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Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates

Pauline Douglas1, Jianing Zhong, Ruiqiong Ye

  • 1Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.

Molecular and Cellular Biology
|January 13, 2010
PubMed

Insights

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruits protein phosphatase 6 (PP6) to DNA damage sites. PP6 dephosphorylates gamma-H2AX, aiding DNA repair and cell cycle checkpoint release after radiation.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is crucial for DNA double-strand break (DSB) repair via nonhomologous end joining (NHEJ).
  • DNA-PKcs activity is regulated by autophosphorylation and dephosphorylation by protein phosphatase 2A (PP2A)-like enzymes, including PP2A, PP4, and PP6.

Purpose of the Study:

  • To investigate the interaction between DNA-PKcs and protein phosphatases involved in DNA damage response.
  • To elucidate the role of PP6 in regulating DNA-PKcs activity and downstream DNA damage signaling pathways.

Main Methods:

  • Co-immunoprecipitation assays to detect protein interactions.
  • Small interfering RNA (siRNA) to silence PP6 catalytic (PP6c) and regulatory (PP6R1) subunits.
  • Western blotting to assess phosphorylation levels of histone H2AX (gamma-H2AX), DNA-PKcs, and ATM.
  • Flow cytometry to analyze cell cycle progression and G(2)/M checkpoint release.

Main Results:

  • DNA-PKcs interacts with PP6 catalytic subunit (PP6c) and PP6 regulatory subunits (PP6R1, PP6R2, PP6R3).
  • Silencing PP6c or PP6R1 using siRNA resulted in sensitivity to ionizing radiation (IR) and delayed G(2)/M checkpoint release.
  • PP6c or PP6R1 silencing led to sustained gamma-H2AX phosphorylation after IR, but did not affect DNA-PKcs or ATM autophosphorylation.
  • DNA-PKcs does not appear to be required for PP6-mediated dephosphorylation of gamma-H2AX.

Conclusions:

  • DNA-PKcs functions to recruit PP6 to sites of DNA damage.
  • PP6 plays a significant role in the dephosphorylation of gamma-H2AX, resolution of IR-induced foci, and timely release from the G(2)/M checkpoint.
  • These findings reveal a novel mechanism by which DNA-PKcs facilitates DNA repair and cell cycle regulation through interaction with PP6.

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