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

Abstract

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 Ser6 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-di-fluorobutanoic acid (F2Pab), in place of phosphoserine. Fmoc-F2Pab was prepared by an improved synthesis and chemically incorporated using solid phase peptide synthesis. Affinity-purified antibodies elicited by immunizing rabbits with an F2Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser6 but not the unphosphorylated peptide or the same peptide phosphorylated at Ser9, Ser15, Ser20, Ser33, or Ser37. Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser6 that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser9 using antibodies raised against a conventional phosphopeptide. Ser9 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 F2Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies.