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TRIB2, a member of the human Tribbles pseudokinase family, functions as a molecular scaffold in diverse signaling pathways. In this issue of Structure, Jamieson et al. report the first high-resolution structure of TRIB2 bound to a nanobody that offers insights into its "active-like" state.

The MET receptor tyrosine kinase (RTK) and its cognate ligand hepatocyte growth factor (HGF) comprise a signaling axis essential for development, wound healing and tissue homeostasis. Aberrant HGF/MET signaling is a driver of many cancers and contributes to drug resistance to several approved therapeutics targeting other RTKs, making MET itself an important drug target. In RTKs, homeostatic receptor signaling is dependent on autoinhibition in the absence of ligand binding and orchestrated set of conformational changes induced by ligand-mediated receptor dimerization that result in activation of the intracellular kinase domains. A fundamental understanding of these mechanisms in the MET receptor remains incomplete, despite decades of research. This is due in part to the complex structure of the HGF ligand, which remains unknown in its full-length form, and a lack of high-resolution structures of the complete MET extracellular portion in an apo or ligand-bound state. A current view of HGF-dependent MET activation has evolved from biochemical and structural studies of HGF and MET fragments and here we review what these findings have thus far revealed.

The development and activity of our immune system are largely controlled by the action of pleiotropic cytokines and growth factors, small secreted proteins, which bind to receptors on the surface of immune cells to initiate an appropriate physiological response. Cytokine signalling is predominantly executed by intracellular proteins known as the Janus kinases (JAKs) and the signal transducers and activators of transcriptions (STATs). Although the 'nuts and bolts' of cytokine-activated pathways have been well established, the nuanced way in which distinct cellular outcomes are achieved and the precise molecular details of the proteins that regulate these pathways are still being elucidated. This is highlighted by the intricate role of the suppressor of cytokine signalling (SOCS) proteins. The SOCS proteins act as negative feedback inhibitors, dampening specific cytokine signals to prevent excessive cellular responses and returning the cell to a homeostatic state. A great deal of study has demonstrated their ability to inhibit these pathways at the receptor complex, either through direct inhibition of JAK activity or by targeting the receptor complex for proteasomal degradation. Detailed analysis of individual SOCS proteins is slowly revealing the complex and highly controlled manner by which they can achieve specificity for distinct substrates. However, for many of the SOCS, a level of detail is still lacking, including confident identification of the full suite of tyrosine phosphorylated targets of their SH2 domain. This review will highlight the general mechanisms which govern SOCS specificity of action and discuss the similarities and differences between selected SOCS proteins, focusing on CIS, SOCS1 and SOCS3. Because of the functional and sequence similarities within the SOCS family, we will also discuss the evidence for functional redundancy.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5′-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

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 preformed 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.

Objective To examine the impact of the gp130 cytokine family on murine articular cartilage and to explore a potential regulatory role of suppressor of cytokine signaling 3 (SOCS-3) in murine chondrocytes. Methods In wild-type (WT) mouse chondrocytes, baseline receptor expression levels and gp130 cytokine–induced JAK/STAT signaling were determined by flow cytometry, and expression of SOCS-3 was assessed by quantitative polymerase chain reaction. The role of endogenous SOCS-3 was examined in cartilage explants and chondrocytes from mice with conditional deletion of Socs3 driven by the Col2a1 promoter in vitro (Socs3Δ/Δcol2) and from mice during CD4+ T cell–dependent inflammatory monarthritis. Bone erosions in the murine joints were analyzed by micro–computed tomography. Results On chondrocytes from WT mice, gp130 and the oncostatin M (OSM) receptor were strongly expressed, whereas the transmembrane interleukin-6 (IL-6) receptor was expressed at much lower levels. Compared to other gp130 cytokines, OSM was the most potent activator of the JAK/STAT pathway and of SOCS-3 induction. Treatment of Socs3Δ/Δcol2 mouse cartilage explants and chondrocytes with gp130 cytokines prolonged JAK/STAT signaling, enhanced cartilage degradation, increased the expression of Adamts4, Adamts5, and RANKL, and elevated the production of IL-6, granulocyte colony-stimulating factor, CXCL1, and CCL2. Socs3Δ/Δcol2 mice developed exacerbated inflammation and joint damage in response to gp130 cytokine injections, and these histopathologic features were also observed in mice with inflammatory monarthritis. Conclusion The results of this study highlight a key role for SOCS-3 in regulating chondrocyte responses during inflammatory arthritis. Within the gp130 cytokine family, OSM is a potent stimulus of chondrocyte responses, while IL-6 probably signals via trans-signaling. The gp130 cytokine–driven production of RANKL in chondrocytes may link chondrocyte activation and bone remodeling during inflammatory arthritis. Thus, these findings suggest that the inhibition of OSM might reduce the development and severity of structural joint damage during inflammatory arthritis.

Influenza virus infections have a significant impact on global human health. Individuals with suppressed immunity, or suffering from chronic inflammatory conditions such as COPD, are particularly susceptible to influenza. Here we show that suppressor of cytokine signaling (SOCS) five has a pivotal role in restricting influenza A virus in the airway epithelium, through the regulation of epidermal growth factor receptor (EGFR). Socs5-deficient mice exhibit heightened disease severity, with increased viral titres and weight loss. Socs5 levels were differentially regulated in response to distinct influenza viruses (H1N1, H3N2, H5N1 and H11N9) and were reduced in primary epithelial cells from COPD patients, again correlating with increased susceptibility to influenza. Importantly, restoration of SOCS5 levels restricted influenza virus infection, suggesting that manipulating SOCS5 expression and/or SOCS5 targets might be a novel therapeutic approach to influenza. DOI: http://dx.doi.org/10.7554/eLife.20444.001, eLife digest Influenza, commonly referred to as the flu, is a highly contagious disease caused by a virus. When an infected person coughs or sneezes, droplets containing the virus are released into the air. Other individuals nearby may breathe in the virus, which then enters the cells lining the lungs and multiplies. Some people are more susceptible to the influenza virus than others. In particular, individuals with chronic obstructive pulmonary disease (COPD) often suffer much worse flu symptoms and are more likely to be admitted to hospital. COPD results from smoke exposure, including cigarette smoke and, in developing countries, the smoke from cooking fires, but it is not clear why individuals with COPD are more susceptible to the influenza virus. The influenza virus gains entry to lung cells by manipulating receptors on the cell surface. A protein called SOCS5 is present inside these cells and has been suggested as a potential regulator of these receptors. Here, Kedzierski et al. reveal that SOCS5 plays a critical role in protecting lung cells in mice and humans from the virus. The experiments show that mice lacking the gene that encodes SOCS5 were more susceptible to infection by the influenza virus, had more severe symptoms of disease and increased amounts of virus in their lungs. Further experiments in lung cells collected from human volunteers show that SOCS5 levels increased in both healthy smokers and non-smokers in response to influenza infection. Conversely, SOCS5 levels in lung cells of smokers with COPD remained low after infection. This suggests that SOCS5 might be an important factor in the susceptibility of these patients to influenza. The next is step is to understand exactly how SOCS5 works, which may make it possible to develop new treatments that boost SOCS5 activity in influenza patients. DOI: http://dx.doi.org/10.7554/eLife.20444.002

Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish(-/-) mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function.

The suppressor of cytokine signalling (SOCS) box was first identified in the SH2-containing SOCS box family (cytokine-inducible SH2-containing protein, SOCS1-7) and is a 40-amino acid motif, which functions to recruit an E3 ubiquitin ligase complex consisting of the adapter proteins elongins B and C, Rbx2 and the scaffold protein Cullin5. The SOCS box is found in a diverse array of intracellular signalling molecules, many of which contain different protein interaction domains such as SPRY and WD40 domains, leucine and ankyrin repeats or other functional domains such as GTPases. In general, the SOCS box-containing proteins are thought to act as substrate-recognition modules to mediate the polyubiquitination and subsequent degradation of substrate proteins by the 26S proteasome.

The suppressor of cytokine signaling (SOCS) family of proteins are key negative regulators of cytokine and growth factor signaling. They act at the receptor complex to modulate the intracellular signaling cascade, preventing excessive signaling and restoring homeostasis. This regulation is critical to the normal cessation of signaling, highlighted by the complex inflammatory phenotypes exhibited by mice deficient in SOCS1 or SOCS3. These two SOCS proteins remain the best characterized of the eight family members (CIS, SOCS1-7), and in particular, we now possess a sound understanding of the mechanism of action for SOCS3. Here, we review the mechanistic role of the SOCS proteins and identify examples where clear, definitive data have been generated and discuss areas where the information is less clear. From this functional viewpoint, we discuss how the SOCS proteins achieve exquisite and specific regulation of cytokine signaling and highlight outstanding questions regarding the function of the less well-studied SOCS family members.

To examine the impact of the gp130 cytokine family on murine articular cartilage and to explore a potential regulatory role of suppressor of cytokine signaling 3 (SOCS-3) in murine chondrocytes.In wild-type (WT) mouse chondrocytes, baseline receptor expression levels and gp130 cytokine-induced JAK/STAT signaling were determined by flow cytometry, and expression of SOCS-3 was assessed by quantitative polymerase chain reaction. The role of endogenous SOCS-3 was examined in cartilage explants and chondrocytes from mice with conditional deletion of Socs3 driven by the Col2a1 promoter in vitro (Socs3(Δ/Δcol2) ) and from mice during CD4+ T cell-dependent inflammatory monarthritis. Bone erosions in the murine joints were analyzed by micro-computed tomography.On chondrocytes from WT mice, gp130 and the oncostatin M (OSM) receptor were strongly expressed, whereas the transmembrane interleukin-6 (IL-6) receptor was expressed at much lower levels. Compared to other gp130 cytokines, OSM was the most potent activator of the JAK/STAT pathway and of SOCS-3 induction. Treatment of Socs3(Δ/Δcol2) mouse cartilage explants and chondrocytes with gp130 cytokines prolonged JAK/STAT signaling, enhanced cartilage degradation, increased the expression of Adamts4, Adamts5, and RANKL, and elevated the production of IL-6, granulocyte colony-stimulating factor, CXCL1, and CCL2. Socs3(Δ/Δcol2) mice developed exacerbated inflammation and joint damage in response to gp130 cytokine injections, and these histopathologic features were also observed in mice with inflammatory monarthritis.The results of this study highlight a key role for SOCS-3 in regulating chondrocyte responses during inflammatory arthritis. Within the gp130 cytokine family, OSM is a potent stimulus of chondrocyte responses, while IL-6 probably signals via trans-signaling. The gp130 cytokine-driven production of RANKL in chondrocytes may link chondrocyte activation and bone remodeling during inflammatory arthritis. Thus, these findings suggest that the inhibition of OSM might reduce the development and severity of structural joint damage during inflammatory arthritis.

The discovery of the Suppressor of Cytokine Signaling (SOCS) family of proteins has resulted in a significant body of research dedicated to dissecting their biological functions and the molecular mechanisms by which they achieve potent and specific inhibition of cytokine and growth factor signaling. The Australian contribution to this field has been substantial, with the initial discovery of SOCS1 by Hilton, Starr and colleagues (discovered concurrently by two other groups) and the following work, providing a new perspective on the regulation of JAK/STAT signaling. In this review, we reflect on the critical discoveries that have lead to our current understanding of how SOCS proteins function and discuss what we see as important questions for future research.

