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See detailOncostatin M up-regulates the ER chaperone Grp78/BiP in liver cells
Vollmer, Stefan UL; Haan, Claude UL; Behrmann, Iris UL

in Biochemical Pharmacology (2010), 80(12), 2066-2073

OSM, a cytokine of the IL-6-type cytokine family, regulates inflammatory processes (like the acute phase response), tissue remodeling, angiogenesis, cell differentiation and proliferation. Inflammation is ... [more ▼]

OSM, a cytokine of the IL-6-type cytokine family, regulates inflammatory processes (like the acute phase response), tissue remodeling, angiogenesis, cell differentiation and proliferation. Inflammation is discussed to favor carcinogenesis and the inflammatory cytokine OSM was lately described to up-regulate HIF-1α, whose up-regulation is also observed in many cancers. In this study we demonstrate that OSM, and to a lesser degree IL-6, induces the expression of Grp78/BiP, an ER chaperone associated with tumor development and poor prognosis in cancer. In contrast, IFN-γ or TNF-α had no effect on Grp78 expression. The up-regulation seems to be specific to liver cells, as it occurs in hepatocytes and hepatoma cells but not in prostate, melanoma, breast or kidney cells. OSM does not lead to up-regulation of Grp94, enhanced XBP-1 mRNA splicing or phosphorylation of eIF2α, indicating that it is not associated to a general ER stress response. Analysis of the underlying mechanism showed that Grp78 is up-regulated by transcriptional processes which are to the greater part, though not completely, dependent on MEK/Erk activation. © 2010 Elsevier Inc. [less ▲]

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See detailCross-regulation of cytokine signalling: Pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation
Radtke, S.; Wüller, S.; Yang, X.-P. et al

in Journal of Cell Science (2010), 123(6), 947-959

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 ... [more ▼]

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-1b, TNFa 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. [less ▲]

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See detailSignal transduction, receptors, mediators and genes: Younger than ever - The 13th meeting of the Signal Transduction Society focused on aging and immunology
Entschladen, F.; Altschmied, J.; Baumgrass, R. et al

in Cell Communication and Signaling (2010), 8

The 13th meeting of the Signal Transduction Society was held in Weimar, from October 28 to 30, 2009. Special focus of the 2009 conference was "Aging and Senescence", which was co-organized by the SFB 728 ... [more ▼]

The 13th meeting of the Signal Transduction Society was held in Weimar, from October 28 to 30, 2009. Special focus of the 2009 conference was "Aging and Senescence", which was co-organized by the SFB 728 "Environmentally-Induced Aging Processes" of the University of Düsseldorf and the study group 'Signal Transduction' of the German Society for Cell Biology (DGZ). In addition, several other areas of signal transduction research were covered and supported by different consortia associated with the Signal Transduction Society including the long-term associated study groups of the German Society for Immunology and the Society for Biochemistry and Molecular Biology, and for instance the SFB/Transregio 52 "Transcriptional Programming of Individual T Cell Subsets" located in Würzburg, Mainz and Berlin. The different research areas that were introduced by outstanding keynote speakers attracted more than 250 scientists, showing the timeliness and relevance of the interdisciplinary concept and exchange of knowledge during the three days of the scientific program. This report gives an overview of the presentations of the conference. © 2010 Entschladen et al; licensee BioMed Central Ltd. Entschladen, F.; Institute of Immunology, Witten/Herdecke UniversityGermany; email: entschladen@uni-wh.de [less ▲]

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See detailPerspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases.
Haan, Claude UL; Behrmann, Iris UL; Haan, Serge UL

in Journal of Cellular & Molecular Medicine (2010), 14(3), 504-27

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase ... [more ▼]

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets. [less ▲]

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See detailSOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.
Haan, Serge UL; Wuller, S.; Kaczor, Jakub UL et al

in Oncogene (2009), 28(34), 3069-80

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS ... [more ▼]

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia. [less ▲]

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See detailAn unusual insertion in Jak2 is crucial for kinase activity and differentially affects cytokine responses
Haan, Claude UL; Kroy, Daniela C.; Wüller, Stefan et al

in Journal of Immunology (2009), 182(5), 2969-2977

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated ... [more ▼]

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-gamma and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks. [less ▲]

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See detailHypoxia-inducible factor 1alpha is up-regulated by oncostatin M and participates in oncostatin M signaling
Vollmer, Stefan UL; Kappler, Valérie; Kaczor, Jakub UL et al

in Hepatology (Baltimore, Md.) (2009), 2009(3),

The interleukin-6-type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen-activated protein kinases and is ... [more ▼]

