Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Structural and functional characterisation of the pseudokinase scaffold SgK269 (#201)

Ashleigh Kropp 1 , Weiwen Dai 1 , Michael DW Griffin 2 , James M Murphy 1 , 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. Previous studies have shown that SgK223 and SgK269 homo-and hetero- associate [1] and recently the structures of SgK223 and SgK269 were solved [2, 3]. These structures revealed a dimerisation interface composed of the helices that flank the pseudokinase domain (PK domain). Additionally, the SgK223 structure revealed an oligomerisation interface formed by the interaction of the aG helix of the PK domain with the N-lobe of the PK domain. This study identified energetic hotspots that are important for dimerisation of SgK223. In addition, mutagenesis within the aG helix of SgK223 and SgK269 disrupted the ability of these proteins to oligomerise.

In this study, 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. Single alanine mutants at the dimerisation interface of SgK269 failed to disrupt dimerisation. Future studies will involve using double/triple mutants or replacing hydrophobic residues to charged residues at the dimerisation interface. Single mutations within the N-lobe of SgK223 disrupted homo- and hetero-oligomerisation. However, single mutations within the N-lobe of SgK269 failed to disrupt oligomerisation, hence it is likely oligomerisation of SgK269 may occur through a different interface that does not involve the N-lobe of the PK domain.

The future directions on this project will be to functionally characterise the interactions of SgK223 and SgK269 in a cellular setting. Microscopy techniques such as super-resolution and lattice light sheet microscopy will be used to identify the localisation of these proteins, and how they alter cell morphology and migration.

  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.
  3. Ha, B.H. and T.J. Boggon, The crystal structure of pseudokinase PEAK1 (Sugen Kinase 269) reveals an unusual catalytic cleft and a novel mode of kinase fold dimerization. J Biol Chem, 2017.