TTBK2 kinase substrate specificity and the impact of spinocerebellar ataxia-causing mutations on expression, activity, localisation and development
Résumé
Mutations that truncate the C-terminal, non-catalytic moiety of the Tau tubulin Kinase 2 (TTBK2) cause the inherited, autosomal dominant inherited spinocerebellar ataxia type 11 (SCA11) movement disorder. In this study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate priming-groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localisation. We generate an SCA11-mutation-carrying knock-in mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that in homozygosity, the SCA11 mutation causes embryonic lethality at E10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations impact on protein expression, catalytic activity, localisation and development. We hope these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.
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