The interleukin-6-type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen-activated protein kinases and is known to critically regulate processes such as liver development and regeneration, hematopoiesis, and angiogenesis, which are also determined by hypoxia with the hypoxia-inducible factor 1alpha (HIF1alpha) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1alpha under normoxic and hypoxic conditions. Furthermore, the OSM-dependent HIF1alpha increase is mediated via Janus kinase/signal transducer and activator of transcription 3 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 pathways. OSM-mediated HIF1alpha up-regulation did not result from an increase in HIF1alpha protein stability but from increased transcription from the HIF1alpha gene. In addition, we show that the OSM-induced HIF1alpha gene transcription and the resulting enhanced HIF1alpha protein levels are important for the OSM-dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. Conclusion: HIF1alpha levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1alpha contributes to OSM downstream signaling events, pointing to a cross-talk between cytokine and hypoxia signaling in processes such as liver development and regeneration. (HEPATOLOGY 2009.). [less ▲]

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See detailZelluläre Signalprozesse
Friedrich, Karlheinz; Behrmann, Iris UL

in Biochemie und Molekularbiologie des Menschen (2009)

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See detailDual role of the Jak1 FERM and kinase domains in cytokine receptor binding and in stimulation-dependent Jak activation
Haan, Serge UL; Margue, Christiane UL; Engrand, A. et al

in Journal of Immunology (2008), 180(2), 998-1007

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1 ... [more ▼]

Jak1 is a tyrosine kinase that noncovalently forms tight complexes with a variety of cytokine receptors and is critically involved in signal transduction via cytokines. Jaks are predicted to have a 4.1, ezrin, radixin, moesin (FERM) domain at their N terminus. FERM domains are composed of three structurally unrelated subdomains (F1, F2, and F3) which are in close contact to one another and form the clover-shaped FERM domain. We generated a model structure of the Jak1 FERM domain, based on solved FERM structures and the alignments with other FERM domains. To destabilize different subdomains and to uncover their exact function, we mutated specific hydrophobic residues conserved in FERM domains and involved in hydrophobic core interactions. In this study, we show that the structural integrity of the F2 subdomain of the FERM domain of Jak1 is necessary to bind the IFN-gammaRalpha. By mutagenesis of hydrophobic residues in the hydrophobic core between the three FERM subdomains, we find that the structural context of the FERM domain is necessary for the inhibition of Jak1 phosphorylation. Thus, FERM domain mutations can have repercussions on Jak1 function. Interestingly, a mutation in the kinase domain (Jak1-K907E), known to abolish the catalytic activity, also leads to an impaired binding to the IFN-gammaRalpha when this mutant is expressed at endogenous levels in U4C cells. Our data show that the structural integrity of both the FERM domain and of the kinase domain is essential for both receptor binding and catalytic function/autoinhibition. [less ▲]

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See detailCell density dependent increase of constitutive signal transducers and activators of transcription 3 activity in melanoma cells is mediated by Janus kinases
Kreis, Stephanie UL; Munz, G. A.; Haan, Serge UL et al

in Molecular Cancer Research (2008), 5(12), 1331-41

Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signaling. Moreover, these transcription factors play a crucial role in oncogenic signaling where inappropriate and ... [more ▼]

Signal transducers and activators of transcriptions (STAT) are key mediators of cytokine signaling. Moreover, these transcription factors play a crucial role in oncogenic signaling where inappropriate and sustained activation of STATs, especially STAT3, is a trait of many different cancers and their derived cell lines. Constitutively active STAT3 has been reported to prevent programmed cell death and enhance cell proliferation, whereas the disruption of STAT3 signaling can inhibit tumor growth. The physiologic activation of STAT3 by cytokines has been well established; however, little is known about altered, stimulation-independent STAT3 activation. Here, we show that, in most but not all melanoma cell lines, STAT3 phosphorylation increased substantially with cell density and that this STAT3 was able to bind to DNA and to activate transcription. Inhibitor studies showed that the cell density-dependent STAT3 activation relies on Janus kinases (JAK) rather than Src kinases. Using a specific JAK inhibitor, sustained STAT3 activation was completely abrogated in all tested melanoma lines, whereas inhibition of Src or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 had no effect on constitutively tyrosine-phosphorylated STAT3 levels. Although STAT3 activation was completely blocked with JAK inhibitor I and to a lesser extent with the common JAK inhibitor AG490, only the latter compound markedly decreased proliferation and induced apoptosis. Taken together, variations in cell density can profoundly modify the extent of JAK-mediated persistent STAT3 phosphorylation; however, STAT3 activation was not sufficient to provide critical growth and survival signals in melanoma cell lines. [less ▲]

