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Related Experiment Videos

Conformation-dependent phosphorylation of p53

V Adler1, M R Pincus, T Minamoto

  • 1Molecular Carcinogenesis Program, American Health Foundation, Valhalla, NY 10595, USA.

Proceedings of the National Academy of Sciences of the United States of America
|March 4, 1997
PubMed
Summary

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Phosphorylation of the p53 tumor suppressor protein is influenced by its conformation. Different kinases like protein kinase A (PKA) and jun-N-kinase (JNK) phosphorylate p53, with specific regions like amino acids 97-117 being critical.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cancer Research

Background:

  • The p53 tumor suppressor protein's function is modulated by phosphorylation.
  • Understanding p53 phosphorylation is crucial for cancer biology and therapeutic development.

Purpose of the Study:

  • To investigate the phosphorylation of human p53 by various kinases in vitro.
  • To determine the influence of p53 conformation and specific amino acid regions on kinase interactions and phosphorylation patterns.

Main Methods:

  • Utilized bacterially produced glutathione S-transferase (GST)-p53 fusion protein and baculovirus-expressed histidine-tagged p53 ((His)p53).
  • Assessed p53 phosphorylation by purified jun-N-kinase (JNK), protein kinase A (PKA), casein kinase II beta subunit (CKIIbeta), and whole cell extracts (WCEs).

Related Experiment Videos

  • Employed truncated p53 forms, mutant p53 (p53(249), p53(273)), and peptide fragments (97-117) for detailed analysis, including computer modeling.

    • Main Results:

    • PKA effectively phosphorylates p53 in a conformation- and concentration-dependent manner.
    • JNK interacts with and phosphorylates p53, particularly truncated forms.
    • Wild-type p53 (p53wt) is phosphorylated more efficiently than mutant forms (p53(249), p53(273)) by CKIIbeta and WCE kinases.
    • Phosphorylation is dependent on amino acids 97-155 within the DNA-binding domain; residues 97-117 modulate kinase activity.
    • Mutant p53(249) shows conformational changes in the 97-117 region, impacting phosphorylation.

    Conclusions:

    • p53 protein conformation is a critical determinant for its substrate availability and phosphorylation patterns by various kinases.
    • Specific regions, notably amino acids 97-117, play a key role in regulating p53 phosphorylation by different kinase systems.
    • These findings provide insights into the regulation of p53 function and potential therapeutic targeting in cancer.