Growth Hormone Receptor-associated Tyrosine Kinase Research Articles

Growth hormone signaling pathways

Over 20years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein α, a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1β is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GH-dependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight.

Phosphorylation of the adaptor protein SH2B1βregulates its ability to enhance growth hormone (GH)-dependent macrophage motility

Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1β to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicating SH2B1β in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1β releases SH2B1β from the plasma membrane. Here, we examined the role of SH2B1β in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cultures of peritoneal and bone marrow-derived macrophages. SH2B1β overexpression enhances, whereas SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1β-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1β are phosphorylated. Mutating these tyrosines in SH2B1β decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1β or creating the phosphomimetics SH2B1β(S161E) or SH2B1β(S165E), all of which release SH2B1β from the plasma membrane, enhances macrophage motility. Conversely, SH2B1β(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serine residues does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1β. Mutating tyrosines 439 and 494 does not affect localization of SH2B1β at the plasma membrane or movement of SH2B1β into focal adhesions. Taken together, these results suggest that SH2B1β enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1β on tyrosines 439 and 494 and movement of SH2B1β out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).

Signal transduction pathways in directed migration of human monocytes induced by human growth hormone in vitro

The human growth hormone (GH) was shown to modulate leukocyte functions such as stimulating directed migration of human monocytes in vitro. Dimerisation of GH-receptors leads to the activation of various signalling mechanisms. As transduction of GH signals to monocytes is unknown, we investigated GH signalling mechanisms in monocyte migration using a modified Boyden chamber chemotaxis assay. Inhibition of tyrosyl phosphorylation of GH receptor-associated tyrosine kinase by tyrphostin-23 or staurosporine blocked GH-stimulated monocyte migration down to random levels. Furthermore, pre-incubation with effective concentrations of 4B-phorbol-12-myristate-13-acetate (PMA), staurosporine and bisindolylmaleimide I, inhibitors of protein kinase C, significantly decreased GH-induced migration, suggesting that PKC is involved in the signalling cascade. Additionally, phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) activation seems to be required. This study revealed signalling pathways in monocyte movement toward GH in vitro.

Interaction of growth hormone-activated STATs with SH2-containing phosphotyrosine phosphatase SHP-1 and nuclear JAK2 tyrosine kinase.

Growth hormone (GH) rapidly stimulates tyrosine phosphorylation followed by serine/threonine phosphorylation of multiple cytoplasmic STAT transcription factors, including one, STAT5b, that is uniquely responsive to the temporal pattern of plasma GH stimulation in rat liver and is proposed to play a central role in the activation of male-expressed liver genes by GH pulses in vivo (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K. (1995) J. Biol. Chem. 270, 13262-13270). We now show that JAK2, the GH receptor-associated tyrosine kinase, is present both in the cytosol and in the nucleus in cultured liver cells and in rat liver in vivo and that GH-activated STAT3 but not STAT5b becomes associated with nuclear JAK2. GH is also shown to activate by 3-4-fold SHP-1, a phosphotyrosine phosphatase that contains two src homology 2 (SH2) domains. GH also induces nuclear translocation and binding of SHP-1 to tyrosine-phosphorylated STAT5b, suggesting that this GH-activated phosphatase may play a role in dephosphorylation leading to deactivation of nuclear STAT5b following the termination of a plasma GH pulse in male rat liver in vivo. No such association of SHP-1 with GH-activated STAT3 was detected, a finding that could help explain the marked desensitization of STAT3, but not STAT5b, to subsequent GH pulses following an initial GH activation event.

Dexamethasone-induced antagonism of growth hormone (GH) action by down-regulation of GH binding in 3T3-F442A fibroblasts.