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See detailIL-24: a classic cytokine and/or a potential cure for cancer?
Kreis, Stephanie UL; Philippidou, Demetra UL; Margue, Christiane UL et al

in Journal of Cellular & Molecular Medicine (2008)

IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL-24 receptor chains (IL-20R1/IL-20R2 and IL-22R/IL ... [more ▼]

IL-24, a member of the IL-10 family of cytokines, is produced by monocytes and Th2 cells. Interestingly, immune cells do not appear to express specific IL-24 receptor chains (IL-20R1/IL-20R2 and IL-22R/IL-20R2), it is therefore unlikely that IL-24 has classical immune-modulating properties. Skin, on the other hand, seems to represent a major target tissue for IL-24 and related cytokines such as IL-19, -20, and -22. However, the initial interest in IL-24 did not arise from its physiological signalling properties through its cognate receptors but rather because of its tentative ability to selectively kill different cancer cells. In an attempt to further investigate the signalling events underlying the IL-24-induced cancer cell death, we found that melanoma cell lines did not react in the expected and previously described way. Using several different forms and delivery modes of IL-24, we were unable to detect any apoptosis-inducing properties of this cytokine in melanoma cells. In the present "Point of view" we will briefly summarise these findings and put them in context of published reports stating that IL-24 might be a long sought after treatment for several types of cancer. [less ▲]

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See detailRecombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells
Kreis, Stephanie UL; Philippidou, Demetra UL; Margue, Christiane UL et al

in PLoS ONE (2007), 2(12), 1300

IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th(2) cells as well as by activated monocytes. Binding of IL-24 to ... [more ▼]

IL-24, also known as melanoma differentiation antigen 7 (mda-7), is a member of the IL-10 family of cytokines and is mainly produced by Th(2) cells as well as by activated monocytes. Binding of IL-24 to either of its two possible heterodimeric receptors IL-20R1/IL-20R2 and IL-22R/IL-20R2 activates STAT3 and/or STAT1 in target tissues such as lung, testis, ovary, keratinocytes and skin. To date, the physiological properties of IL-24 are still not well understood but available data suggest that IL-24 affects epidermal functions by increasing proliferation of dermal cells. In stark contrast to its "normal" and physiological behaviour, IL-24 has been reported to selectively and efficiently kill a vast variety of cancer cells, especially melanoma cells, independent of receptor expression and Jak-STAT signalling. These intriguing properties have led to the development of adenovirally-expressed IL-24, which is currently being evaluated in clinical trials. Using three different methods, we have analysed a large panel of melanoma cell lines with respect to IL-24 and IL-24 receptor expression and found that none of the investigated cell lines expressed sufficient amounts of functional receptor pairs and therefore did not react to IL-24 stimulation with Jak/STAT activation. Results for three cell lines contrasted with previous studies, which reported presence of IL-24 receptors and activation of STAT3 following IL-24 stimulation. Furthermore, evaluating four different sources and modes of IL-24 administration (commercial recombinant IL-24, bacterially expressed GST-IL-24 fusion protein, IL-24 produced from transfected Hek cells, transiently over-expressed IL-24) no induction or increase in cell death was detected when compared to appropriate control treatments. Thus, we conclude that the cytokine IL-24 itself has no cancer-specific apoptosis-inducing properties in melanoma cells. [less ▲]

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See detailA cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background
Haan, Claude UL; Behrmann, Iris UL

in Journal of Immunological Methods (2007), 318(1-2), 11-9

We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol ... [more ▼]

We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other non-commercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested. [less ▲]

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See detailConstitutive suppressor of cytokine signaling 3 expression confers a growth advantage to a human melanoma cell line
Komyod, W.; Böhm, M.; Metze, D. et al

in Molecular Cancer Research (2007), 5(3), 271-81

The growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, whereas late stage melanoma cells have often been reported to be "multi-cytokine-resistant." Here, we analyzed ... [more ▼]

The growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, whereas late stage melanoma cells have often been reported to be "multi-cytokine-resistant." Here, we analyzed the melanoma cell line 1286, resistant towards the growth-inhibitory effects of interleukin 6 (IL-6), and oncostatin M (OSM), to better understand the mechanisms underlying cytokine resistance. Although the relevant receptors gp130 and OSMR are expressed at the cell surface of these cells, cytokine stimulation hardly led to the activation of Janus kinase 1 and signal transducer and activator of transcription (STAT)3 and STAT1. We found a high-level constitutive expression of suppressors of cytokine signaling 3 (SOCS3) that did not further increase after cytokine treatment. Importantly, upon suppression of SOCS3 by short interfering RNA, cells became susceptible towards OSM and IL-6: they showed an enhanced STAT3 phosphorylation and a dramatically increased STAT1 phosphorylation. Moreover, suppression of SOCS3 rendered 1286 cells sensitive to the antiproliferative action of IL-6 and OSM, but not of IFN-alpha. Interestingly, SOCS3-short interfering RNA treatment also increased the growth-inhibitory effect in cytokine-sensitive WM239 cells expressing SOCS3 in an inducible way. Thus, SOCS3 expression confers a growth advantage to these cell lines. Constitutive SOCS3 mRNA expression, although at lower levels than in 1286 cells, was found in nine additional human melanoma cell lines and in normal human melanocytes, although at the protein level, SOCS3 expression was marginal at best. However, in situ analysis of human melanoma specimens revealed SOCS3 immunoreactivity in 3 out of 10 samples, suggesting that in vivo SOCS3 may possibly play a role in IL-6 resistance in at least a fraction of tumors. [less ▲]

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See detailSTAT1 regulates p73-mediated Bax gene expression
Soond, S. M.; Carroll, C.; Townsend, P. A. et al

in FEBS Letters (2007), 581(6), 1217-26

Although signal transducer and activator of transcription 1 (STAT1) mediated regulation of p53 transcription and apoptosis has been previously reported, modulation of other members of the p53 family of ... [more ▼]

Although signal transducer and activator of transcription 1 (STAT1) mediated regulation of p53 transcription and apoptosis has been previously reported, modulation of other members of the p53 family of transcription factors remains poorly understood. In this study, we found that STAT1 and TA-p73 can interact directly and that p73-mediated Bax promoter activity was observed to be reduced by STAT1 expression in a p53-independent manner for which STAT1 Tyrosine-701 and Serine-727 are key residues. This study presents the first report physically linking STAT1 and TA-p73 signalling and highlights the modulation of the Bax promoter in the context of IFN-gamma stimulation. [less ▲]

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See detailJaks and cytokine receptors - an intimate relationship
Haan, Claude UL; Kreis, Stephanie UL; Margue, Christiane UL et al

in Biochemical Pharmacology (2006), 72(11), 1538-46

Most cytokine receptors lack intrinsic kinase activity and many of them signal via Janus kinases (Jaks). These tyrosine kinases are associated with cytokine receptor subunits, they become activated upon ... [more ▼]

Most cytokine receptors lack intrinsic kinase activity and many of them signal via Janus kinases (Jaks). These tyrosine kinases are associated with cytokine receptor subunits, they become activated upon receptor triggering and subsequently activate downstream signalling events, e.g. the phosphorylation of STAT transcription factors. The successful interplay between cytokines, their receptors and the connected Jaks not only determines signalling competence but is also vital for intracellular traffic, stability, and fate of the cognate receptors. Here, we will discuss underlying mechanisms as well as some structural features with a focus on Jak1 and two of the signal transducing receptor subunits of interleukin (IL)-6 type cytokines, gp130 and OSMR. Regions that are critically involved in Jak-binding have been identified for many cytokine receptor subunits. In most cases the membrane-proximal parts comprising the box1 and box2 regions within the receptor are involved in this association while, within Jaks, the N-terminal FERM domain, possibly together with the SH2-like domain, are pivotal for binding to the relevant receptors. The exclusive membrane localisation of Jaks depends on their ability to associate with cytokine receptors. For gp130 and Jak1, it was shown that the cytokine receptor/Jak complex can be regarded as a receptor tyrosine kinase since both molecules have the same diffusion dynamics and are virtually undissociable. Furthermore, Jaks take an active role in the regulation of the surface expression of at least some cytokine receptors, including the OSMR and this may provide a quality control mechanism ensuring that only signalling-competent receptors (i.e. those with an associated Jak) would be enriched at the cell surface. [less ▲]

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See detailBiological responses to PDGF-BB versus PDGF-DD in human mesangial cells
van Roeyen, C. R.; Ostendorf, T.; Denecke, B. et al

in Kidney International (2006), 69(8), 1393-402

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more ... [more ▼]

