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Endocrinology Vol. 141, No. 9 3328-3336
Copyright © 2000 by The Endocrine Society

ARTICLES

Insulin Receptor Substrate-1-Mediated Enhancement of Growth Hormone-Induced Mitogen-Activated Protein Kinase Activation1

Liang Liang, Jing Jiang and Stuart J. Frank

From the Department of Medicine, Division of Endocrinology and Metabolism (L.L., J.J., S.J.F.) and the Department of Cell Biology (S.J.F.), University of Alabama at Birmingham, and the Veterans Affairs Medical Center (S.J.F.), Birmingham, Alabama 35294

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, Room 756, DERB UAB Station, 1808 7th Avenue South, Birmingham, Alabama 35294. E-mail: frank{at}endo.dom.uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 ~ 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 µM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
GH EXERTS a range of growth promoting and metabolic effects in a wide variety of vertebrate species by specifically interacting with the GH receptor (GHR) (1). The GH-GHR interaction and consequent GHR dimerization leads to activation of the GHR-associated cytoplasmic tyrosine kinase, JAK2 (2, 3, 4). Activation of the GHR-JAK2 complex results in variable engagement of several intracellular signaling systems, including the signal transducer and activator of transcription (STAT, principally STAT5), Ras/Raf/MEK1/ERK (MAP kinase), and insulin receptor substrate (IRS)/PI3 kinase (PI3K) pathways (5, 6, and references therein).

Depending on the target cell type or tissue in question and the experimental system studied, GH-induced engagement of particular pathways has been linked to some of the biological effects of GH. For example, STAT5B knockout mice lack the GH-dependent sexual dimorphism of hepatic gene expression characteristic of rodents; these animals also manifest defects in body growth and adipocyte differentiation (7). Likewise, the ability of GH to promote differentiation of 3T3-F442A preadipocytes into adipocytes is dependent on its ability to activate STAT5B (8).

Engagement of pathways leading to activation of MAP kinases ERK1 and ERK2 is also important in signaling certain GH-induced biological and biochemical outcomes. While not required for GH priming of adipocyte differentiation, later stages in the differentiation process, for example, are blocked in the presence of MAP kinase antisense oligonucleotides (8). MAP kinase activation also appears to be critical in allowing GH-induced c-fos, egr-1, and junB gene transcription and activation of the Elk-1 transcription factor (9). Further, we recently observed in 3T3-F442A cells a GH-induced serine/threonine phosphorylation of ErbB-2, which renders ErbB-2 less activatable by EGF and correlates with GH’s antagonism of EGF-induced mitogenesis in that cell type (10, 11). The ErbB-2 serine/threonine phosphorylation is blocked when the cells are treated before GH addition with PD98059, an inhibitor of MEK1 that prevents GH-induced activation of ERK1 and ERK2, implicating a role for the MAP kinase pathway also in this potentially important GH-mediated cross-talk with the EGF signaling system.

While GH induction of the MAP kinase pathway may thus facilitate some GH actions, the determinants that allow GHR engagement to result in MAP kinase activation are not yet fully understood. Previous work indicates that physical and functional association of the GHR with a catalytically competent JAK2 is required for GH-induced MAP kinase activation (12). Correspondingly, region(s) in the proximal cytoplasmic domain of the GHR involved in association with and activation of JAK2 are also required for coupling to MAP kinase activity (13, 14, 15). However, not all cells that express GHRs and JAK2 and respond to GH with JAK2 activation exhibit significant GH-induced MAP kinase activation (16). While the reason(s) for this variable access of GH signaling to MAP kinase activation is uncertain, GH induction of MAP kinase activity, when operative, very likely involves the Ras/Raf/MEK1 pathway upstream of ERKs-1 and -2 (17, 18, 19).

Using the IRS-1 and -2 deficient, factor-dependent, murine 32D promonocytic cell line, we recently demonstrated that reconstitution with the rabbit (rb) GHR and IRS-1 compared with rbGHR alone conferred marked enhancement in GH-induced cell proliferation (20). Further, 32D-rbGHR-IRS-1 cells exhibited substantially increased GH-induced MAP kinase activation compared with 32D-rbGHR cells (20). In this study, we extended this observation and showed in the 32D system that both IRS-1-independent and IRS-1-enhanced GH-induced ERK1 and ERK2 activation were largely blocked by wortmannin, an inhibitor of PI3K activity. This sensitivity of GH-induced MAP kinase activation to wortmannin was correlated with sensitivity of GH-induced MEK1 activation to the same inhibitor. However, GH-induced MAP kinase activation was either less sensitive (in IRS-1-containing cells) or insensitive (in cells lacking IRS-1) to another PI3K inhibitor, LY294002, suggesting that other wortmannin-sensitive enzymes in addition to PI3K might be involved in mediation of GH-induced MAP kinase activation. IRS-1 expression in this system also enhanced both basal and GH-induced Ras activation, indicating that at least part of IRS-1’s positive influence on GH-induced MAP kinase activation might reflect its ability to lessen the threshold for productive Ras pathway engagement. In contrast to Ras and MAP kinase activation, IRS-1 expression in this system did not affect GH-induced STAT5 tyrosine phosphorylation, suggesting a selective influence of IRS-1 on access to GH-induced signaling pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Materials
Recombinant hGH was kindly provided by Eli Lilly & Co. (Indianapolis, IN). Routine reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted.

