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Identification of the proteome complement of humanTLK1 reveals it binds and phosphorylates NEK1 regulating its

Vibha Singh1, Zachary M Connelly1, Xinggui Shen2

  • 1a Department of Biochemistry and Molecular Biology , Louisiana State University Health Sciences Center , Shreveport , LA , USA.

Cell Cycle (Georgetown, Tex.)
|April 21, 2017
PubMed

Insights

Tousled Like Kinase 1 (TLK1) interacts with NEK1, a key player in DNA repair. This interaction, regulated by DNA damage, activates the ATR-Chk1 pathway, crucial for cell cycle control following DNA damage.

Area of Science:

  • Cellular Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The DNA Damage Response (DDR) is essential for maintaining genomic stability.
  • Tousled Like Kinases (TLKs) are implicated in the DDR, but their substrates and precise roles are not fully understood.
  • NEK1 is known to function upstream of ATR and Chk1 in the DDR.

Purpose of the Study:

  • To identify novel substrates and interaction partners of TLK1.
  • To elucidate the functional relationship between TLK1 and NEK1 in the context of DNA damage.
  • To characterize the role of the TLK1-NEK1 interaction in regulating the DDR pathway.

Main Methods:

  • Proteomic screening to identify TLK1 interacting proteins.
  • Co-immunoprecipitation (coIP) to confirm TLK1-NEK1 interaction.
  • Immunofluorescence to observe protein localization following DNA damage.
  • In vitro kinase assays and Western blotting to assess protein phosphorylation and pathway activation.
  • Cell cycle analysis and overexpression studies using NEK1 mutants.

Main Results:

  • A novel proteomic screen identified 165 human proteins interacting with TLK1, including NEK1.
  • TLK1 and NEK1 interact, and their binding is enhanced by hydrogen peroxide (H2O2).
  • Both proteins colocalize to nuclear repair foci (γH2AX) after doxorubicin treatment.
  • TLK1 phosphorylates NEK1 at T141, increasing its kinase activity.
  • Inhibition of TLK1 or expression of a NEK1-T141A mutant impairs ATR and Chk1 activation.
  • A TLK1>NEK1>ATR>Chk1 pathway was proposed.
  • Overexpression of NEK1-T141A mutant alters cell cycle progression under oxidative stress, bypassing G1 arrest and activating an intra-S-phase checkpoint.

Conclusions:

  • The study identifies NEK1 as a novel substrate and interaction partner of TLK1 within the DDR.
  • TLK1-mediated phosphorylation of NEK1 is critical for the activation of the ATR-Chk1 signaling cascade.
  • This pathway plays a significant role in regulating cell cycle progression and DNA repair following genotoxic stress.
Keywords:
ATRNek1TLK1cell cycle checkpoint activationregulation of DNA damage responsereplication stress

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