Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.     

Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling

In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is tyrosyl-phosphorylated following stimulation of 3T3-F442A fibroblasts with growth hormone (GH), leukemia inhibitory factor and interferon-gamma. In response to GH and leukemia inhibitory factor, IRS-2 is immediately phosphorylated, with maximal phosphorylation detected at 15 min; the signal is substantially diminished by 60 min. In response to interferon-gamma, tyrosine phosphorylation of IRS-2 was prolonged, with substantial signal still detected at 60 min. Characterization of the mechanism of signaling utilized by GH indicated that tyrosine residues in GH receptor are not necessary for tyrosyl phosphorylation of IRS-2; however, the regions of GH receptor necessary for IRS-2 tyrosyl phosphorylation are the same as those required for JAK2 association and tyrosyl phosphorylation. The role of IRS-2 as a signaling molecule for GH is further demonstrated by the finding that GH stimulates association of IRS-2 with the 85-kDa regulatory subunit of phosphatidylinositol 3'-kinase and with the protein-tyrosine phosphatase SHP2. These results are consistent with the possibility that IRS-2 is a downstream signaling partner of multiple members of the cytokine family of receptors that activate JAK kinases.     

Effects of tyrosine kinase inhibitors on tyrosine phosphorylations and the insulin-like effects in response to human growth hormone in isolated rat adipocytes

Recent data suggest involvement of the Janus tyrosine kinase-2 (JAK2) in human GH-induced tyrosine phosphorylation of the GH receptor and the insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), leading to activation of the phosphatidylinositol 3-kinase and the acute insulin-like effects in primary rat adipocytes. To investigate the functional role of this kinase, we screened a number of tyrosine kinase inhibitors for their ability to inhibit three rapid effects of GH on primary adipocytes: increased lipogenesis, inhibition of noradrenaline-induced lipolysis, and promotion of JAK2 tyrosine phosphorylation. Only staurosporine was found to inhibit all three effects. The inhibition of lipogenesis and antilipolysis exhibited the same staurosporine dose dependency (IC50, approximately 40 nM) as inhibition of JAK2 and IRS-1 tyrosine phosphorylation as well as binding of the p85 subunit of phosphatidylinositol 3-kinase to IRS-1 and IRS-2. The unidentified cytosolic tyrosine-phosphorylated protein pp95, in contrast, was not affected, suggesting that it is not phosphorylated primarily by JAK2. Protein kinase C does not seem to be directly involved in the insulin-like effects, because the selective protein kinase C inhibitor calphostin C had no effect at levels up to 100 nM above which unspecific cellular effects occurred. Methyl-2,5-dihydroxy cinnamate inhibited GH-induced lipogenesis from [3-3H]glucose and nonstimulated lipogenesis from [2-14C]-pyruvate and [3H]acetate, but was without effect on GH-induced 2-deoxy-D-[1-3H]glucose uptake, JAK2 phosphorylation and antilipolysis, suggesting unspecific effects on mitochondrial metabolism rather than a direct effect on the GH-mediated signal. Tyrphostin 25 and herbimycin A had no effect on any of the parameters studied, except for a slight increase in JAK2 phosphorylation in response to tyrphostin 25. In summary, these data support the role for JAK2 in mediating the insulin-like effects of GH in adipocytes.     

Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.     

Tissue-specific regulation of IRS-1 in unilaterally nephrectomized rats

Insulin stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate 1 (IRS-1). IRS-1 is also a substrate for different peptides and growth factors, and a transgenic mouse "knockout" for this protein does not have normal growth. However, the role of IRS-1 in kidney hypertrophy and/or hyperplasia was not investigated. In the present study we investigated IRS-1 protein and tyrosine phosphorylation levels in the remnant kidney after unilateral nephrectomy (UNX) in 6-week-old male Wistar rats. After insulin stimulation the levels of insulin receptor and IRS-1 tyrosine phosphorylation were reduced to 79 +/- 5% (P < 0.005) and 58 +/- 6% (P < 0.0001), respectively, of the control (C) levels, in the remnant kidney. It is possible that a circulating factor and/or a local (paracrine) factor playing a role in kidney growth can influence the early steps of insulin action in parallel. To investigate the hypothesis of a circulating factor, we studied the early steps of insulin action in liver and muscle of unilateral nephrectomized rats. There was no change in pp185 tyrosine phosphorylation levels in liver (C 100 +/- 12% vs UNX 89 +/- 9%, NS) and muscle (C 100 +/- 22% vs UNX 91 +/- 17%, NS), and also there was no change in IRS-1 phosphorylation levels in both tissues. These data demonstrate that after unilateral nephrectomy there is a decrease in insulin-induced insulin receptor and IRS-1 tyrosine phosphorylation levels in kidney but not in liver and muscle. It will be of interest to investigate which factors, probably paracrine ones, regulate these early steps of insulin action in the contralateral kidney of unilaterally nephrectomized rats.     

Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes

In the present study we have investigated the contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling pathways in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulated IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation was barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha revealed a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain showed functional differences. While a strong association between IRS-1/Grb-2 was found, IRS-2/Grb-2 association was virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increased the amount of Grb-2-associated SHC proteins by the same extent. Our results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation that leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2 leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation.     

Divergent signaling capacities of the long and short isoforms of the leptin receptor

Leptin receptors include a long form (OBRl) with 302 cytoplasmic residues that is presumed to mediate most or all of leptins signaling, and several short forms, including one (OBRs) that has 34 cytoplasmic residues, is widely expressed, and is presumed not to signal but to mediate transport or clearance of leptin. We studied the abilities of these two receptor isoforms to mediate signaling in transfected cells. In response to leptin, OBRl, but not OBRs, underwent tyrosine phosphorylation that was enhanced by co-expression with JAK2. In cells expressing receptors and JAK2, both OBRs and OBRl mediated leptin-dependent tyrosine phosphorylation of JAK2, and this was abolished with OBRs when the Box 1 motif was mutated. In cells expressing receptors, JAK2 and IRS-1, leptin induced tyrosine phosphorylation of IRS-1 through OBRs and OBRl. In COS cells expressing hemagglutinin-ERK1 and receptors, leptin increased ERK1 kinase activity through OBRl, with the magnitude increased by co-expression of JAK1 or JAK2, and to a lesser degree through OBRs, despite greater receptor expression. In stable Chinese hamster ovary cell lines expressing OBRs or OBRl, leptin stimulated endogenous ERK2 phosphorylation. Whereas leptin stimulated tyrosine phosphorylation of hemagglutinin-STAT3 and induction of a c-fos luciferase reporter plasmid through OBRl, OBRs was without effect in these assays. In conclusion, OBRl is capable of signaling to IRS-1 and mitogen-activated protein kinase via JAK, in addition to activating STAT pathways. Although substantially weaker than OBRl, OBRs is capable of mediating signal transduction via JAK, but these activities are of as yet unknown significance for leptin biology in vivo.     

Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay

The high-affinity receptor (R) for IL-5 consists of a unique alpha chain (IL-5R alpha) and a beta chain (beta c) that is shared with the receptors for IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF). We defined two regions of IL-5R alpha for the IL-5-induced proliferative response, the expression of nuclear proto-oncogenes, and the tyrosine phosphorylation of cellular proteins including beta c, SH2/SH3-containing proteins and JAK2 kinase. In the studies described here, we demonstrate that IL-5, IL-3 or GM-CSF stimulation induces the tyrosine phosphorylation of JAK2, and to a lesser extent JAK1, and of STAT5. Mutational analysis revealed that one of the proline residues, particularly Pro352 and Pro355, in the membrane-proximal proline-rich sequence (Pro352-Pro353-X-Pro355) of the cytoplasmic domain of IL-5R alpha is required for cell proliferation, and for both JAK1 and JAK2 activation. In addition, transfectants expressing chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c responded to IL-5 for proliferation and tyrosine phosphorylation of JAK1. Intriguingly, electrophoretic mobility shift assay analysis revealed that STAT5 was activated in cells showing either JAK1 or JAK2 tyrosine phosphorylation. These results indicate that activation of JAK1, JAK2 and STAT5 is critical to coupling IL-5-induced tyrosine phosphorylation and ultimately mitogenesis, and that Pro352 and Pro355 in the proline-rich sequence appear to play more essential roles in cell growth and in both JAK1/STAT5 and JAK2/STAT5 activation than Pro353 does.     

