Claude Haan

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.

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.

Long term association of the cytokine receptor gp130 and the Janus kinase Jak1 revealed by FRAP analysis

Signal transduction through cytokine receptors is mediated mainly by non-covalently associated Jak tyrosine kinases. By confocal microscopy, the cytokine receptor gp130 and Jak1, fused with either yellow (YFP) or cyan (CFP) fluorescent protein, were found to be colocalized predominantly at intracellular vesicular structures and at the plasma membrane. Quantitative fluorescence recovery after photobleaching (FRAP) analysis at the plasma membrane revealed equal mobilities for gp130-YFP and Jak1-YFP. Thus, Jak1-YFP diffuses like a transmembrane protein indicating that membrane-bound Jak1 does not exchange rapidly with cytosolic Jaks. Applying a novel dual-color FRAP approach we found that immobilization of gp130-CFP by a pair of monoclonal antibodies led to a corresponding immobilization of co-transfected Jak1-YFP. We conclude from these findings that Jak1, once bound to a gp130 molecule, does not exchange between different receptors at the plasma membrane neither via the cytoplasmic compartment nor via a membrane-associated state.

Interleukin‐27 displays interferon‐γ–like functions in human hepatoma cells and hepatocytes

Interleukin‐27 (IL‐27) is a cytokine belonging to the IL‐6/IL‐12 cytokine family. It is secreted by antigen‐presenting cells, strongly acts on T cells, and also stimulates innate immune cells. In most studies, the effects of IL‐27 on T cells were investigated; however, not much is known about possible effects of IL‐27 on other cell types. IL‐27 signals via the common IL‐6–type cytokine receptor chain gp130 and the IL‐27–specific chain WSX‐1. Given the importance of gp130 in regulating liver responses such as the acute phase response or liver regeneration, we investigated whether IL‐27 could also have a function in liver cells. We find that IL‐27 stimulates hepatoma cells and hepatocytes by inducing a sustained signal transducer and activator of transcription (STAT)1 and STAT3 activation. Whereas the STAT3 mediated responses to IL‐27 (γ‐fibrinogen and hepcidin induction) are not detectable, we observe an interferon‐gamma (IFN‐γ)–like STAT1 response leading to the induction of interferon‐regulated proteins such as STAT1, STAT2, interferon response factor (IRF)‐1, IRF‐9, myxovirus resistance A and guanylate binding protein 2. Conclusion: Our study provides evidence for a function of IL‐27 in hepatoma cells and hepatocytes and shows that IL‐27 responses are not restricted to the classical immune cells. Our results suggest that IL‐27 exerts IFN‐like functions in liver cells and that it can contribute to the antiviral response in these cells. (HEPATOLOGY 2009.)

Dual Role of the Jak1 FERM and Kinase Domains in Cytokine Receptor Binding and in Stimulation-Dependent Jak Activation

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-γRα. 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-γRα 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.

Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases

•  Introduction ‐  Jaks in disease ‐  Jak2‐V617F signal transduction •  Structure/function: the potential interest of the Jak domains as drug targets ‐  The FERM domain ‐  The FERM domain as drug target ‐  The SH2 domain ‐  The SH2 domain as drug target ‐  The pseudokinase domain ‐  The pseudokinase domain as drug target ‐  The kinase domain ‐  The kinase domain as drug target ‐  Summary •  The growing family of ATP‐competitive nanomolar Jak inhibitors •  Analogue‐sensitive kinases and possible applications to Jaks ‐  Chemical genetics to characterize kinases ‐  Chemical genetics in practice: possible pitfalls and requirements ‐  Advantages and possible applications of the chemical genetics approach ‐  Investigation of specific kinase‐mediated effects ‐  Specific and flexible pharmacologic intervention allows target validation of compounds from drug screens ‐  Identification of direct substrates •  Structure‐based interpretation of the Jak mutations ‐  Mutations within the FERM and SH2 domains ‐  Mutations within the kinase‐like domain ‐  Mutations within the kinase domain •  Perspectives

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling

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.

Hypoxia‐inducible factor 1α is up‐regulated by oncostatin M and participates in oncostatin M signaling

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 1α (HIF1α) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1α under normoxic and hypoxic conditions. Furthermore, the OSM‐dependent HIF1α 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 HIF1α up‐regulation did not result from an increase in HIF1α protein stability but from increased transcription from the HIF1α gene. In addition, we show that the OSM‐induced HIF1α gene transcription and the resulting enhanced HIF1α protein levels are important for the OSM‐dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. Conclusion: HIF1α levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1α 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.)

Recombinant interleukin-24 lacks apoptosis-inducing properties in melanoma cells.

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 Th2 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.

Oncostatin M-induced and constitutive activation of the JAK2/STAT5/CIS pathway suppresses CCL1, but not CCL7 and CCL8, chemokine expression.

The recruitment of leukocytes to injured tissue is crucial for the initiation of inflammatory responses as well as for immune surveillance to fight tumor progression. In this study, we show that oncostatin M, a member of the IL-6-type cytokine family and potent proinflammatory cytokine stimulates the expression of the chemokines CCL1, CCL7, and CCL8 in primary human dermal fibroblasts at a faster kinetic than IL-1β or TNF-α. The production of CCL1 and CCL8 is important for migration of monocytes, while specific Abs against CCL1 additionally inhibit the migration of T lymphocytes. We identify the mitogen-activated protein kinases ERK1/2 and p38 as crucial factors for the enhanced expression of CCL1 and CCL8. Depletion of the ERK1/2 target genes c-Jun or c-Fos strongly decrease CCL1 and CCL8 expression, while p38 MAPK prolongs the half-life of CCL1, CCL7, and CCL8 mRNA through inhibition of tristetraprolin. None of the STAT transcription factors STAT1, STAT3, or STAT5 stimulate transcription of CCL1 or CCL8. However, we identify a negative regulatory function of activated STAT5 for the gene expression of CCL1. Importantly, not STAT5 itself, but its target gene cytokine inducible SH2-domain containing protein is required for the STAT5 inhibitory effect on CCL1 expression. Finally, we show that constitutive activation of STAT5 through a mutated form of JAK2 (JAK2 V617F) occurring in patients with myeloproliferative disorders similarly suppresses CCL1 expression. Taken together, we identify novel important inflammatory target genes of OSM which are independent of STAT signaling per se, but depend on MAPK activation and are partly repressed through STAT5-dependent expression of cytokine inducible SH2-domain containing protein.