Cell culture and generation of stable transfectants
The generation of 32D-rbGHR and 32D-rbGHR-IRS-1 cells has been previously described (20, 21). In brief, factor-dependent murine promonocytic 32D cells were cotransfected by electroporation with the rabbit GHR complementary DNA (cDNA) in the pSX eukaryotic expression vector and with a vector [pRc/CMV (Invitrogen, Rockville, MD)] that encodes the neomycin resistance marker. Stably transfected cells were selected by growth in G418 (0.8 mg/ml, Life Technologies, Inc., Gaithersburg, MD) without IL-3-containing medium and were previously referred to as 32D-rGHR (21). To generate the cells used in studies of IRS-1’s effects on GH signaling (Ref. 20 and the current study) these 32D-rGHR cells were used as a target for either IRS-1 or vector control transfection. Pools of 32D cells stably coexpressing the rabbit GHR and either rat IRS-1 (32D-rbGHR-IRS-1) or no IRS-1 (histidinol resistance vector only, now referred to as 32D-rbGHR) were similarly generated by electroporation of 32D-rGHR cells (2 x 107/ml in complete medium; 250 V, 960 µF in a GenePulser (Bio-Rad Laboratories, Inc., Richmond, CA) electroporator) with the pSX-driven IRS-1 cDNA and a vector (pCMV) that encodes the histidinol resistance marker. Coselection of cells expressing the rabbit GHR and IRS-1 protein was carried out in G418 and histidinol (2 mM, Sigma). Both 32D-rbGHR and 32D-rbGHR-IRS-1 cells were cultured in RPMI 1640 medium supplemented with 7% FBS, 2 mM histidinol, 0.8 mg/ml G418, and 50 µg/ml gentamicin sulfate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all Biofluids, Carlsbad, CA).

Antibodies
Anti-MAPK affinity-purified rabbit antibody (directed at residues 333–367 of rat ERK1; recognizes both ERK1 and ERK2) and anti-p85 rabbit serum were purchased from Upstate Biotechnology, Inc., Lake Placid, NY (UBI). Antiactivated (anti-phospho-) MAPK affinity-purified rabbit antibody (recognizing the dually phosphorylated Thr183 and Tyr185 residues that correspond to the active forms of ERK1 and ERK2) was purchased from Promega Corp. (Madison, WI). Affinity purified anti-MEK1/2 antibody and anti-phospho-MEK1/2 rabbit antibody (specifically recognizing MEK1 and MEK2 that are phosphorylated at residues Ser217 and Ser221), anti-Akt, and anti-phospho-Akt (specifically recognizing Akt phosphorylated at Ser473) affinity purified rabbit antibodies were purchased from New England Biolabs, Inc. (Beverly, MA). Rabbit polyclonal anti-Raf-1 (C-12) and anti-SHP2 (anti-SH-PTP2) (C-18) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-SHP-2 serum (21) (for immunoprecipitation) was a gift from Dr. G.-S. Feng. Anti-Ras monoclonal antibody and anti-STAT5 monoclonal antibody (which recognizes both STAT5A and STAT5B) were purchased from Transduction Laboratories, Inc. (Lexington, KY). Rabbit antiphosphotyrosine-STAT5 polyclonal antibody (raised against a phosphopeptide surrounding phosphorylated Tyr694 of murine STAT5A, which is conserved in both STAT5A and STAT5B) was obtained from Zymed Laboratories, Inc. (San Francisco, CA).

Cell stimulation, protein extraction, and immunoblotting
32D-rbGHR and 32D-rbGHR-IRS-1 cells were serum starved in DMEM by substitution of 0.5% (wt/vol) BSA (fraction V, Roche Molecular Biochemicals, Indianapolis, IN) for serum in the culture medium for 4–6 h before experiments and the cells were resuspended for experiments in binding buffer (BB, consisting of 25 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 0.1% (wt/vol) BSA, and 1 mM dextrose) at 37 C. Pretreatments with PD98059 (100 µM, New England Biolabs, Inc.), GF109203X (500 nM, Calbiochem, San Diego, CA), wortmannin, and LY294002 (at indicated concentrations), or vehicle control (0.1–0.2% DMSO) were for 15 min before GH stimulation, as indicated. hGH (GH) was used at a final concentration of 500 ng/ml in BB for 15 min at 37 C. Stimulated cells were collected by centrifugation (800 x g for one minute at 4 C) and aspiration of the BB. The pelleted cells were solubilized for 15 min at 4 C in fusion lysis buffer (1% (vol/vol) Triton X-100, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, 10 µg/ml aprotinin), as indicated. After centrifugation at 15,000 x g for 15 min at 4 C, the detergent extracts were resolved under reduced conditions by SDS-PAGE. Western transfer of proteins and blocking of Hybond-ECL membranes (Amersham Pharmacia Biotech, Arlington Heights, IL) with 2% BSA were performed as previously described (4, 11, 20, 21). Membranes were immunoblotted with 1 µg/ml or the indicated dilutions of antibodies against MAPK, phospho-MAPK (1:20,000), MEK1/2 (1:1000), phospho-MEK1/2 (1:1000), Raf-1, Ras (1:1000), STAT5 (1:1000), phosphotyrosine-STAT5 (1:5000), Akt (1:1000), phospho-Akt (1:1000), SHP-2 (1:500), or p85 (1:2000). Detection by ECL detection reagents (all from Amersham Pharmacia Biotech) and stripping and reprobing of blots were accomplished according to the manufacturer’s suggestions.

