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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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Isoform specific phosphorylation of p53 by protein kinase CK1

Andrea Venerando1, Oriano Marin, Giorgio Cozza

  • 1Venetian Institute of Molecular Medicine (VIMM), Via G. Orus, 2, 35129, Padova, Italy.

Cellular and Molecular Life Sciences : CMLS
|December 31, 2009
PubMed

Insights

Protein kinase CK1 isoforms alpha and delta phosphorylate the p53 protein, but not the gamma isoform. This phosphorylation, particularly at S20, is crucial for p53 regulation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • Protein kinase CK1 (casein kinase 1) is involved in various cellular processes.
  • The tumor suppressor protein p53 plays a critical role in maintaining genomic stability.
  • Phosphorylation of p53 regulates its stability, localization, and transcriptional activity.

Purpose of the Study:

  • To investigate the isoform-specific phosphorylation of the p53 N-terminal region by protein kinase CK1.
  • To identify the specific sites and mechanisms of CK1-mediated p53 phosphorylation.

Main Methods:

  • Utilized recombinant p53 protein and a synthetic peptide (p53 1-28) as substrates.
  • Assessed phosphorylation activity of CK1 isoforms alpha, gamma(1), and delta.
  • Employed kinetic analysis (K(m) determination) and mutational analysis.

Main Results:

  • CK1alpha and CK1delta efficiently phosphorylated p53, while CK1gamma(1) did not.
  • Full-length p53 showed a significantly higher affinity for CK1 than the N-terminal peptide.
  • Phosphorylation preferentially occurred at S20, dependent on E17, while S6 was unaffected.
  • A remote docking site (K(221)RQK(224) loop) was identified as crucial for CK1 binding and phosphorylation.

Conclusions:

  • CK1alpha and CK1delta exhibit isoform-specific phosphorylation of p53.
  • Phosphorylation at S20 is a key regulatory event mediated by CK1, involving both local and remote interactions.
  • These findings provide insights into the regulation of p53 function by CK1.

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