Poster abstracts
Poster number 20 submitted by Jose Gabriel Javellana
Understanding the role of autism-linked TAOK2β mutants in translational control
Jose Gabriel Javellana (Department of Biological Chemistry and Pharmacology), Michael G. Kearse (Department of Biological Chemistry and Pharmacology)
Abstract:
Dysregulation of translation elongation is a common molecular phenotype of neurological diseases, including autism spectrum disorder (ASD), intellectual disability, and schizophrenia. Elongation is inhibited by phosphorylation of eukaryotic elongation factor 2 (eEF2) at Thr56, which is predominantly facilitated by the eEF2 kinase, eEF2K. TAOK2β was recently identified as a second inhibitory eEF2 kinase. Importantly, two de novo TAOK2β mutations have been identified in ASD patients. The A135P substitution in the kinase domain is reported to cause reduced kinase activity. The P1022* mutation that results in a truncated C-terminal regulatory domain causes increased kinase activity. When overexpressed in Neuro2A cells, both TAOK2β mutants cause an increase in polysomes compared to wild-type TAOK2β, suggesting dysregulated translation. However, the mechanisms by which the A135P and P1022* mutations affect TAOK2β remain unclear. Our preliminary data demonstrate that recombinant TAOK2β A135P has reduced eEF2 kinase activity in vitro, compared to wild type. Additionally, isogenic Flp-In T-REx 293 cells overexpressing wild-type TAOK2β show increased p-eEF2. Moving forward, we will use these in vitro and cell-based approaches to fully dissect the biochemical and molecular impact of each ASD-linked TAOK2β mutation on protein synthesis. Through in vitro kinase assays, we will determine the rate of eEF2 phosphorylation by wild-type and ASD-mutant TAOK2β. To directly measure changes in elongation rate caused by mutant TAOK2β, we will perform translation run-off assays with harringtonine treatment and polysome profiling at different time points. To determine structural changes caused by the mutations that lead to dysregulated translation, we will perform circular dichroism. We will also identify changes in intramolecular interactions between the mutants and wild type by crosslinking mass spectroscopy. This work will provide new insights into the second inhibitory kinase of eEF2 and will shed light on the molecular basis of ASD-linked human mutations and translational control.
Keywords: Translational control, Translation elongation, TAOK2