James M Murphy

University of Melbourne, Melbourne, Victoria, Australia

Are you James M Murphy?

Claim your profile

Publications (57)362.41 Total impact

  • James M. Murphy · James E. Vince ·

    F1000 Research 11/2015; DOI:10.12688/f1000research.7046.1
  • [Show abstract] [Hide abstract]
    ABSTRACT: CCAAT-enhancer binding proteins (C/EBPs) are transcription factors that play a central role in the differentiation of myeloid cells and adipocytes. Tribbles pseudokinases govern levels of C/EBPs by recruiting them to the COP1 ubiquitin ligase for ubiquitination. Here, we present the first crystal structure of a Tribbles protein, which reveals a catalytically inactive TRIB1 pseudokinase domain with a unique adaptation in the αC helix. A second crystal structure and biophysical studies of TRIB1 with its C-terminal extension, which includes the COP1-binding motif, show that the C-terminal extension is sequestered at a site formed by the modified TRIB1 αC helix. In addition, we have identified and characterized the TRIB1 substrate-recognition sequence within C/EBPα, which is evolutionarily conserved in C/EBP transcription factors. Binding studies indicate that C/EBPα recruitment is weaker in the presence of the C-terminal COP1-binding motif, but the magnitude of this effect suggests that the two bind distinct rather directly overlapping binding sites.
    Structure 10/2015; DOI:10.1016/j.str.2015.08.017 · 5.62 Impact Factor
  • Peter E Czabotar · James M Murphy ·
    [Show abstract] [Hide abstract]
    ABSTRACT: Recently, the programmed necrosis or "necroptosis" cell death pathway has attracted much interest because of its implication in multiple pathologies, including inflammatory diseases and the cell death arising from ischemia reperfusion injuries. Pharmacologically, necroptosis is an attractive target, because, unlike the counterpart pathway, apoptosis, it is dispensable for mammalian development. In particular, the most terminal-known obligate effector in the necroptosis pathway, the pseudokinase MLKL (mixed lineage kinase domain-like), holds particular appeal because, thus far, its only known function is as a mediator of necroptotic cell death. Here, we review the current understanding and gaps in knowledge relating to how MLKL can be activated by RIPK3 downstream of TNF receptor 1:RIPK1, Toll like receptor-3:TRIF and viral DNA:DAI/ZBF1. We also discuss the potential mechanism(s) by which MLKL induces necroptotic cell death, with particular emphasis on insights arising from structural studies of mouse and human MLKL. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 09/2015; DOI:10.1111/febs.13504 · 4.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The pseudokinase, MLKL (mixed lineage kinase domain-like), has recently emerged as a critical component of the necroptosis cell death pathway. While it is clear that phosphorylation of the activation loop in the MLKL pseudokinase domain by the upstream protein kinase, RIPK3 (receptor interacting protein kinase-3), is crucial to trigger MLKL activation, it has remained unclear whether other phosphorylation events modulate MLKL function. By reconstituting Mlkl(-/-) , Ripk3(-/-) and Mlkl(-/-)Ripk3(-/-) cells with MLKL phospho-site mutants, we compared the function of known MLKL phosphorylation sites in regulating necroptosis with three phospho-sites that we identified by mass spectrometry, S158, S228 and S248. Expression of a phospho-mimetic S345D MLKL activation loop mutant induced stimulus-independent cell death in all knockout cells, demonstrating that RIPK3 phosphorylation of the activation loop of MLKL is sufficient to induce cell death. Cell death was also induced by S228A, S228E and S158A MLKL mutants in the absence of death stimuli, but was most profound in Mlkl (-/-) Ripk3(-/-) double knockout fibroblasts . These data reveal a potential role for RIPK3 as a suppressor of MLKL activation and indicate that phosphorylation can fine-tune the ability of MLKL to induce necroptosis. Copyright 2015 The Author(s).
    Biochemical Journal 08/2015; 471(2). DOI:10.1042/BJ20150678 · 4.40 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements and NOE analysis, we demonstrate the presence of pre-formed structural elements in the JIR of mouse SOCS5 (mSOCS5175-244), consisting of an α-helix encompassing residues 224-233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis.
    Biochemistry 07/2015; 54(30). DOI:10.1021/acs.biochem.5b00619 · 3.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.
    Proceedings of the National Academy of Sciences 06/2015; 112(27). DOI:10.1073/pnas.1504232112 · 9.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-κB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-κB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-κB activation downstream of NOD engagement. Despite only delaying NF-κB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.
    Nature Communications 03/2015; 6. DOI:10.1038/ncomms7442 · 11.47 Impact Factor
  • Joanne M Hildebrand · Isabelle S Lucet · James M Murphy ·
    [Show abstract] [Hide abstract]
    ABSTRACT: The pseudokinase domain of the necroptosis effector mixed lineage kinase domain-like (MLKL) functions as a latch to restrain the unleashing of its N-terminal 4-helix bundle (4HB) domain. Cell death mediated by the 4HB domain relies on membrane association and oligomerization, which can be inhibited by an ATP-mimetic small molecule that binds the pseudokinase domain of MLKL.
    01/2015; 2(3):00-00. DOI:10.4161/23723556.2014.985550
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyer's patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyer's patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN.
    eLife Sciences 12/2014; 3(3). DOI:10.7554/eLife.03464 · 9.32 Impact Factor

