Ubiquitination and degradation of SIK2 by DNA-PKcs deficiency promote radiation-induced mitotic catastrophe.
Ubiquitination and degradation of SIK2 by DNA-PKcs deficiency promote radiation-induced mitotic catastrophe.
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DNA-PKcs 缺陷引起的 SIK2 泛素化和降解促进辐射诱导的有丝分裂灾难
DOI：
10.1016/j.gendis.2022.02.004
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发表时间：
2023-03
期刊：
影响因子：
6.8
通讯作者：
Gu, Yongqing
中科院分区：
文献类型：
--
作者：
Zhu, Jiaojiao;Zhang, Ying;Yan, Ziyan;Wang, Jianxiao;Wang, Ping;Liang, Xinxin;Liu, Yuhao;Ao, Xingkun;Zhu, Maoxiang;Zhou, Pingkun;Gu, Yongqing
盐诱导激酶2（SIK 2）是AMP激活的丝氨酸/苏氨酸激酶家族的成员。据报道，抑制SIK 2可以增强紫杉醇的细胞毒性，1促进有丝分裂前凋亡，并导致细胞周期停滞在中期。因此，靶向SIK 2可能是癌症药物和放射治疗抗性的治疗策略。有丝分裂灾难是在电离辐射（IR）诱导的细胞损伤过程中发现的一种以多极纺锤体和多核化为特征的导致细胞死亡的异常有丝分裂。[3]然而，有丝分裂灾难的机制还不清楚。本研究旨在评估SIK 2敲低对IR诱导的有丝分裂灾难的影响。在IR刺激后，SIK 2蛋白含量增加，这取决于IR的时间和剂量（图S1 A，B）。为了进一步阐明SIK 2在由IR诱导的灾难性有丝分裂中的作用，我们在用60 Co γ射线照射的同时进行了SIK 2的抑制（图S1 C），并且在HeLa细胞中检测到异常纺锤体如三极和四极纺锤体的存在。此外，与单独IR处理组相比，IR处理与同时敲减SIK 2组显示细胞中异常纺锤体的比例从约30%增加到50%（图1A;图1D）。多核细胞的数量也显著增加（图1 B;图S1 E）。总之，这些数据表明，敲低SIK 2促进IR诱导的细胞有丝分裂灾难。为了进一步验证SIK 2调控有丝分裂灾难的特异性机制，进行了酵母双杂交试验，结果表明SIK 2可能与DNA-PKcs相互作用（图S2 A）。DNA-PKcs在修复DNA双链断裂（DSB）中发挥着重要作用。DNA-PKcs还被证明可以调节辐射损伤细胞的有丝分裂过程，并参与维持中心体和纺锤体结构的稳定性。4与先前的研究一致，IR刺激48 h的DNA-PKcs缺陷使多极纺锤体的数量从30%增加到50%，多核细胞的数量从10%增加到20%（图S2 BeD）。这些发现表明，DNA-PKcs缺陷促进IR诱导的细胞有丝分裂灾难的程度与SIK 2敲低相当。接下来，通过免疫共沉淀（CO-IP）测定在HeLa细胞中评估SIK 2和DNA-PKcs之间的相互作用，并且GST下拉测定进一步表明SIK 2和DNA-PKcs可以直接相互作用（图1C;图S2 E）。DNA-PKcs T2609和DNA-PKcs S2056与SIK 2共定位在中心体处，如在免疫荧光分析中观察到的（图S2 F）。我们接下来阐明了SIK 2是否可以通过其与DNA-PKcs的相互作用影响IR诱导的有丝分裂灾难。DNA-PKcs的敲低或失活降低了SIK 2蛋白的含量（图1D;图S3 A）。此外，在DNA-PKcs稳定敲低的HeLa细胞系中，与对照组相比，通过过表达SIK 2，异常细胞纺锤体的数量从50%降低到30%，多核细胞的比例从17%降低到8%（图S3 BeD）。总的来说，敲低DNA-PKcs可以通过降低SIK 2的蛋白质含量来促进IR诱导的有丝分裂灾难。GST pulldown分析显示GST-SIK 2（1 e926）、GST-SIK 2（280 e926）和GST-SIK 2（400 e926）与DNA-PKcs相互作用，提示SIK 2与DNA-PKcs相互作用的区域。相反，GST-SIK 2（700 e926）不与DNA-PKcs相互作用。这些结果表明，存在…
Salt-inducible kinase 2 (SIK2) is a member of the AMP-activated serine/threonine kinase family. It has been reported that inhibition of SIK2 can enhance the cytotoxicity of paclitaxel, 1 promote premitotic apoptosis, and lead to cell cycle arrest in the metaphase. 2 Thus, targeting SIK2 may be a therapeutic strategy for cancers drug and radiotherapy resistance. Mitotic catastrophe is a type of abnormal mitosis leading to cell death characterized by the multipolar spindle and multinucleation, which was first discovered during an ionizing radiation (IR)-induced cell damage. 3 However, the mechanism of mitotic catastrophe is not well understood. The present study aimed to assess the effect of the knockdown of SIK2 on IR-induced mitotic catastrophe. SIK2 protein content increased following IR stimulation, depending on the time and dose of IR (Fig. S1A, B). To further clarify the role of SIK2 in mitosis catastrophic induced by IR, we performed inhibition of SIK2 while irradiating with 60Co g-rays (Fig. S1C) and the presence of abnormal spindles such as tripole and quadrupole spindles was detected in HeLa cells. Furthermore, compared to the IR treatment alone group, the IR treatment with simultaneous