Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Structural mapping of the PEAK pesudokinase interactome identifies a molecular switch regulating PEAK3/CrkII signalling (#355)

Michael J Roy 1 2 , Joshua M Hardy 1 2 , Minglyanna Surudoi 1 2 , Lung-Yu Liang 1 2 , Tom R Cotton 1 2 , Bernhard C Lechtenberg 1 2 , Weiwen Dai 1 2 , Isabelle S Lucet 1 2 , Onisha Patel 1 2
  1. Walter & Eliza Hall Institute , Parkville, VIC, Australia
  2. Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

PEAK pseudokinases (PEAK1, PEAK2 and PEAK3) are important and intriguing protein scaffolds that regulate cell migration, invasion and proliferation by recruiting signalling effectors at the cytoskeleton.1 Despite lacking catalytic activity, alteration in the expression level of PEAK family members is associated with several type of aggressive cancers. An emerging thread of PEAK family function is the ability to utilise homo- and hetero-dimerisation to integrate and diversify cellular signals.

Our lab was the first to structurally characterise PEAK2, revealing a conserved mechanism by which PEAKs dimerise via a unique alpha-helical domain.2 In addition to this unique dimerisation domain, all PEAK pesudokinases possess an N‑terminal intrinsically disordered region that harbours multiple sites for protein-protein interactions and enables dynamic recruitment of signalling partners. However, a detailed understanding of how PEAK pseudokinases and their associated network orchestrate oncogenic signals is lacking.

This dearth of knowledge is particularly pronounced for PEAK3, which was only very recently identified.3,4 An important interactor of PEAK3 has been identified as the signalling adapter protein CrkII.4 In addition, two recent pre-print manuscripts have described additional PEAK3 interactors including the adapter protein Grb2, epidermal growth factor receptor (EGFR), the integrin-associated tyrosine kinase PYK2 and Arf-GAP family member ASAP1. 5, 6

In this presentation, we report a detailed biochemical and structural characterisation of the PEAK pseudokinase signalling network. Importantly, we have uncovered and characterised a novel site on PEAK3, conserved in other PEAK proteins, that modulates a high-affinity protein interaction and functions as a molecular switch for PEAK3/CrkII signalling. By characterizing the key interactors that drive the formation of PEAK functional networks, we rationalize why dimerization has a crucial function in PEAK pseudokinase signal transduction and demonstrate how signal specificity is achieved within the PEAK family.

  1. 1. Patel, O., Roy, M. J., Murphy, J. M., and Lucet, I. S. (2020) The PEAK family of pseudokinases, their role in cell signalling and cancer, FEBS J 287, 4183-4197.
  2. 2. Patel, O., Griffin, M. D. W., Panjikar, S., Dai, W., Ma, X., Chan, H., Zheng, C., Kropp, A., Murphy, J. M., Daly, R. J., and Lucet, I. S. (2017) Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association, Nat Commun 8, 1157.
  3. 3. Lecointre, C., Simon, V., Kerneur, C., Allemand, F., Fournet, A., Montarras, I., Pons, J. L., Gelin, M., Brignatz, C., Urbach, S., Labesse, G., and Roche, S. (2018) Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation, Structure 26, 1563.
  4. 4. Lopez, M. L., Lo, M., Kung, J. E., Dudkiewicz, M., Jang, G. M., Von Dollen, J., Johnson, J. R., Krogan, N. J., Pawlowski, K., and Jura, N. (2019) PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization, Proc Natl Acad Sci U S A 116, 15495-15504.
  5. 5. Hou, J., Nguyen, E. V., Surudoi, M., Roy, M. J., Patel, O., Lucet, I. S., Ma, X., and Daly, R. J. (2021) PEAK3 pseudokinase represents a pro-migratory and -invasive signalling scaffold. BioRxiv. Preprint. doi: https://doi.org/10.1101/2021.02.17.431740.
  6. 6. Ounoughene, Y., Fourgous, E., Boublik, Y., Saland, E., Guiraud, N., Recher, C., Urbach, S., Fort, P., Sarry, J.-E., Fesquet, D., and Roche, S. (2021) SHED-dependent oncogenic signalling of the PEAK3 pseudo-kinase. BioRxiv. Preprint. doi: https://doi.org/10.1101/2021.08.30.457780.