Assay for detection of activated Ras
Activated Ras interaction assays were performed as described previously (22, 23). A glutathione S-transferase (GST) fusion protein containing the Ras binding domain (RBD) of Raf-1 (residues 1–149 of Raf-1), which binds only GTP-bound Ras, was prepared from the pGEX-RBD plasmid (kindly provided by Drs. R. Carter and L. Xiaoli, UAB). Induction and affinity purification of GST-RBD on glutathione-agarose beads (Amersham Pharmacia Biotech) were performed as described previously (20). For affinity precipitation of activated Ras with GST-RBD, 20 million cells/sample (treated with or without GH, as indicated) were solubilized in a lysis buffer consisting of 0.5% (vol/vol) Nonidet P-40, 0.1% (vol/vol) deoxycholate, 150 mM NaCl, 10% (vol/vol) glycerol, 50 mM HEPES (pH 7.5), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovandate, 10 mM benzamidine, and 10 µg/ml aprotinin. Purified GST-RBD (20 µg per sample) bound to glutathione-agarose beads was incubated with clarified detergent extracts (90% of the total extract) for 30 min at 4 C. The beads were washed extensively with lysis buffer and the bound proteins were eluted in reduced SDS sample buffer and resolved by SDS-PAGE, as was the remaining 10% of each extract. Both the relative abundance of Ras bound to the GST-RBD and total Ras present in unprecipitated extracts were detected by anti-Ras immunoblotting.

Densitometric analysis
Densitometry of ECL immunoblots was performed using a solid state video camera (Sony 77, Sony Corp., Tokyo, Japan) and a 28 mm MicroNikkor lens over a lightbox of variable intensity (Northern Light Precision 890, Imaging Research, Inc., Toronto, Canada). Quantification was performed using a Macintosh II-based image analysis program (Image 1.61, developed by W. S. Rasband, Research Services Branch, NIMH, Bethesda, MD). Basal and GH-induced MAPK and MEK1/2 activities were estimated for 32D-rbGHR and 32D-rbGHR-IRS-1 by normalizing the relative total ERK (ERK1 plus ERK2) or MEK1/2 densitometric signals of each sample’s antiactivated MAPK or anti-phospho-MEK1 blot by that of the stripped and reprobed anti-MAPK or anti-MEK1/2 blot (that is, normalizing the activated ERK1 and ERK2 for ERK1 and ERK2 abundance and the phosphorylated MEK1/2 for MEK1/2 abundance), as previously (20).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The insulin receptor substrate (IRS) molecules IRS-1 and -2 were first described as prominent proximal tyrosine phosphorylated targets of insulin (IRS-1) and interleukin-4 (IRS-2) signaling (24, 25). When tyrosine phosphorylated, these proteins form docking sites for SH2-containing signaling molecules including Grb-2, SHP-2, and the p85 regulatory subunit of PI3K (26); indeed, IRS-1 (and the other IRS family members, which now include IRS-3 and -4 (27, 28) in addition to IRS-2) is a principal mediator of insulin-induced PI3K activation. GH also promotes tyrosine phosphorylation of IRS-1, -2, and -3, their association with p85, and the consequent activation of PI3K (29, 30, 31, 32, 33). In studying the involvement of IRS-1 in GH signaling, we recently described a specific nonphosphotyrosine-dependent association between JAK2 and IRS-1, which mapped to regions in the amino terminus of IRS-1 (20). Further, we showed that reconstitution of 32D cells with IRS-1 and rbGHR (32D-rbGHR-IRS-1) resulted in enhanced GH-induced proliferation when compared with cells expressing rbGHR alone (32D-rbGHR) (20).

Though 32D-rbGHR-IRS-1 and 32D-rbGHR exhibited similar cell surface 125I-hGH binding and GHR abundance by immunoblotting and possessed comparable JAK2 protein levels and GH-induced JAK2 tyrosine phosphorylation, 32D-rbGHR-IRS-1 cells responded to GH with more robust MAP kinase activation (20). This phenomenon was explored in more detail in the experiment shown in Fig. 1Go. The level of activation of MAP kinases ERK1 and ERK2 in 32D-rbGHR and 32D-rbGHR-IRS-1 induced by varying duration of exposure to GH was estimated by immunoblotting of detergent cell extracts from each cell with an antibody that specifically recognizes the phosphorylated threonine-183 and tyrosine-185 residues in the MAP kinase molecules that correlate to their enzymatic activation. In both cells, GH promoted similarly transient activation of ERK1 and ERK2 within 15 min that, in concert with our previous findings, was substantially (roughly 3.4-fold) more robust in 32D-rbGHR-IRS-1 than in 32D-rbGHR (Fig. 1Go, upper panel, lanes 9, 10 vs. 2, 3). Similar abundance of ERK1 and ERK2 in each cell and comparable protein loading for each sample were verified by blotting of the same extracts with anti-MAPK (Fig. 1Go, lower panel).



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  		Figure 1. Time course of GH-promoted activation of MAP kinases (ERK1 and ERK2) in 32D-rbGHR and 32D-rbGHR-IRS-1 cells. 32D-rbGHR and 32D-rbGHR-IRS-1 cells were treated with 500 ng/ml GH for indicated durations. Detergent extracts (2 million cells per condition) were resolved by SDS-PAGE and sequentially immunoblotted with anti-phospho-MAPK (pMAPK) (upper panel) and anti-MAPK (lower panel) antibodies. The positions of MAPK (ERK1/ERK2 and pERK1/pERK2) are indicated. The experiment shown is representative of three such experiments.

