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NUCLEAR PROTEIN-ANCHORING FUNCTION OF CYTOSKELETON AND ITS ANTI-APOPTOTIC EFFECTS AGAINST P53-DEPENDENT APOPTOSIS.

细胞骨架的核蛋白锚定功能及其对 P53 依赖性细胞凋亡的抗凋亡作用。

基本信息

批准号：
11670008
负责人：
NISHIO Koji
金额：
$ 2.3万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670008/
关键词：
cytoskeleton vimentin p53 apoptosis anchoring function nuclear translocation functional domain 核移行反応

项目摘要

To reveal the molecular mechanism of anchoring of p53 and S1protein on the vimentin cytoskeleton, We constructed the expression vectors that produce the GFP-chimera of the full length, amino (N)-terminal or carboxyl (C)-terminal truncated p53. The N-terminal 20 or 40 amino acid and C-terminal 33 or 63 amino acid were truncated by PCR.The truncated p53 cDNA were subcloned into pEGFP vectors. Vimentin expressing cells, Cos7 and human fibroblasts or vimentin knockout Vim-/- cells were transfected with the truncated or full-length p53-GFP expression vectors. The wild type p53-GFP localized in the nuclei and occasionally in the cytoplasm. The truncated wild type p53-GFP localized in the nuclei in most cells. The localization of class III mutant p53V143A-GFP is cytoplasmic and dependent on the presence of vimentin. In contrast, the truncated mutant p53V143A-GFP (mp53V143AN41 and mp53V143AC330) significantly localized in the nuclei of Vim +/+ cells as like as wild type p53. These results indicates that both of the N-terminal and C-terminal domains of p53 involve with the anchoring on the vimentin cytoskeleton. We also examined GFP-chimera of class I (functional in both G1-arrest and apoptosis), class II (G1-arrest but not in apoptosis), and other class III mutants of p53 (defective in G1-arrest and apoptosis). Class I and class II mutants localized in the nuclei, in contrast to class III mutants, which significantly localized in the cytoplasm of Cos-7 cells. These results suggest that the conformation of p53 or interaction with other cellular component (s) is important in its cytoplasmic anchoring, which may have an inhibitory effect against p53-dependent apoptosis. P53 with mutant type conformation involves with cytoplasmic anchoring and may have an inhibitory effect against p53-dependent apoptosis. Biochemical investigation of apoptosis revealed that cleavage of nuclear lamin B2 was necessary for the DNA fragmentation.

为了揭示p53和S1蛋白锚定在波形蛋白细胞骨架上的分子机制，我们构建了全长、氨基（N）端或羧基（C）端截短的p53的GFP嵌合体表达载体。用PCR方法截短N端20或40个氨基酸和C端33或63个氨基酸，将截短的p53 cDNA亚克隆到pEGFP载体中。用截短的或全长p53-GFP表达载体转染表达波形蛋白的细胞、Cos 7和人成纤维细胞或波形蛋白敲除的Vim-/-细胞。野生型p53-GFP定位于细胞核，偶尔定位于细胞质。截短的野生型p53-GFP定位于大多数细胞的细胞核中。III类突变体p53 V143 A-GFP的定位是细胞质的，并依赖于波形蛋白的存在。相反，截短的突变体p53 V143 A-GFP（mp53 V143 AN 41和mp53 V143 AC 330）与野生型p53一样显著定位于Vim +/+细胞的细胞核中。这些结果表明，p53的N端和C端结构域都参与了锚定在波形蛋白细胞骨架上。我们还研究了GFP嵌合体的I类（功能在G1-逮捕和细胞凋亡），II类（G1-逮捕，但不凋亡），和其他III类突变体的p53（缺陷G1-逮捕和细胞凋亡）。I类和II类突变体定位在细胞核中，相反，III类突变体，这显着定位在Cos-7细胞的细胞质中。这些结果表明，p53的构象或与其他细胞成分的相互作用是重要的，在其细胞质锚定，这可能对p53依赖性细胞凋亡的抑制作用。突变型P53蛋白参与细胞质锚定，可能对p53依赖的细胞凋亡具有抑制作用。细胞凋亡的生化研究表明，核纤层蛋白B2的切割是必要的DNA片段化。