Keywords

N-terminal, Phosphorylation, Mechanism, IDR, PNAS

Reference

DOI: 10.1073/pnas.1013078107

Notes

This paper examines how multisite phosphorylation of p53 regulates its interaction with CREB-binding protein (CBP/p300), crucial for p53-mediated transcription. While p53 acetylation by CBP is known to be essential for its activation, this study focuses on how different phosphorylation patterns fine-tune p53’s recruitment of CBP/p300.

Core idea:

  • Single phosphorylation (at Thr18) works as an on/off switch, especially for p53-HDM2 interaction.
  • Multiple phosphorylation events create a graded (rheostat-like) enhancement of p53’s binding to CBP.

Experimental Methods

  • ITC (Isothermal Titration Calorimetry):
    To measure binding affinity between phosphorylated p53 peptides and HDM2, showing how Thr18 phosphorylation disrupts p53-HDM2 interaction.

  • NMR (Nuclear Magnetic Resonance):
    To study phosphorylated p53 binding to KIX and NCBD domains of CBP, revealing molecular details of interaction modes.

  • Fluorescence Anisotropy:
    To quantify binding affinity of phosphorylated p53 to TAZ1 and TAZ2 domains of CBP, demonstrating how additional phosphorylation sites increase affinity in a graded way.

Main Findings

  1. Thr18 phosphorylation functions as an on/off switch:

    • Disrupts p53-HDM2 interaction, stabilizing p53.
    • Slightly enhances p53-CBP interaction.

  2. Multisite phosphorylation (e.g., Ser15, Thr18, Ser20) synergistically enhances p53 binding to CBP/p300:

    • The more phosphorylation sites added, the stronger the affinity for TAZ1, TAZ2, KIX domains.
    • Functions like a rheostat to fine-tune p53 transcriptional response.
    • This graded increase allows p53 to compete more effectively for limiting amounts of CBP/p300, outcompeting other transcription factors under high-stress conditions.

  3. Mechanistic insight:

    • The enhanced interaction with CBP is proposed to result primarily from electrostatic effects, not just specific site recognition.
    • This challenges simpler models of “site-specific” interaction and emphasizes additive charge-based effects in IDRs (intrinsically disordered regions).

Why It’s Interesting

  • The concept of single phosphorylation as a binary switch and multisite phosphorylation as a rheostat is elegant and offers a broader framework to understand IDR-mediated regulation in other systems.
  • Shows how gradual accumulation of phosphorylation events allows p53 to scale its activity with the level of stress, rather than being a simple “on/off” mechanism.
  • Highlights electrostatic modulation as a key factor in protein-protein interactions involving IDRs — an idea that might be applicable to many signaling pathways.

Take-home message

Multisite phosphorylation of p53 provides a flexible, tunable mechanism to regulate its interaction with CBP/p300, balancing between degradation (via HDM2) and transcriptional activation.

  • Thr18 phosphorylation is a binary switch for HDM2 binding.
  • Multiple phosphorylations work additively, acting like a rheostat to strengthen CBP recruitment in response to DNA damage intensity.
  • This mechanism explains how p53 fine-tunes transcriptional responses under different cellular stress levels and highlights the importance of dynamic PTM patterns in IDR-mediated interactions.