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.
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Research Article|
June 14 2011
TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development
Michale Bouskila;
Michale Bouskila
1
*MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Noor Esoof;
Noor Esoof
*MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Laurie Gay;
Laurie Gay
2
*MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Emily H. Fang;
Emily H. Fang
†Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Maria Deak;
Maria Deak
*MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Michael J. Begley;
Michael J. Begley
‡Department of Systems Biology, Harvard Medical School, Boston, MA 02115, U.S.A.
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Lewis C. Cantley;
Lewis C. Cantley
‡Department of Systems Biology, Harvard Medical School, Boston, MA 02115, U.S.A.
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Alan Prescott;
Alan Prescott
§Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Kate G. Storey;
Kate G. Storey
†Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
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Dario R. Alessi
Dario R. Alessi
3
*MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
3To whom correspondence should be addressed (email d.r.alessi@dundee.ac.uk).
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Publisher: Portland Press Ltd
Received:
February 14 2011
Revision Received:
May 03 2011
Accepted:
May 06 2011
Accepted Manuscript online:
May 06 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 437 (1): 157–167.
Article history
Received:
February 14 2011
Revision Received:
May 03 2011
Accepted:
May 06 2011
Accepted Manuscript online:
May 06 2011
Citation
Michale Bouskila, Noor Esoof, Laurie Gay, Emily H. Fang, Maria Deak, Michael J. Begley, Lewis C. Cantley, Alan Prescott, Kate G. Storey, Dario R. Alessi; TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development. Biochem J 1 July 2011; 437 (1): 157–167. doi: https://doi.org/10.1042/BJ20110276
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