Molecular Structure and Function of Janus Kinases: Implications for the Development of Inhibitors

Abstract Cytokines can trigger multiple signalling pathways, including Janus tyrosine kinases [JAK] and signal transducers and activators of transcription [STATS] pathways. JAKs are cytoplasmic proteins that, following the binding of cytokines to their receptors, transduce the signal by phosphorylating STAT proteins which enter the nuclei and rapidly target gene promoters to regulate gene transcription. Due to the critical involvement of JAK proteins in mediating innate and adaptive immune responses, these family of kinases have become desirable pharmacological targets in inflammatory diseases, including ulcerative colitis and Crohn’s disease. In this review we provide an overview of the main cytokines that signal through the JAK/STAT pathway and the available in vivo evidence on mutant or deleted JAK proteins, and discuss the implications of pharmacologically targeting this kinase family in the context of inflammatory diseases.

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Direct Interaction of Jak1 and v-Abl Is Required for v-Abl-Induced Activation of STATs and Proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6795 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6795-6804 ◽

Cited By ~ 39

Author(s):

Nika N. Danial ◽

Julie A. Losman ◽

Tianhong Lu ◽

Natalie Yip ◽

Kartik Krishnan ◽

...

Keyword(s):

Tyrosine Kinases ◽

Leukemia Virus ◽

Direct Interaction ◽

Janus Kinase ◽

Signaling Proteins ◽

Interaction Domain ◽

Signal Transducers ◽

Stat Proteins ◽

Immature B Cells

ABSTRACT In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl–Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abloncogene.

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Activation and association of Stat3 with Src in v-Src-transformed cell lines.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1595 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1595-1603 ◽

Cited By ~ 264

Author(s):

X Cao ◽

A Tay ◽

G R Guy ◽

Y H Tan

Keyword(s):

Growth Factor ◽

Cell Lines ◽

Tyrosine Kinases ◽

New Members ◽

Signal Transducers ◽

Stat Proteins ◽

Transformed Cell ◽

Transformed Cell Lines

STAT proteins are a group of latent cytoplasmic transcription factors which function as signal transducers and activators of transcription. Stat1 and -2 were originally identified to function in interferon signaling, and Stat1 was also found to be activated by epidermal growth factor (EGF) and other cytokines. New members of the STAT gene family are identified. Among them, Stat3 has 52.5% amino acid sequence homology with Stat1 and is activated by platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), EGF, interleukin-6, and other cytokines. Treatment of cells with EGF activates Stat1 and Stat3, which become phosphorylated on tyrosine residues to form homo - or heterodimers and translocate into the nucleus, binding to the sis-inducible element (SIE) in the c-fos promoter. Somatic cell genetic analyses demonstrated that Jaks, a family of nontransmembrane protein tyrosine kinases, are required for the activation of Stat1 and Stat2 in interferon-treated cells. However, little is known about the activation of Stat3 by growth factors. Here we report that in all v-Src-transformed cell lines examined, Stat3 is constitutively activated to bind to DNA and the phosphorylation of tyrosine on Stat3 is enhanced by the induction of v-Src expression. We also report that Src is shown to be associated with Stat3 in vivo, as well as in vitro, and phosphorylates Stat3 in vitro. Stat3 is also activated by CSF-1, possibly through CSF-1 receptor-c Src association in NIH 3T3 cells overexpressing CSF-1 receptors. Together, the data suggest that Src is involved in activation of Stat3 in growth factor signal transduction.

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Extracellular vesicles as mediators and markers of acute organ injury: current concepts

European Journal of Trauma and Emergency Surgery ◽

10.1007/s00068-021-01607-1 ◽

2021 ◽

Author(s):

Birte Weber ◽

Niklas Franz ◽

Ingo Marzi ◽

Dirk Henrich ◽

Liudmila Leppik

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Diseases ◽

Organ Injury ◽

Isolation And Characterization ◽

Communication Processes ◽

Incidence And Mortality ◽

New Biomarkers ◽

And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

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Abstract P153: Role of Nonreceptor Tyrosine Kinases in Cardiac Fibrosis in a Mouse Model of Pressure Overload Hypertrophy

Circulation Research ◽

10.1161/res.109.suppl_1.ap153 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Sundaravadivel Balasubramanian ◽

Harinath Kasiganesan ◽

Lakeya Quinones ◽

Yuhua Zhang ◽

Amy Bradshaw ◽

...

