Simone Radtke

Novel Role of Janus Kinase 1 in the Regulation of Oncostatin M Receptor Surface Expression

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex that mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and transcription factors of the signal transducers and activators of transcription (STAT) family. Upon heterologous expression of the OSMR in several cell lines, we observed that its surface expression was significantly enhanced by coexpression of the Janus kinases Jak1, Jak2, and Tyk2 but not Jak3. Chimeric receptors consisting of the extracellular region of the interleukin-5 receptor β chain and the transmembrane and intracellular part of the OSMR were similarly up-regulated on the plasma membrane when Jak1 was coexpressed. The overall expression level of these constructs did not change significantly, but Jak1 coexpression increased the amount of endoglycosidase H-resistant, fully processed OSMR chimeras. Using mutated receptor and Jak1 constructs, we were able to demonstrate that association of Jak1 with the membrane proximal region of the receptor, but not its kinase activity, is necessary for this effect. Moreover, deletion of the OSMR box1/2 region also resulted in an improved surface expression indicating that this region may contain a signal preventing efficient receptor surface expression in the absence of associated Jaks. Finally we demonstrate that in Jak1-deficient cells, the endogenous OSMR is significantly down-regulated, an effect that can be reversed by transient expression of Jak1 in these cells.

Characterization of the signaling capacities of the novel gp130-like cytokine receptor.

The gp130-like receptor (GPL) is a recently cloned member of the family of type I cytokine receptors. The name reflects its close relationship to gp130, the common receptor subunit of the interleukin (IL)-6-type cytokines. Indeed, the recently proposed ligand for GPL, IL-31, is closely related to the IL-6-type cytokines oncostatin M, leukemia inhibitory factor, and cardiotrophin-1. The second signal transducing receptor for IL-31 seems to be the oncostatin M receptor β (OSMRβ). The present study characterizes in depth the molecular mechanisms underlying GPL-mediated signal transduction. GPL is a strong activator of STAT3 and STAT5, whereas STAT1 is only marginally tyrosine-phosphorylated. We identify tyrosine residues 652 and 721 in the cytoplasmic region of the longest isoform of GPL (GPL745) as the major STAT5- and STAT3-activating sites, respectively. Additionally, we demonstrate Jak1 binding to GPL and its activation in heteromeric complexes with the OSMRβ but also in a homomeric receptor complex. Most interesting, unlike OSMRβ and gp130, GPL is insufficient to mediate ERK1/2 phosphorylation. We propose that this is due to a lack of recruitment of the tyrosine phosphatase SHP-2 or the adaptor protein Shc to the cytoplasmic domain of GPL.

Cross-regulation of cytokine signalling: Pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1β, TNFα or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

Non-redundant signal transduction of interleukin-6-type cytokines. The adapter protein Shc is specifically recruited to rhe oncostatin M receptor.

The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr861 of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr861 is essential for activation of ERK1/2 and for full activation of the α2-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.

Signal transduction of IL-6, leukemia-inhibitory factor, and oncostatin M: structural receptor requirements for signal attenuation.

Stimulation of the IL-6R complex leads to Src homology domain containing tyrosine phosphatase 2 (SHP2) recruitment to the receptor subunit gp130 and its subsequent tyrosine phosphorylation. SHP2 is a two-SH2 domain-containing protein tyrosine phosphatase that is activated by many cytokines and growth factors. SHP2 counteracts the activation of transcription factors of the STAT family and the induction of IL-6-responsive genes. Tyrosine 759 of gp130, the signal transducing subunit of the IL-6R complex, is essential for the phosphorylation of SHP2. Mutation of tyrosine 759 to phenylalanine leads to an enhanced inducibility of IL-6-dependent genes. Here we demonstrate that no further tyrosines in the cytoplasmic part of gp130 are required for the phosphorylation of SHP2. We also tested whether the tyrosine 759 motifs in both subunits of the gp130 dimer are required for SHP2 association and tyrosine phosphorylation. Interestingly, one SHP2-recruiting phosphotyrosine motif in a single chain of the gp130 dimer is sufficient to mediate SHP2 association to the gp130 receptor subunit and its tyrosine phosphorylation as well as to attenuate IL-6-dependent gene induction. Furthermore, we show that repression of gene induction via Y759 does not require the presence of the SHP2 and STAT recruitment sites within the same receptor subunit, but within the same receptor complex. The Y759 motif in gp130 also attenuates gene induction mediated by the oncostatin M and leukemia inhibitory factor receptor complexes, which both contain gp130 as the shared subunit.

