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Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
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DNA damaging agent-induced apoptosis is regulated by MCL-1 phosphorylation and degradation mediated by the

Wataru Nakajima1,2, Kanika Sharma1, June Young Lee1

  • 1Phillips Institute for Oral Health Research, School of Dentistry, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia, USA.

Oncotarget
|May 12, 2016
PubMed

Insights

The pro-apoptotic protein Noxa triggers cell death by targeting MCL-1 for degradation. DNA damaging agents induce Noxa, leading to MCL-1 phosphorylation and apoptosis via the Noxa/MCL-1/CDK2 complex.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Noxa is a BH3-only protein that induces apoptosis by interacting with anti-apoptotic BCL-2 family proteins.
  • MCL-1 is an anti-apoptotic protein that plays a critical role in cell survival.
  • DNA damaging agents can trigger apoptosis through various pathways.

Purpose of the Study:

  • To elucidate the mechanism by which Noxa induces apoptosis.
  • To investigate the role of MCL-1 phosphorylation in Noxa-mediated apoptosis.
  • To identify the key regulators involved in the Noxa-MCL-1 interaction.

Main Methods:

  • Western blotting to detect protein levels and phosphorylation.
  • Immunoprecipitation to analyze protein complexes.
  • Site-directed mutagenesis to study the function of specific phosphorylation sites.
  • Cell viability assays to assess apoptosis.

Main Results:

  • DNA damaging agents (cisplatin, etoposide) upregulate Noxa expression.
  • Noxa is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, leading to its proteasome-mediated degradation and apoptosis.
  • MCL-1 phosphorylation occurs at the mitochondria and is regulated by CDK2, forming a stable complex with MCL-1.
  • Noxa binds to the MCL-1/CDK2 complex, facilitating MCL-1 phosphorylation.
  • Mutating Ser64/Thr70 of MCL-1 to alanine renders it resistant to phosphorylation and degradation, inhibiting Noxa- or cisplatin-induced apoptosis.

Conclusions:

  • Noxa-mediated phosphorylation of MCL-1 at Ser64/Thr70, followed by its degradation, is crucial for apoptosis induced by DNA damaging agents.
  • The Noxa/MCL-1/CDK2 complex is a key regulatory hub in this apoptotic pathway.
  • Targeting this complex may offer therapeutic strategies for cancer treatment.
Keywords:
CDK2MCL-1Noxachemotherapyphosphorylation

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