| Abstract No.: | A-A1006 |
| Country: | Canada |
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| Title: | EXPRESSION AND FUNCTIONAL IMPORTANCE OF TES KINASE IN NEURONS |
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| Authors/Affiliations: | 1 Horia Pribiag*; 1 Alyson Fournier;
1 Montreal Neurological Institute; QC, Canada
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| Content: | Background: TES kinase (TESK) is a dual-specificity serine/threonine and tyrosine kinase homologous to LIM Kinase expressed in various tissues including brain. TESK phosphorylates cofilin and is involved in integrin-mediated re-organization of the actin cytoskeleton and in focal adhesion formation. In non-neuronal cells, TESK1 activity is down-regulated by interactions with the binding partners 14-3-3β, sprouty4, actopaxin and spred1. Furthermore, the interaction of TESK1 with sprouty2 abrogates inhibition of the ERK/MAPK signaling pathway, and its interaction with MARKK inhibits MARK-induced collapse of the microtubule cytoskeleton. To date, no studies have identified a functional role for TESK in neurons. Based on the known functions of TESK in non-neuronal cells we hypothesize that it may play important roles in regulating dynamics of the neuronal cytoskeleton.
Objectives: (1) To characterize TESK1 and TESK2 protein expression in the central nervous system (2) To determine whether the kinase activity of TESK is important in regulating neuronal phenotype
(3) To determine whether TESK regulates neurite outgrowth on myelin-associated inhibitory substrates
Materials and Methods: Protein expression was characterized by western blotting and immunohistochemistry in rat and mouse brain tissues using antibodies directed against TESK1 and TESK2. The role of TESK activity in regulating neuronal phenotype was assessed by overexpressing TESK or dominant negative TESK and examining morphological changes to the actin and microtubule cytoskeletons in N1E-115 neuroblastoma cells, and by performing neurite outgrowth assays using primary neurons. Neurite outgrowth assays were performed on myelin substrates to examine the role of TESK during myelin inhibition.
Results: TESK1 protein expression is detected by western blotting in rat dorsal root ganglia (DRG), hippocampus, and cerebellum. Immunostaining reveals expression in retinal ganglion neurons, various regions of the cerebral cortex, hippocampus, and cerebellum. In N1E-115 neuroblastoma cells, overexpressed TESK1 localizes to vesicles and puncta, while TESK2 localizes to the nucleus and cytosol. TESK overexpression leads to an increase in F-actin stress fibers which correlates with an increase in cofilin phosphorylation. Outgrowth of rat DRG and hippocampal neurons is inhibited by overexpression of TESK, while dominant negative kinase dead forms have no effect. Outgrowth on myelin is enhanced by overexpression of wild-type and kinase dead forms of TESK.
Conclusions: TESK is expressed in several regions of the central nervous system. Differential sub-cellular localization of TESK1 and TESK2 suggests differing functions for each isoform. TESK is involved in regulation of the neuronal actin cytoskeleton.
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