Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase which plays a key role in several signaling pathways and its homologues have been identified in most eukaryotes. Since GSK3£]is an essential protein kinase that regulates numerous functions within the cell, an effort to survey possible GSK3£]- interacting proteins from a human testis cDNA library using the yeast two-hybrid system is made. Two interesting candidates are chosen to characterize their functions in this study. One is a centrosomal protein, hNinein, and the other is a novel inhibitor of GSK3£], designated as GSKIP (GSK3£] interaction protein).
In the first part of the present thesis we describe the identification of four diverse CCII-termini of human hNinein isoforms, including a novel isoform 6, by differential expression in a tissue-specific manner. In a kinase assay, the CCII region of hNinein isoforms provides a differential phosphorylation site by GSK3£]. In addition, either N-terminal or CCIIZ domain disruption may cause hNinein conformational change which recruits £^-tubulin to centrosomal or non-centrosomal hNinein-containing sites. Further, depletion of all hNinein isoforms caused a significant decrease in the £^-tubulin signal in the centrosome. In domain swapping, it clearly shows that the CCIIX-CCIIY region provides docking sites for £^-tubulin. Moreover, nucleation of microtubules from the centrosome is significantly affected by the overexpression of either the full-length hNinein or CCIIX-CCIIY region. Taken together, these results show that the centrosomal targeting signals of hNinein have a role not only in regulating hNinein conformation, resulting in localization change, but also provide docking sites to recruit £^-tubulin at centrosomal and non-centrosomal sites.
In the second part of the thesis we describe another candidate, GSK3£]interaction protein (GSKIP), to characterize its functions in neuron differentiation. We use human neuroblastoma SH-SY5Y cells as a model of neuronal cell differentiation. When overexpression of GSKIP prevents neurite outgrowth from RA-mediated differentiation, this result is similar to the presence of LiCl or SB415286, an inhibitor of GSK3£]. Further, GSKIP regulates the activity of GSK3£] through protein-protein interactions rather than post-modulation and GSKIP may affect GSK3£] on neurite outgrowth via inhibiting the specific phosphorylation site of tau. In addition to inhibition of neurite outgrowth, GSKIP overexpressed in SH-SY5Y cells also promotes cell cycle progression by analyzing cell proliferation with cell growth and MTT assay. Furthermore, GSKIP raises the level of £]-catenin and cyclin D1 through inhibition of GSK3£] activity in RA-mediated differentiation SH-SY5Y cells. Taken together, the data suggest that GSKIP, a dual functional molecule, is able to inhibit neurite outgrowth and promote cell proliferation via negative regulation of GSK3£] activity in RA-mediated differentiation of SH-SY5Y cells.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0910107-105059 |
| Date | 10 September 2007 |
| Creators | Lin, Ching-chih |
| Contributors | Chung-Lung Cho, Yi-Ren Hong, Pei-Jung Lu, Wen-Tsan Chang, Ching-Mei Hsu, Bei-Chang Yang, De-Ching Chang |
| 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-0910107-105059 |
| Rights | campus_withheld, Copyright information available at source archive |
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