knockdown of the SIK2 group showed an increased proportion of abnormal spindles from approximately 30% e 50% in cells (Fig. 1 A; Fig. S1D). The number of multinuclear cells also significantly increased (Fig. 1 B; Fig. S1E). To summarize, these data suggest that the knockdown of SIK2 promotes IR-induced cellular mitotic catastrophe. The yeast two-hybrid assay was performed to verify further the specific mechanism by which SIK2 regulates mitotic catastrophe, and the results showed that SIK2 might interact with DNA-PKcs (Fig. S2A). DNA-PKcs is well known for playing a key role in repairing DNA double-strand breaks (DSBs). DNA-PKcs has also been documented to regulate the mitotic process of radiation-damaged cells and is involved in maintaining the stability of the centrosome and spindle structure. 4 Consistent with previous studies, the deficiency of DNA-PKcs with IR stimulation for 48 h increased the number of multipolar spindles from 30% to 50%, and the number of multinuclear cells increased from 10% to 20%(Fig. S2BeD). These findings suggested that DNA-PKcs deficiency promotes IR-induced cellular mitotic catastrophe to a degree comparable to SIK2 knockdown. Next, the interaction between SIK2 and DNA-PKcs was assessed in the HeLa cells by co-immunoprecipitation (CO-IP) assay, and GST pulldown assay further suggested that SIK2 and DNA-PKcs can interact directly (Fig. 1 C; Fig. S2E). DNA-PKcs T2609 and DNA-PKcs S2056 were colocalized with SIK2 at the centrosomes, as observed in immunofluorescence analysis (Fig. S2F). We next elucidated whether SIK2 can affect IR-induced mitotic catastrophe through its interaction with DNA-PKcs. The knockdown or inactivation of DNA-PKcs decreased the content of the SIK2 protein (Fig. 1 D; Fig. S3A). Moreover, in HeLa cell lines with stably knockdown of DNA-PKcs, compared to the control group, the number of abnormal cellular spindles decreased from 50% to 30% by overexpression of SIK2, and the proportion of multinuclear cells decreased from 17% to 8%(Fig. S3BeD). Collectively, the knockdown of DNA-PKcs can promote IR-induced mitotic catastrophe by decreasing the protein content of SIK2. The GST pulldown assay showed that GST-SIK2 (1e926), GST-SIK2 (280e926), and GST-SIK2 (400e926) interacted with DNA-PKcs, indicating the region in which SIK2 interacts with DNA-PKcs. In contrast, GST-SIK2 (700e926) did not interact with DNA-PKcs. These results suggest the presence …
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影响因子：
50.3
作者：
Ahmed AA;Lu Z;Jennings NB;Etemadmoghadam D;Capalbo L;Jacamo RO;Barbosa-Morais N;Le XF;Australian Ovarian Cancer Study Group;Vivas-Mejia P;Lopez-Berestein G;Grandjean G;Bartholomeusz G;Liao W;Andreeff M;Bowtell D;Glover DM;Sood AK;Bast RC Jr
通讯作者：
Bast RC Jr
影响因子：
14.9
作者：
Zhu S;Fisher LA;Bessho T;Peng A
通讯作者：
Peng A
DOI：
10.1007/978-3-030-28151-9_18
发表时间：
2019-01-01
期刊：
MACROMOLECULAR PROTEIN COMPLEXES II: STRUCTURE AND FUNCTION
影响因子：
--
作者：
Curtis, Natalie L.;Bolanos-Garcia, Victor M.
通讯作者：
Bolanos-Garcia, Victor M.
影响因子：
5.2
作者：
Raab M;Rak M;Tesch R;Gasimli K;Becker S;Knapp S;Strebhardt K;Sanhaji M
通讯作者：
Sanhaji M