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Updated: Jan 11, 2026

Purification of Ubiquitinated p53 Proteins from Mammalian Cells
10:55

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Published on: March 21, 2022

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Regulation of cellular states via targeted phosphorylation of p53 using a nanobody-coupled kinase system

Hoe Eun Lim1, Hae Yong Yoo2,3

  • 1Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, Republic of Korea.

Cell Death Discovery
|November 10, 2025
PubMed

Insights

Researchers developed a target protein phosphorylation (TPP) system to control cell behavior by phosphorylating key proteins like p53. This system shows promise for cancer therapy by inhibiting tumor growth and enhancing drug efficacy.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Phosphorylation is crucial for cell signaling, affecting processes like proliferation, differentiation, and apoptosis.
  • Understanding phosphorylation's regulatory mechanisms is key for developing new therapeutic strategies.
  • Dysregulated phosphorylation is implicated in various diseases, highlighting the need for precise control mechanisms.

Purpose of the Study:

  • To develop and validate a novel target protein phosphorylation (TPP) system for precise control of protein phosphorylation.
  • To investigate the TPP system's ability to phosphorylate target proteins and modulate cellular characteristics.
  • To explore the therapeutic potential of the TPP system in cancer models.

Main Methods:

  • Development of a TPP system comprising a nanobody fused to a kinase domain.
  • Verification of the system's phosphorylation activity using GFP as a model substrate.
  • Analysis of p53 phosphorylation sites and downstream effects on cell cycle and growth.
  • In vitro combination studies with chemotherapeutic drugs (5-Fluorouracil, Oxaliplatin).
  • In vivo evaluation using a doxycycline-inducible TPP system in a colorectal cancer xenograft mouse model.

Main Results:

  • The TPP system successfully phosphorylated GFP and specifically targeted p53 at identified sites.
  • p53 phosphorylation led to protein stabilization, increased p21 expression, cell cycle delay, and suppressed cell growth.
  • Combined TPP system and chemotherapy significantly enhanced cytotoxicity in colorectal cancer cells.
  • The TPP system induced a DNA-damaged cellular state without external stimuli.
  • In vivo, doxycycline-induced TPP expression markedly inhibited tumor growth in a xenograft model.

Conclusions:

  • The TPP system provides a novel method for targeted protein phosphorylation and cellular state modulation.
  • This technology offers potential applications in studying phosphorylation-dependent pathways and developing therapies for phosphorylation-related diseases.
  • The TPP system demonstrates significant potential for cancer treatment, both as a standalone therapy and in combination with existing chemotherapeutics.

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