TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development.

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.

Investigation of the hepatotoxicity profile of chemical entities using Liverbeads and WIF-B9 in vitro models.

The cytotoxicity profile of various chemical entities was evaluated using two in vitro hepatocyte models. Liverbeads is a cryopreserved model consisting of primary hepatocytes entrapped in alginate beads. WIF-B9 is a hybrid cell line obtained by fusion of rat hepatoma (Fao) and human fibroblasts (WI38). Various reference hepatotoxicants were tested and ranked according to their equivalent concentration 50 (EC50) for various biochemical endpoints (lactate dehydrogenase (LDH) release, 3-(4,5 dimethylthiazol 2yl)-2,5-diphenyl-2H tetrazolium bromure (MTT) activity, adenosine triphosphate (ATP) and glutathione (GSH) levels). The ranking obtained was comparable in both models and consistent with previously published results on hepatocyte monolayers. Ketoconazole, erythromycin estolate, retinoic acid, telithromycin and alpha-naphthyl-isothiocyanate were among the most toxic chemicals in both models, with an EC50 < 200 microM. Troleandomycin, spiramycin, erythromycin, diclofenac, taurodeoxycholate, warfarin, galactosamine, valproic acid and isoniazid were found to be less toxic. Few marked differences, potentially linked to metabolism pathways, were observed between EC50s in the two models for compounds such as cyclosporine A (10 and > 831 microM) and warfarin (5904 and 1489 microM) in WIF-B9 and Liverbeads, respectively. The results obtained indicate that Liverbeads and WIF-B9 cells are reliable in vitro models to evaluate the hepatotoxic potential of a wide range of chemicals, irrespective of structure and pharmaceutical class.

Allosteric regulation of glycogen synthase controls glycogen synthesis in muscle.

Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ∼80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation in vivo.

Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle.

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6-P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3 alpha and -beta genes are replaced with mutant forms (GSK3 alpha/beta S21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 alpha/beta S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6-P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6-P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.