 
We sought to characterize further the biochemical enhancement of GH-induced MAP kinase activation in the IRS-1-containing cells. We first determined the degree to which potential upstream activation pathways that could lead to MAP kinase activation might be affected by IRS-1 overexpression. PD98059 is an inhibitor of MAP kinase activation that traps the upstream activator of ERKs, MEK1, in its inactive conformation, thus preventing its ability to efficiently phosphorylate ERK1 and ERK2 (34). As expected, pretreatment with PD98059 (PD) substantially reduced GH-induced ERK1 and ERK2 phosphorylation to similar levels in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells (Fig. 2AGo, lanes 3, 4 vs. 1, 2 and 9, 10 vs. 7, 8) (49% and 68% reduction by PD98059 for 32D-rbGHR and 32D-rbGHR-IRS-1, respectively, in this particular experiment). It is not yet certain whether residual ERK1 and ERK2 phosphorylation in the presence of PD98059 reflects its incomplete blockade of MEK1 in these cells or the possibility that a component of MAP kinase activation is not mediated by MEK1. (We have similarly noted a small degree of residual GH-induced (11) and platelet-derived growth factor-induced (Guan, R., Y. Zhang, J. Jiang, C. A. Baumann, R. A. Black, G. Baumann, and S. J. Frank, manuscript in preparation) ERK1 and ERK2 phosphorylation in the presence of PD98059 in 3T3-F442A preadipocytes.)



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  		Figure 2. GH-induced MAP kinase activation is sensitive to a MEK1, but not a PKC, inhibitor in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. A, 32D-rbGHR and 32D-rbGHR-IRS-1 cells were pretreated with the MEK1 inhibitor, PD98059 (PD, 100 µM), the PKC inhibitor, GF109203X (GF, 500 nM), or the DMSO vehicle (-, 0.2% final) for 15 min before treatment with GH (+) or its vehicle (-) for an additional 15 min. Detergent extracts (2 million cells per condition) were resolved by SDS-PAGE and sequentially immunoblotted with anti-phospho-MAPK (pMAPK) (upper panel) and anti-MAPK (lower panel) antibodies. The experiment shown is representative of five such experiments. B, Cells were pretreated with the PKC inhibitor, GF109203X (GF, +, 500 nM) or DMSO vehicle (-, 0.2% final) for 15 min before treatment with PMA (1 µg/ml) for an additional 15 min. Detergent extracts (2 million cells per condition) were resolved by SDS-PAGE and immunoblotted with anti-phospho-MAPK (pMAPK) antibodies. Note the similarity of PMA-induced pMAPK signal in 32D-rbGHR and 32D-rbGHR-IRS-1 cells and its inhibition by GF109203X in both cells.

 
Protein kinase C activity and/or particular protein kinase C isozymes have been implicated by some in GH-induced MAP kinase activation in some cell types (35); others have not detected such a PKC dependency (36, 37). We therefore tested whether the IRS-1-augmentation of GH-induced MAP kinase activation in 32D-rbGHR cells might be affected by inhibition of PKC. In contrast to the effects of PD98059, pretreatment of 32D-rbGHR and 32D-rbGHR-IRS-1 cells with GF109203X (GF), an inhibitor of most PKC isozymes, had no effect in either cell type on the abundance of phosphorylated ERK1 and ERK2 observed in response to GH (Fig. 2AGo, lanes 5, 6 vs. 1, 2 and 11, 12 vs. 7, 8). As a control, we tested the effects of the phorbol ester, PMA, on MAP kinase activation (Fig. 2BGo). In contrast to the effects of GH, PMA elicited nearly identical acute activation of ERK1 and ERK2 phosphorylation in 32D-rbGHR and 32D-rbGHR-IRS-1 cells (Fig. 2BGo, lane 1 vs. 3), indicating a lack of difference in MAP kinase activatability when PKC is pharmacologically activated. The efficacy of GF109203X was further revealed in this experiment in that PMA-induced ERK1 and ERK2 phosphorylation was nearly completely blocked in both cells when GF109203X was present (Fig. 2BGo, lanes 2 vs. 1 and 4 vs. 3). In other experiments, PD98059 (100 µM) also substantially and equivalently inhibited PMA-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1, as expected (not shown). Collectively, these data indicate that in 32D cells, ERK1 and ERK2 abundance and pharmacologic activatability are not affected by IRS-1 expression, but GH-induced MAP kinase activation, which occurs most likely via a nonPKC-dependent (or at least nonGF109203X-inhibitable) pathway, is more robust in IRS-1-expressing cells.

