Protein phosphatase PP6 is required for homology-directed repair of DNA double-strand breaks

Abstract

DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially-produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2, and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6, and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.

PP6 Controls T Cell Development and Homeostasis by Negatively Regulating Distal TCR Signaling

“…The holoenzyme of PP6 is composed of a catalytic subunit, a SAPS domain subunit, and an ankyrin repeat subunit (10). PP6 regulates chromosome segregation in mitosis through inhibiting Aurora A activity (11) and facilitating the repair of DNA double-strand breaks by activating DNA-activated protein kinase (DNA-PK) and dephosphorylating g-H2AX in HeLa cells (12,13). Involvement of PP6 in modulating NF-kB activity is suggested by findings that the PP6 protein interacts with and protects IkBε from TNF-a-induced degradation in Cos7 cells (14).…”

Serine/threonine protein phosphatases in DNA damage response

“…This appears contradictory to the findings of other reports [99][100][101], in which inhibition of PP6c or PP6R1 expression by siRNA impairs DNA-PK activation in response to IR, though all reports demonstrated that preventing PP6c expression sensitizes tumor cells to IR. Our laboratory further demonstrated that depletion of PP6c or PP6R2, but not PP6R1, compromised repair of CPT-induced DSBs or I-SceI-induced DSB, suggesting that DNA lesions induced by different agents may require different PP6 holoenzymes [102].…”

“…PP2A is mainly responsible for H2AX in response to exogenous DNA damage [83]. We and our collaborators have also revealed that H2AX is dephosphorylated by PP6 both in vitro and in vivo [99,102]. Silencing of either PP6c or PP6R1 led to sustained H2AX levels after IR, whereas inhibition of PP6c or PP6R2 expression, but not PP6R1 expression, resulted in sustained H2AX levels after CPT treatment [102].…”

“…We and our collaborators have also revealed that H2AX is dephosphorylated by PP6 both in vitro and in vivo [99,102]. Silencing of either PP6c or PP6R1 led to sustained H2AX levels after IR, whereas inhibition of PP6c or PP6R2 expression, but not PP6R1 expression, resulted in sustained H2AX levels after CPT treatment [102]. It is very interesting that H2AX generated under different circumstances is dephosphorylated by different phosphatases, and if by the same phosphatase, the requirement of regulatory/scaffolding subunits is different.…”

“…Our laboratory also examined expression status of PP6c and its regulatory subunits in breast tumor samples derived from a cohort of approximately 200 breast cancer patients [102]. It was unexpected to find that expression of PP6c, PP6R2, and PP6R3 was lowered in breast cancer samples in comparison with benign breast lesions, suggesting that PP6 may have a protective role during breast tumorigenesis.…”