Impaired insulin signaling in skeletal muscles from transgenic mice expressing kinase-deficient insulin receptors

Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.     

Effect of aging on insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of rats

Insulin stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1), which, in turn, associates with phosphatidylinositol 3-kinase (PI 3-kinase), thereby activating the latter. Aging is associated with insulin resistance, but the exact molecular mechanism is unknown. In the present study, we examined the levels and phosphorylation status of the insulin receptor and IRS-1 as well as the association between IRS-1 and PI 3-kinase in the liver and muscle of 2-, 5-, 12-, and 20-month-old rats. There were no changes in the insulin receptor concentration in the liver and muscle of rats 2-. 5-, 12-, and 20-month rats. There were no changes in the insulin receptor concentration in the liver and muscle of rats 2-20 months old, as determined by immunoblotting using antibody to the COOH-terminus of the receptor. However, insulin stimulation of receptor autophosphorylation, as determined by immunoblotting with antiphosphotyrosine antibody was reduced by 25% (P < 0.05) in the liver and muscle of rats at 20 months. Interestingly, IRS-1 protein levels decrease at an early stage (5 months) by 58 +/- 9%, (P < 0.01) and remained at low levels thereafter in muscle, but not in liver. In samples previously immunoprecipitated with anti-IRS-1 antibody and blotted with antiphosphotyrosine antibody, there were 60 +/- 9% (P < 0.001) and 92 +/- 4% (P < 0.001) decreases in the insulin-stimulated IRS-1 association with PI 3-kinase was decreased by 70 +/- 2% in the liver and muscle, respectively, of 20-month rats. The insulin-stimulated IRS-1 association with PI 3-kinase was decreased by 70 +/- 2% in the liver (P < 0.001) and by 98 +/- 3% (P < 0.001) in the muscle of 20-month-old rats, with no change in the PI 3-kinase protein levels. The phosphotyrosine-associated PI 3-kinase activity after insulin stimulation was dramatically reduced in liver and muscle of 20-month-old rats compared to that in 2-month-old rats. Finally, by immunoprecipitation, the detection of insulin-stimulated IRS-2 phosphorylation followed the same pattern as that for IRS-1 in both liver of 2- and 20-month-old rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance observed in old animals.     

Enhanced insulin-stimulated activation of phosphatidylinositol 3-kinase in the liver of high-fat-fed rats

Insulin receptor substrate (IRS)-1 and IRS-2, which mediate phosphatidylinositol (PI) 3-kinase activation, play essential roles in insulin-induced translocation of GLUT4 and in glycogen synthesis. In this study, we investigated the process of PI 3-kinase activation via binding with IRS-1 and -2 in liver, muscle, and fat of high-fat-fed rats, a model of insulin-resistant diabetes. In the liver of high-fat-fed rats, insulin increased the PI 3-kinase regulatory subunit p85alpha and the PI 3-kinase activities associated with IRS-1 3.6- and 2.4-fold, and with IRS-2, 4.7- and 3.0-fold, respectively, compared with those in control rats. The tyrosine phosphorylation levels of IRS-1 and IRS-2 were not significantly altered, however. In contrast with the liver, tyrosine phosphorylation levels and associated PI 3-kinase proteins and activities were decreased in the muscle and adipose tissue of high-fat-fed rats. Thus, high-fat feeding appears to cause insulin resistance in the liver by a mechanism different from the impaired PI 3-kinase activation observed in muscle and adipose tissue. Taking into consideration that hepatic PI 3-kinase activation is severely impaired in obese diabetic models such as Zucker fatty rats, it is possible that the mechanism by which a high-fat diet causes insulin resistance is quite different from that associated with obesity and overeating due to abnormality in the leptin system. This is the first report to show increased PI 3-kinase activation by insulin in an insulin-resistant diabetic animal model. These findings may be important for understanding the mechanism of insulin resistance in human NIDDM, since a high-fat diet is considered to be one of the major factors exacerbating insulin insensitivity in humans.