Jak2 and proteasome activities control the availability of cell surface growth hormone receptors during ligand exposure

Several mechanisms participate in the down-regulation of growth hormone receptor (GHR) signalling under ligand exposure. In CHO cells expressing GHR, we show that ligand stimulation induces degradation of the total cell GHR content. Experiments with 125I-hGH indicate that ligand-bound internalized receptors are not immediately replaced. Using cell surface biotinylation, we demonstrate for the first time that, concomitantly with the degradation of cell surface receptors, GHRs from the intracellular compartments are also degraded. We thus suggest that under prolonged ligand exposure, some GHRs are targeted to the cell surface, while others are routed to degradation compartments. Inhibitors of Jak2 and of the proteasome partially inhibited degradation of cell surface receptors, while these compounds completely inhibit the degradation of intracellular GHRs, resulting in their accumulation. We therefore propose that Jak2 and proteasome activities control the amount of intracellular GHRs, and thus the availability of receptors at the cell surface, during ligand exposure.     

Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.     

Jak2 acts as both a STAT1 kinase and as a molecular bridge linking STAT1 to the angiotensin II AT1 receptor

Angiotensin II activates the Jak-STAT pathway via the AT(1) receptor. We studied two mutant AT(1) receptors, termed M5 and M6, that contain Y to F substitutions for the tyrosine residues naturally found in the third intracellular loop and the carboxyl terminus. After binding ligand, both the M5 and M6 AT(1) receptors trigger STAT1 tyrosine phosphorylation equivalent to that observed with the wild type receptor, indicating that angiotensin II-mediated phosphorylation of STAT1 is independent of these receptor tyrosine residues. In response to angiotensin II, Jak2 autophosphorylates on tyrosine, and Jak2 and STAT1 physically associate, a process that depends on the SH2 domain of STAT1 in vitro. Evaluation of the wild type, M5, and M6 AT(1) receptors showed that angiotensin II-dependent AT(1) receptor-Jak2-STAT1 complex formation is dependent on catalytically active Jak2, not on the receptor tyrosine residues in the third intracellular loop and carboxyl tail. Immunodepletion of Jak2 virtually eliminated the ligand-dependent binding of STAT1 to the AT(1) receptor. These data indicate that the association of STAT1 with the AT(1) receptor is not strictly bimolecular; it requires Jak2 as both a STAT1 kinase and as a molecular bridge linking STAT1 to the AT(1) receptor.     

The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression

The four mammalian Jak tyrosine kinases are non-covalently associated with cell surface receptors binding helical bundled cytokines. In the type I interferon receptor, Tyk2 associates with the IFNAR1 receptor subunit and positively influences ligand binding to the receptor complex. Here, we report that Tyk2 is essential for stable cell surface expression of IFNAR1. In the absence of Tyk2, mature IFNAR1 is weakly expressed on the cell surface. Rather, it is localized into a perinuclear endosomal compartment which overlaps with that of recycling transferrin receptors and with early endosomal antigen-1 (EEA1) positive vesicles. Conversely, co-expressed Tyk2 greatly enhances surface IFNAR1 expression. Importantly, we demonstrate that Tyk2 slows down IFNAR1 degradation and that this is due, at least in part, to inhibition of IFNAR1 endocytosis. In addition, Tyk2 induces plasma membrane relocalization of the R2 subunit of the interleukin-10 receptor. These results reveal a novel function of a Jak protein on internalization of a correctly processed cytokine receptor. This function is distinct from the previously reported effect of other Jak proteins on receptor exit from the endoplasmic reticulum.     

Reactive oxygen species generated by hematopoietic cytokines play roles in activation of receptor-mediated signaling and in cell cycle progression

Hematopoietic cytokines, including interleukin (IL)-3 and erythropoietin (Epo), regulate hematopoiesis by stimulating their receptors coupled with the Jak2 tyrosine kinase to induce receptor tyrosine phosphorylation and activate mainly the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways. Here we demonstrate that IL-3 or Epo induces a rapid and transient (peaking at 30 min) as well as late progressive increase in reactive oxygen species (ROS) in a hematopoietic progenitor model cell line, 32Dcl3, and its subclone expressing the Epo receptor (EpoR), 32D/EpoR-Wt. The cytokine-induced ROS generation was not affected in 32Dcl3 cells depleted of mitochondrial DNA. The antioxidant N-acetyl-L-cysteine (NAC) inhibited IL-3-induced tyrosine phosphorylation of Jak2, IL-3 receptor betac subunit (IL-3Rbetac), and STAT5 as well as activation-specific phosphorylation of Akt, MEK, and ERK, while treatment of cells with H2O2 activated these signaling events. NAC also inhibited the EpoR-induced transphosphorylation of IL-3Rbetac. Moreover, NAC treatment reduced the expression levels of c-Myc, Cyclin D2, and Cyclin E, and induced expression of p27, thus inhibiting the G1 to S phase transition of cells cultured with IL-3. Further studies have shown that the degradation of c-Myc was facilitated or inhibited by treatment of cells with NAC or H2O2, respectively. These data indicate that the rapid generation of ROS by cytokine stimulation, which is at least partly independent of mitochondria, may play a role in activation of Jak2 and the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways as well as in transactivation of cytokine receptors. The cytokine-induced ROS generation was also implicated in G1 to S progression, possibly through stabilization of c-Myc and induction of G1 phase Cyclin expression leading to suppression of p27.     