Because IRS proteins are prominently linked to the PI3K pathway and others have shown inhibition of GH-induced MAP kinase activation in 3T3-F442A cells by pretreatment with the PI3K inhibitor, wortmannin (10–500 nM) (9, 38), we tested whether IRS-1’s enhancement of GH-induced MAP kinase activation in our cells was similarly sensitive to such inhibition. We first tested whether wortmannin was effective at inhibiting PI3K activation in 32D-rbGHR and 32D-rbGHR-IRS-1 cells by tracking the GH-induced activation of Akt, a serine/threonine kinase downstream of PI3K (Fig. 3Go). The relative level of activation of Akt was assessed by immunoblotting of cell extracts with a state-specific anti-Akt antibody that detects only Akt phosphorylated at residue Ser473; this phosphorylation correlates to activation of the molecule (39). GH induced Akt phosphorylation in both cells, but substantially more so in 32D-rbGHR-IRS-1 than 32D-rbGHR (Fig. 3Go, upper panel, lanes 2 vs. 1 and 7 vs. 6), whereas Akt levels were similar in the two cell types (Fig. 3Go, lower panel). Pretreatment with wortmannin (wort) at concentrations of 10 nM or greater inhibited GH-induced Akt phosphorylation in both cells (Fig. 3Go, upper panel, lanes 2–5 and 7–10). As expected, neither PD98059 nor GF109203X pretreatment had any effect on GH-induced Akt activation (data not shown), consistent with the lack of known effects of either of these two drugs on PI3K activity. Because 32D cells do not express IRS-1, -2, -3, or -4 (40, 41, 42), the GH-induced activation of Akt phosphorylation and its inhibition by wortmannin in the absence of reconstituted IRS-1 in 32D-rbGHR indicates that GH can couple to a PI3K inhibitor-sensitive pathway in the absence of IRS proteins, but that IRS-1 substantially augments this coupling. Whether IRS-independent coupling of GH activation to this pathway is via other IRS-like adapters, such as Gab-1 (43) or -2 (44) or via JAK2 (which has two YXXM consensus sites (45) that, when phosphorylated, could form binding sites for the p85 SH2 domains) is not yet known.



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  		Figure 3. GH-induces Akt phosphorylation that is sensitive to the PI3K inhibitor, wortmannin. 32D-rbGHR and 32D-rbGHR-IRS-1 cells were pretreated with the indicated concentrations (nM) of wortmannin or its vehicle (-) for 15 min before treatment with GH (+) or its vehicle (-) for an additional 15 min. Detergent extracts (2 million cells per condition) were resolved by SDS-PAGE and sequentially immunoblotted with anti-phospho-Akt (pAkt) (upper panel) and anti-Akt (lower panel) antibodies. The positions of Akt and pAkt are indicated. The experiment shown is representative of six such experiments.

 
Interestingly, as seen in Fig. 4AGo, pretreatment with wortmannin inhibited GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, similarly to its inhibition of Akt phosphorylation in Fig. 3Go. Like MAP kinase activation, GH-induced activation of MEK1 (as assessed by immunoblotting with a state-specific antiphosphorylated MEK1 antibody in Fig. 4BGo, upper panel) was also detectable in both cells, augmented in the IRS-1-containing cells, and sensitive to wortmannin with a similar concentration dependence. A difference in MEK1 abundance could not explain the augmentation of MEK1 activation brought about in the 32D-rbGHR-IRS-1 cells as both cells had similar MEK1 levels (Fig. 4BGo, lower panel). The results of several experiments such as those in Fig. 4Go, A and B, are summarized in Fig. 5Go, A and B, in which the immunoblotting signals for activated MAP kinases (Fig. 5AGo) and phosphorylated MEK1 (Fig. 5BGo), each normalized for the abundance of the particular protein, are plotted. These data indicate that GH-induced MEK1 and MAP kinase activation, though augmented in the presence of IRS-1, are inhibited by wortmannin with very similar concentration dependencies in the presence or absence of IRS-1. In both cells, 500 nM wortmannin nearly completely inhibited GH-induced ERK1 and ERK2 activation and MEK1 activation.



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  		Figure 4. GH-induced MAP kinase and MEK1 phosphorylation is sensitive to wortmannin in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. A and B, Cells were pretreated with wortmannin or vehicle control and then stimulated with GH as in Fig. 3Go. Detergent extracts (2 million cells per condition) were resolved by SDS-PAGE and sequentially immunoblotted with anti-phospho-MAPK (A, upper panel) and anti-MAPK (A, lower panel) antibodies or anti-phospho-MEK1/2 (B, upper panel) and anti-MEK1/2 (B, lower panel) antibodies. The positions of MAPK (ERK1/ERK2 and pERK1/pERK2) and MEK and pMEK are indicated. The experiments shown are each representative of six such experiments.

 


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  		Figure 5. Quantitative comparison of wortmannin sensitivity of GH-induced MAP kinase and MEK1 phosphorylation and LY294002 sensitivity of GH-induced MAP kinase phosphorylation in 32D-rbGHR and 32D-rbGHR-IRS-1 cells. A and B, Densitometric assessment of wortmannin sensitivity of GH-induced MAP kinase (A) and MEK1/2 (B) phosphorylation, as described in Materials and Methods from six independent experiments. In each case, the signals within each experiment are plotted with respect to the GH-stimulated 32D-rbGHR-IRS-1 sample (the maximum in each experiment) for comparability purposes. Error bars are SEM. P values of unpaired t tests for indicated comparisons are displayed. C, Densitometric assessment of sensitivity of GH-induced MAP kinase phosphorylation to 50 µM LY294002, as in A, from three independent experiments. By unpaired t test: P < 0.001 for comparison of 32D-rbGHR-IRS-1 cells, - vs. + LY294002; NS for comparison of 32D-rbGHR cells, - vs. + LY294002.

