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

Investigating how the EphB6 pseudokinase mediates cell-cell communication using Cryo-ET (#151)

Lung-Yu Liang 1 2 , Andrew Leis 1 2 , Niall Geoghegan 1 , Michael Mlodzianoski 1 , Kelly Rogers 1 2 , Debnath Ghosal 3 4 , James Murphy 1 2 , Isabelle 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. Department of Biochemistry & Pharmacology, University of Melbourne, Parkville, VIC, Australia
  4. Bio21, University of Melbourne, Parkville, VIC, Australia

Eph receptors are the largest receptor tyrosine kinase (RTK) family members. Their ligands, ephrins, are also membrane tethered. Eph receptors are activated through an extracellular interaction with ephrins in trans. The ephrin-ligated Eph receptors dimerise and further oligomerise at the plasma membrane, followed by autophosphorylation of their intracellular domains1. The oligomerised and autophosphorylated Eph receptors serve as docking sites for Src Homology 2 (SH2) domain-containing proteins, leading to the cytoplasmic signal transduction1. Aberrant expression of Eph receptors has been widely linked to oncogenesis, and small molecules and monoclonal antibodies have been developed to target their kinase activity. Interestingly, EphB6, a poorly characterised Eph receptor, is predicted to be a pseudokinase, as it only has a pseudokinase domain unable to catalyse ATP. Whether EphB6 is able to bind ephrins and oligomerise at the plasma membrane was completely unknown. Moreover, how EphB6 void of kinase activity functions as a membrane receptor was an outstanding question.

 

Capitalising on my recent work characterising the intracellular regions of the EphB6 pseudokinase2, I further used an integrative approach to investigate the functions of the full-length EphB6 protein. By employing a co‑culture system, I used the cutting-edge lattice light-sheet microscope to identify that ephrinB1 is a ligand of EphB6. Upon ligation to ephrinB1, EphB6 oligomerised and clustered at the plasma membrane. Strikingly, the EphB6/ephrinB1 clusters promoted formation of tubular structures interconnecting the EphB6 and ephrinB1 expressing cells. In order to dissect these tubular structures at a nanometre resolution, I established a correlative light and electron microscopy (CLEM) workflow and performed Cryo-electron tomography (Cryo-ET). By Cryo-ET, I discovered that these tubular structures residing in the EphB6/ephrinB1 clusters possess an unprecedented double-membrane morphology. My findings indicate that the EphB6 pseudokinase is functionally competent, by mediating a novel approach of cell-cell communication.

  1. Eph receptor signalling: from catalytic to non-catalytic functions. Liang, LY et al., Oncogene 38, 6567–6584 (2019).
  2. The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs. Liang, LY et al., Biochem J 17 September 2021; 478 (17): 3351–3371.