Abstract
Abnormal p53 cellular localization has been considered to be one of the mechanisms that could inactivate p53 function. To understand the regulation of p53 cellular trafficking, we have previously identified two p53 domains involved in its localization. A basic domain, Lys(305)-Arg(306), is required for p53 nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic sequestration domain (CSD) from residues 326-355, could block the nuclear import of Lys(305) or Arg(306) mutated p53. To characterize further the function of these two domains, we demonstrate in this report that the previously described major nuclear localization signal works together with Lys(305)-Arg(306) to form a bipartite and functional nuclear localization sequence (NLS) for p53 nuclear import. The CSD could block the binding of p53 to the NLS receptor, importin alpha, and reduce the efficiency of p53 nuclear import in MCF-7, H1299, and Saos-2 cells. The blocking effect of the CSD is not due to the enhancement of nuclear export or oligomerization of the p53. These results indicate that the CSD can regulate p53 nuclear import by controlling access of the NLS to importin alpha binding.
Highlights
The p53 tumor suppressor is a nuclear phosphoprotein whose activities have been linked to cell cycle control, apoptotic cell death, DNA repair, stress responses, and genomic stability [1,2,3]. p53 is thought to regulate cell growth and viability through transcriptional dependent and independent pathways [3, 4]
We have identified two cis-acting domains important for p53 subcellular localization as follows: a basic domain (Lys305Arg306), in addition to the known NLSI, that is required for p53 nuclear import, and a carboxyl-terminal cytoplasmic sequestration domain (CSD, residues 326 –355) that interacts with this Lys-Arg domain to regulate p53 nuclear import [23, 24]
Nuclear Export Is Not Associated with Cytoplasmic Sequestration of Lys305-mutated p53—Due to the fact that p53 cellular localization is a dynamic equilibrium between rapid nuclear import and ongoing export, it is possible that the cytoplasmic sequestration effect of the Lys305 or Arg306 mutations is a result of enhancement of p53 nuclear export, probably by increasing the interaction with CRM1
Summary
Cell Culture—The MCF-7 breast cancer cell line, which expresses wild-type p53, and two p53-null cell lines, H1299 lung adenocarcinoma and Saos-2 osteosarcoma, were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) containing 10% (v/v) fetal bovine serum at 37 °C in a humidified 5% CO2 atmosphere. To analyze the relationship between p53 oligomerization and nuclear import, the FLAG-tagged DNA fragment corresponding to p53 residues 325–369 was amplified by PCR with a 5Ј primer containing FLAG sequences, and the PCR product was ligated to the BamHI and EcoRI sites of the pcDNA3 plasmid. In Vitro Binding Assay of p53 and Importin ␣—The wild-type or mutated p53 DNA fragments were ligated into the BamHI and EcoRI sites of vector pGEX-2T (Amersham Pharmacia Biotech) and transformed into the Escherichia coli strain DH5␣. One-twentieth of the beads were removed to analyze the amount of immobilized GST fusion proteins by immunoblotting with the anti-p53 pAb122 hybridoma supernatant (ATCC TIB116) or the anti-GST mouse IgG1 monoclonal antibody (Santa Cruz Biotechnology). The relative intensity of importin ␣ was measured by NIH Image software
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