic mice lacking IRS-1 expression indicates that a protein migrating above the position for IRS-1 and designated IRS-2 exists in adipocytes, liver, and muscle cells with functions similar to those of IRS-1(48, 49) . The tissue distribution of these proteins has not yet been established, but the pp180 protein found here might be identical or related to 4PS or IRS-2.

FOOTNOTES

*

Financial support was given by the Swedish Medical Research Council (Project 8689), the Medical Faculty, University of Lund, Swedish Diabetic Association, and the Påhlsson, Novo Nordisk, Crafoord and Bergwall Foundations. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Medical and Physiological Chemistry, Section of Molecular Signaling, P. O. Box 94, S-221 00 Lund, Sweden. Fax: 46-46-10-40-22.

()

The abbreviations used are: GH, growth hormone; PI 3-kinase, phosphatidylinositol 3-kinase; IRS-1, insulin receptor substrate-1; IGF-I, insulin-like growth factor-1; SH2, Src homology 2; IL-4, interleukin 4; PAGE, polyacrylamide gel electrophoresis.

()

H. Eriksson, M. Ridderstråle, and H. Tornqvist, manuscript in preparation.

ACKNOWLEDGEMENTS

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