Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents

To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser(6) using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic, l-2-amino-4-phosphono-4, 4-difluorobutanoic acid (F(2)Pab), in place of phosphoserine. Fmoc-F(2)Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F(2)Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser(6) but not the unphosphorylated peptide or the same peptide phosphorylated at Ser(9), Ser(15), Ser(20), Ser(33), or Ser(37). Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser(6) that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser(9) using antibodies raised against a conventional phosphopeptide. Ser(9) was phosphorylated by casein kinase 1 in vitro in a phosphoserine 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F(2)Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.