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

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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.