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In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

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The circadian E3 ligase complex SCFFBXL3+CRY targets TLK2

Stephanie Papp Correia1, Alanna B Chan1, Megan Vaughan1

  • 1Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.

Scientific Reports
|January 19, 2019
PubMed

Insights

Cryptochromes (CRY1/2) act as cofactors for SCFFBXL3, a protein complex that regulates c-MYC and TLK2. This study identifies new substrates and links circadian and cell cycle regulation.

Area of Science:

  • Molecular Biology
  • Chronobiology
  • Cell Cycle Regulation

Background:

  • The circadian clock protein CRY2 was identified as a cofactor for SCFFBXL3-mediated ubiquitination of c-MYC.
  • CRY2's role in recruiting phosphorylated substrates to SCFFBXL3 was unexpected, prompting further investigation into its broader function.

Purpose of the Study:

  • To investigate the scope of CRY1 and CRY2 as cofactors for SCFFBXL3 by identifying additional substrates.
  • To establish a molecular link between the circadian and cell cycle regulatory systems.

Main Methods:

  • Affinity purification mass spectrometry (APMS) screen to identify potential SCFFBXL3+CRY1/2 substrates.
  • Analysis of TLK2 (Tousled-like kinase 2) interaction with SCFFBXL3 in the presence of CRY1/2.

Main Results:

  • Over one hundred potential SCFFBXL3+CRY1/2 substrates were identified, including TLK2.
  • Both CRY1 and CRY2 recruit TLK2 to SCFFBXL3, dependent on TLK2 kinase activity.
  • CRY1/2 overexpression or deletion modulated TLK2 protein abundance, indicating CRY-dependent regulation.

Conclusions:

  • CRYs serve as essential cofactors for SCFFBXL3, expanding the known substrates of this complex.
  • A molecular connection is established between circadian and cell cycle oscillators through CRY-modulated TLK2 turnover.

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