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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
09:47

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

Direct role for proliferating cell nuclear antigen in substrate recognition by the E3 ubiquitin ligase CRL4Cdt2

Courtney G Havens1, Nadia Shobnam, Estrella Guarino

  • 1Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

The Journal of Biological Chemistry
|February 4, 2012
PubMed

Insights

The CRL4(Cdt2) E3 ligase targets cell cycle proteins for destruction via a PIP degron mechanism involving PCNA. A key acidic residue on PCNA is crucial for recruiting CRL4(Cdt2) to chromatin for ubiquitylation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The E3 ubiquitin ligase Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) regulates the cell cycle and responds to DNA damage.
  • CRL4(Cdt2) targets proteins like Cdt1, p21, and Set8 for degradation.
  • Substrate recognition involves a PIP degron interacting with proliferating cell nuclear antigen (PCNA).

Purpose of the Study:

  • To investigate the role of PCNA in CRL4(Cdt2) substrate ubiquitylation.
  • To identify molecular determinants on PCNA essential for CRL4(Cdt2) function.

Main Methods:

  • Experiments were conducted using Xenopus egg extracts.
  • Analysis of protein-protein interactions and ubiquitylation.
  • Site-directed mutagenesis of PCNA.

Main Results:

  • An acidic residue in PCNA, adjacent to the PIP degron binding site, was identified as essential for CRL4(Cdt2) substrate destruction.
  • This PCNA residue is not required for substrate binding to PCNA but is critical for CRL4(Cdt2) recruitment.
  • CRL4(Cdt2) recruitment to chromatin depends on determinants in both the substrate degron and PCNA.

Conclusions:

  • CRL4(Cdt2) substrate ubiquitylation requires specific molecular interactions involving both the substrate's PIP degron and a critical acidic residue on PCNA.
  • This highlights a mechanism where E3 ligase recruitment is coupled to protein-protein interaction interfaces.
  • The findings provide insights into the regulation of cell cycle progression and DNA damage response pathways.

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