Constant rate of p53 tetramerization in response to DNA damage controls the p53 response

Abstract

The dynamics of the tumor suppressor protein p53 have been previously investigated in single cells using fluorescently tagged p53. Such approach reports on the total abundance of p53 but does not provide a measure for functional p53. We used fluorescent protein‐fragment complementation assay ( PCA ) to quantify in single cells the dynamics of p53 tetramers, the functional units of p53. We found that while total p53 increases proportionally to the input strength, p53 tetramers are formed in cells at a constant rate. This breaks the linear input–output relation and dampens the p53 response. Disruption of the p53‐binding protein ARC led to a dose‐dependent rate of tetramers formation, resulting in enhanced tetramerization and induction of p53 target genes. Our work suggests that constraining the p53 response in face of variable inputs may protect cells from committing to terminal outcomes and highlights the importance of quantifying the active form of signaling molecules in single cells. image Quantification of the dynamics of p53 tetramers in single cells using a fluorescent protein‐fragment complementation assay reveals that, while total p53 increases proportionally to the DNA damage strength, p53 tetramers are formed at a constant rate. A fluorescent protein‐fragment complementation assay is developed and used to quantify p53 tetramers in single living cells. In response to DNA damage, p53 total levels increase proportionally to the strength of the damage; however, p53 tetramers are formed at a constant rate across damage doses. The protein ARC is a key component of the “molecular throttle” that controls the rate of p53 tetramer formation and breaks the linear relationship between the input strength ( DNA damage) and cellular output (active p53). In the absence of ARC , the rate of p53 tetramerization becomes dose dependent and the expression of p53 target genes is enhanced.