The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members

Genetic and biochemical studies have shown that Ser²⁰ phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser²⁰ phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser²⁰ kinases. Phosphorylation of a p53 transactivation domain fragment at Ser²⁰ by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser²⁰ kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser²⁰ phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser²⁰ kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser²⁰, (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser²⁰ phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Δp53 spliced variant lacking the BOX-V motif is refractory to Ser²⁰ phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.