Protein kinases play a key role in numerous cell signaling pathways. They have emerged as an important class of enzymes since aberrant kinase activity is associated with human diseases such as cancer and inflammatory disorders such as rheumatoid arthritis. While the human kinome is comprised of over 500 members, only a fraction have been exploited as attractive drug targets for therapeutic intervention. Thus, many members of the kinase superfamily and their role in human disease remain poorly understood. This can largely be attributed to the fact they are in low cellular abundance in complex protein samples and their detection is often reliant on the quality of available phosphoantibodies. In recent years, efforts have been made to develop immobilized small-molecule kinase inhibitors as kinase-capturing reagents. This kinase enrichment step coupled with highly sensitive mass spectrometry analysis allows for the comprehensive analysis of the kinome [1] . In this study, we have used two kinase-capturing reagents to profile the kinome from various cellular sources including HEK293Ts, human G-CSF mobilized neutrophils and murine bone marrow-derived macrophages (BMDMs). Kinome enrichment was achieved using CTx-0294885 resin (KiNET-1 beads, Synkinase) and a pan-JAK1/2 inhibitor Cyt387 (Cytopia) coupled to NHS-Sepharose beads. In this way we have identified a number of novel kinases within specific cell types, some of which may play important roles in the context of disease. This kinome profiing strategy was performed alongside a phosphopeptide enrichment strategy for quantitative mass spectrometry analysis. Using this approach we identified a number of novel phosphopeptide sites within clinically-relevant kinases including the JAK protein tyrosine kinases. We identified 40 phosphorylation sites in murine JAK2 (23 novel and 17 known sites), while 43 phosphorylation sites were identified in murine JAK1 (32 novel and 11 known sites). Mutational studies are currently underway to screen a selection of these evolutionarily-conserved candidate phosphorylation sites for functional relevance. The authors would like to acknowledge Leanne Daly from Synkinase for supplying the KiNET-1 beads used in this study.

Mutations of the reelin gene cause severe defects in cerebral cortex development and profound intellectual impairment. While many aspects of the reelin signaling pathway have been identified, the molecular and ultimate cellular consequences of reelin signaling remain unknown. Specifically, it is unclear if termination of reelin signaling is as important for normal cortical neuron migration as activation of reelin signaling. Using mice that are single or double deficient, we discovered that combined loss of the suppressors of cytokine signaling, SOCS6 and SOCS7, recapitulated the cortical layer inversion seen in mice lacking reelin and led to a dramatic increase in the reelin signaling molecule disabled (DAB1) in the cortex. The SRC homology domains of SOCS6 and SOCS7 bound DAB1 ex vivo. Mutation of DAB1 greatly diminished binding and protected from degradation by SOCS6. Phosphorylated DAB1 was elevated in cortical neurons in the absence of SOCS6 and SOCS7. Thus, constitutive activation of reelin signaling was observed to be equally detrimental as lack of activation. We hypothesize that, by terminating reelin signaling, SOCS6 and SOCS7 may allow new cycles of reelin signaling to occur and that these may be essential for cortical neuron migration.

SPRY/B30.2 domain-containing proteins are found in 103 human proteins that regulate numerous cellular processes including RNA processing, histone methylation, red blood cell membrane integrity, terminal erythroid cell differentiation and innate immune responses. Despite an increased understanding of SPRY/B30.2 domain function, some confusion remains as to the precise domain boundaries, the number and location of binding sites, and importantly how activation of these domains contribute to signal transduction. The B30.2 domain of tripartite motif 25 (TRIM25) interacts with the viral RNA sensor retinoic acid-inducible gene I (RIG-I), facilitating the formation of a RIG-I tetramer, which is required for activation of the innate immune response against RNA viruses such as influenza A, measles, HIV and hepatitis C. Here we have investigated the biochemical and structural changes associated with the activation of TRIM25 in the context of RIG-I binding. We solved a 1.8 Å crystal structure of the TRIM25 B30.2 domain and identified a putative RIG-I-binding site, which was confirmed by mutagenesis and functional analyses. Further mutagenesis identified a second site on the opposing face of the B30.2 domain that also specifically interacts with RIG-I. While the CARD1 domain of RIG-I is known to interact with the B30.2 domain of TRIM25, our data suggest that a conformational change within the CARD2 domain of RIG-I enables it to also interact with the B30.2 domain of TRIM25. We propose that both RIG-I CARDs interact with sites on opposing sides of the TRIM25 B30.2 domain to form a TRIM25/RIG-I complex that facilitates RIG-I tetramerisation and activation. The characterization of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family. Disease-associated polymorphisms of Pyrin, involved in Familial Mediterranean Fever, and mutational analyses of TRIM5a, required for HIV restriction, suggest that a second binding site may be a characteristic feature of other B30.2-containing TRIM proteins, and may facilitate the formation of large oligomeric complexes that are important for inflammasome function and HIV capsid destruction. Disclosures No relevant conflicts of interest to declare.