Browsing by Subject "TYROSINE KINASE"

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  • Raivola, Juuli; Haikarainen, Teemu; Silvennoinen, Olli (2020)
    The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-gamma (IFN gamma) and interferon-alpha (IFN alpha) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFN gamma signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the alpha C-helix. Mutating L633 at the alpha C reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators.
  • Dietrich, Sascha; Oles, Malgorzata; Lu, Junyan; Sellner, Leopold; Anders, Simon; Velten, Britta; Wu, Bian; Huellein, Jennifer; Liberio, Michelle da Silva; Walther, Tatjana; Wagner, Lena; Rabe, Sophie; Ghidelli-Disse, Sonja; Bantscheff, Marcus; Oles, Andrzej K.; Slabicki, Mikolaj; Mock, Andreas; Oakes, Christopher C.; Wang, Shihui; Oppermann, Sina; Lukas, Marina; Kim, Vladislav; Sill, Martin; Benner, Axel; Jauch, Anna; Sutton, Lesley Ann; Young, Emma; Rosenquist, Richard; Liu, Xiyang; Jethwa, Alexander; Lee, Kwang Seok; Lewis, Joe; Putzker, Kerstin; Lutz, Christoph; Rossi, Davide; Mokhir, Andriy; Oellerich, Thomas; Zirlik, Katja; Herling, Marco; Nguyen-Khac, Florence; Plass, Christoph; Andersson, Emma; Mustjoki, Satu; von Kalle, Christof; Ho, Anthony D.; Hensel, Manfred; Duerig, Jan; Ringshausen, Ingo; Zapatka, Marc; Huber, Wolfgang; Zenz, Thorsten (2018)
    As new generations of targeted therapies emerge and tumor genome sequencing discovers increasingly comprehensive mutation repertoires, the functional relationships of mutations to tumor phenotypes remain largely unknown. Here, we measured ex vivo sensitivity of 246 blood cancers to 63 drugs alongside genome, transcriptome, and DNA methylome analysis to understand determinants of drug response. We assembled a primary blood cancer cell encyclopedia data set that revealed disease-specific sensitivities for each cancer. Within chronic lymphocytic leukemia (CLL), responses to 62% of drugs were associated with 2 or more mutations, and linked the B cell receptor (BCR) pathway to trisomy 12, an important driver of CLL. Based on drug responses, the disease could be organized into phenotypic subgroups characterized by exploitable dependencies on BCR, mTOR, or MEK signaling and associated with mutations, gene expression, and DNA methylation. Fourteen percent of CLLs were driven by mTOR signaling in a non-BCR-dependent manner. Multivariate modeling revealed immunoglobulin heavy chain variable gene (IGHV) mutation status and trisomy 12 as the most important modulators of response to kinase inhibitors in CLL. Ex vivo drug responses were associated with outcome. This study overcomes the perception that most mutations do not influence drug response of cancer, and points to an updated approach to understanding tumor biology, with implications for biomarker discovery and cancer care.
  • Chmielarz, Piotr; Er, Safak; Konovalova, Julia; Bandres, Laura; Hlushchuk, Irena; Albert, Katrina; Panhelainen, Anne; Luk, Kelvin; Airavaara, Mikko; Domanskyi, Andrii (2020)
    Background Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded alpha-synuclein (alpha-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded alpha-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. Objectives We studied the effects of GDNF on misfolded alpha-syn accumulation in DA neurons. Methods Lewy pathology progression was modeled by the application of alpha-syn preformed fibrils in cultured DA neurons and in the adult mice. Results We discovered that GDNF prevented accumulation of misfolded alpha-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced alpha-syn accumulation. Conclusions For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded alpha-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. (c) 2020 International Parkinson and Movement Disorder Society
  • Raivola, Juuli; Hammaren, Henrik M.; Virtanen, Anniina T.; Bulleeraz, Vilasha; Ward, Alister C.; Silvennoinen, Olli (2018)