Supraphysiological levels of glucocorticoids, whether endogenous (Cushing's syndrome) or exogenous (glucocorticoid therapy), inhibit growth in children and immature animals. This effect has long been suspected to be due to glucocorticoid antagonism of GH action at the level of peripheral tissues. In the present study we demonstrate direct antagonism of GH action at the cellular level by the artificial glucocorticoid dexamethasone. Dexamethasone was found to inhibit the ability of GH to elicit several early events in GH signaling in 3T3-F442A fibroblasts. Dexamethasone (100 nM) for 24 h decreases by 50-75% GH-induced tyrosyl phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, the transcription factor Stat3/APRF, the GH receptor-associated tyrosine kinase JAK2, and the GH receptor. These effects appear to be specific to GH. Dexamethasone does not inhibit induction of tyrosyl phosphorylation of ERK proteins by epidermal growth factor or phorbol myristate acetate, nor does it block induction of tyrosyl phosphorylation of Stat3/APRF by leukemia inhibitory factor or interleukin-6, or induction of JAK2 by leukemia inhibitory factor or interferon-gamma. Dexamethasone does not decrease the expression of ERK1 or -2, Stat3, or JAK2 proteins. Rather, the effects of dexamethasone on GH action appear to be due to a decrease in the number of GH receptors in the plasma membrane. Twenty-four-hour treatment with dexamethasone leads to a 50% decrease i GH binding, which Scatchard analysis suggests is due to a decrease in GH receptor number. These findings suggest that glucocorticoids antagonize cellular GH action by decreasing GH binding, suggesting a mechanism by which systemic glucocorticoids could antagonize GH action in peripheral tissues.

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

Insulin receptor substrate-1 (IRS-1) is tyrosine-phosphorylated in response to insulin resulting in association with and activation of phosphatidylinositol 3-kinase (PI 3-kinase), thereby initiating some of the effects of insulin. We have recently shown that the insulin-like effects of growth hormone (GH) in adipocytes can be inhibited by the selective PI 3-kinase inhibitor wortmannin (Ridderstråle, M., and Tornqvist, H. (1994) Biochem. Biophys. Res. Commun. 203, 306-310), suggesting a similar role for PI 3-kinase in GH action. Here we show that IRS-1 is tyrosine-phosphorylated in a time- and dose-dependent manner in response to GH in primary rat adipocytes. This phosphorylation coincided with the extent of interaction between IRS-1 and the 85-kDa subunit of PI 3-kinase as evidenced by coimmunoprecipitation. Stimulation with 23 nM GH increased the PI 3-kinase activity associated with IRS1 4-fold. Our data suggest that GH-induced tyrosine phosphorylation of IRS-1 and the subsequent docking of PI 3-kinase are important postreceptor events in GH action. The mechanism for the phosphorylation of IRS-1 induced by GH is unknown, but involvement of JAK2, the only known GH receptor-associated tyrosine kinase, seems possible.

The identification of JAK2 tyrosine kinase as a signaling molecule for growth hormone.

The intracellular pathways by which the binding of growth hormone (GH) to its receptor elicits its diverse effects have eluded investigators for many years. Studies showing that GH rapidly stimulates tyrosyl phosphorylation of cellular proteins, and that tyrosine kinase activity co-purifies with GH-GH receptor complexes, led us to hypothesize that activation by GH of a receptor-associated tyrosine kinase is an important early, and perhaps, initiating step in signal transduction by GH. Here, we review the work identifying JAK2 as a GH receptor-associated tyrosine kinase that is rapidly activated by ligand binding.

Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase

Growth hormone receptor (GHR) forms a complex with a tyrosine kinase, suggesting involvement of a ligand-activated tyrosine kinase in intracellular signaling by growth hormone (GH). Here we identify JAK2, a nonreceptor tyrosine kinase, as a GHR-associated tyrosine kinase. Immunological approaches were used to establish GH-dependent complex formation between JAK2 and GHR, activation of JAK2 tyrosine kinase activity, and tyrosyl phosphorylation of both JAK2 and GHR. The JAK2-GHR and JAK2-erythropoietin receptor interactions described here and in the accompanying paper provide a molecular basis for involvement of tyrosyl phosphorylation in physiological responses to these ligands and suggest a shared signaling mechanism among members of the cytokine/hematopoietin receptor family.

Evidence for involvement of the growth hormone receptor-associated tyrosine kinase in actions of growth hormone.

