碩士 / 長庚大學 / 基礎醫學研究所 / 92 / Abstract
Pinin was discovered as a protein occurring in several tissues. Pinin was found to exist at cell-cell contact and thought to play a key role in reorganization and stabilization of the desmosome-intermediate filament complex in epithelial cells. In addition to its cell peripheral location, pinin has been discovered in nuclear speckle domain and participate in pre-mRNA splicing. Although the desmosome-form ( d-form ) and the nucleus-form ( n-form ) pinin share identical amino acid sequences as demonstrated by cDNA library screening and DNA sequencing, they exhibit remarkably different biochemical properties, reflecting the apparent different multiprotein cellular location. Owing to dual intracellular location, we suggest that pinin be a moonlighting protein with more than one function within cells. It’s therefore intriguing to unravel the mechanism underlying nucleocytoplasmic transport to pinin. Utilizing heterokaryon assay, I found that a significant portion of n-form pinin displays nucleocytoplasmic shuttling upon fusion of donor cells ( Cos-7 ) which contain exogenously expressed pinin with recipient cells ( NIH3T3 ). Since Pinin contains several potential NLS ( nuclear localization signal ) and NES ( nuclear export signal ) , I generated a series of pinin deletion mutants to systematically investigate their roles in pinin nucleocytoplasmic distribution. I found that not only NLS and NES but also coil-coiled domain and spatial folding co-operatively control the pinin subcellular localization and protein stability. In addition, I demonstrated that accumulation of pinin at cell-cell contact sites, is mediated by protein kinase A ( PKA ) stimulation of n-form pinin, reflecting the potential involvement of n-form pinin in regulating d-form pinin biogenesis. Pinin will interact with TSG101 by yeast two-hybrid screening. By immunofluorescent microscopy and Western blotting analysis, I found that overexpressed TSG101 rendered pinin degradation. But the TSG101 induced degradation of pnn is depend on the p53 status of cells examined, because in p53 null cell, such as SKOV-3, nuclear pnn is not affected by TSG101 over-expression. Using immunoprecipitation assay, we found that pinin can interact with ubiquitin when cells were pretreated with proteosome inhibitor. Hence, the interaction betwenn pinin, TSG101, and ubiquitin cooperatively regulate the pnn degradation. In addition, over-expression of ubiquitin also renders pinin degradation, and this effect can be counteracted by transfection of p53 or TSG101 SiRNA constructs. Overall, we suggest that pinin degradation through ubiquitination may be mediated by TSG101 and regulated by p53.
| Identifer | oai:union.ndltd.org:TW/092CGU00325006 |
| Date | January 2004 |
| Creators | Chun-Lun Lin, 林群倫 |
| Contributors | Pin Ouyang, Kwang-Huei Lin, Jau-Song Yu, 歐陽品, ---, --- |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 127 |
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