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Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Phosphorylation of ATR-interacting protein on Ser239 mediates an interaction with breast-ovarian cancer

Monica Venere1, Andrew Snyder, Omar Zgheib

  • 1Wistar Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Cancer Research
|July 10, 2007
PubMed

Insights

Breast-ovarian cancer susceptibility 1 (BRCA1) directly interacts with ATR-interacting protein (ATRIP), a key partner in ATR signaling. This interaction is crucial for maintaining the G(2)-M checkpoint, essential for DNA damage response.

Area of Science:

  • Cellular biology
  • Molecular genetics
  • Biochemistry

Background:

  • DNA damage and replication stress activate signaling pathways involving kinases like ATM and ATR.
  • Proteins with BRCA1 COOH-terminal (BRCT) domains are implicated in these pathways, interacting with ATM/ATR but their precise role is unclear.
  • Understanding these interactions is vital for comprehending DNA repair mechanisms and cancer development.

Purpose of the Study:

  • To elucidate the molecular mechanism of interaction between BRCA1 and the ATR signaling complex.
  • To investigate the functional significance of the BRCA1-ATRIP interaction in DNA damage response.
  • To determine if a direct physical link exists between BRCA1 and ATRIP.

Main Methods:

  • Co-immunoprecipitation assays to detect protein interactions.
  • Site-directed mutagenesis to investigate the role of specific residues (e.g., Ser239 of ATRIP).
  • Cell cycle checkpoint analysis (G2-M) to assess DNA damage response functionality.

Main Results:

  • Demonstrated a direct physical interaction between BRCA1 and ATRIP, an ATR-binding protein.
  • Identified that the BRCT domains of BRCA1 and Serine 239 (Ser239) of ATRIP are critical for this interaction.
  • Showed that phosphorylation of ATRIP at Ser239 occurs in both normal and irradiated cells.
  • Observed a G(2)-M checkpoint defect upon mutation of ATRIP Ser239 to Alanine, indicating impaired DNA damage response.

Conclusions:

  • A direct physical interaction between BRCA1 and ATRIP is established.
  • This interaction, involving BRCA1's BRCT domains and ATRIP's Ser239, is essential for the checkpoint function mediated by ATR.
  • BRCA1 plays a direct role in ATR-dependent DNA damage signaling and checkpoint control.

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