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 ␤ 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. The oncostatin M receptor (OSMR)1 belongs to the family of class I cytokine receptors and is one of three signal transducing receptor subunits of the interleukin (IL)-6-type cytokines. Its closest relatives are the receptor for leukemia inhibitory factor and the glycoprotein (gp) 130, which represents the common signal transducing receptor chain of all IL-6-type cytokines (1). This family consists of seven members: IL-6, IL-11, leukemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor, cardiotrophin-1, and the novel neurotrophin-1/B-cell stimulatory factor-3/cardiotrophin-like cytokine (2-4). Like the other IL-6-type cytokines, OSM is involved in various systemic and local responses, such as regulation of the acute phase reaction, hematopoiesis, bone remodeling, and homeostasis of the extracellular matrix, and can act as a mediator for both the proliferation and the growth arrest of various cell lines (3, 5-7). In contrast to all other members of this family, which transduce their signals either via a gp130 homodimer or a gp130/ leukemia inhibitory factor receptor heterodimer, OSM has the unique property to signal via a receptor complex comprising gp130 and the OSMR (1, 3).As the signal transducing receptor chains of IL-6-type cytokines do not contain an intrinsic kinase activity, a property shared with most other cytokine receptors, signaling is mediated by the Janus family of tyrosine kinases. These cytoplasmic kinases contain a kinase and a kinase-like domain in their C-terminal region, ...

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.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is centrally involved in diverse processes including haematopoiesis, immunity and cancer progression. In response to cytokine stimulation, STAT3 is activated through phosphorylation of a single tyrosine residue. The phosphorylated STAT3 dimers are stabilized by intermolecular interactions between SH2 domains and phosphotyrosine. These activated dimers accumulate in the nucleus and bind to specific DNA sequences, resulting in target gene expression. We analysed and compared the structural organizations of the unphosphorylated latent and phosphorylated activated STAT3 dimers using Fö rster resonance energy transfer (FRET) in fixed and living cells. The latent dimers are stabilized by homotypic interactions between the N-terminal domains. A somatic mutation (L78R) found in inflammatory hepatocellular adenoma (IHCA), which is located in the N-terminal domain of STAT3 disturbs latent dimer formation. Applying intramolecular FRET, we verify a functional role of the SH2 domain in latent dimer formation suggesting that the protomers in the latent STAT3 dimer are in a parallel orientation, similar to activated STAT3 dimers but different from the antiparallel orientation of the latent dimers of STAT1 and STAT5. Our findings reveal unique structural characteristics of STAT3 within the STAT family and contribute to the understanding of the L78R mutation found in IHCA.

Janus kinase 1 (Jak1) is a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors. Here we show that the in vitro translated N-terminal domains of Jak1 are sufficient for binding to a biotinylated peptide comprising the membrane-proximal 73 amino acids of gp130, the signal-transducing receptor chain of interleukin-6-type cytokines. By the fold recognition approach amino acid residues 36 -112 of Jak1 were predicted to adopt a ␤-grasp fold, and a structural model was built using ubiquitin as a template. Substitution of Tyr 107 to alanine, a residue conserved among Jaks and involved in hydrophobic core interactions of the proposed ␤-grasp domain, abrogated binding of full-length Jak1 to gp130 in COS-7 transfectants. By further mutagenesis we identified the loop 4 region of the Jak1 ␤-grasp domain as essential for gp130 association and gp130-mediated signal transduction. In Jak1-deficient U4C cells reconstituted with the loop 4 Jak1 mutants L80A/Y81A and ⌬(Tyr 81 -Ser 84 ), the interferon-␥, interferon-␣, and interleukin-6 responses were similarly impaired. Thus, loop 4 of the ␤-grasp domain plays a role in the association of Jak1 with both class I and II cytokine receptors. Taken together the structural model and the mutagenesis data provide further insight into the interaction of Janus kinases with cytokine receptors.Cytokines are involved in a variety of biological processes including hematopoiesis and the regulation of the immune system. Many cytokines signal via tyrosine kinases of the Janus family (Jaks) 1 and STAT (signal transducer and activator of transcription) transcription factors. Jaks are large enzymes (molecular mass, 120 -140 kDa) that are cytoplasmically preassociated with signal-transducing cytokine receptor subunits (1). Upon cytokine-induced receptor aggregation, Jaks are activated likely by auto-and transphosphorylation. Tyrosine residues within the cytoplasmic tail of the receptor are subsequently phosphorylated by the kinases, providing docking sites for Src homology 2 domain-containing signaling proteins including STATs, tyrosine phosphatases, and suppressors of cytokine signaling. Tyrosine-phosphorylated STATs homo-or heterodimerize and translocate to the nucleus where they bind to specific DNA sequences in the promoter regions of their respective target genes (2, 3).Whereas the structure/function relationship for the interaction between cytokines and the extracellular parts of their receptors is reasonably well understood and the structures of STATs bound to enhancer sequences have been solved (4 -7), no structural information is available on the interaction of the cytoplasmic parts of the signal-transducing subunits of cytokine receptors with the associated Janus kinases. Structural information on the receptor-kinase complex, however, is crucial to understand the binding specificity and the activation process of Janus kinases, which is the initial event of the intracellular signal transduction cascade after ligand binding to the extracellular part of...

We established a system of receptor chimeras that enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor ␣ and ␤ chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent interleukin-5-inducible STAT1 activation for which the presence of both the ␣ and the ␤ chain chimera was needed. No STAT activity was detected if one of the cytoplasmic tails of the receptor complex was deleted, indicating that STAT activity resulted from a receptor dimer rather than from higher receptor aggregates.We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.A detailed analysis of interferon (IFN) 1 signaling events first provided insight into a general signaling mechanism, the Jak-STAT pathway, by which many cytokines lead to an altered pattern of gene expression. Jak (Janus kinase) refers to a family of cytoplasmic tyrosine kinases that comprises four known members in mammals: Jak1, Jak2, Jak3, and Tyk2 (1). STAT (signal transducers and activators of transcription) refers to a family of transcription factors with seven known members (2). Cytokine-induced dimerization of receptor components leads to the activation of Jaks, which are constitutively associated with the cytoplasmic parts of the respective receptors. One substrate of the Jaks is the receptor itself. Upon phosphorylating specific tyrosine residues of the cytoplasmic tail of the receptor STAT factors and other proteins with "matching" SH2 domains can be recruited to the receptor where they become activated by tyrosine phosphorylation. Subsequently, the STATs dissociate from the receptor and translocate as homo-or heterodimers to the nucleus where they bind to enhancer elements of target genes and influence transcriptional activity (2, 3).The events leading to STAT factor dimerization are not well understood. Due to receptor dimerization, many cytokines lead to the formation of a paired set of STAT docking sites, e.g. IFN␥ signals through a homodimer of STAT1 that binds to ␥-interferon-activated sequence elements of IFN␥-regulated genes (4, 5). A single tyrosine residue (Tyr-440) in the ␣ chain of the IFN␥ receptor serves as a docking site for STAT1 (6 -8). Since IFN␥ receptor ␣ chains dimerize upon binding of IFN␥ (9), phosphorylation of Tyr-440 forms two juxtaposed docking sites for latent STAT1. Dimerization of STAT1 monomers phosphorylated on a specific tyrosine residue (Tyr-701) might be favored b...