Linkage between alpha(1) adrenergic receptor and the Jak/STAT signaling pathway in vascular smooth muscle cells

The Jak/STAT pathway is activated following stimulation of the type I angiotensin II receptor. To examine whether this pathway is shared among other G-protein-coupled receptors, we studied the linkage between the alpha(1) adrenergic receptor and this pathway. The alpha(1) agonist phenylephrine induced tyrosine phosphorylation of Jak2, Tyk2, and STAT1 in vascular smooth muscle cells. The phosphorylation of Jak2 was prevented by the alpha(1) receptor antagonists prazosin and chloroethylclonidine, but not by WB4101, and that of STAT1 was inhibited by prazosin and the Jak2 inhibitor AG490. After stimulation with phenylephrine, Jak2 and STAT1 were found to associate with alpha(1B) receptor. Phenylephrine stimulated the DNA binding activity of STAT1. Protein synthesis promoted by phenylephrine was inhibited by prazosin, AG490, and the introduction of a decoy oligonucleotide for STAT1. These results suggested that alpha(1) receptor is linked to the Jak/STAT pathway and that this pathway mediates alpha(1) agonist-induced smooth muscle hypertrophy.     

Jak2 is a negative regulator of ubiquitin-dependent endocytosis of the growth hormone receptor

Background

Length and intensity of signal transduction via cytokine receptors is precisely regulated. Degradation of certain cytokine receptors is mediated by the ubiquitin ligase SCF(βTrCP). In several instances, Janus kinase (Jak) family members can stabilise their cognate cytokine receptors at the cell surface.

Principal Findings

In this study we show in Hek293 cells that Jak2 binding to the growth hormone receptor prevents endocytosis in a non-catalytic manner. Following receptor activation, the detachment of phosphorylated Jak2 induces down-regulation of the growth hormone receptor by SCF(βTrCP). Using γ2A human fibroblast cells we show that both growth hormone-induced and constitutive growth hormone receptor endocytosis depend on the same factors, strongly suggesting that the modes of endocytosis are identical. Different Jak2 RNA levels in HepG2, IM9 and Hek293 cells indicate the importance of cellular concentration on growth hormone receptor function. Both Jak2 and βTrCP bind to neighbouring linear motifs in the growth hormone receptor tail without the requirement of modifications, indicating that growth hormone sensitivity is regulated by the cellular level of non-committed Jak2.

Conclusions/Significance

As signal transduction of many cytokine receptors depends on Jak2, the study suggests an integrative role of Jak2 in cytokine responses based on its enzyme activity as well as its stabilising properties towards the receptors.     

Janus kinase (Jak) subcellular localization revisited: the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases.     

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.     

Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.     

Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha

Interleukin-7 (IL-7) receptor signaling begins with activation of the Janus tyrosine kinases Jak1 and Jak3, which are associated with the receptor complex. To identify potential targets of these kinases, we examined Pyk2 (a member of the focal adhesion kinase family) using an IL-7-dependent murine thymocyte line, D1. We demonstrate that stimulation of D1 (or normal pro-T) cells by IL-7 rapidly increased tyrosine phosphorylation and enzymatic activity of Pyk2, with kinetics slightly lagging that of Jak1 and Jak3 phosphorylation. Conversely, IL-7 withdrawal resulted in a marked decrease of Pyk2 phosphorylation. Pyk2 was found to be physically associated with Jak1 prior to IL-7 stimulation and to increase its association with IL-7Ralpha chain following IL-7 stimulation. Pyk2 appeared to be involved in cell survival, because antisense Pyk2 accelerated the cell death process. Activation of Pyk2 via the muscarinic and nicotinic receptors using carbachol or via intracellular Ca(2+) rise using ionomycin/phorbol myristate acetate promoted survival in the absence of IL-7. These data support a role for Pyk2 in coupling Jak signaling to the trophic response.     

Activation of nuclear receptors: a perspective from structural genomics

Crystal structures of more than two dozen different nuclear receptor ligand binding domains have defined a simple paradigm of receptor activation, in which agonist binding induces the activation function-2 (AF-2) helix to form a charge clamp for coactivator recruitment. Recent structural studies present a surprising contrast. Activation of the mouse LRH-1 receptor is independent of a bound agonist despite its large ligand binding pocket, whereas the activation of the Drosophila DHR38 receptor is dependent on ecdysteroids even though the receptor lacks a ligand binding pocket. These new findings shed light on the diverse structural mechanisms that nuclear receptors have evolved for activation, and have important implications in their respective signaling pathways.