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Acta Cryst. (2014). A70, C440
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The RET receptor tyrosine kinase is crucial for embryonic and adult development, with mutations in both the extracellular and kinase domains leading to several types of cancer. In order to understand the mechanisms of RET activation in more detail, we have investigated how RET interacts with its bipartite ligand comprising of a glial cell line derived neurotrophic factor (GDNF) family ligand and a GDNF family receptor (GFRalpha). To visualise this interaction, we have reconstituted two vertebrate RET ternary complexes containing both ligand and co-receptor and have determined a pseudo-atomic model for a mammalian RET ternary complex using electron microscopy. Our structures reveal the basis for ligand recognition and will be presented. As RET is a validated anti-cancer target, we are actively investigating RET chemical inhibitors in collaboration with several chemistry laboratories. We have determined structures of a diverse set of chemical scaffolds bound to RET leading to an improved RET pharmacophore based on crystallographic, biochemical and cell-based data. As current FDA-approved drugs for RET-dependent metastatic thyroid cancer suffer from off-target dose-dependent toxicity and lack of specificity, we hope our data will usefully contribute to the design of second generation RET chemical inhibitors.

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Acta Cryst. (2014). A70, C1168
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Actin dynamics control many aspects of cell shape and cell motility through regulatory interactions with a large variety of actin-binding proteins. Signalling to these actin regulators frequently involves a Rho GTPase-stimulated pathway that leads to a dramatic fluctuation in the levels of monomeric actin (G-actin) following polymerisation to F-actin. Recent studies have identified a molecular G-actin sensor called the RPEL domain that links RPEL-containing proteins and their subcellular localisation to actin dynamics. The RPEL domain contains a tandem array of typically three RPEL motifs, each of which is competent to bind a G-actin molecule [1]. The domain is present in two otherwise unrelated protein families; the MRTF family of serum response factor (SRF) transcriptional co-activator proteins and the Phactr family of actin and PP1 phosphatase-binding proteins. We have begun to investigate how the RPEL domain operates in both of these protein contexts and how it modulates subcellular localisation, transcriptional regulation and actomyosin contractility. To define the molecular basis for the sensor we have reconstituted pentameric and trimeric G-actin complexes with the RPEL domain from both MRTF-A and Phactr and used crystallography to reveal discrete supramolecular assemblies with repetitive arrangements of the G-actin subunits around the "crankshaft"-shaped RPEL domain [2,3]. These arrangements are quite different from F-actin intermolecular contacts and are quite unexpected. Our crystal structures reveal cooperative loading of G-actin onto the RPEL domain that we show by several cell-based reporter assays to be of functional importance. These structures explain how G-actin interaction alters the subcellular localisation of both MRTF-A and Phactr by inhibiting nuclear import through competing with importin alpha-beta binding [2,3].

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Acta Cryst. (2014). A70, C1168
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Actin dynamics control many aspects of cell shape and cell motility through regulatory interactions with a large variety of actin-binding proteins. Signalling to these actin regulators frequently involves a Rho GTPase-stimulated pathway that leads to a dramatic fluctuation in the levels of monomeric actin (G-actin) following polymerisation to F-actin. Recent studies have identified a molecular G-actin sensor called the RPEL domain that links RPEL-containing proteins and their subcellular localisation to actin dynamics. The RPEL domain contains a tandem array of typically three RPEL motifs, each of which is competent to bind a G-actin molecule [1]. The domain is present in two otherwise unrelated protein families; the MRTF family of serum response factor (SRF) transcriptional co-activator proteins and the Phactr family of actin and PP1 phosphatase-binding proteins. We have begun to investigate how the RPEL domain operates in both of these protein contexts and how it modulates subcellular localisation, transcriptional regulation and actomyosin contractility. To define the molecular basis for the sensor we have reconstituted pentameric and trimeric G-actin complexes with the RPEL domain from both MRTF-A and Phactr and used crystallography to reveal discrete supramolecular assemblies with repetitive arrangements of the G-actin subunits around the "crankshaft"-shaped RPEL domain [2,3]. These arrangements are quite different from F-actin intermolecular contacts and are quite unexpected. Our crystal structures reveal cooperative loading of G-actin onto the RPEL domain that we show by several cell-based reporter assays to be of functional importance. These structures explain how G-actin interaction alters the subcellular localisation of both MRTF-A and Phactr by inhibiting nuclear import through competing with importin alpha-beta binding [2,3].

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Acta Cryst. (2014). A70, C1282
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The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. Deregulation of PKCι signalling has multiple effects including aberrant cell polarity, which is a hallmark of aggressive cancers. PKCι associates with two discrete polarity complexes; one containing the polarity proteins Par3 and Par6 (the PAR complex) and the other contains Crumbs, Stardust and PatJ (the Crbs complex). Both complexes are found in vertebrates and invertebrates where they are crucial for maintaining apical-basal polarity. We are interested in how these two complexes recruit Par6-aPKC to the cell membrane and how aPKC activity is stimulated once within the PAR complex. Several substrates of the PAR complex are also able to inhibit its catalytic activity suggesting a complex regulatory mechanism. Our structural, biochemical and in vivo results from studying the PAR complex will be presented. Our data indicate a hierarchy among PAR complex substrates. In parallel, we have characterised somatic mutations found in PKCι in human cancer, indicating that perturbing a substrate-specific recruitment site selectively disrupts the polarizing activity of PKCι. Finally, a series of ATP-competitive thieno[3,2-d]pyrimidine- based PKCι inhibitors that show potent and selective inhibition of PKCι in biochemical, cellular and in vivo models will be presented.
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