Folia Biologica
Journal of Cellular and Molecular Biology, Charles University 

The p53 tumour suppressor protein is a nuclear phosphoprotein which plays a key role in cell-cycle regulation. The p53 protein protects cells from undergoing tumorigenic alterations by inducing either the cell growth arrest or programmed cell death in response to a variety of cellular stress signals such as DNA damage, hypoxia, heat shock, oncogene activation or metabolic changes (Levine, 1997). One of the critical issues of p53 response to cellular stress or DNA damage is the way of its activation. It has been suggested that posttranslational modification involving phosphorylation of the p53 protein is most likely the mechanism through which the activity of the p53 protein may be regulated. p53 is phosphorylated on several serine residues within the N- and C-terminal regions by several cellular kinases. The N-terminal part of p53 is phosphorylated using different protein kinases including casein kinase I (CKI) (Milne et al., 1992), DNA-PK (Lees-Miller et al., 1992), Chk1, Chk2, MAP kinase (Milne et al., 1994) and c-Jun kinase (Milne et al., 1995). There are also at least three phosphorylation sites on the C-terminus of human p53 at amino acids 315, 378 and 392. Serine³¹⁵ of p53 is a target for cdk1 and cdk2 (Price et al., 1995). Serine³⁷⁸ is phosphorylated by PKC (Baudier et al., 1992). Serine³⁹² is phosphorylated by purified casein kinase II (CKII) in vitro (Blaydes and Hupp, 1998). Phosphorylation of the human p53 at Ser³⁹² has been shown to enhance p53 sequence-specific DNA binding in vitro (Hupp and Lane, 1994), which is responsible for transcriptional activation of the p53 protein. Recently, it has also been shown that phosphorylation of Ser³⁹² is important for p53-mediated transcriptional activation in vivo (Hao et al., 1996). Phosphorylation of different proteins in cells can be studied using a range of different methods, but the primary technique for determining phosphate incorporation into the specific sites in target proteins involves labelling of cells with [³²P] phosphate followed by phospho-amino-acid analysis or sequencing of the protein of interest (Van der Geer et al., 1993). The main problem associated with this technique is the incubation of cells with a radioactive precursor [³²P], which itself can activate growth arrest and stress-responsive signalling pathways, obviously perturbing protein phosphorylation (Yeargin and Haas, 1995; Dover et al., 1994). Preparation of monoclonal antibodies that are specific to either phosphorylated or non-phosphorylated epitopes within the target protein provides a powerful alternative to the above techniques.

« Previous article