  • 2nd Annual Meeting of the International-Cytokine-and-Interferon-Society; 11/2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Necroptosis is considered to be complementary to the classical caspase-dependent programmed cell death pathway, apoptosis. The pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) is an essential effector protein in the necroptotic cell death pathway downstream of the protein kinase Receptor Interacting Protein Kinase-3 (RIPK3). How MLKL causes cell death is unclear, however RIPK3-mediated phosphorylation of the activation loop in MLKL trips a molecular switch to induce necroptotic cell death. Here, we show that the MLKL pseudokinase domain acts as a latch to restrain the N-terminal four-helix bundle (4HB) domain and that unleashing this domain results in formation of a high-molecular-weight, membrane-localized complex and cell death. Using alanine-scanning mutagenesis, we identified two clusters of residues on opposing faces of the 4HB domain that were required for the 4HB domain to kill cells. The integrity of one cluster was essential for membrane localization, whereas MLKL mutations in the other cluster did not prevent membrane translocation but prevented killing; this demonstrates that membrane localization is necessary, but insufficient, to induce cell death. Finally, we identified a small molecule that binds the nucleotide binding site within the MLKL pseudokinase domain and retards MLKL translocation to membranes, thereby preventing necroptosis. This inhibitor provides a novel tool to investigate necroptosis and demonstrates the feasibility of using small molecules to target the nucleotide binding site of pseudokinases to modulate signal transduction.
    Proceedings of the National Academy of Sciences 10/2014; 111(42). DOI:10.1073/pnas.1408987111 · 9.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of hematopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a β-subunit, the principal signalling entity. In the mouse, two β-subunits have co-evolved: a common β-subunit (βc) shared between IL-3 and the related cytokines, IL-5 and GM-CSF; and an IL-3-specific β-subunit (βIL-3). βIL‑3 differs from βc in its specificity for IL-3 and its capacity to bind IL-3 directly in the absence of an α-subunit and, in the absence of structural information, the basis for these properties has remained enigmatic. Here, we present the crystal structure of the βIL-3 ectodomain at 3.45 Å resolution. This structure provides the first evidence that βIL-3 adopts an arch-shaped, intertwined homodimer with similar topology to the paralogous βc structure. In contrast to apo-βc, however, the ligand-binding interface of βIL‑3 appears to pre-exist in a conformation receptive to IL-3 engagement. Molecular modelling of the IL-3:βIL‑3 interface, in conjunction with previous mutational studies, suggests that divergent evolution of both βIL‑3 and IL-3 underlies their unique capacity for direct interaction and specificity.
    Biochemical Journal 08/2014; 463(3). DOI:10.1042/BJ20140863 · 4.40 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors.
    Biochemical Journal 08/2014; 462(1):1-13. DOI:10.1042/BJ20140712 · 4.40 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.
    Cell 05/2014; 157(5). DOI:10.1016/j.cell.2014.04.019 · 32.24 Impact Factor
  • Source
    James M Murphy · John Silke ·
    [Show abstract] [Hide abstract]
    ABSTRACT: When our time comes to die most people would probably opt for a quick, peaceful and painless exit. But the manner and timing are rarely under our direct control. Hence the Ars moriendi, literally, "The Art of Dying", two texts written in Latin around the 15th century that offered advice on how to die well according to the Christian ideals of the time. In contrast, for individual cells, the death process is frequently under their control and several signaling pathways that cause cell death, including apoptosis, pyroptosis and necroptosis, have been described. Furthermore the manner in which cells die can have good or bad consequences for the organism. In this review we will discuss how cells die via the necroptotic signaling pathway, with emphasis on recent structural work and place this work in a biological context by discussing relevant studies with knock-out animals.
    EMBO Reports 02/2014; 15(2). DOI:10.1002/embr.201337970 · 9.06 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Loss of Inhibitor of Apoptosis (IAP) proteins, particularly cIAP1, can promote production of tumor necrosis factor (TNF) and sensitize cancer cell lines to TNF induced necroptosis by promoting formation of a death inducing signaling complex containing receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1, RIPK3). To define the role of IAPs in myelopoiesis, we generated a mouse with cIAP1, cIAP2 and XIAP deleted in the myeloid lineage. Loss of cIAPs and XIAP in the myeloid lineage caused over-production of many pro-inflammatory cytokines, resulting in granulocytosis and severe sterile inflammation. In vitro differentiation of macrophages from bone marrow in the absence of cIAPs and XIAP led to detectable levels of TNF and resulted in reduced numbers of mature macrophages. The cytokine production and consequent cell death caused by IAP depletion was attenuated by loss or inhibition of TNF or TNFR1. The loss of RIPK1 or RIPK3, but not the RIPK3 substrate MLKL, attenuated TNF secretion and thereby prevented apoptotic cell death and not necrosis. Our results demonstrate that cIAPs and XIAP together restrain RIPK1 and RIPK3 dependent cytokine production in myeloid cells to critically regulate myeloid homeostasis.
    Blood 02/2014; 123(16). DOI:10.1182/blood-2013-06-510743 · 10.45 Impact Factor
  • Nufail Khan · Kate E Lawlor · James M Murphy · James E Vince ·
    [Show abstract] [Hide abstract]
    ABSTRACT: Necroptosis describes a pro-inflammatory form of cell death governed by the kinases RIP1 and RIP3. Necroptosis can occur following stimulation of the DNA receptor, DAI, or activation of death receptor, Toll-like receptor, T-cell antigen receptor, or interferon receptor signaling. Analysis of RIP3 deficient mice has implicated necroptosis in several inflammatory-driven diseases, including atherosclerosis, alcoholic liver disease and retinal degeneration. Although studies have demonstrated that mixed lineage kinase domain-like (MLKL) is the only substrate of RIP3 kinase that is essential for necroptotic death, the molecular determinants acting downstream of MLKL remain ambiguous. In addition, RIP3 can signal necroptosis independent of RIP1, may induce apoptosis, and can directly promote pro-inflammatory cytokine production. Therefore it will be important to determine if non-necroptotic RIP3 signaling influences RIP3 dependent pathologies.
    Current opinion in immunology 02/2014; 26C(1):76-89. DOI:10.1016/j.coi.2013.10.017 · 7.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Activation of the cell surface receptor, c-Mpl, by the cytokine, thrombopoietin (TPO), underpins megakaryocyte and platelet production in mammals. In humans, mutations in c-Mpl have been identified as the molecular basis of Congenital Amegakaryocytic Thrombocytopenia (CAMT). Here, we show that CAMT-associated mutations in c-Mpl principally lead to defective receptor presentation on the cell surface. In contrast, one CAMT mutant c-Mpl, F104S, was expressed on the cell surface, but showed defective TPO binding and receptor activation. Using mutational analyses, we examined which residues adjacent to F104 within the membrane-distal cytokine receptor homology module (CRM) of c-Mpl comprise the TPO-binding epitope, revealing residues within the predicted Domain 1 E-F and A-B loops and Domain 2 F'-G' loop as key TPO-binding determinants. These studies underscore the importance of the c-Mpl membrane-distal CRM to TPO-binding and suggest that mutations within this CRM that perturb TPO binding could give rise to CAMT.
    Growth factors (Chur, Switzerland) 01/2014; 32(1). DOI:10.3109/08977194.2013.874347 · 3.39 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: JAK2 (Janus kinase 2) initiates the intracellular signalling cascade downstream of cell surface receptor activation by cognate haematopoietic cytokines, including erythropoietin and thrombopoietin. The pseudokinase (JH2) domain of JAK2 negatively regulates the catalytic activity of the adjacent tyrosine kinase (JH1) domain and mutations within the pseudokinase domain underlie human myeloproliferative neoplasms, including polycythaemia vera and essential thrombocytosis. To date, the mechanism of JH2-mediated inhibition of JH1 kinase activation as well as the susceptibility of pathological mutant JAK2 to inhibition by the physiological negative regulator, SOCS3, have remained unclear. Here, using recombinant, purified JAK2JH1-JH2 proteins, we demonstrate that, when activated, wild-type and myeloproliferative neoplasm-associated mutants of JAK2 exhibit comparable enzymatic activity and inhibition by SOCS3 in in vitro kinase assays. Small angle X-ray scattering (SAXS) showed that JAK2JH1-JH2 exists in an elongated configuration in solution with no evidence for interaction between JH1 and JH2 domains in cis. Collectively, these data are consistent with a model in which JAK2's pseudokinase domain binds and inhibits the activation of the tyrosine kinase domain of a neighbouring JAK2 molecule within a cytokine receptor complex, but does not influence the activity of JAK2 once it has been activated. Our data indicate that, in the absence of the N-terminal FERM domain and thus cytokine receptor association, the wild-type and pathological mutants of JAK2 are enzymatically equivalent and equally susceptible to inhibition by SOCS3.
    Biochemical Journal 12/2013; 458(2). DOI:10.1042/BJ20131516 · 4.40 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The pseudokinase, Mixed lineage kinase domain-like (MLKL), was recently identified as an essential checkpoint in the programmed necrosis or "necroptosis" cell death pathway. Here, we present the crystal structure of the human MLKL pseudokinase domain at 1.7 Å resolution and probe its nucleotide-binding mechanism by performing structure-based mutagenesis. By comparing the structures and nucleotide binding determinants of human and mouse MLKL orthologues, our study provides insights into the evolution of nucleotide binding mechanisms amongst pseudokinases and their mechanistic divergence from conventional, catalytically-active protein kinases.
    Biochemical Journal 11/2013; 457(3). DOI:10.1042/BJ20131270 · 4.40 Impact Factor

Publication Stats

903 Citations
362.41 Total Impact Points


  • 2011-2015
    • University of Melbourne
      Melbourne, Victoria, Australia
  • 2009-2014
    • The Walter and Eliza Hall Institute of Medical Research
      • Division of Cancer and Haematology
      Melbourne, Victoria, Australia
  • 2008-2014
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 2013
    • University of Liverpool
      Liverpool, England, United Kingdom
  • 2001-2010
    • Australian National University
      • Molecular Bioscience Department
      Canberra, Australian Capital Territory, Australia
  • 2007-2009
    • Samuel Lunenfeld Research Institute
      Toronto, Ontario, Canada
  • 2006
    • University of Toronto
      • Department of Molecular Genetics
      Toronto, Ontario, Canada