Previous observations have led to the speculation that activation of a growth hormone (GH) receptor-associated tyrosine kinase is an early, perhaps initiating, event in transmembrane signaling by GH. To test this hypothesis further, a Western blotting assay employing antibodies to phosphotyrosine was used to determine whether proteins other than the GH receptor might serve as substrates of the GH receptor-associated tyrosine kinase. The ability of inhibitors of the GH receptor-associated kinase to block actions of GH was also investigated. Over a physiologically relevant range of concentrations, GH was found to promote, in 3T3-F442A fibroblasts, rapid changes in the level of tyrosyl phosphorylation of more than 13 proteins. At the highest GH concentration employed (500 ng/ml), increased tyrosyl phosphorylation of two proteins, pp121 and pp97, was clearly visible at 1 min, the earliest time tested. Increased tyrosyl phosphorylation of a number of other proteins (pp250, pps160-180, pps140-160, pp130, pp90, pp75, pp45, pp42, pp39, and pp36) and decreased tyrosyl phosphorylation of a 140-kDa protein were apparent after 5-10 min of incubation with GH. Staurosporine, herbimycin A, and tyrphostin were identified as inhibitors of the GH receptor-associated kinase. When added to anti-GH antibody immunoprecipitates from GH-treated cells, they inhibited incorporation of 32P from [gamma-32P]ATP into tyrosyl residues in GH receptor complexes. When added to cells, all three inhibitors blocked all GH-dependent increases in tyrosyl phosphorylation of cellular proteins. Inhibitors of the GH receptor-associated tyrosine kinase also abolished GH-dependent activation of microtubule-associated protein (MAP) kinase. Consistent with these inhibitors inhibiting the GH receptor-associated tyrosine kinase, they had little or no effect on activation of MAP kinase by epidermal growth factor. In contrast, genistein and hydroxy-(2-naphthyl)-methylphosphonic acid, tyrosine kinase inhibitors lacking specificity for the GH receptor-associated kinase, decreased GH-dependent tyrosyl phosphorylation of only a subset of GH-responsive bands and partially blocked GH-dependent activation of MAP kinase. These data show that increased tyrosyl phosphorylation of specific cellular proteins is a very rapid response to the binding of GH by the cell and most likely involves multiple tyrosine kinases. Furthermore, inhibition of the GH receptor-associated tyrosine kinase blocks at least two actions of GH, the stimulation of tyrosyl phosphorylation of multiple proteins and MAP kinase activation. These results are consistent with the GH receptor-associated kinase playing an important, perhaps initiating, role in trans-membrane signaling by GH.

Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase.

The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present at low levels in the COS-7 cells. To test whether a higher level of GH receptor phosphorylation would be observed when the GH receptor was expressed in a different cell line, GH receptor cDNAs were stably transfected into mouse L and CHO cells, which have few or no endogenous GH receptors, and RIN5-AH cells, which do express endogenous GH receptors. In vivo tyrosyl phosphorylation of the cloned GH receptor in mouse L cells and in vitro phosphorylation of the cloned GH receptor in both L and CHO cells were higher than in transfected COS-7 cells but still substantially lower than in untransfected 3T3-F442A cells. Significantly increased 32P incorporation into tyrosyl residues in GH receptors in the in vitro kinase assay was demonstrated for GH receptors isolated from the transfected RIN5-AH cells. These studies show that the cloned liver GH receptor can be tyrosyl phosphorylated when expressed in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.

Stimulation by growth hormone (GH) of GH receptor-associated tyrosine kinase activity.

