Regulation of DNA binding and transactivation in p53 by nuclear localization and phosphorylation.
Compelling evidence indicates that p53 acts as a transcription factor and that this activity is regulated by several factors including subcellular localization and phosphorylation status of the protein. To learn more about how these two processes determine whether p53 becomes activated, we studied the temperature sensitive murine p53, p53val135. At nonpermissive temperatures, p53val135 remains sequestered in the cytoplasm of cells which express it. Electrophoretic mobility shift assays demonstrated that, under these conditions, the protein lacked DNA binding activity. However, by shifting to the permissive temperature, p53val135 became concentrated in the nucleus, hyperphosphorylated, and had acquired the ability to bind DNA in a sequence specific manner. This was accompanied by the induction of two p53 regulated genes, mdm2 and p21waf1, which indicated that p53val135 had become an active transcription factor. Two dimensional gel electrophoresis and tryptic peptide mapping showed that entry into the nucleus resulted in the appearance of new phosphorylated isoforms and that the protein had become extensively phosphorylation at the N-terminus. Notably, phosphorylation at the N-terminus occurred only in the nucleus, whereas phosphorylation at the C-terminus could occur in both the cytoplasm and the nucleus. Based on these observations, we suggest that phosphorylation of p53's N-terminus is compartmentally restricted.
Martinez
J D
JD
Department of Radiation Oncology, University of Arizona, Tucson 85724, USA.
Craven
M T
MT
Joseloff
E
E
Milczarek
G
G
Bowden
G T
GT
D000818:Animals; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D009687:Nuclear Proteins; D010449:Peptide Mapping; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D012313:RNA / Q000378:metabolism; D051381:Rats; D013696:Temperature; D013997:Time Factors; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D012313:RNA; D004247:DNA; C497208:Mdm2 protein, rat; D051736:Proto-Oncogene Proteins c-mdm2