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

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。用聚合酶链式反应截短N端20或40个氨基酸和C端33或63个氨基酸，将截短的p53基因亚克隆到pEGFP载体中。将截短或全长的p53-GFP表达载体分别导入表达波形蛋白的细胞、COS7和人成纤维细胞或波形蛋白基因敲除的Vim-/-细胞。野生型P53-GFP定位于胞核，偶见胞浆。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p53V143A-GFP的定位是细胞质的，并依赖于波形蛋白的存在。截短的突变体p53V143A-GFP(mp53V143AN41和mp53V143AC330)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端结构域都与波形蛋白细胞骨架上的锚定有关。我们还检测了GFP嵌合体的I类(在G1期停滞和凋亡中都有功能)、II类(G1期停滞但不在凋亡中)，以及其他III类突变体(G1期停滞和凋亡中存在缺陷)。I类和II类突变体定位于细胞核，而III类突变体显著定位于Cos-7细胞的细胞质。这些结果表明，P53的构象或与其他细胞成分(S)的相互作用在其细胞质锚定中起重要作用，这可能对P53依赖的细胞凋亡具有抑制作用。突变型P53与细胞质锚定有关，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，核层蛋白B2的断裂是DNA断裂所必需的。

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.