 
We also tested the effect of another PI3K inhibitor, LY294002, on GH-induced MAP kinase activation in 32D-rbGHR and 32D-rbGHR-IRS-1 cells (Fig. 5CGo). Pretreatment with 50 µM LY294002 caused on average a statistically significant 72% decline in GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells. Pretreatment with 5 µM LY294002 also caused a significant decline (29% on average) in relative MAP kinase activation in these cells (not shown). In contrast to the findings in 32D-rbGHR-IRS-1 cells, 50 µM LY294002 pretreatment resulted in only a modest (25% on average) decline in GH-induced MAP kinase activation that did not achieve statistical significance (Fig. 5CGo) and no inhibition was observed when 5 µM LY294002 was used (not shown). Further, pretreatment with 5 µM LY294002 resulted in nearly complete inhibition of GH-induced Akt phosphorylation in both cells (not shown). Thus, while there is substantial and significant LY294002 inhibition of GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, this is not the case for 32D-rbGHR cells and LY294002 appears to more potently inhibit PI3K (as inferred from Akt activation) than MAP kinase activation in the IRS-1 containing cells. This is in distinction to our findings for wortmannin (Figs. 4Go and 5Go, A and B) which exhibits nearly superimposable inhibitory dose dependencies for GH-induced MAP kinase, MEK1/2, and Akt phosphorylation/activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells.

We are at this point uncertain as to how to entirely explain the discrepancy in the dose dependencies of the two PI3K inhibitors. We note that it has previously been observed by Scheid and Duronio (46) in studying GM-CSF-induced MAP kinase activation in a factor-dependent cell line that wortmannin substantially inhibited MAP kinase activation at the same concentration (100 nM) that it nearly completely inhibited GM-CSF-induced PI3K activity; however, in that system, LY294002 (25 µM) only minimally (by roughly 16%) inhibited GM-CSF-induced MAP kinase activation, but completely inhibited PI3K activation. These authors cited other studies in which LY294002 inhibited MAP kinase activation by various ligands only at concentrations higher than those expected to be required for PI3K inhibition, but that wortmannin more often inhibited PI3K and MAP kinase activation with similar dose dependencies (discussed in Ref. 46). It was concluded that wortmannin and LY294002 might be differentially inhibiting enzymes other than PI3K that function upstream of MAP kinase activation.

While those observations are relevant for interpretation of our results, it should be recognized that our data are, however, somewhat different in that LY294002 even at 5 µM did significantly inhibit GH-induced MAP kinase activation partially in 32D-rbGHR-IRS-1 cells, but not in 32D-rbGHR cells. Ours are the only data of which we are aware in which the involvement of PI3K in GH-induced MAP kinase activation has been tested using LY294002 in addition to wortmannin. As stated above, pretreatment with wortmannin (in a concentration range that typically inhibits PI3K) blocks GH-induced MAP kinase activation in the 3T3-F442A cell line (9, 38), a line in which GH-induced IRS-1 tyrosine phosphorylation occurs (31). From our data and that available in the GH signaling literature, we can conservatively conclude that wortmannin, in addition to inhibiting PI3K activity, may be inhibiting another as yet undefined aspect of GH-induced MAP kinase activation and that IRS-1 substantially augments access to wortmannin-sensitive MAP kinase activation pathway(s) in the 32D cell system.

Our previous work indicated that proximal aspects of GH signaling such as GHR and JAK2 abundance and GH-induced JAK2 tyrosine phosphorylation were similar in 32D-rbGHR and 32D-rbGHR-IRS-1 cells (20). To determine further whether the presence of IRS-1 differentially affected GH signaling pathways other than the PI3K/Akt and MAP kinase pathways in these cells, we examined GH-induced STAT5 phosphorylation, using an antibody (antiphosphotyrosine-STAT5) that specifically detects the tyrosine phosphorylated form of STAT5 (Fig. 6AGo). The level of anti-STAT5-reactive proteins (two predominant forms, presumably STAT5A and STAT5B) detected by immunoblotting was similar in the two cells (Fig. 6AGo, lower panel, lane 1 vs. lane 3). Likewise, the level of tyrosine phosphorylated STAT5 detected in extracts of each cell in response to GH was very similar (Fig. 6AGo, upper panel, lane 1 and 2 vs. 3 and 4), indicating that, though IRS-1 confers enhanced GH-induced activation of PI3K (reflected by phosphorylated Akt) and MAP kinase (ERK1 and ERK2 phosphorylation), activation of the STAT5 tyrosine phosphorylation is relatively unaffected by IRS-1 expression. This is an important result in that it indicates a selectivity in the pathways differentially accessed in response to GH via IRS-1. Consistent with these findings, wortmannin at concentrations of 10–500 nM had little effect on the ability of GH to induce STAT5 tyrosine phosphorylation in either cell (data not shown).



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  		Figure 6. Lack of difference in STAT5 activation or of SHP2, p85, or Raf 1 levels between 32D-rbGHR and 32D-rbGHR-IRS-1 cells. A, 32D-rbGHR and 32D-rbGHR-IRS-1 cells were treated with GH (+) or vehicle control (-) for indicated 15 min. Detergent extracts (2 million cells per condition) were resolved by SDS-PAGE and sequentially immunoblotted with antiphosphotyrosine-STAT5 (upper panel) and anti-STAT5 (lower panel) antibodies. The positions of tyrosine phosphorylated STAT5 (pSTAT5) and STAT5 are indicated. [Though we are not certain, based on relative migration in SDS-PAGE and a typically more substantial shift in migration of STAT5B in response to GH, we assume the lower of the STAT5 bands is STAT5B and the upper is STAT5A (our unpublished observations). The dominant GH-induced phospho-STAT5 form in the upper panel exactly comigrates with the shifted band in the lower panel.] The experiment shown is representative of six such experiments. B–D, Detergent extracts (2 million cells per lane) of each cell were resolved by SDS-PAGE and immunoblotted with anti-SHP2 (B), anti-p85 (C), and anti-Raf 1 (D) antibodies. The positions of each protein are indicated. The experiments shown are representative of three (B), or four (C, D) such experiments.

