Characterize the mechanisms of nucleocytoplasmic shuttling of the PP2A B56γ3 regulatory subunit

• Main Authors: Chia-Te Lin, 林佳德
• Other Authors: Chung-Liang Ho
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/58657072288228379076

碩士 === 國立成功大學 === 分子醫學研究所 === 96 === Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme which consists of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit. There are at least four subfamilies of the B subunit, named B, B’, B’’, and B’’’. The B subunit has been thought to determine the substrate specificity and subcellular localization of the enzyme. B56γ3, one of the B’ isoform, has been shown to have roles in suppressing cell transformation and tumorigenesis. Consistent with previous findings, we have observed B56γ3 in NIH3T3 and HeLa cells existing in both nucleus and cytoplasm in cells transiently and stably expressing exogenous B56γ3. The mechanisms underlying the subcellular localization of B56γ3 are not clear. We have found four putative nuclear localization signals (NLSs) and three putative nuclear export signals (NESs) within B56γ3 by sequence analyses using software. We serially truncated the four putative NLSs from the C terminus of B56γ3 and investigated the subcellular localization of these NLS truncation mutants of B56γ3. Surprisingly, none of the serially NLS-truncated B56γ3 mutants showed impaired nuclear localization in both transient and stable expression experiments in both NIH3T3 and HeLa cells. Nevertheless, we found a significant increase of nuclear localization of B56γ3 after truncations of putative NLS 4, NLS 3, and NLS 2. However, the phenotype was diminished in NIH3T3 cells but not in HeLa cells when the linker segment between putative NLS 1 and NLS 2 was deleted. In addition, fluorescence microscopy analysis of transiently expressed yellow fluorescent protein (YFP)-fused full-length and NLS truncated mutants showed that all forms of B56γ3 were capable of locating to the nucleus. Furthermore, to examine whether these truncation mutants of B56γ3 can associate with the AC core enzyme to form a functional trimeric holoenzyme, we performed co-immunoprecipitation analysis and found that the truncated B56γ3 mutants without the linker segment between NLS 1 and 2 greatly lost the ability to associate with the AC core enzyme. Our findings reveal that a noncanonical NLS may be involved in regulating B56γ3 nuclear localization and demonstrate that a motif near the C terminus is critical for B56γ3 association with the AC core enzyme.