Oncostatin M Receptor-mediated Signal Transduction Is Negatively Regulated by SOCS3 through a Receptor Tyrosine-independent Mechanism

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr759 in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR). OSM leads to a much stronger and prolonged induction of SOCS3 in HepG2 hepatoma cells and murine embryonal fibroblasts (MEF) compared with IL-6. A negative effect of SOCS3 on OSM signaling was confirmed using MEF cells lacking SOCS3. We can show that the OSMR-mediated signaling is inhibited by SOCS3 to a similar extent as previously described for gp130. However, the inhibition occurs independent of tyrosine motifs within the OSMR. Instead, SOCS3 interacts directly with JAK1 in a stimulation-dependent manner, a mechanism so far only known for SOCS1.

Box 2 region of the oncostatin M receptor determines specificity for recruitment of Janus kinases and STAT5 activation.

Human and murine oncostatin M (OSM) induce their bioactivities through a heterodimeric receptor complex consisting of gp130 and the OSM receptor (OSMR), which initiates a signaling pathway involving Janus kinases (JAKs) and transcription factors of the signal transducers and activators of transcription (STAT) family. In contrast to the signal transducing receptor subunit gp130, the OSMR allows strong activation of STAT5B. The underlying molecular mechanism, however, remained unclear. Here we demonstrate that the human and murine OSM receptors use distinct mechanisms for STAT5B activation. The human receptor contains a STAT5B recruiting tyrosine motif (Tyr837/Tyr839) C-terminal to the box 1/2 region, which is absent in the mouse receptor. In contrast, the murine receptor initiates STAT5 activation directly via the receptor bound Janus kinases. Intriguingly, the murine receptor preferentially recruits JAK2, whereas the human receptor seems to have a higher affinity for JAK1. We identify a single amino acid (Phe820) in the human receptor that is responsible for this preference. Exchange by the murine counterpart (Cys815) allows recruitment of JAK2 by the human receptor and consequently activation of STAT5B independently of receptor tyrosine motifs. STAT5B interacts directly with JAK2 only in response to activation of the murine OSMR or the mutated human OSMR. Additionally, we show that OSM-induced STAT1 phosphorylation occurs independently of receptor tyrosine motifs and is mediated directly by Janus kinases, whereas the two C-terminally located tyrosine residues Tyr917/Tyr945 of the OSMR are crucial for STAT3 activation.

Three Dileucine-like Motifs within the Interbox1/2 Region of the Human Oncostatin M Receptor Prevent Efficient Surface Expression in the Absence of an Associated Janus Kinase

The oncostatin M receptor (OSMR) is part of receptor complexes for oncostatin M and interleukin-31. Signaling events are triggered by Jaks (Janus kinases) that constitutively bind to membrane-proximal receptor regions. Besides their established role in signaling, Jaks are involved in the regulation of the surface expression of several cytokine receptors. Here, we analyzed the structural requirements within the human OSMR that underlie its limited surface expression in the absence of associated Jaks. We identified three dileucine-like motifs within the Jak-binding region of the OSMR that control receptor surface and overall expression. A receptor mutant in which all three motifs were mutated to alanine displayed markedly increased surface expression. Although the surface half-life of this mutant was increased compared with that of the wild-type receptor, no difference in the internalization rate was detectable, implying that these receptors differ in their post-endocytic fate. The protein stability of the wild-type receptor was markedly lower than that of mutant receptors, but could be strongly increased in the presence of the lysosomal inhibitor chloroquine. Our data are consistent with the dileucine motifs being involved in destabilization of receptors devoid of associated Jaks as part of a quality control ensuring signaling competence of OSMRs.

Differential inhibition of IL-6-type cytokine-induced STAT activation by PMA

Prior activation of mitogen‐activated protein kinases by phorbol 13‐myristate 12‐acetate (PMA) results in an inhibition of interleukin (IL)‐6‐induced activation of the Janus kinase/signal transducer and activator of transcription (STAT) signaling pathway which is most likely mediated by the induction of suppressor of cytokine signaling‐3 and requires the specific SHP2 binding site Y759 of the IL‐6 signal transducer gp130. In this study, we demonstrate that PMA inhibits STAT activation by IL‐6 and the related cytokine leukemia inhibitory factor (LIF) but not by oncostatin M (OSM). Since the LIF receptor also contains an SHP2 recruitment site whereas the OSM receptor lacks such a module, we propose that two SHP2 binding modules within a homo‐ or heterodimeric receptor are necessary to mediate the PMA inhibitory effect.