DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation

Abstract

Abundance and activity of p53 are predominantly regulated posttranslationally. Structural disturbance in transcribed genes induced by radiation, e.g. DNA damage, or by transcriptional inhibitors cause p53 protein stabilization by a yet unknown mechanism. Using stable and transient transfections for the analysis of p53 mutant proteins, we have ruled out a role in stabilization by UV, gamma irradiation or actinomycin C for the following putative phosphorylation sites in the p53 protein: serines 6, 9, 15, 33, 315 and 392, and threonine 18. By double mutation combinations of phosphorylations were also ruled out; 6,9; 15,18; 15,37. These mutations eliminate modi®cations by casein kinases I and II, DNA-PK, ATM, CDK and JNK. Also the 30 carboxyterminal amino acids are not required for induced p53 stabilization. Thus neither phosphorylations of individual amino acids nor interactions of the carboxyterminus of p53 with cellular macromolecules appear to play a role in the stabilization process. The only single prerequisite for induced stabilization of p53 is its prior destabilization by Mdm2. However, the level of active Mdm2 must be controlled carefully: overexpression of Mdm2 inhibits UV induced p53 stabilization.

Activation and activities of the p53 tumour suppressor protein

“…These activities of MDM2 are probably related to its interaction with other proteins, including regulators of the cell cycle and cell fate such as MTBP , Numb (Juven-Gershon et al, 1998), the retinoblastoma tumour suppressor protein (pRB) (Hsieh et al, 1999), E2F1/DP1 (Loughran and La Thangue, 2000) and SMAD transcription factors (Yam et al, 1999). E2F-1 has been shown to be susceptible to proteasome-mediated degradation and, like p53, it can be stabilized by various forms of cellular stress (Blattner et al, 1999;O'Connor and Lu, 2000). However whether MDM2 participates in these processes remains unclear Loughran and La Thangue, 2000).…”

ATM: A mediator of multiple responses to genotoxic stress

“…It seems plausible, therefore, that Ser-15 of p53 is a physiological target of ATM. The role of Ser-15 phosphorylation in vivo is not clear, however, since it is dispensable for IR-induced p53 activation and stabilization (Blattner et al, 1999;Chehab et al, submitted;Khosravi et al, submitted).…”

Mechanisms of switching on p53: a role for covalent modification?

“…While many of these studies have provided evidence supporting a role for modi®cation, others have suggested that p53 function is not impaired by preventing modi®cation of key sites in vivo. For example, mutation of a series of key phosphorylation sites in the N-and C-termini of human p53, including the residues important for blocking the p53-MDM2 interaction and stimulating interaction with 14-3-3, has no e ect on the ability of UV or ionizing radiation to induce stabilization of the mutant p53 proteins in cultured cells (Blattner et al, 1999). Similarly, collective mutation of the N-or C-terminal phosphorylation sites, or all of the known phosphorylation sites simultaneously, shows only subtle e ects on stabilization following DNA damage, transcriptional function or interaction with MDM2 (Ashcroft et al, 1999).…”

“…Similarly, collective mutation of the N-or C-terminal phosphorylation sites, or all of the known phosphorylation sites simultaneously, shows only subtle e ects on stabilization following DNA damage, transcriptional function or interaction with MDM2 (Ashcroft et al, 1999). In contrast the level of MDM2 has a profound e ect on the ability to induce these proteins (Blattner et al, 1999). Moreover, disruption of the interaction between endogenous p53 and MDM2 proteins in cells expressing only wild type p53 is su cient in itself to stabilize the p53 and elicit a measurable p53 response in terms of activation of a p53-responsive reporter gene and the onset of cell cycle arrest (Bottger et al, 1997).…”