PHOSPHORYLATION OF THE C-TERMINAL SITES OF HUMAN P53 REDUCES NON-SEQUENCE-SPECIFIC DNA-BINDING AS MODELED WITH SYNTHETIC PEPTIDES

Phosphorylation of the tumor suppressor p53 is generally thought to mo dify the properties of the protein in four of its five independent dom ains. We used synthetic peptides to directly study the effects of phos phorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino ac ids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) s ite, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombi nant p53 protein or the synthetic peptides bound to two unrelated targ et DNA fragments. Phosphorylation of the peptide at the PKC or the CKI I sites clearly decreased DNA binding, and addition of a second phosph ate group almost completely abolished binding. Circular dichroism spec troscopy showed that the peptides assumed identical unordered structur es in aqueous solutions. The unmodified peptide, unlike the Ser378 pho sphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detec ted when circular dichroism and nuclear magnetic resonance spectra wer e: taken in trifluoroethanol-water mixtures. A single or double phosph orylation destabilized the helix around the phosphorylated Ser378 resi due but stabilized the helix downstream in the sequence.