Investigation of the physiological function of TTBK2 (tau tubulin kinase 2), a protein kinase mutated in spinocerebellar ataxia type 11 (SCA11)
TTBK2 was first identified based on its physical association with microtubules and its ability to phosphorylate microtubule-associated proteins (tau, tubulin, MAP2) in vitro (Takahashi et al., 1995, Tomizawa et al., 2001). Since this discovery by Japanese scientists in 1995, considerable work has been invested in examining TTBK2’s relationship with aberrant tau phosphorylation and neurodegeneration in Alzheimer’s disease. However, these studies have never been substantiated in vivo. The identification of TTBK2 truncating mutations as the cause of spinocerebellar ataxia type 11 (SCA11), in 2007, emphasised that TTBK2 has a prominent physiological role in the nervous system. When I started my PhD, there was little known about TTBK2 and many fundamental questions about TTBK2 remained unanswered. In Chapter 3, I describe an initial analysis of TTBK2 substrate specificity by using an unbiased positional scanning peptide library and show that it has a conspicuous preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. This information was then exploited to develop an optimised peptide substrate, named TTBKtide, to assess TTBK2 catalytic activity. TTBKtide was used to demonstrate that SCA11 truncating mutations lead to marked inhibition of TTBK2 kinase activity. The identification of TTBK2’s interacting proteins was of paramount importance to advance our understanding of both biological and disease processes. Identifying and characterising the key targets (substrates) of TTBK2 in the nervous system would provide vital new insights into the physiological role of TTBK2, which in turn, may uncover the molecular pathogenic mechanism underpinning the development of spinocerebellar ataxia type 11 (SCA11). To this end, I identified a number of TTBK2 interactors by co-immunoprecipitation of endogenous TTBK2 from mouse brain homogenates (Chapter 4). This screen provided tantalising evidence that TTBK2 operates in a distinct step of the synaptic vesicle cycle. An analysis of potential protein substrates identified synaptic vesicle glycoprotein 2A (SV2A) as a substrate of TTBK2. Phosphopeptide mapping revealed that SV2A is phosphorylated at two clusters of three highly-conserved residues by TTBK2. These two phosphorylation clusters were then shown to mediate the phospho-dependent interactions of SV2A with AP2 clathrin adaptors and synaptotagmin 1. SV2A and synaptotagmin 1 are both synaptic vesicle membrane proteins. Their interaction is both physical and functional. In chapter 5, I describe the characterisation of the SV2A/synaptotagmin 1 interaction using biophysical and functional methods. I have used two different biophysical methods: Isothermal titration calorimetry (ITC) and fluorescence polarisation, for quantitative analyses of the SV2A/synaptotagmin 1 phosphospecific interaction. Also, in collaboration with Prof. Michael Cousin’s laboratory (University of Edinburgh), the role of SV2A phosphorylation and the effect of disrupting the SV2A/synaptotagmin 1 interaction were examined via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from wild-type mice.
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