Keyword(s):

Tyrosine Kinases ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Heart Diseases ◽

Pressure Overload ◽

In Vivo Studies ◽

Survival Pathways ◽

And Function ◽

Nonreceptor Tyrosine Kinases

During prolonged hypertrophic insult to the myocardium, while the function of cardiomyocytes needs to be protected, the hyperactivation of cardiac fibroblasts has to be curbed to prevent fibrosis. Previously, we showed that integrin-mediated non-receptor tyrosine kinase (NRTK) activation is required for normal functioning of both cardiac fibroblasts and cardiomyocytes. We hypothesized that inhibition of NRTKs in cardiac fibroblasts without affecting cardiomyocytes would be beneficial to the stressed myocardium. Our initial studies using kinase inactive forms of Src, Pyk2 and FAK expressed adenovirally in isolated primary cardiac fibroblasts showed that the pro-fibrotic signaling events as studied by fibronectin and collagen deposition are downregulated. Our in vivo studies in mouse transverse aortic constriction (TAC) model suggest that dasatinib, a multikinase NRTK inhibitor administration via a peritoneally implanted mini-osmotic pump is able to preserve ventricular geometry and function and reduce the accumulation of fibrotic extracellular matrix (ECM) proteins upon 4 wk pressure overload. Data obtained from cell culture experiments with kinase inactive NRTKs and dasatinib suggest that NRTK inhibition is able to reduce the proliferation, migration and mitogenic signaling in cardiac fibroblasts without affecting the cell survival pathways in cardiomyocytes. These data indicate that NRTKs play a significant pro-fibrotic role in cardiac fibroblasts and curbing the activity of NRTKs could be a potential therapeutic approach to treat fibrosis in hypertrophic heart diseases.

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Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets

Blood ◽

10.1182/blood.v87.2.439.bloodjournal872439 ◽

1996 ◽

Vol 87 (2) ◽

pp. 439-446 ◽

Cited By ~ 126

Author(s):

Y Miyakawa ◽

A Oda ◽

BJ Druker ◽

H Miyazaki ◽

M Handa ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Human Platelet ◽

Blood Platelets ◽

Human Platelets ◽

Genetically Engineered ◽

Hematopoietic Growth Factors ◽

Signal Transducers ◽

Stat Proteins ◽

Human Blood Platelets

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c- Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.

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Expression and Function of Murine Receptor Tyrosine Kinases, TIE and TEK, in Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v89.12.4317 ◽

1997 ◽

Vol 89 (12) ◽

pp. 4317-4326 ◽

Cited By ~ 71

Author(s):

Michihiro Yano ◽

Atsushi Iwama ◽

Hitoshi Nishio ◽

Junko Suda ◽

Goro Takada ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Tyrosine Kinases ◽

Receptor Tyrosine Kinases ◽

Stem Cell Marker ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

And Function

Abstract Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit+ and Sca-1+ (Lin− c-Kit+ Sca-1+) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin− c-Kit+ Sca-1+ cells were further divided by the expression of TIE and TEK. TIE+ and TEK+ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE+ and TEK+ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.

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In Vivo Targeting of the Growth Hormone Receptor (GHR) Box1 Sequence Demonstrates that the GHR Does Not Signal Exclusively through JAK2

Molecular Endocrinology ◽

10.1210/me.2009-0233 ◽

2010 ◽

Vol 24 (1) ◽

pp. 204-217 ◽

Cited By ~ 45

Author(s):

Johanna L. Barclay ◽

Linda M. Kerr ◽

Leela Arthur ◽

Jennifer E. Rowland ◽

Caroline N. Nelson ◽

...

Keyword(s):

Tyrosine Kinases ◽

Growth Hormone Receptor ◽

Target Genes ◽

Postnatal Growth ◽

Signal Transducers ◽

Phenotypic Differences ◽

Protein Levels ◽

Igf Binding ◽

Transcript Profiles

Abstract GH is generally believed to signal exclusively through Janus tyrosine kinases (JAK), particularly JAK2, leading to activation of signal transducers and activators of transcription (STAT), ERK and phosphatidylinositol 3-kinase pathways, resulting in transcriptional regulation of target genes. Here we report the creation of targeted knock-in mice wherein the Box1 motif required for JAK2 activation by the GH receptor (GHR) has been disabled by four Pro/Ala mutations. These mice are unable to activate hepatic JAK2, STAT3, STAT5, or Akt in response to GH injection but can activate Src and ERK1/2. Their phenotype is identical to that of the GHR−/− mouse, emphasizing the key role of JAK2 in postnatal growth and the minimization of obesity in older males. In particular, they show dysregulation of the IGF-I/IGF-binding protein axis at transcript and protein levels and decreased bone length. Because no gross phenotypic differences were evident between GHR−/− and Box1 mutants, we undertook transcript profiling in liver from 4-month-old males. We compared their transcript profiles with our 391-GHR truncated mice, which activate JAK2, ERK1/2, and STAT3 in response to GH but not STAT5a/b. This has allowed us for the first time to identify in vivo Src/ERK-regulated transcripts, JAK2-regulated transcripts, and those regulated by the distal part of the GHR, particularly by STAT5.

