The Effects Of SIK2in The Cellular Responses To Ionizing Radiation-induced Damage

Salt-induced kinase2(SIK2) is a serine/threonine protein kinase belonging to adenosine monophosphate-activated protein kinase (AMPK) family. In addition to its role in the metabolisms of lipid and glucose, SIK2was recently reported to act as a regulator of the initiating and maintaining of normal cell mitosis, and also participates in regulating the fusion and maturation of phagosome and lysosome as well as autophagy process. DNA-dependent kinase catalytic subunit (DNA-PKcs), a member of phosphatidylinositol-3-kinase-like kinase (PIKK) family, is an important molecule in DNA damage signaling and repair in response to ionizing radiation-induced DNA double strand break. Our laboratory has previously identified that SIK2interacted with DNA-PKcs. This study was designed to investigate the functions and primary mechanisms of SIK2on the cellular response to ionizing radiation, including the G2/M checkpoint and protective autophagy.First, we found that the exoression level of SIK2protein changes with cell cycle, and reaches its peak in the G2/M phase. Employing the lentiviral vector-mediated RNA interference technology, we have generated a SIK2-knockdown HeLa cell model. Using the synchronizated cells, we found the ratio of pH3SerlO positive mitotic cells decreased in the SIK2knockdown cells. Meanwhile, the phosphorylated Cdc25C/S216and Cdc2/Tyr15levels increased, which may result in a delaying of G2/M transition. The expression level of SIK2protein and mRNA was found increased in HepG2and Hela cells after y-rays radiation. Cell colony formation assay indicated that the radiosensitivity of SIK2knockdown cells increased as compared to control cells, especially at4Gy irradiation. The level of yH2AX level associated with G2/M arrest induced by y-rays was decreased in the SIK2knockdown cells. SIK2knockdown cells could initiated the G2/M checkpoint properly as that in control HeLa cells after4Gy irradiation. Demonstrated by the mitotic marker of pH3Ser10labelling, however, the G2/M arrest of SIK2knockdown cells released earlier that that of control cells, which may resulte in the "premature" entry of cells with unrepaired DNA damage into mitosis, and consequently increase the radiosenstivity and genomic instability. The western blot analysis indicated that the irradiation-induced phosphorylated Chk2/T68in SIK2knock-down cells faded away faster than that in control cells. Meanwhile, we further observed that the depletion of SIK2decreased the radiation-induced phosphorylation of Cdc25C/Ser216and Cdc2/Tyr15. It can be inferred that the depression of SIK2may affect the ATM/Chk2/Cdc25C/Cdc2signaling pathway.We also studied the effect of SIK2on the radiation-induced autophagy. The autophagy inhibitor3-Methyladenine (3-MA) promoted cell apoptosis after4Gy irradiation, which suggested that autophagy may play a protective role in radiation response. The autophagy marker p62was induced post irradiation in control cells while this induction was attenuated in SIK2-depleted cells. It is likely that SIK2knockdown impacts the radiation-induced autophagy and leads to the increase of radiosenstivity.This study is the first report about the role of SIK2in cellular response to ionizing radiation damage, including the regulation of normal G2/M checkpoint function. The depression of SIK2resulted in the abnormal of G2/M checkpoint, a deffect of protective autophagy, and increased radiosensitivity. These research will deepen the understanding of mechanisms of cellular response to radiation injury.