A role for protein phosphatase 4 in regulating non-homologous end-joining.

DNA double strand breaks (DSBs) are the most deleterious form of DNA damage as unrepaired or misprocessed DBSs can lead to genomic instability and ultimately cancer. To counter the deleterious nature of DSBs, cells have developed a number of pathways which repair DSBs with the two most prominent being non-homologous end joining (NHEJ) and homologous recombination (HR).The cellular response to DSBs requires efficient recognition of the damaged DNA, signal transduction pathways, activation of cell cycle checkpoint controls and repair pathway selection. At the heart of the cellular response to DSBs is the DNA damage response (DDR), which mediates DNA damage-induced signal transduction via activation of a large number of protein kinases. Phosphorylation mediated by these protein kinases is required for the transmission, and coordination of the DDR and the direct targets include the sensors of the DSBs, DDR signaling mediators, checkpoint control transducers and effectors, repair proteins, histones and chromatin modifiers.

While the role of protein kinases on the repair process have been well-documented, the role of serine/threonine protein phosphatases (PPs) in the DDR and the DSB repair process have only recently started to be uncovered. A number of PPs have been implicated in regulating the phosphorylation status of DDR proteins and HR, including PP1, PP2A, PP4, PP6 and Wip1. Direct substrates include initial sensors of the DSB (Ku70/Ku80), DDR signaling mediators (BRCA1), checkpoint control transducers and effectors (CHK1, CHK2 and p53), repair proteins (RPA), histones (γH2AX) and the DSB activated kinases (ATM, DNA-PKcs and ATR) implicating that reversible phosphorylation mediated by PPs plays an important role in the cellular response to DSBs.

In a report by Liu et al. in Cell Cycle, the Xu group continued their studies on the role that protein phosphatase 4 (PP4) plays in DSB repair as they had previously shown that PP4 is required for HR., As a number of NHEJ factors are phosphorylated in response to DSBs, in this study the Xu group determined if PP4 played a role in NHEJ. Using established in vivo NHEJ assays, the authors showed that the PP4 catalytic subunit (PP4c) and its catalytic activity are required for NHEJ. The PP4 holoenzyme occurs in different assemblies of the catalytic subunit with one or more regulatory subunits. Knockdown of each of the PP4 regulatory subunits with specific siRNAs showed that PP4R2, but not PP4R1, PP4R3α or PP4R3β-containing, holoenzymes are involved in the regulation of NHEJ.

Using a large-scale immunoprecipitation assay, followed by mass spectrometry analysis to identify the PP4 substrate which plays a role in NHEJ, the authors identified KAP1 (KRAB-associated protein (1) as an interactor of PP4c as well as PP4R2. KAP1 was identified as a transcriptional co-repressor but was later found to be rapidly phosphorylated by ATM at serine 824 (S824) in response to DSBs. Phosphorylation of KAP1 at S824 impacts repair of DSBs within heterochromatin by promoting chromatin relaxation to allow repair proteins access to the DSB. The authors next assessed if KAP1 was a substrate of PP4. Knockdown of PP4c or PP4R2 resulted in an increase in KAP1 phosphorylation at S824 following DNA damage. PP4 dephosphorylated KAP1 in vitro further implicating that KAP1 is a PP4 substrate. Finally, knockdown of KAP1 resulted in a decrease in NHEJ but co-depletion of PP4 and KAP1did not have a synergistic effect on NHEJ suggesting that PP4 and KAP1 are in the same NHEJ epistasis group. Together, the data implicates that PP4’s ability to regulate NHEJ is through its ability to regulate KAP1 phosphorylation.

Many new interesting questions are raised by this study: (1) does PP4 play a role in all end-joining pathways or a specific one; (2) KAP1 dephosphorylation by PP4 should result in chromatin condensation, is this important for NHEJ; (3) a recent manuscript also found that PP4 dephosphorylates KAP1 at S824 but this was due to a holoenzyme with PP4R3β8, is dephosphorylation of KAP1 by different PP4 holoenzymes regulated; (4) PP4c knockdown results in a greater decrease in NHEJ than KAP1 implicating that PP4 may have other NHEJ substrate(s), what are these substrates and what role do they play in regulating NHEJ. Together, it will be of great interest to continue to identify the role(s) that PPs play in regulating NHEJ.