Activation and control of p53 tetramerization in individual living cells

Abstract

Homo-oligomerization is found in many biological systems and has been extensively studied in vitro. However, our ability to quantify and understand oligomerization processes in cells is still limited. We used fluorescence correlation spectroscopy and mathematical modeling to measure the dynamics of the tetramers formed by the tumor suppressor protein p53 in single living cells. Previous in vitro studies suggested that in basal conditions all p53 molecules are bound in dimers. We found that in resting cells p53 is present in a mix of oligomeric states with a large cell-to-cell variation. After DNA damage, p53 molecules in all cells rapidly assemble into tetramers before p53 protein levels increase. We developed a model to understand the connection between p53 accumulation and tetramerization. We found that the rapid increase in p53 tetramers requires a combination of active tetramerization and protein stabilization, however tetramerization alone is sufficient to activate p53 transcriptional targets. This suggests triggering tetramerization as a mechanism for activating the p53 pathway in cancer cells. Many other transcription factors homo-oligomerize, and our approach provides a unique way for probing the dynamics and functional consequences of oligomerization.H omo-oligomerization, the formation of a protein complex out of identical components, is extremely common in nature; in Escherichia coli it is estimated that 35% of proteins form homo-oligomers (1), with an average of four subunits per complex. In yeast and human cells many transcription factors undergo homo-oligomerization, which has been shown to be crucial for their function (2). The molecular dynamics of oligomerization have been studied for some proteins in vitro, but no study has quantified a discrete number of oligomers in a dynamic oligomerization process in live single cells. Here we focus on the homo-tetramers formed by the tumor suppressor p53 and quantify the fraction, dynamics, and function of homo-oligomers in single living cells in response to DNA damage.p53 is a stress-response transcription factor that orchestrates cell fate decisions such as cell-cycle arrest, senescence, and apoptosis. Tetramerization of p53 is required for its direct binding to DNA (3,4). Mutations in the p53 tetramerization domain (326-356 aa) lead to a reduction in, or loss of, its transcriptional activity in cells (5) and were shown to cause early cancer onset, known as Li-Fraumeni syndrome (6, 7).In in vitro studies, p53 first assembles into homo-dimers with a K d of ∼1 nM (8), and these dimers then come together in tetramers with a K d of ∼100 nM-1 μM (8-11). The K d of tetramerization in vitro can be lowered by specific posttranslational modifications (10-12). Based on these measurements and the estimated p53 concentration in cells of 140 nM (13), it has been proposed that p53 should be primarily dimeric in basal conditions and that it forms tetramers in stressed conditions (14). However, there is currently no direct experimental evidence for this in cells.We used...

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