DNA damage-induced phosphorylation of p53 at serine 20 correlates with p21 and Mdm-2 induction in vivo

Abstract

We investigated the induction and physiological role of Ser20 phosphorylation of p53 in response to DNA damage caused by ionizing radiation (IR) or ultraviolet radiation (UV). A polyclonal antibody that speci®cally recognizes a p53 peptide containing phosphorylated Ser20 was generated and used to detect p53 phosphorylation at Ser20. Western blot analyses of p53 in four cell lines with this antibody revealed that the p53 protein was phosphorylated at Ser20 to a dierent extent after treatment with IR or UV. The phosphorylation of Ser20 of wild-type p53 correlated with enhanced induction of the p53 downstream target genes p21 WAF1/Cip1 (p21) and mdm-2. These results suggest that DNA damage-induced phosphorylation of p53 at Ser20 enhances the transactivation function of p53 for p21 and mdm-2 in vivo.

Mdm-2 binding and TAFII31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21

“…Note, the only other site phosphorylated in vivo within the amphipathic a-helix is Ser20 (Chehab et al, 1999;Shieh et al, 1999;Unger et al, 1999a;Jabbur et al, 2000). Interestingly, Ser20 has a positively charged residue, Lys24, within proximity for potentially chargeattractive effects mediated by Ser20 phosphorylation.…”

“…The phosphorylation of p53 at Ser15, Ser33 and Ser37 enhances the recruitment of p300 and CBP (Lambert et al, 1998;Sakaguchi et al, 1998), whereas phosphorylation at Ser15, Ser20 and Ser37 disrupts the interaction of p53 with Mdm-2 Chehab et al, 1999;Unger et al, 1999a;Jabbur et al, 2001). In addition, the phosphorylation of p53 at Ser15, Ser20 and Ser37 modulates the transactivation function of p53 Jabbur et al, 2000Jabbur et al, , 2001, whereas Ser20 and Ser33 phosphorylation affects apoptotic function in a cell-type specific manner (Bulavin et al, 1999;Unger et al, 1999a,b). Hence, the stress-induced phosphorylation of p53 within the transactivation domain bestows p53 with transactivation attributes consistent with its ability to repel Mdm-2 and to interact with p300 and CBP.…”

“…DNA damage caused by treatment with ionizing radiation (IR) or ultraviolet radiation (UV) has been reported to result in the de novo phosphorylation of p53 within the transactivation domain at Ser6 , Ser9 , Ser15 (Siliciano et al, 1997;Shieh et al, 1997), Thr18 Sakaguchi et al, 2000; this manuscript), Ser20 (Chehab et al, 1999;Unger et al, 1999a;Jabbur et al, 2000), Ser33 and Ser37 Sakaguchi et al, 1998). In vitro experiments attribute these events to cellular kinases, including CKI Sakaguchi et al, 2000;Higashimoto et al, 2000), p38 MAPK (Bulavin et al, 1999), ATM (Banin et al, 1998;Canman et al, 1998), ATR (Tibbetts et al, 1999), DNA-PK (Woo et al, 1998), Cds1/Chk2 (Shieh et al, 2000), Chk1 (Chehab et al, 2000;Hirao et al, 2000;Shieh et al, 2000), JNK1 (Milne et al, 1995) and CAK (Ko et al, 1997).…”

The complexity of p53 stabilization and activation

“…75 In another study, when the same site was substituted with Asp (S20D) it led to a constitutively activated form of p53 but this change did not affect p53 stability. 76 In yet another report, S20A substitutions alone or in combination with S15A and T18A failed to alter the degree of p53 stabilization in response to ionizing radiation. 77 Hirao et al 78 showed that Chk2-deficient mice are unable to stabilize p53 in response to radiation exposure.…”

Role of DNA damage in atherosclerosis—Bystander or participant?

“…This prevents entry of Cdc25 into the nucleus blocking interaction with its substrate Cyclin E-Cdk2 [56]. Following DNA damage ATM activation also phosphorylates 'the guardian of the genome' p53 at residues Ser 15 and Ser 20 leading to accumulation of this protein in the nucleus [57,58]. Active p53 induces the cyclin-dependent kinase inhibitor p21 that inhibits the CyclinD1-Cdk2 and CyclinE-Cdk4 kinase complexes preventing the cell from moving to the next stage of the cell cycle [59].…”