WW domain-containing oxidoreductase WWOX, a putative tumor suppressor, has been suggested to be involved in the hyperphosphorylation of Alzheimer¡¦s tau. Tau is a microtubule-associated protein which plays an important role in microtubule assembly and stability. GSK-3b regulates tau by phosphorylation as well as by regulating splice variance. Hyperphosphorylated tau has less affinity toward the microtubules and disrupting microtubule stability. By bioinformatics analysis, we found that WWOX has three GSK3£] binding motifs, FXXXLI/VXRLE. The results of immunoprecipitation demonstrated that WWOX interacted physically with GSK3£], which was supported by the colocalization of WWOX and GSK3£] in immunofluorescence study. Using in vitro GST pull down assay, we demonstrated that WWOX can bind directly to GSK3£] through its ADH/SDR domain. The site-direct mutagenesis was performed to locate the GSK3£] binding sequences within the ADH/SDR domain precisely. By in vitro kinase assay, we found that WWOX can inhibit Ser396 and Ser404 phosphorylation of Tau by GSK3£] in a dose- and time- dependent manner, indicating that WWOX may be involved in regulating GSK3£] activity in cells. It has been demonstrated that inhibition of GSK3£] plays an essential role during neuron differentiation. By in vitro MT formation assay, we showed that GSK3£] hyperphospohorylated Tau failed to promote tubulin polymerization. Together with the Tau hyperphosphorylation inhibited its function to promote microtubule formation and neurite outgrowth, we investigated the effect of WWOX in neuron differentiation. Interestingly, overexpression of WWOX enhanced the SH-SY5Y differentiation with or without retinoic acid treatment. SH-SY5Y cells increase expression of WWOX and decrease expression of pTau S396 as they differentiate in culture. RNAi-mediated knockdown of WWOX in RA-differentiated SH-SY5Y cells caused a decrease in neurite outgrowth. Furthermore, inhibition of GSK3 by Bio enhanced SH-SY5Y differentiation and overexpression of GSK3£] caused a decrease in neurite outgrowth. We concluded that WWOX may be involved in regulating GSK3£] activity to reduce the level of phosphorylated Tau that subsequently promoted the neurite outgrowth during neuron differentiation.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0804108-231134 |
| Date | 04 August 2008 |
| Creators | Juo, Liang-I |
| Contributors | Nan-Shan Chang, Yi-Ren Hong, Jiin-Tsuey Cheng, Pei-jung Lu |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0804108-231134 |
| Rights | not_available, Copyright information available at source archive |
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