    Janus kinase 3 (JAK3) tyrosine kinase has a central role in the control of lymphopoiesis, and mutations in JAK3 can lead to either severe combined immunodeficiency or leukemia and lymphomas. JAK3 associates with the common gamma chain (yc) receptor and functions in a heteromeric signaling pair with JAK1. In IL-2 signaling JAK1 is the effector kinase for STAT5 phosphorylation but the precise molecular regulatory mechanisms of JAK1 and JAK3 and their individual domains are not known. The pseudokinase domain (JAK homology 2, JH2) of JAK3 is of particular interest as approximately half of clinical JAK3 mutations cluster into it. In this study, we investigated the role of JH2s of JAK1 and JAK3 in IL-2R signaling and show that STAT5 activation requires both JH1 and JH2 of JAK1, while both JH1 and JH2 in JAK3 are specifically required for the cytokine-induction of cellular signaling. Characterization of recombinant JAK3 JH2 in thermal shift assay shows an unstable protein domain, which is strongly stabilized by ATP binding. Unexpectedly, nucleotide binding to JAK3 JH2 was found to be cation-independent. JAK3 JH2 showed higher nucleotide binding affinity in MANT-ATP and fluorescent polarization competition assays compared to the other JAK JH2s. Analysis of the functional role of ATP binding in JAK3 JH2 in cells and in zebrafish showed that disruption of ATP binding suppresses ligand-independent activation of clinical JAK3 gain-of-function mutations residing in either JH2 or JH1 but does not inhibit constitutive activation of oncogenic JAK1. ATP-binding site mutations in JAK3 JH2 do not, however, abrogate normal IL-2 signaling making them distinct from JH2 deletion or kinase-deficient JAK3. These findings underline the importance of JAK3 JH2 for cellular signaling in both ligand-dependent and in gain-of-function mutation-induced activation. Furthermore, they identify the JH2 ATP-binding site as a key regulatory region for oncogenic JAK3 signaling, and thus a potential target for therapeutic modulation.
  • Vorkapic, Emina; Dugic, Elma; Vikingsson, Svante; Roy, Joy; Mäyränpää, Mikko; Eriksson, Per; Wagsater, Dick (2016)
    Background: Abdominal aortic aneurysm (AAA) is characterized by vascular remodeling with increased infiltration of inflammatory cells and apoptosis/modulation of vascular smooth muscle cells (SMCs). Imatinib is a selective inhibitor of several tyrosine kinases, including PDGF receptors, Abl, and c-kit. The objective of this study was to characterize the potential protective role of imatinib on AAA development and the molecular mechanisms involved. Methods: Male ApoE(-/)-mice were infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Daily treatment with 10 mg/kg imatinib, or tap water as control, was provided via gavage for 4 weeks. Results: Treatment with imatinib was found to decrease the aortic diameter and vessel wall thickness, mediated by multiple effects. Imatinib treatment in AngII infused mice resulted in a reduced cellular infiltration of CD3 epsilon positive T lymphocytes by 86% and reduced gene expression of mast cell chymase by 50% compared with AngII infused mice lacking imatinib. Gene expression analysis of SMC marker SM22a demonstrated an increase by 48% together with a more intact medial layer after treatment with imatinib as evaluated with SM22 alpha immunostaining. Conclusion: Present findings highlight the importance of tyrosine kinase pathways in the development of AAA. Our results show, that imatinib treatment inhibits essential mast cell, T lymphocyte and SMC mediated processes in experimental AAA. Thus, our results support the idea that tyrosine kinase inhibitors may be useful in the treatment of pathological vascular inflammation and remodeling in conditions like AAA. (C) 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
  • Gadina, Massimo; Le, Mimi T.; Schwartz, Daniella M.; Silvennoinen, Olli; Nakayamada, Shingo; Yamaoka, Kunihiro; O'Shea, John J. (2019)
    Cytokines are critical mediators of diverse immune and inflammatory diseases. Targeting cytokines and cytokine receptors with biologics has revolutionized the treatment of many of these diseases, but targeting intracellular signalling with Janus kinase (JAK) inhibitors (jakinibs) now represents a major new therapeutic advance. We are still in the first decade since these drugs were approved and there is still much to be learned about the mechanisms of action of these drugs and the practical use of these agents. Herein we will review cytokines that do, and just as importantly, do not signal by JAKs, as well as explain how this relates to both efficacy and side effects in various diseases. We will review new, next-generation selective jakinibs, as well as the prospects and challenges ahead in targeting JAKs.
  • Kleino, Iivari; Jarviluoma, Annika; Hepojoki, Jussi; Huovila, Ari Pekka; Saksela, Kalle (2015)
    A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.
  • Salonen, Johanna; Ronnholm, Gunilla; Kalkkinen, Nisse; Vihinen, Mauno (2013)
  • Jmaeff, Sean; Sidorova, Yulia; Lippiatt, Hayley; Barcelona, Pablo F.; Nedev, Hinyu; Saragovi, Lucia M.; Hancock, Mark A.; Saarma, Mart; Saragovi, H. Uri (2020)
    Glial cell line-derived neurotrophic factor (GDNF) binds the GFR alpha 1 receptor, and the GDNF-GFR alpha 1 complex binds to and activates the transmembrane RET tyrosine kinase to signal through intracellular Akt/Erk pathways. To dissect the GDNF-GFR alpha 1-RET signaling complex, agents that bind and activate RET directly and independently of GFR alpha 1 expression are valuable tools. In a focused naphthalenesulfonic acid library from the National Cancer Institute database, we identified small molecules that are genuine ligands binding to the RET extracellular domain. These ligands activate RET tyrosine kinase and afford trophic signals irrespective of GFR alpha 1 coexpression. However, RET activation by these ligands is constrained by GFR alpha 1, likely via an allosteric mechanism that can be overcome by increasing RET ligand concentration. In a mouse model of retinitis pigmentosa, monotherapy with a small-molecule RET agonist activates survival signals and reduces neuronal death significantly better than GDNF, suggesting therapeutic potential. SIGNIFICANCE STATEMENT A genuine ligand of RET receptor ectodomain was identified, which acts as an agonist. Binding and agonism are independent of a coreceptor glial cell line-derived neurotrophic factor family receptor a, which is required by the natural growth factor glial cell line-derived neurotrophic factor, and are selective for cells expressing RET. The lead agent protects neurons from death in vivo. This work validates RET receptor as a druggable therapeutic target and provides for potential leads to evaluate in neurodegenerative states. We also report problems that arise when screening chemical libraries.
  • Siddiqui, Arafat; Tumiati, Manuela; Joko, Alia; Sandholm, Jouko; Roering, Pia; Aakko, Sofia; Vainionpää, Reetta; Kaipio, Katja; Huhtinen, Kaisa; Kauppi, Liisa; Tuomela, Johanna; Hietanen, Sakari (2021)
    Critical DNA repair pathways become deranged during cancer development. This vulnerability may be exploited with DNA-targeting chemotherapy. Topoisomerase II inhibitors induce double-strand breaks which, if not repaired, are detrimental to the cell. This repair process requires high-fidelity functional homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). If either of these pathways is defective, a compensatory pathway may rescue the cells and induce treatment resistance. Consistently, HR proficiency, either inherent or acquired during the course of the disease, enables tumor cells competent to repair the DNA damage, which is a major problem for chemotherapy in general. In this context, c-Abl is a protein tyrosine kinase that is involved in DNA damage-induced stress. We used a low-dose topoisomerase II inhibitor mitoxantrone to induce DNA damage which caused a transient cell cycle delay but allowed eventual passage through this checkpoint in most cells. We show that the percentage of HR and NHEJ efficient HeLa cells decreased more than 50% by combining c-Abl inhibitor imatinib with mitoxantrone. This inhibition of DNA repair caused more than 87% of cells in G2/M arrest and a significant increase in apoptosis. To validate the effect of the combination treatment, we tested it on commercial and patient-derived cell lines in high-grade serous ovarian cancer (HGSOC), where chemotherapy resistance correlates with HR proficiency and is a major clinical problem. Results obtained with HR-proficient and deficient HGSOC cell lines show a 50-85% increase of sensitivity by the combination treatment. Our data raise the possibility of successful targeting of treatment-resistant HR-proficient cancers.
  • Nilsson, Ingrid; Bahram, Fuad; Li, Xiujuan; Gualandi, Laura; Koch, Sina; Jarvius, Malin; Soderberg, Ola; Anisimov, Andrei; Kholova, Ivana; Pytowski, Bronislaw; Baldwin, Megan; Yla-Herttuala, Seppo; Alitalo, Kari; Kreuger, Johan (2010)
  • Saarenpää, Tuulia; Kogan, Konstantin; Sidorova, Yulia; Mahato, Arun Kumar; Tascon, Igor; Kaljunen, Heidi; Yu, Liying; Kallijärvi, Jukka; Jurvansuu, Jaana; Saarma, Mart; Goldman, Adrian (2017)
    Glial cell line-derived neurotrophic factor ( GDNF) is a ligand that activates, through coreceptor GDNF family receptor alpha-1 (GFR alpha 1) and receptor tyrosine kinase "RET ", several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRa1 and GDNF, and Drosophila melanogaster RET and two isoforms of coreceptor GDNF receptor-like. Our results report high-level insect cell expression of posttranslationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRa1 and GDNF are comparably active as mammalian cell- produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFR alpha 1-GDNF binding RET is 5.9 mu M. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRa1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRa1 and GDNF cannot bind and activate non-mammalian RET and vice versa.