We have shown previously that GH receptors (GHRs) become phosphorylated on tyrosyl residues when GH-responsive cells are exposed to GH. In this work we investigate the molecular mechanism by which GH binding stimulates tyrosyl phosphorylation of GHR. To test whether in the presence of GH, GHR and the tyrosine kinase responsible for GHR phosphorylation are tightly associated in a complex or form a transient enzyme-substrate complex, the rate of in vitro phosphorylation of GHR was determined as a function of receptor concentration. GH-GHR complexes were purified from 3T3-F442A fibroblasts by immunoadsorption to and elution from immobilized phosphotyrosyl-binding antibody. The rate of in vitro tyrosyl phosphorylation of GH-GHR complexes was not substantially reduced when the GHR preparation was diluted 1:10 or 1:100 before phosphorylation, consistent with a tight association between kinase and substrate (GHR). When cells were labeled metabolically with 35S, and GHRs containing phosphorylated tyrosyl residues were isolated by immunoadsorption to and elution from phosphotyrosyl-binding antibody, the ability to immunoprecipitate 35S-labeled GHR with GHR antibodies was only evident when the cells had been incubated with GH. This indicates that in vivo, GHRs are phosphorylated on tyrosyl residues only when GH is bound. Additionally, when anti-GHR antibodies were used to immunoprecipitate GHR from solubilized cells, only GHRs isolated from GH-treated cells were phosphorylated when subjected to an in vitro kinase assay. The increased tyrosyl phosphorylation of GHR detected after incubation of cells with GH is consistent either with GH increasing tyrosine kinase activity associated with the GHR or with GH inducing a conformational change in GHR that renders it susceptible to tyrosyl phosphorylation. To discern whether GH binding increased GHR-associated kinase activity, we tested the ability of anti-GHR antibody immunoprecipitates to phosphorylate the synthetic substrate poly(Glu4,Tyr). GH treatment of cells resulted in a 2-fold increase in the rate of phosphorylation of poly(Glu4,Tyr). The increase in kinase activity was dose dependent, with half-maximal stimulation between 15-20 ng/ml GH. These results provide strong evidence that GH actually increases tyrosine kinase activity associated with the GHR. This would be consistent with GH-dependent complex formation between a constitutively activated kinase and GHR and/or activation of a constitutively associated or intrinsic kinase.

Stimulation by growth hormone (GH) of GH receptor-associated tyrosine kinase activity

Stimulation by growth hormone (GH) of GH receptor-associated tyrosine kinase activity

Demonstration of Growth Hormone (GH) Receptor- Associated Tyrosine Kinase Activity in Multiple GHResponsive Cell Types*

Highly purified GH-receptor preparations from 3T3-F442A fibroblasts, whose differentiation into adipocytes is promoted by GH, have been shown to contain a tyrosine kinase capable of phosphorylating GH receptors. In the current work, characteristics of the tyrosine kinase responsible for the in vitro phosphorylation of GH receptors from cultured 3T3-F442A fibroblasts were examined, and the presence of this GH receptor-associated tyrosine kinase activity was demonstrated in multiple cell types. GH-receptor complexes from GH-treated cells were partially purified by immunoprecipitation using anti-GH antibodies and then incubated as an immune complex with [gamma 32P] ATP. Incorporation of 32P into the GH receptor from 3T3-F442A fibroblasts was apparent within 1 min at 30 C after the addition of [gamma 32P]ATP (5-10 microM). A divalent cation was requisite for the phosphorylation; Mn2+ was significantly more effective than Mg2+ and Co2+; Ba2+, Ca2+, or Zn2+ had no effect. Excess unlabeled ATP, but not cytosine triphosphate, GTP, or uridine triphosphate, abolished 32P incorporation into the GH receptor and [gamma 32P]GTP could not replace [gamma 32P]ATP as a source of 32P. At 5.5 mM Mn2+, phosphorylation exhibited a biphasic dose response to ATP, with maximal phosphorylation occurring at a concentration of 10 microM ATP. At more physiological concentrations of ATP (1 mM), phosphorylation of the GH receptor was also stimulated by lower concentrations of Mn2+ (as low as 500 nM). Optimal reaction conditions determined for the phosphorylation reaction in 3T3-F442A fibroblasts were used to demonstrate incorporation of 32P from [gamma 32P]ATP into partially purified GH receptors from cultured human IM-9 lymphocytes, murine 3T3-F442A adipocytes, rat H-35 hepatoma cells, and freshly isolated rat adipocytes. The 32P was shown to be incorporated into tyrosyl residues in receptors from the two cell types tested (IM-9 lymphocytes and rat adipocytes). Cross-linked [125I] hGH-receptor complexes solubilized from the four cell types (IM-9 lymphocytes, 3T3-F442A adipocytes, H-35 hepatoma cells, and freshly isolated rat adipocytes) bound to and could be eluted from phosphotyrosyl antibody, suggesting that tyrosyl phosphorylation of GH receptors in all of these cells occurs in vivo. The presence of tyrosine kinase activity associated with GH receptors in multiple cell types from different species is consistent with tyrosine kinase activity playing a role in the actions of GH.

Phosphorylation of Highly Purified Growth Hormone Receptors by a Growth Hormone Receptor-associated Tyrosine Kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.