title: TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development.
abstract: Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue 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 localization. We generate an SCA11-mutation-carrying knockin 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 embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.
authors.affiliation: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
authors.forename: Michale
authors.initials: M
authors.lastname: Bouskila
authors.forename: Noor
authors.initials: N
authors.lastname: Esoof
authors.forename: Laurie
authors.initials: L
authors.lastname: Gay
authors.forename: Emily H
authors.initials: EH
authors.lastname: Fang
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Begley
authors.forename: Lewis C
authors.initials: LC
authors.lastname: Cantley
authors.forename: Alan
authors.initials: A
authors.lastname: Prescott
authors.forename: Kate G
authors.initials: KG
authors.lastname: Storey
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D020754:Spinocerebellar Ataxias / Q000235:genetics* / Q000378:metabolism; D013132:Spinocerebellar Degenerations; D013379:Substrate Specificity
chemical_list: C095903:tau-tubulin kinase; D017346:Protein Serine-Threonine Kinases