Figure 1 | Oncogene

Figure 1

From: The ataxia-telangiectasia related protein ATR mediates DNA-dependent phosphorylation of p53

Figure 1

Identification of a novel DNA-activated protein kinase that phosphorylates p53 on serine-15. (a) Antisera raised against phospho-serine-15 of p53 recognize p53 after it is phosphorylated by DNA-PK. Reactions contained either wild-type or Ser15->Ala mutant p53, as indicated. Reactions were performed with p53 alone, or p53 and DNA-PK in the presence or absence of 25 ng of sheared genomic DNA, as indicated. Proteins were separated by SDS – PAGE and p53 serine-15 phosphorylation detected by Western blotting using phospho-specific antibodies raised against serine-15 of p53. (b) Fractionation of HeLa nuclear extract by ion-exchange chromatography reveals two peaks of activity capable of phosphorylating serine-15 of p53. HeLa nuclear extract was fractionated over Q-sepharose and fractions employed in kinase reactions in the presence of 25 ng of sheared genomic DNA using p53 as a substrate. p53 phosphorylation was detected as in (a). (c) Q-sepharose derived SFK2 is not inhibited by low concentrations of wortmannin that inhibit SFK1 (DNA-PK). Q-sepharose derived SFK1 (corresponding to DNA-PK) or SFK2 was employed in kinase reactions using p53 as a substrate in either the presence or absence of 1 μM wortmannin. Phosphorylated p53 was detected as in (a). (d) Q-sepharose derived SFK2 is stimulated by DNA. Kinase assays were performed in the absence (−) or presence of 1, 10, 50, 100 and 250 ng of either sheared genomic, circular plasmid or linear plasmid DNA as indicated. Phosphorylated p53 was detected as in (a)

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