Platelet-derived growth factor (PDGF)-BB and PDGF-DD mediate mesangial cell proliferation in vitro and in vivo. While PDGF-BB is a ligand for the PDGF alpha- and beta-receptor chains, PDGF-DD binds more selectively to the beta-chain, suggesting potential differences in the biological activities. Signal transduction and regulation of gene expression induced by PDGF-BB and -DD were compared in primary human mesangial cells (HMCs), which expressed PDGF alpha- and beta-receptor subunits. The growth factor concentrations used were chosen based on their equipotency in inducing HMCs proliferation and binding to the betabeta-receptor. Both growth factors, albeit at different concentrations induced phosphorylation and activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2. In addition, PDGFs led to the phosphorylation and activation of signal transducers and activators of transcription 1 (STAT1) and STAT3. HMCs proliferation induced by either PDGF-BB or -DD could be blocked by signal transduction inhibitors of the mitogen-activated protein kinase-, Janus kinase (JAK)/STAT-, or phosphatidyl-inositol 3-kinase pathways. Using a gene chip array and subsequent verification by real-time reverse transcriptase (RT)-polymerase chain reaction, we found that in HMC genes for matrix metalloproteinase 13 (MMP-13) and MMP-14 and, to a low extent, cytochrome B5 and cathepsin L were exclusively regulated by PDGF-BB, whereas no exclusive gene regulation was detected by PDGF-DD. However, at the protein level, both MMP-13 and -14 were equally induced by PDGF-BB and -DD. PDGF-BB and -DD effect similar biological responses in HMCs albeit at different potencies. Rare apparently differential gene regulation did not result in different protein expression, suggesting that in HMCs both PDGFs exert their biological activity almost exclusively via the PDGF beta-receptor. [less ▲]

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See detailThree 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
Radtke, S.; Jörissen, A.; de Leur, H. S. et al

in Journal of Biological Chemistry (2006), 281(7), 4024-34

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 ... [more ▼]

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. [less ▲]

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See detailMultiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation
Haan, Serge UL; Keller, J. F.; Behrmann, Iris UL et al

in Cellular Signalling (2005), 17(12), 1542-50

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and ... [more ▼]

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and fertility. We found that IL-6-type cytokine stimulation of cell lines and primary human macrophages results in a different distribution of the DNA-binding competent STAT dimer species in the cytosol and nucleus as demonstrated by electrophoretic mobility shift assays. In the absence of detergent, STAT3/STAT3, STAT1/STAT3 were the predominant species in the cytoplasm while STAT3/STAT3 was predominant in the nucleus. However, in detergent containing total cellular lysates and nuclear fractions prepared with detergent containing buffers, the STAT1/STAT1 homodimer was as prominent or even more prominent than STAT3/STAT3 and STAT1/STAT3. We were interested in the cause of this discrepancy since STAT1-regulated genes have not been described to be expressed upon IL-6-type cytokine stimulation. In addition to the more transient STAT1 activation, IL-6-type cytokines such as IL-6 and OSM lead to a much less efficient STAT1 activation compared to the potent STAT1 activators IFNgamma and IFNalpha. Studies with STAT1-deficient cells revealed that STAT1 activation does not seem to be an important competitive process to STAT3 activation arguing again for a very inefficient STAT1 activation upon IL-6-type cytokine stimulation. We also describe that pY-STAT3 is much more efficiently shuttled into the nucleus than pY-STAT1. [less ▲]

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See detailAre STATS arginine-methylated?
Komyod, W.; Bauer, U. M.; Heinrich, P. C. et al

in Journal of Biological Chemistry (2005), 280(23), 21700-5

Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by ... [more ▼]

Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by phosphorylation on tyrosine and serine residues. Recent evidence suggested that STATs are methylated on a conserved arginine residue within the N-terminal region. STAT arginine methylation has been described to be important for STAT function and loss of arginine methylation was discussed to be involved in interferon resistance of cancer cells. Here we provide several independent lines of evidence indicating that the issue of arginine methylation of STATs has to be reassessed. First, we show that treatment of melanoma and fibrosarcoma cells with inhibitors used to suppress methylation (N-methyl-2-deoxyadenosine, adenosine, dl-homocysteine) had profound and rapid effects on phosphorylation of STAT1 and STAT3 but also on p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Second, we show that anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Third, we show that mutation of Arg(31) to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg(31). Finally, purified catalytically active protein arginine methyltransferases (PRMT1, -2, -3, -4, and -6) did not methylate STAT proteins, and cotransfection with PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, our data suggest the absence of arginine methylation of STAT1 and STAT3. [less ▲]

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