MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, U.K.
Biochem J. 2011 Jul 1;437(1):157-67. doi: 10.1042/BJ20110276.
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.
截断 TTBK2（微管相关蛋白 tau 激酶 2）C 端非催化结构域的突变导致遗传性、常染色体显性遗传的 SCA11（脊髓小脑共济失调 11 型）运动障碍。在本研究中，我们首先评估了 TTBK2 的底物特异性，并证明它对磷酸化位点+2 位的磷酸酪氨酸残基具有异常偏好。我们详细阐述了一种可用于量化 TTBK2 激酶活性的肽底物（TTBKtide，RRKDLHDDEEDEAMSIYpA）。通过建模和突变分析，我们在 TTBK2 激酶结构域内鉴定出一个假定的磷酸预启动沟。我们证明，SCA11 截断突变促进 TTBK2 蛋白表达，抑制激酶活性，并导致核定位增强。我们生成了携带 SCA11 突变的 knockin 小鼠，并表明这导致内源性 TTBK2 蛋白激酶活性受到抑制。最后，我们发现，在纯合状态下，SCA11 突变导致胚胎在胚胎第 10 天致死。这些发现首次深入了解了 TTBK2 的一些内在特性，并揭示了 SCA11 致病突变如何影响蛋白表达、催化活性、定位和发育。我们希望这些发现将有助于未来对 TTBK2 的调节和功能及其在 SCA11 中的作用的研究。