 
In principle, the selective influence of IRS-1 on GH-induced activation of the MEK1/MAP kinase pathway in 32D-rbGHR-IRS-1 vs. 32D-rbGHR cells could be explained by differences in expression and/or activation of various components of the PI3K or Ras/Raf/MEK1/MAP kinase cascades. The data presented in Figs. 1–4GoGoGoGo indicate no difference in abundance of either ERK1, ERK2, MEK1, or Akt between the two cells. The SH2-containing protein tyrosine phosphatase, SHP-2, is tyrosine phosphorylated in response to GH in 3T3-F442A preadipocytes and in 32D-rbGHR cells (21, 47). We have identified SHP-2 as a positive regulator of GH-induced c-fos transcriptional activation (21) and others have shown that fibroblasts with a targeted disruption of SHP-2 responded less well than those with normal SHP-2 to various growth factors with regard to induction of activation of MAP kinase(s) (48). We thus compared the expression of SHP-2 in 32D-rbGHR and 32D-rbGHR-IRS-1 cells and found no difference in immunodetectable SHP-2 abundance between them (Fig. 6BGo). In other experiments (not shown), we also found that GH promoted very similar degrees of SHP-2 tyrosine phoshorylation and coimmunoprecipitation of Grb-2 in both cells. Further, we also detected no difference in the abundance of the p85 regulatory subunit of PI3K (Fig. 6CGo) or Raf-1 (Fig. 6DGo) between the cells. Thus, a difference in the level of these key proteins in the PI3K and MAP kinase pathways does not underlie the differentially enhanced access afforded by IRS-1 to GH-stimulated pathways that cause phosphorylation of ERK1 and ERK2.

Previous work clearly implicates a role for Ras activation in GH-induced MAP kinase activation (18, 19). In response to growth factors, IRS-1 can bind several potential Ras-activating proteins. However, the relationship between IRS-1 and Ras activation pathways as it pertains to GH signaling has not yet been investigated. We tested GH’s effect on the activation state of Ras in 32D-rbGHR and 32D-rbGHR-IRS-1 cells by assessing the ability of a GST fusion protein containing the Ras binding domain of Raf-1 to precipitate Ras from extracts of cells previously stimulated or not with GH. This assay makes use of the findings of others that activated (GTP-bound) Ras binds with much more avidity to the Ras binding domain of Raf than does inactive (GDP-bound) Ras (49).

The results of three independent experiments are graphically displayed in Fig. 7Go. In each experiment, anti-Ras immunoblotting was used to determine the relative abundance of GST-Raf-1-bound (active) Ras normalized for total Ras content in the cell extract from the same sample. GH promoted a 2.7-fold increase in Ras binding in 32D-rbGHR cells. In 32D-rbGHR-IRS-1 cells, basal (non-GH-dependent) Ras binding was increased by 2.3-fold in comparison to basal Ras binding in 32D-rbGHR cells. We note that the IRS-1 expressed in 32D-rbGHR-IRS-1 cells, in addition to being inducibly tyrosine phosphorylated in response to GH, is somewhat tyrosine phosphorylated basally (20). A degree of basal tyrosine phosphorylation of IRS-1 was also observed by others in 3T3-F442A cells and CHO cells transfected with the GHR (31). It is conceivable that this basal tyrosine phosphorylation in 32D-rbGHR-IRS-1 was related to the enhanced basal Ras activation in these cells, though we have no direct evidence of this possibility. GH treatment of 32D-rbGHR-IRS-1 promoted further Ras binding such that it achieved on average a 7.5-fold increase over the non-GH-treated 32D-rbGHR cell control (roughly 3.3-fold increase over the non-GH-treated 32D-rbGHR-IRS-1 cells). Though the Ras activation achieved in these cells in response to GH was robust, the increased basal Ras activation causes us to conclude that we cannot attribute the entirety of IRS-1’s augmentation of GH-induced MAP kinase activation to an effect at or upstream of Ras. However, it is possible that IRS-1, by increasing basal Ras activation, lessens the threshold for GH activation of this pathway.



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  		Figure 7. GH-induced Ras activation in 32D-rbGHR and 32D-rbGHR-IRS-1 cells. Cells (20 million per condition) were stimulated with GH (+) or its vehicle control (-) for 15 min and detergent extracts were prepared, as in Materials and Methods. Ninety percent of each extract was affinity precipitated with GST-RBD. Eluates of the precipitates and the remaining 10% of the each extract were resolved by SDS-PAGE and each immunoblotted with anti-Ras antibody. The ratio of densitometrically determined GST-RBD-bound (active) Ras signal to total (unprecipitated) Ras signal was determined for each sample and compared within each experiment to that for the non-GH-treated 32D-rbGHR sample. The relative Ras binding (average ± SEM) so determined for three independent experiments is plotted. By unpaired t test: P < 0.005 for 32D-rbGHR, -GH vs. +GH; P < 0.02 for 32D-rbGHR-IRS-1, -GH vs. +GH; P < 0.02 for 32D-rbGHR --GH vs. 32D-rbGHR-IRS-1 --GH; P < 0.03 for 32D-rbGHR + GH vs. 32D-rbGHR-IRS-1 + GH.

