PHOSPHORYLATION OF SERINE-392 STABILIZES THE TETRAMER FORMATION OF TUMOR-SUPPRESSOR PROTEIN-P53

Tumor suppressor protein p53 is a tetrameric phosphoprotein that activ ates transcription from several cell cycle regulating genes in respons e to DNA damage. Tetramer formation is critical to p53's ability to ac tivate transcription; however, posttranslational modifications and pro tein stabilization also contribute to p53's ability to activate transc ription. To determine if phosphorylation affects tetramer formation, w e synthesized phosphopeptides corresponding to residues 303-393 of hum an p53, which includes the domain responsible for tetramer formation, Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association const ant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, bu t phosphorylation of Ser315 largely reversed the effect of phosphoryla tion at Ser392. Analyses by calorimetry demonstrated that phosphorylat ion may influence subunit affinity (and, in turn, DNA binding) by an e nthalpy-driven process, possibly between the C-terminal residues and t he region immediately adjacent to Ser315. The K-d for the tetramer-mon omer transition of the unphosphorylated p53 C-terminal domain was dete rmined to be similar to 1-10 mu M. Thus, in normal, undamaged cells p5 3 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p5 3 as a transcription factor in response to DNA damage.