A novel system to investigate the phosphorylation of the p53 tumor suppressor protein by the protein kinase CK2.
The tumor suppressor protein p53 is phosphorylated at a C-terminal residue (serine 386 in mouse p53) by the protein kinase CK2. Phosphorylation by CK2 activates the specific DNA binding function of p53 and stimulates its ability to suppress cellular growth. Previous reports have suggested that phosphorylation of p53 at the CK2 site is stimulated in cells expressing the large tumor antigen (T antigen) of simian virus 40 (SV40). To test this idea, we have expressed a C-terminal p53 "mini-protein" which comprises amino acids 154-387 of mouse p53 and therefore lacks the heavily phosphorylated N-terminus. In addition, the serine 309 phosphorylation site (targeted by cyclin-dependent kinases) has been mutated to encode alanine. We have expressed the p53 mini-protein in mammalian cells and shown by phosphopeptide mapping that it is phosphorylated at a single physiological phosphorylation site, serine 386. Using this mini-protein as a cellular target for CK2, we have shown that phosphorylation of p53 by CK2 is not affected by the presence of T antigen. The p53 mini-protein is likely to be a useful tool with which to probe the regulation of p53 phosphorylation by CK2 in response to other factors which influence cell growth.
McKendrick
L
L
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, UK.
Meek
D W
DW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology; D047390:Casein Kinase II; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013539:Simian virus 40 / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases