Structural and functional characterisation of the molecular assembly of two pseudokinase scaffolds
AuthorKropp, Ashleigh Kate
Document TypeMasters Research thesis
Access StatusThis item is embargoed and will be available on 2021-11-18.
© 2019 Ashleigh Kate Kropp
The human pseudokinase SgK269, and its structurally related homologue SgK223, are oncogenic interacting scaffolds that promote the assembly of specific tyrosine kinase signalling pathways. SgK223 and SgK269, as well as the recently discovered PEAK3, belong to the PEAK family of protein pseudokinases. They are large, multidomain proteins that are comprised of an N-terminal region of unknown structure and function, a large unstructured PEST region containing tyrosine phosphorylation sites and a C-terminal domain comprised of a pseudokinase domain flanked by regulatory helices. SgK223 and SgK269 have been shown to localise to focal adhesions and their overexpression leads to increased cell migration and changes in cell morphology, hallmarks of cancerous cells. Recent studies from our lab and others have provided structural insight into the C-terminal domain and flanking alpha helices of SgK223 and SgK269. These structures highlighted a conserved mechanism of dimerisation that drives homo- and hetero-association of SgK223 and SgK269 and plays an important role in cell migration. Additionally, SgK223 and SgK269 were demonstrated to undergo homo- and hetero-oligomerisation through their pseudokinase domains. In contrast to the C-terminal domain, little is known about the function of the N-terminal domains of SgK223 and SgK269, although there is sequence conservation between them. In this study, we have begun characterising the N-terminal domains of SgK223 and SgK269 using biophysical and biochemical techniques, initially demonstrating that these domains are monomeric and appear to have no defined secondary structure. To further investigate SgK223 and SgK269 homo- and hetero-association we carried out single site alanine mutagenesis to determine the energetic hotspots at the dimerisation interface of SgK269. Furthermore, we carried out mutagenesis within the N-lobe of SgK223 and SgK269, to investigate the role of this interface in homo- and hetero-oligomerisation. Additionally, we characterised the PEAK family interactions with the critical interacting signalling adaptor protein, CrkII, using biophysical assays and X-ray crystallography. We found that each member of the PEAK family has a proline-rich motif within their PEST linker that interacts with CrkII N-SH3 domain with ~1-3 uM affinity. The crystal structure of the CrkII N-SH3 domain bound to the SgK269 proline-rich motif demonstrated the critical consensus residues for the PEAK family interaction with CrkII. To further investigate the role of SgK223 and SgK269 homo- and hetero-association in cells, these studies were complemented with localisation microscopy techniques. Utilising mutants of SgK223 and SgK269 that can no longer dimerise or oligomerise, we investigated the importance of SgK223 and SgK269 associations for their localisation and thus, role in signalling. Insights into the scaffolding functions of SgK223 and SgK269 will inform how they contribute to the assembly of signalling pathways and hence their role in cancer.
KeywordsPseudokinase; Structural biology; Signal transduction
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