Division of Signalling Biology, Ontario Cancer Institute (University Health Network), 610 University Avenue, Toronto, Ontario M5G 2M9, Canada.
Nucleic Acids Res. 2013 Apr;41(7):4080-92. doi: 10.1093/nar/gkt134. Epub 2013 Feb 28.
APLF is a forkhead associated-containing protein with poly(ADP-ribose)-binding zinc finger (PBZ) domains, which undergoes ionizing radiation (IR)-induced and Ataxia-Telangiectasia Mutated (ATM)-dependent phosphorylation at serine-116 (Ser(116)). Here, we demonstrate that the phosphorylation of APLF at Ser(116) in human U2OS cells by ATM is dependent on poly(ADP-ribose) polymerase 3 (PARP3) levels and the APLF PBZ domains. The interaction of APLF at sites of DNA damage was diminished by the single substitution of APLF Ser(116) to alanine, and the cellular depletion or chemical inhibition of ATM or PARP3 also altered the level of accumulation of APLF at sites of laser-induced DNA damage and impaired the accumulation of Ser(116)-phosphorylated APLF at IR-induced γH2AX foci in human cells. The data further suggest that ATM and PARP3 participate in a common signalling pathway to facilitate APLF-Ser(116) phosphorylation, which, in turn, appears to be required for efficient DNA double-strand break repair kinetics and cell survival following IR. Collectively, these findings provide a more detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that Ser(116)-APLF phosphorylation facilitates APLF-dependent double-strand break repair.
APLF 是一种具有多聚（ADP-核糖）结合锌指（PBZ）结构域的叉头相关蛋白，可被电离辐射（IR）诱导，并依赖共济失调毛细血管扩张突变（ATM）在丝氨酸-116（Ser（116））处发生磷酸化。在这里，我们证明 ATM 在人 U2OS 细胞中通过 Ser（116）对 APLF 的磷酸化依赖于多聚（ADP-核糖）聚合酶 3（PARP3）水平和 APLF PBZ 结构域。APLF 在 DNA 损伤部位的相互作用通过 APLF Ser（116）到丙氨酸的单一取代而减弱，并且 ATM 或 PARP3 的细胞耗竭或化学抑制也改变了 APLF 在激光诱导的 DNA 损伤部位的积累水平，并损害了人细胞中 IR 诱导的 γH2AX 焦点处 Ser（116）磷酸化的 APLF 的积累。数据进一步表明，ATM 和 PARP3 参与共同的信号通路以促进 APLF-Ser（116）磷酸化，这反过来似乎是 IR 后有效 DNA 双链断裂修复动力学和细胞存活所必需的。总之，这些发现提供了对导致 DNA 损伤后 APLF 磷酸化的分子途径的更详细理解，并表明 Ser（116）-APLF 磷酸化促进了 APLF 依赖性双链断裂修复。