 
In this work we have established a model reconstitution system for biochemically assessing the role of IRS-1 in various GH signaling pathways. The selective enhancement of GH-induced Akt and MEK1/MAP kinase activation and increase in both basal and GH-induced Ras activation conferred by expression of IRS-1 were not accompanied by augmented STAT5 tyrosine phosphorylation. We have not yet assessed whether STAT5-dependent gene expression, however, might be affected by the enhanced MAP kinase activation; this is a worthy topic for investigation. Despite the power of the 32D-rbGHR system for evaluation of IRS protein function, our data do not allow complete understanding of several issues. Though IRS-1-enhanced GH-induced MEK1/MAP kinase activation was wortmannin sensitive, we cannot conclude that only PI3K is involved because the LY294002 dose dependence for inhibition of GH-induced MAP kinase activation in the IRS-1-containing cells did not perfectly correlate with the effect of the drug on GH-induced Akt activation (taken as an accurate proxy for PI3K activation). The wortmannin sensitivity of GH-induced MAP kinase activation correlates well to the findings of others in 3T3-F442A cells (9, 38), but our work illustrates the importance of characterizing more completely the sensitivity to more than one PI3K inhibitor before concluding the sole involvement of that enzyme in these processes. The nature of the wortmannin-sensitive enzymes involved in both the IRS-1-independent and IRS-1-dependent MAP kinase activation is currently unknown to us.

Along these same lines, we cannot yet know the degree to which or the mechanisms whereby IRS-1’s enhancement of GH-induced MAP kinase activation is due to enhancement of Ras activation, but it is clear that both basal and GH-induced Ras activation were increased in the IRS-1-containing cells. Although SHP-2 abundance, SHP-2 tyrosine phosphorylation, and SHP-2/Grb-2 association were not significantly altered in the IRS-1-containing cells, it is quite possible that SHP-2, which we and others (21, 46) have implicated as a positive regulator of pathways related to MAP kinase activation, could still be exerting effects via IRS-1 on the GH-induced activation of the Ras/Raf/MEK1/MAP kinase pathway. Such a positive effect of SHP-2 on EGF-induced MAP kinase activation, independent of SHP-2 tyrosine phosphorylation, has recently been shown to be dependent on EGF-induced association of SHP-2 with the tyrosine phosphorylated Gab1 docking protein (50). Gab1 is a PH domain-containing member of the IRS-1 family of docking proteins that associates with SHP-2, Grb-2, and PI3K p85 in response to EGF and insulin (43). Gab1 or other as yet unknown similar docking proteins may tentatively be considered as candidates to be mediators of IRS-1-independent GH-induced MAP kinase activation in the 32D-rbGHR and other systems. This possibility should be explored.

We believe that the data presented herein provide a point of departure for studies that dissect the pathways mediating wortmannin-sensitive IRS-1-dependent and IRS-1-independent activation of MAP kinase by GH. Such studies may best be designed to compare the effects of overexpression of IRS-1 in the setting of cells that are rendered deficient (either genetically or by a dominant negative overexpression strategy) in possible mediators of Ras activation such as SHP-2, Grb-2/SOS, or PI3K. It will be interesting and important to identify the molecule(s) that mediates this IRS-1 enhancement and to determine if a differential ability to functionally couple GH stimulation to IRS-1 activation may underlie the variable degree of GH-induced MAP kinase activation observed in different cell types.


   Acknowledgments
 
The authors gratefully acknowledge Drs. W. Wood, J. Pierce, A. Kraft, R. Carter, L. Xiaoli, J. Bonifacino, and K. Arai for contribution of cells, plasmids, and reagents and Eli Lilly & Co. for providing the hGH. We appreciate helpful conversations with Drs. J. Kudlow, A. Paterson, E. Chin, J. Messina, A. Theibert, E. Benveniste, G. Fuller, S.-O. Kim, Y. Zhang, and R. Guan.


   Footnotes
 
1 This work was supported in part by NIH Grants DK-46395 (to S.J.F.) and T32 HL-07631 (to L.L.). Back

Received February 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Y. Huang, S.-O. Kim, J. Jiang, and S. J. Frank
Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells: MODULATION OF EGF-INDUCED TRAFFICKING AND SIGNALING
J. Biol. Chem., May 23, 2003; 278(21): 18902 - 18913.
[Abstract] [Full Text] [PDF]

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 EndocrinologyHome page
J. A. Bush, S. R. Kimball, P. M. J. O'Connor, A. Suryawan, R. A. Orellana, H. V. Nguyen, L. S. Jefferson, and T. A. Davis
Translational Control of Protein Synthesis in Muscle and Liver of Growth Hormone-Treated Pigs
Endocrinology, April 1, 2003; 144(4): 1273 - 1283.
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S.-O. Kim, K. Loesch, X. Wang, J. Jiang, L. Mei, J. M. Cunnick, J. Wu, and S. J. Frank
A Role for Grb2-Associated Binder-1 in Growth Hormone Signaling
Endocrinology, December 1, 2002; 143(12): 4856 - 4867.
[Abstract] [Full Text] [PDF]

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S. Ji, S. J. Frank, and J. L. Messina
Growth Hormone-induced Differential Desensitization of STAT5, ERK, and Akt Phosphorylation
J. Biol. Chem., August 9, 2002; 277(32): 28384 - 28393.
[Abstract] [Full Text] [PDF]