Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Understanding the structure and function of doublecortin-like kinases (#222)

Joshua M Hardy 1 2 , Hanadi Hoblos 1 , Minglyanna G Surudoi 1 , Andrew Leis 1 2 , Hariprasad Venugopal 3 , Marija Dramicanin 1 2 , Michael J Roy 1 2 , Onisha Patel 1 2 , Isabelle S Lucet 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia
  3. Ramaciotti Centre for Cryo Electron Microscopy, Monash University, Clayton, VIC, Australia

The human doublecortin family of proteins includes microtubule-associated proteins (MAPs) that play crucial roles in neurogenesis and neuronal migration. MAPs dynamically regulate the assembly and turnover of microtubules in response to intracellular and extracellular cues. MAPs thus play fundamental roles in cell division and migration, and mutations or changes in their expression can promote tumour growth and metastasis. Doublecortin proteins typically contain one or two doublecortin domains (DCs) that bind to tubulin and stabilise microtubules. One subset of this family, known as the doublecortin-like kinases (DCLK1/DCLK2), additionally harbours a C-terminal serine/threonine kinase domain, providing an opportunity to develop a selective therapeutic approach over other members of this family1.

Our lab previously determined the crystal structure of the DCLK1 kinase domain2 but due to the conformational flexibility of the DC domains we are still lacking a complete model of full-length DCLK proteins. To address this knowledge gap, we are pursuing a structure of DCLK1 by cryo-electron microscopy (cryo-EM) using a combination of stabilising interacting proteins and nanobodies. We have also generated multiple constructs of DCLK1 to understand the role of the N- and C-terminal regions in the regulation of its kinase and microtubule-binding functions.

The 14-3-3 family of proteins binds to phosphorylated sites on cellular proteins to regulate their conformation and activity, but the role of 14-3-3 in modulating DCLK function is unknown. We have used mass-spectrometry and bioinformatic approaches to identify 14-3-3 binding sites within DCLK1 and characterised their binding by surface plasmon resonance (SPR). Complexes of DCLK1-14-3-3 have been purified for structural and functional studies.

Through enzyme assays and imaging by cryo-EM, we have shown that DCLK1 is able to polymerise and stabilise microtubules in vitro with the majority of microtubules being composed of 13 protofilaments. We have also established mammalian HEK293T cell lines expressing fluorescently-tagged DCLK1 and found that it co-localises with microtubule networks. Importantly, DCLK1-microtubule co-localisation was observed at cellular protrusions, centrosomes and bipolar spindles during cell division, underscoring its role in cancer and metastasis.

Together, our work provides a platform for the structural and functional characterisation of DCLK and rational design of targeted therapies.

  1. Patel, O., Roy, M.J., Kropp, A., Hardy, J.M., Dai, W., Lucet, I.S. Structural basis for small molecule targeting of Doublecortin Like Kinase 1 with DCLK1-IN-1. Commun Biol. 2021;4(1):1105.
  2. Patel, O. et al. Biochemical and Structural Insights into Doublecortin-like Kinase Domain 1. Structure. 2016;24(9):1550-61.