Posttranslational Modification of Mutant p53 Protein in Different Tumor Cell-Lines after UV Light Damage
Tumor suppressor protein p53 represents one of the most important factors regulating cell proliferation, differentiation and programmed cell death in normal cells. Human p53 is a nuclear phospho-protein consisting of 393 amino acids. Function of p53 lies on its ability to bind the DNA consensus sequences and operate as a sequence-specific transcription factor. Proportionally to the extent of DNA damage, p53 induces G1-cell arrest allowing DNA reparation before next DNA synthesis or initiates mechanisms of apoptosis if DNA reparation is not complete. Disturbance of these processes (due to mutations or deffective posttranslational modifications) leads usually to malignant transformation of the cell. Transformation of latent form of p53 to the active form by means of posttranslational modification includes mainly phosphorylation and acetylation of specific sites. One of the modifications at the casein kinase II (CK II) phosphorylation site in the C-terminal domain of p53 activates the latent specific DNA-binding function of p53 protein in vitro.
The aim of this study was to characterize the level of phosphorylation of p53 mutant forms in the different tumour cell lines as a response to UV light irradiation. Using immunochemistry and immunocytochemistry we analysed posttranslational modification of p53 protein at serine 392 after UV light damage in eight tumor cell lines with known p53 status (MCF-7, HS913T, HOS, BT549, SK-LMS-1, BT474, SK-UT-1 and T47D). For the characterization of phosphorylation at this site we developed a panel of novel phospho-specific monoclonal antibodies recognizing p53 protein phosphorylated at Ser392 which exhibit different affinities towards the target epitope.
Response of total level of p53 as well as phosphorylated portion of p53 differed among different tumor cell lines with defined p53 mutation status after UV light damage. We also detected the posttranslationally modified mutant forms of p53 (phosphorylated at Ser392) while three cell lines ? BT549 (Arg249Ser), SK-LMS-1 (Gly245Ser) and T47D (Leu194Phe) were shown to increase the level of p53 phosphorylation at Ser392 after UV irradiation. The level of posttranslational modification of different p53 mutant forms vary depending on the type of cell line expressing mutant p53.
This work was supported by grants: IGA MH CR 4783-3; GA CR 312/99/1550 and GA CR 301/00/P094.
K. Kankova, S. Pospisilova, M. Svitakova, B. Vojtesek
Department of Cellular and Molecular Oncology,