Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Structural and functional characterisation of two interacting pseudokinase scaffolds (#260)

Ashleigh Kropp 1 , Michael J Mlodzianoski 1 , Kelly L Rogers 1 , Michael DW Griffin 2 , Roger J Daly 3 , Onisha Patel 1 , Isabelle S Lucet 1
  1. Department of Medical Biology, Walter and Eliza Hall Institute of Medical Research, Parkville
  2. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville
  3. Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton

The human pseudokinase SgK269 and its structurally related homologue SgK223, are oncogenic interacting scaffolds that promote the assembly of specific tyrosine kinase signaling pathways [1]. They are large, multidomain proteins that are comprised of a 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 (PsK) domain flanked by regulatory helices. SgK223 and SgK269 have been shown to localise to focal adhesions (FA) 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 hetero-association of SgK223 and SgK269 and plays an important role in cell migration. Additionally, our study has identified a novel mechanism of open ended oligomerisation of these pseudokinases [2].

To further investigate the impact of dimerisation/oligomerisation on localisation of SgK269 and SgK223 in cells, we have employed structured illumination microscopy (SIM). We show that SgK223 localises to actin and paxillin, a marker of FA, when expressed exogenously in SgK223 and SgK269 knockout cells. We have biochemically characterised mutants that disrupt dimerisation and oligomerisation of SgK223 and SgK269 in vitro. Future experiments involve transfecting these mutants into cells and investigating their effect on SgK223 and SgK269 localisation.

In contrast to the C-terminal domain, little is known about the function of the N-terminal domain. In this study, we have begun biochemically characterising of the N-terminal domains of SgK223 and SgK269 using size exclusion chromatography (SEC), analytical ultracentrifugation (AUC) and circular dichroism (CD), initially demonstrating that these domains are mostly monomeric.

This study will provide insight into the less characterised non-catalytic scaffolding function of these pseudokinases.

  1. Liu, L., et al., Homo- and Heterotypic Association Regulates Signaling by the SgK269/PEAK1 and SgK223 Pseudokinases. J Biol Chem, 2016. 291(41): p. 21571-21583.
  2. Patel, O., et al., Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association. Nat Commun, 2017. 8(1): p. 1157.