Structured DNA promotes phosphorylation of p53 by DNA‐dependent protein kinase at serine 9 and threonine 18

Abstract

Phosphorylation at multiple sites within the N-terminus of p53 promotes its dissociation from hdm2/mdm2 and stimulates its transcriptional regulatory potential. The large phosphoinositide 3-kinase-like kinases ataxia telangiectasia mutated gene product and the ataxia telangectasia and RAD-3-related kinase promote phosphorylation of human p53 at Ser15 and Ser20, and are required for the activation of p53 following DNA damage. DNA-dependent protein kinase (DNA-PK) is another large phosphoinositide 3-kinaselike kinase with the potential to phosphorylate p53 at Ser15, and has been proposed to enhance phosphorylation of these sites in vivo. Moreover, recent studies support a role for DNA-PK in the regulation of p53-mediated apoptosis. We have shown previously that colocalization of p53 and DNA-PK to structured single-stranded DNA dramatically enhances the potential for p53 phosphorylation by DNA-PK. We report here the identification of p53 phosphorylation at two novel sites for DNA-PK, Thr18 and Ser9. Colocalization of p53 and DNA-PK on structured DNA was required for efficient phosphorylation of p53 at multiple sites, while specific recognition of Ser9 and Thr18 appeared to be dependent upon additional determinants of p53 beyond the N-terminal 65 amino acids. Our results suggest a role for DNA-PK in the modulation of p53 activity resultant from the convergence of p53 and DNA-PK on structured DNA.Keywords: DNA-dependent protein kinase; p53; structured single-stranded DNA; phosphorylation.The large phosphatidylinositide 3-kinase (PI3K)-like kinases are broad specificity serine/threonine kinases with essential roles in regulating DNA metabolism and responses to DNA damage. Three of these kinases, DNA-dependent protein kinase (DNA-PK), the ataxia telangiectasia mutated gene product (ATM) and the ataxia telangectasia and RAD-3-related kinase (ATR) [1,2] show a redundant specificity for accessible SQ and TQ motifs in vitro that has hindered definition of their individual roles in DNA repair and metabolism. In particular, while DNA-PK and its associated kinase activity have been shown to be required for double-stranded DNA (dsDNA) break repair through the nonhomologous end-joining pathway, for V(D)J recombination, and to play at least some role in the regulation of other processes including transcription, DNA replication and viral integration, demonstration of a role for DNA-PK in specific protein phosphorylation in vivo has remained elusive [1]. We and others have hypothesized that substrate phosphorylation by DNA-PK in vivo depends to a large extent on mechanisms that promote the recruitment of substrates to DNA-bound, active, DNA-PK [3-6]. p53 is a key regulatory protein that has the potential to be phosphorylated by DNA-PK, ATM and ATR as Ser15 of human p53 is efficiently phosphorylated by all three kinases in vitro [7]. Phosphorylation as well as ubiquitylation and acetylation control the activation status of p53 [8]. A majority of the phosphorylation sites on p53 are clustered within the N-terminal 40 amino acids (se...

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PML enhances the regulation of p53 by CK1 in response to DNA damage

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Novel homeodomain‐interacting protein kinase family member, HIPK4, phosphorylates human p53 at serine 9

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