29 research outputs found
Investigations of the suppressor of cytokine signalling 5
No full text© 2016 Dr. Edmond Michael LinossiThe SOCS family of proteins provide crucial negative regulation of cytokine and growth factor signalling. They achieve this through multiple mechanisms, including ubiquitination and degradation of target substrates by the proteasome, direct inhibition of enzyme activity and steric competition for key binding sites in signalling proteins. Whilst the biology and mechanisms of action have been well defined for a number of SOCS family members, a clear physiological function has not been described for SOCS5. This is due in part to discrepancies in the literature, where exogenous expression of SOCS5 in mice suggests a role in CD4+ T cells that is not supported by analysis of SOCS5- deficient mice. It has also been implicated as a negative regulator of EGFR signalling and its expression is inversely correlated with receptor levels in aggressive hepatocellular carcinoma patients. SOCS5 shares a high degree of sequence homology with Socs36E of Drosophila, which is known to regulate EGFR and JAK signalling in vivo.
In this work, I aimed to identify SOCS5 interacting proteins to better understand its role. I have characterised two specific targets in JAK1 and ShcA and extended the search for interacting proteins using mass spectrometry to broadly define the SOCS5 interactome in HEK293T cells. These studies revealed an extensive network of interactions, likely mediated by the poorly defined N-terminal region of SOCS5, which was heavily modified by phosphorylation. Most interestingly, in the PyMT mouse model of breast cancer, which is driven by many of the identified SOCS5 interacting proteins, SOCS5- deficient mice exhibited accelerated disease onset and tumour growth. These data show for the first time that SOCS5 plays an important protective role in the mammary gland. This was supported by analyses of SOCS5 in human breast cancer that shows patients with low SOCS5 expression exhibit a worse clinical outcome and more aggressive disease
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State of the structure address on MET receptor activation by HGF
No full textThe 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
Author Correction: Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands
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Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.
No full textMET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of the MET intracellular kinase domain in two fusion protein backgrounds: wild-type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase ⍺C-helix, pointing to potential differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for the kinase domain in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain
A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics
No full textOver one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintics. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinases have diverged from vertebrates within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent key targets for the development of new anthelmintic medicines
Author response: Suppressor of cytokine signaling (SOCS)5 ameliorates influenza infection via inhibition of EGFR signaling
No full textMapping kinase domain resistance mechanisms for the MET receptor tyrosine kinase via deep mutational scanning
No full textMutations in the kinase and juxtamembrane domains of the MET Receptor Tyrosine Kinase are responsible for oncogenesis in various cancers and can drive resistance to MET-directed treatments. Determining the most effective inhibitor for each mutational profile is a major challenge for MET-driven cancer treatment in precision medicine. Here, we used a deep mutational scan (DMS) of ~5764 MET kinase domain variants to profile the growth of each mutation against a panel of 11 inhibitors that are reported to target the MET kinase domain. We validate previously identified resistance mutations, pinpoint common resistance sites across type I, type II, and type I ½ inhibitors, unveil unique resistance and sensitizing mutations for each inhibitor, and verify non-cross-resistant sensitivities for type I and type II inhibitor pairs. We augment a protein language model with biophysical and chemical features to improve the predictive performance for inhibitor-treated datasets. Together, our study demonstrates a pooled experimental pipeline for identifying resistance mutations, provides a reference dictionary for mutations that are sensitized to specific therapies, and offers insights for future drug development
Suppressor of cytokine signaling (SOCS) 5 utilises distinct domains for regulation of JAK1 and interaction with the adaptor protein Shc-1
No full textSuppressor 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
Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N‑Terminus of SOCS5
No full textSOCS5
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 (mSOCS5<sub>175–244</sub>), 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
