This is the first biochemical demonstration that heterotrimeric combinations of the catalytic subunit PP6c with a SAPS scaffold subunit and an ankyrin repeat-containing regulatory subunit exhibit phosphatase activity, supporting the hypotheses that the PP6 holoenzyme is a heterotrimeric complex.18
A recent report found that activation of DNA-PK by IR is mediated by PP6c-PP6R1 (other PP6 subunits were not examined). When PP6c and PP6R1 were individually depleted, glioblastoma cells were sensitized to IR.21
This is a reminder of an early report that PP5 is required for ATM/ATR activation after DNA damage.30,31
However, our recent results suggest that DNA-PKcs could be a targeting subunit to recruit PP6 to the DSB sites, rather than a substrate for PP6.11
Our previous results also show that depletion of PP6c or PP6R1 does not obviously compromise repair of IR-induced DSBs in the neutral comet assays.11
However, in the current report, we did find that both PP6c and PP6R2, but not PP6R1 or PP6R3, are required for HDR of CPT-induced DSBs or I-SceI-induced DSBs. This is a clear demonstration that different scaffolding/regulatory/targeting subunits may target PP6c to and dephosphorylate the same substrate generated under different contexts.
It is not a surprise to find that PP6R1 was not enriched at the I-SceI induced DSBs since it is not essential for repair of DSB induced by I-SceI () or ionizing radiation.11
It is interesting to find that PP6c was enriched at the DSB site and surprisingly, at the same time PP6R2 dissociated from the DSB site (). Though PP6c-PP6R2 combinations exhibited enzymatic activity against γ-H2AX in vitro (), these combinations in vivo are apparently not the PP6 holoenzymes responsible for dephosphorylation of γ-H2AX near DSBs. We speculate that the dissociation of PP6R2 from the DSBs may be required for the recruitment/enrichment of PP6c on the DSB sites. Alternatively, PP6R2 dissociation from chromatin may facilitate activation of PP6c present in the nucleoplasm. Nonetheless, we propose that dynamic association of PP6c and PP6R2 with DSBs may play an important role in repair of DSBs.
PP2, PP4, PP6 and PPM1D are all capable of dephosphorylating γ-H2AX. PP2cA was reported to dephosphorylate γ-H2AX mainly generated by IR,7
while PP4c dephosphorylated γ-H2AX mainly generated during replication stress;8
PP6c-PP6R1 is responsible for elimination of γ-H2AX after IR treatment, PP6c and PP6R2 are required for dephosphorylation of γ-H2AX after CPT treatment or after formation of I-SceI-induced DSBs. This suggests that DSBs generated by different stresses (IR, replication, CPT, enzymatic cut by I-SceI) may have distinct DNA ends, thus recruiting different phosphatases or different phosphatase complexes. It is also highly possible that tissue or cell type-specific expression of PP6c and its regulatory subunits will allow a variety of combinations of phosphatase holoenzymatic complexes, thus conferring dephosphorylation of the same substrate by different phosphatase complexes in a tissue or cell type-specific manner.
The type 2A phosphatases are required for HDR of DSBs. Depletion of expression of any of these phosphatases may tip the balance of phosphorylation vs. dephosphorylation or change the phosphorylation status of essential factors for HDR, thus leading to a defective HDR. Though these phosphatases have a common physiological substrate, γ-H2AX, this substrate is generated under different conditions and recognized by different phosphatase complexes. Furthermore, many more physiological substrates of these phosphatases essential for HDR are awaiting further exploration. Therefore, we have many reasons to believe that PP2, PP4 and PP6 have very unique functions in HDR of DSBs, as well as in other signaling pathways.
An intact HDR is essential for high fidelity of cell divisions and maintenance of genome stability, which, when destabilized, can favor tumorigenesis. Therefore, most HDR factors are expected to be involved in tumorigenesis of human cells. In this study, though expression of PP6c and PP6R2 is not a biomarker for breast tumor prognosis in a relative small cohort of breast cancer patients, their expression overall decreases in breast tumors in comparison to that in benign breast diseases. This indicates that PP6 may have a protective role in breast cancer development by modulating HDR. It will be of great interest to determine if and how PP6 is involved in tumorigenesis in animal models.
In summary, we have demonstrated that γ-H2AX is a physiological substrate of PP6, dynamic association of PP6c and PP6R2 with chromatin surrounding DSBs is important for HDR of DSBs and PP6 may play a protective role in the development of breast cancer.