Recent studies have revealed that mutations within the Tau tubulin Kinase 2 (TTBK2) cause a serious autosomal dominant inherited movement disorder termed spinocerebellar ataxias type 11 (SCA11) [1, 2]. SCA11 is characterised by progressive cerebellar ataxia, pyramidal features, peripheral neuropathy, with age of onset from the early teens to the mid 20s [3]. TTBK2 is closely related to a neuronal specific protein kinase termed TTBK1, which was discovered in 1995 as a protein kinase in bovine brain extract that phosphorylated tau and tublin [4].
TTBK2 consists of 1244 residues and apart from an N-terminal serine/threonine protein kinase domain (residues 20 to 280) possesses no distinctive functional domains or motifs. Thus far, four distinct SCA11 families have been identified, each possessing a mutation leading to premature termination of the TTBK2 protein at around residue 450. This leaves the kinase catalytic domain intact, but eliminates most of the non-catalytic portion of the enzyme.
Michale and Noor decided to investigate some of TTBK2's properties as nothing is known about TTBK2 substrate specificity and how SCA11 truncating mutations impact on protein expression, kinase activity, stability and localisation.
Noor started by collaborating with Mike Begley and Lew Cantley at Harvard Medical School to analyse TTBK2 substrate specificity. This surprisingly revealed that TTBK2 possesses a conspicuous preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. Noor then exploited this information to develop an optimised peptide substrate (RRKDLHDDEEDEAMSIYpA) to assess TTBK2 catalytic activity, that he termed TTBKtide. Molecular modelling and mutagenesis analysis undertaken by Noor suggest that the TTBK2 catalytic domain may possess a phosphotyrosine binding substrate-docking site within its catalytic domain.
Michale Bouskila next found that all SCA11 truncating mutations markedly enhanced TTBK2 protein expression, but lead to inhibition of TTBK2 kinase activity as well as promoting nuclear localisation. Michale also generated TTBK2 knock-in mice expressing an SCA11 disease causing mutation and found that this resulted in marked inhibition of endogenous protein kinase activity. Michale in collaboration with Emily Fang and Kate Storey found that in homozygosity the SCA11 mutation caused embryonic lethality at E10. The heterozygous SCA11 animals displayed no obvious movement disorder even after 1 year of age.
These results provide initial insights into substrate specificity of TTBK2 and how SCA11 causing mutations impact on kinase activity and localisation.
In future studies it would be important to define the substrates that TTBK2 phosphorylates and investigate whether reduced phosphorylation of these targets contributes to the development of SCA11. In particular, it would be interesting to determine whether physiological TTBK2 substrates were primed with a +2 phosphotyrosine and how regulation of tyrosine phosphorylation was coupled to the control of these TTBK2 targets.
Identifying the key targets of TTBK2 could provide vital new insights into the molecular mechanism underpinning the development of spinocerebellar ataxia and result in new ideas as to how this debilitating disease might be better treated.
To read a copy of Michale and Noor's paper click here
References
1 Houlden, H., Johnson, J., Gardner-Thorpe, C., Lashley, T., Hernandez, D., Worth, P., Singleton, A. B., Hilton, D. A., Holton, J., Revesz, T., Davis, M. B., Giunti, P. and Wood, N. W. (2007) Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11. Nat Genet. 39, 1434-1436
2 Bauer, P., Stevanin, G., Beetz, C., Synofzik, M., Schmitz-Hubsch, T., Wullner, U., Berthier, E., Ollagnon-Roman, E., Riess, O., Forlani, S., Mundwiller, E., Durr, A., Schols, L. and Brice, A. (2010) Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds. J Neurol Neurosurg Psychiatry. 81, 1229-1232
3 Houlden, H. (2008) Spinocerebellar Ataxia Type 11. In GeneRevews (Pagon, ed.), University of Washington, Seattle
4 Takahashi, M., Tomizawa, K., Sato, K., Ohtake, A. and Omori, A. (1995) A novel tau-tubulin kinase from bovine brain. FEBS Lett. 372, 59-64
TTBK2 consists of 1244 residues and apart from an N-terminal serine/threonine protein kinase domain (residues 20 to 280) possesses no distinctive functional domains or motifs. Thus far, four distinct SCA11 families have been identified, each possessing a mutation leading to premature termination of the TTBK2 protein at around residue 450. This leaves the kinase catalytic domain intact, but eliminates most of the non-catalytic portion of the enzyme.
Michale and Noor decided to investigate some of TTBK2's properties as nothing is known about TTBK2 substrate specificity and how SCA11 truncating mutations impact on protein expression, kinase activity, stability and localisation.
Noor started by collaborating with Mike Begley and Lew Cantley at Harvard Medical School to analyse TTBK2 substrate specificity. This surprisingly revealed that TTBK2 possesses a conspicuous preference for a phosphotyrosine at the +2 position relative to the phosphorylation site. Noor then exploited this information to develop an optimised peptide substrate (RRKDLHDDEEDEAMSIYpA) to assess TTBK2 catalytic activity, that he termed TTBKtide. Molecular modelling and mutagenesis analysis undertaken by Noor suggest that the TTBK2 catalytic domain may possess a phosphotyrosine binding substrate-docking site within its catalytic domain.
Michale Bouskila next found that all SCA11 truncating mutations markedly enhanced TTBK2 protein expression, but lead to inhibition of TTBK2 kinase activity as well as promoting nuclear localisation. Michale also generated TTBK2 knock-in mice expressing an SCA11 disease causing mutation and found that this resulted in marked inhibition of endogenous protein kinase activity. Michale in collaboration with Emily Fang and Kate Storey found that in homozygosity the SCA11 mutation caused embryonic lethality at E10. The heterozygous SCA11 animals displayed no obvious movement disorder even after 1 year of age.
These results provide initial insights into substrate specificity of TTBK2 and how SCA11 causing mutations impact on kinase activity and localisation.
In future studies it would be important to define the substrates that TTBK2 phosphorylates and investigate whether reduced phosphorylation of these targets contributes to the development of SCA11. In particular, it would be interesting to determine whether physiological TTBK2 substrates were primed with a +2 phosphotyrosine and how regulation of tyrosine phosphorylation was coupled to the control of these TTBK2 targets.
Identifying the key targets of TTBK2 could provide vital new insights into the molecular mechanism underpinning the development of spinocerebellar ataxia and result in new ideas as to how this debilitating disease might be better treated.
To read a copy of Michale and Noor's paper click here
References
1 Houlden, H., Johnson, J., Gardner-Thorpe, C., Lashley, T., Hernandez, D., Worth, P., Singleton, A. B., Hilton, D. A., Holton, J., Revesz, T., Davis, M. B., Giunti, P. and Wood, N. W. (2007) Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11. Nat Genet. 39, 1434-1436
2 Bauer, P., Stevanin, G., Beetz, C., Synofzik, M., Schmitz-Hubsch, T., Wullner, U., Berthier, E., Ollagnon-Roman, E., Riess, O., Forlani, S., Mundwiller, E., Durr, A., Schols, L. and Brice, A. (2010) Spinocerebellar ataxia type 11 (SCA11) is an uncommon cause of dominant ataxia among French and German kindreds. J Neurol Neurosurg Psychiatry. 81, 1229-1232
3 Houlden, H. (2008) Spinocerebellar Ataxia Type 11. In GeneRevews (Pagon, ed.), University of Washington, Seattle
4 Takahashi, M., Tomizawa, K., Sato, K., Ohtake, A. and Omori, A. (1995) A novel tau-tubulin kinase from bovine brain. FEBS Lett. 372, 59-64