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Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation

Blood ◽

10.1182/blood.v99.1.102 ◽

2002 ◽

Vol 99 (1) ◽

pp. 102-110 ◽

Cited By ~ 60

Author(s):

Keita Kirito ◽

Koichi Nakajima ◽

Tomoko Watanabe ◽

Mie Uchida ◽

Masaru Tanaka ◽

...

Keyword(s):

Tyrosine Kinases ◽

Transmembrane Domain ◽

Erythropoietin Receptor ◽

Signal Transducers ◽

Chimeric Receptors ◽

Stat Proteins ◽

Human Erythropoietin ◽

Myc Gene ◽

Dependent Cell ◽

Stimulating Factor

Signal transducers and activators of transcription (Stat) proteins play important roles in the regulation of hematopoiesis as downstream molecules of cytokine signal transduction. It was previously demonstrated that erythropoietin (EPO), a major regulator of erythropoiesis, activates 3 different Stat members, Stat1, Stat3, and Stat5, in a human EPO-dependent cell line, UT-7/EPO. To clarify the mechanism by which EPO activates Stat1 and Stat3 via the EPO receptor (EPOR), a series of chimeric receptors was constructed bearing the extracellular domain of the granulocyte colony-stimulating factor receptor linked to the transmembrane domain of EPOR and the full length or several mutants of the cytoplasmic domain of EPOR, and these chimeric receptor complementary DNAs were introduced into UT-7/EPO cells. Tyr432 on human EPOR was important for activation of Stat1 and Stat3 and c-myc gene induction. In addition, Jak2 and Fes tyrosine kinases were involved in EPO-induced activation of Stat1 and Stat3. These results indicate that Stat1 and Stat3 are activated by EPO via distinct mechanisms from Stat5.

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α-Melanocyte Stimulating Hormone Prevents Lipopolysaccharide-Induced Vasculitis by Down-Regulating Endothelial Cell Adhesion Molecule Expression

Endocrinology ◽

10.1210/en.2002-220651 ◽

2003 ◽

Vol 144 (1) ◽

pp. 360-370 ◽

Cited By ~ 63

Author(s):

T. E. Scholzen ◽

C. Sunderkötter ◽

D.-H. Kalden ◽

T. Brzoska ◽

M. Fastrich ◽

...

Keyword(s):

Cell Adhesion ◽

Inflammatory Diseases ◽

Nuclear Factor Κb ◽

Vascular Damage ◽

Adhesion Assay ◽

Melanocyte Stimulating Hormone ◽

And Function ◽

Stimulating Hormone

Abstract The neuroendocrine hormone α-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that α-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNFα in vitro and in vivo. In HDMEC, α-MSH (10−8/10−12m) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNFα as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, α-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, α-MSH effectively inhibited the transcription factor nuclear factor-κB activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of α-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that α-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.

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Molecular mechanisms of the glucocorticoid receptor in steroid therapy – lessons from transgenic mice

BioMolecular Concepts ◽

10.1515/bmc-2011-0033 ◽

2012 ◽

Vol 3 (3) ◽

pp. 241-253 ◽

Cited By ~ 4

Author(s):

Sabine Hübner ◽

Jan Tuckermann

Keyword(s):

Side Effects ◽

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Pathological Condition ◽

Cell Types ◽

Therapeutic Effects ◽

Gene Promoters ◽

Inflammatory Conditions ◽

Anti Inflammatory ◽

And Function

AbstractGlucocorticoids (GCs) are potent anti-inflammatory agents that are used to treat chronic inflammatory diseases, allergic conditions, and some cancers. However, their therapeutic effects are hampered by severe side effects, such as muscle weakness, insulin resistance, fat redistribution, and osteoporosis. GCs act on many cell types that express the GC receptor (GR) via several modes of action. One of them includes GR homodimers recognizing binding sequences in the DNA of gene promoters. Another mode involves the modulation of other DNA-bound transcription factors via dimer-independent mechanisms. To what extent these mechanisms contribute to GC-mediated effects is currently being elucidated from analyses of mice with conditional and function-selective mutations of the GR and is summarized in this review. Whether GR homodimerization or its monomer activity is decisive in the therapeutic effectiveness and associated side effects of GCs for the treatment of inflammatory conditions depends on the type of the pathological condition. Thus, the classic criterion for selective GR modulators, discrimination between GR dimer- and GR monomer-dependent protein-protein interaction, will not help in any condition to avoid side effects and maintain anti-inflammatory activity. Rather, novel criteria for selective GR modulators have to be defined that take into consideration the tissue-specific mechanisms of the GR to achieve optimized anti-inflammatory therapies with reduced side effects. In the case of avoiding osteoporosis as a side effect, a first example of such optimized compounds can be provided.

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