Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein.
Cytoplasmic sequestration of the p53 tumor suppresser protein has been proposed as a mechanism involved in abolishing p53 function. However, the mechanisms regulating p53 subcellular localization remain unclear. In this report, we analyzed the possible existence of cis-acting sequences involved in intracellular trafficking of the p53 protein. To study p53 trafficking, the jellyfish green fluorescent protein (GFP) was fused to the wild-type or mutated p53 proteins for fast and sensitive analysis of protein localization in human MCF-7 breast cancer, RKO colon cancer, and SAOS-2 sarcoma cells. The wild-type p53/GFP fusion protein was localized in the cytoplasm, the nucleus, or both compartments in a subset of the cells. Mutagenesis analysis demonstrated that a single amino acid mutation of Lys-305 (mt p53) caused cytoplasmic sequestration of the p53 protein in the MCF-7 and RKO cells, whereas the fusion protein was distributed in both the cytoplasm and the nucleus of SAOS-2 cells. In SAOS-2 cells, the mutant p53 was a less efficient inducer of p21/CIP1/WAF1 expression. Cytoplasmic sequestration of the mt p53 was dependent upon the C-terminal region (residues 326-355) of the protein. These results indicated the involvement of cis-acting sequences in the regulation of p53 subcellular localization. Lys-305 is needed for nuclear import of p53 protein, and amino acid residues 326-355 can sequester mt p53 in the cytoplasm.
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0936, USA.
S H
SH
Liang
D
D
Hong
M F
MF
Clarke
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins