【作者】 耿礼义；

【导师】 郑树； Randy J. Legerski；

【作者基本信息】 浙江大学 ， 肿瘤学， 2007， 博士

【摘要】 细胞增殖是通过细胞周期(cell cycle)来实现的，而细胞周期的有序运行是通过相关基因的严格监视和调控来保证的。在哺乳动物细胞，细胞周期可分为四个阶段：G1期，S期，G2期和M期。DNA损伤是普遍存在于整个细胞周期中，当细胞受到各种DNA损伤后可以诱发多条细胞应答通路的激活如细胞周期检查点(checkpoint)的激活，损伤DNA的修复和凋亡(Apoptosis)等。在真核细胞，DNA损伤应答主要由PIKKs激酶家族所介导，包括ATM，ATR和DNA-PK激酶。目前已发现有多个蛋白能够被ATM和ATR所激活，如Artemis，p53，Brca1，Nbs1，Smc1，Chk1，Chk2，53BP1，Mdc1和Rad17。这些蛋白除了转导或介导细胞周期checkpoint信号外，也可能直接参与DNA损伤修复。Artemis是SNM1基因家族的成员，除Artemis外，该基因家族还包括SNM1、SNM1B、CPSF73和ELAC2，该基因家族成员的共同特点是拥有一个保守的结构域—SNM1结构域，该结构域由metallo-β-lactamase折叠和β-CASP结构域组成，其中metallo-β-lactamase具有核酸酶的活性，而β-CASP结构域则具有与DNA结合的功能。除了SNM1结构域外，这些蛋白的序列在酵母和人之间是不同的。研究发现，酵母的Snm1具有双链断裂DNA的修复功能。Artemis蛋白除了上述两个结构域外，在其羧基端还有一个SCD(S／TQ cluster domain)结构域，该结构域中的SQ／TQ位点易被PIKK激酶所磷酸化。Artemis是首先通过连锁分析的方法分离得到。在放射线敏感的人类重症联合免疫缺陷病(RS-SCID)的病人中，Artemis发生突变或缺失，该病的主要特征是由于V(D)J重排缺陷导致B淋巴细胞和T淋巴细胞成熟障碍。生物化学研究发现，Artemis具有5′-3′外切核酸酶的活性，当其与DNA-PKcs形成复合物并被DNA-PK所磷酸化，其活性发生转化，由原先的外切核酸酶转变为内切核酸酶，对发夹结构的DNA和5′端或3′端的突出具有切割作用。在T细胞抗原受体(TCR)和免疫球蛋白的成熟过程中须要发生V(D)J重排，在这一过程中，重组酶RAG1和RAG2复合物识别位于V区、D区和J片段侧翼的重组信号识别序列并进行切割，形成一个具有发夹结构的编码联接(coding joint)，而Artemis与DNA-PKcs形成复合物将这一发夹结构打开，从而在DNA修复体系的作用下完成V(D)J重排。Riballo和Ma等报道，Artemis参与了非同源性末端连接(NHEJ)，对双链断裂的DNA进行修复，且Artemis这一功能需要ATM和／或DNA-PK对其的激活。Zhang等研究发现，Artemis参与了对细胞周期的调控，当缺乏Artemis的人类肿瘤细胞受到DNA损伤后，细胞的G2／M阻滞明显缩短。先前的研究表明Artemis蛋白在体外和体内均能被DNA-PK、ATM和ATR所磷酸化。不同的DNA损伤会导致不同的激酶参与对Artemis的磷酸化，当细胞受到离子辐射(IR)后，DNA-PK和ATM参与对Artemis的磷酸化，而在紫外线刺激后则由ATR对Artemis进行磷酸化。但是DNA损伤诱导Artemis的磷酸化的详细情况以及Artemis的磷酸化在DNA损伤后的细胞应答中的作用目前均未阐明。为此，本课题试图回答这些问题并将研究结果报告如下：1．Artemis蛋白的磷酸化位点及相应激酶的鉴定在HEK293细胞中稳定表达GST-Artemis并用IR处理细胞，然后用GST柱子将GST-Artemis纯化并进行质谱分析。发现在IR处理后，Artemis第516位丝氨酸(S516)被磷酸化，这是一个SQ位点。Artemis蛋白的SCD结构域中除了SQ516外，还有6个SQ位点，分别为SQ534，SQ538，SQ548，SQ553，SQ562和SQ645。本课题通过定点突变的方法分别将这些SQ位点的丝氨酸(S)突变成丙氨酸(A)，然后将这些突变的Artemis与GST融合并在HEK293细胞中表达，用蛋白凝胶电泳迁移试验进行分析。结果显示：在IR处理后，S645A突变体蛋白及S534AS538A联合突变体的迁移速率均较野生型的Artemis蛋白快。然后分别合成针对S516、S534、S538和S645四个位点的磷酸化特异性抗体并进行Western blot分析，进一步证实在IR处理后，Artemis蛋白中的S516、S534、S538和S645四个位点发生磷酸化。SO位点是PIKK激酶家族的识别序列，故本课题首先用PIKK激酶的特异性化学抑制剂caffeine(caffeine能够抑制ATM和ATR，但不能抑制DNA-PK)和wortmannin(wortmannin能够抑制ATM、ATR和DNA-PK)处理HEK293细胞。发现在小剂量IR(3Gy)处理后，caffeine和wortmannin均能抑制这四个SQ位点的磷酸化；而当用大剂量IR(10Gy)处理后，wortmannin能完全抑制这四个SQ位点的磷酸化，但caffeine的抑制效果不明显。然后用ATM、ATR和DNA-PKcs特异性的siRNA分别敲除细胞中这些激酶，发现：ATM被敲除后，在细胞用小剂量IR处理后这四个位点磷酸化水平明显降低；当DNA-PKcs被敲除后，在细胞用大剂量的IR处理后这四个位点磷酸化水平明显降低，但对小剂量的IR处理没有明显效果。综上所述，在小剂量IR处理后，ATM是这四个位点磷酸化的主要激酶；在大剂量IR处理后，DNA-PK也参与了这四个位点的磷酸化。在以下的所有实验中均采用小剂量IR(3Gy)处理细胞。2．DNA损伤诱导Artemis的磷酸化在细胞周期调控中的作用研究首先对上述已经被鉴定的四个磷酸化位点在IR处理后的磷酸化动力学进行分析，发现S534和S538两个位点在IR处理后的30分钟内被快速磷酸化，在2小时后又被快速的去磷酸化；而S516和S645两个位点在IR处理后的30分钟内被磷酸化，且其磷酸化一直维持在较高水平，直到24小时后才缓慢下降。本课题首先将上述四个位点的丝氨酸残基(S)分别突变成丙氨酸(A)构建四个位点的单突变体(S516A，S534A，S538A和S645A)，并在HEK293细胞中稳定表达，经IR处理后，然后进行细胞周期的分析，结果并没有发现分别表达四个单突变体的细胞的细胞周期发生改变。由于S516和S645，S534和S538具有相似的磷酸化动力学，本课题又分别构建S516-645A和S534-538A两个双突变体，并在HEK293细胞中稳定表达，然后进行细胞周期的分析。与表达野生型Artemis的HEK293细胞相比，表达S516-645A双突变体的HEK293细胞在IR处理后12小时至24小时之间，G2／M期细胞明显聚集。当将这两个丝氨酸残基同时突变成天冬氨酸(S516-645D)来模拟这两个位点的磷酸化时，其表型与野生型的完全相同。这表明上述的G2／M期阻滞是由于S516-645A双突变体Artemis不能被磷酸化所致。同时磷酸化组蛋白H3(phospho-H3)染色结果显示，相比与野生型和S516-645D，S516-645A双突变体的细胞存在G2向M期转换的阻滞。另外BrdU脉沖标记试验显示，在IR处理后9小时，BrdU标记的表达S516-645D的细胞已经进入下一个细胞周期的G1期，而BrdU阳性的表达S516-645A的细胞即使在12小时也不能进入到G1期。在本研究中并没有观察到S534-538A双突变体的表达对细胞周期的影响。以上结果表明，Artemis蛋白S516和S645位点的联合突变是导致细胞经IR处理后G2期向M期转换阻滞所必需的。S516-645A导致G2／M阻滞延长的原因可能有两种，一是由于细胞对双链断裂DNA的修复能力的降低导致G2／M checkpoint延长，二是由于G2／M checkpoint recovery的缺陷。为了明确S516-645A导致G2／M阻滞延长的原因，首先利用Gamma-H2AX抗体对细胞经IR处理后Gamma-H2AX的表达水平进行分析，表达S516-645A的HEK293细胞与表达野生型Artemis的HEK293细胞系和表达S516-645D的HEK293细胞系之间的Gamma-H2AX的表达水平及持续时间没有差异。另外，应用Artemis特异性的siRNA敲除HEK293细胞内的Artemis，并用FACS结合phospho-H3染色和BrdU标记试验，结果显示：与对照组相比，在IR处理后，Artemis被敲除的细胞其G2向M期转换加速，这与S516-645A的表型完全相反。此外，Gamma-H2AX的表达水平分析显示Artemis被敲除的HEK293细胞系的Gamma-H2AX的表达水平及持续时间与对照组没有区别。这些结果表明S516-645A导致G2／M阻滞延长不是由于细胞对双链断裂DNA的修复能力的降低导致G2／M checkpoint延长所致，而是由于S516-645A导致G2／M checkpoint recovery的缺陷。3．Artemis蛋白S516和S645位点的磷酸化在G2／M checkpoint recovery中的作用的分子机制的研究Cdk1的磷酸化状态影响着Cdk1的活性，从而实施对细胞由G2向M期转换的调控。利用Western blot对Cdk1的磷酸化状态进行分析，结果显示，在IR处理后，与表达野生型Artemis和表达S516-645D的HEK293细胞系相比，表达S516-645A的HEK293细胞系的Cdk1的第14位苏氨酸和第15位酪氨酸(该两个位点为抑制性磷酸化位点)的磷酸化水平较高且维持时间长，在IR处理后18小时，表达野生型Artemis和表达S516-645D的HEK293细胞系的Cdk1均已去磷酸化，而在表达S516-645A的HEK293细胞系中Cdk1仍然处于高抑制性磷酸化状态。这一结果不仅进一步证实了前面研究得到的细胞周期的结果，同时表明DNA损伤后，Artemis是Cdk1的调节蛋白。由于Cdk1的磷酸化是受Cdk1抑制性磷酸化激酶Myt1／Wee1和活化性酪氨酸磷酸酶Cdc25C之间的动态平衡精确调节的。利用Western blot分析表达S516-645A的HEK293细胞系和表达野生型Artemis的HEK293细胞系中Wee1和Cdc25C蛋白表达水平，结果显示：Wee1和Cdc25C蛋白表达水平在两株细胞之间没有差异，或有差异，但该差异与G2／M阻滞延长的预期方向相反。同时，Cdc25C在两株细胞内的分布也没有差异。这一结果表明Artemis对Cdk1磷酸化状态的调节不是通过Wee1和Cdc25C调节通路。当细胞完成对损伤DNA的修复后，Plk1通过磷酸化Wee1并促使其降解，使得Cdk1第15位酪氨酸去磷酸化，细胞从G2／M阻滞恢复，重新进入正常的细胞周期。利用Western blot对Plk1的蛋白表达水平进行分析发现，在表达S516-645A的HEK293细胞系和表达野生型Artemis的HEK293细胞系中Plk1的蛋白表达水平的差异与G2／M阻滞延长的预期方向相反。这表示Plk1并没有参与S516和S645位点的磷酸化对G2／M checkpoint recovery的调控。Cyclin B的Western blot分析结果显示：相比于表达野生型Artemis和表达S516-645D的HEK293细胞系，在表达S516-645A的HEK293细胞系中cyclin B比较稳定，在IR处理后24小时，仍然维持较高的水平；对IR处理后cyclin B的细胞定位和磷酸化状态的分析发现，与表达野生型Artemis的细胞系相比，表达S516-645A的细胞系其细胞浆中的cyclin B明显增加，且磷酸化水平降低。另外，用cyclin B、Cdk1以及Artemis的抗体进行免疫共沉淀发现：与表达野生型Artemis的HEK293细胞系相比，表达S516-645A的HEK293细胞系经IR处理后，其cyclin B和Cdk1的相互作用显著增强，并且是与抑制性磷酸化的Cdk1的相互作用；同时S516-645A使Wee1蛋白与cyclin B-Cdk1复合物的结合明显增加：此外，在IR处理后，Artemis能够与cyclin B发生结合，但这种结合在表达S516-645A的HEK293细胞系中明显增强。这些结果表明，Artemis通过影响cyclin B的稳定性、磷酸化和细胞内定位实施对Cdk1磷酸化的调控；S516-645A增加了无活性Cdk1-cyclin B复合物的积聚。同时，应用Western blot对Artemis被敲除的HEK293细胞系中cyclin B的表达水平及Cdk1的磷酸化状态进行分析，结果显示，与对照组相比，在Artemis被敲除的HEK293细胞系中cyclin B的表达水平明显降低；同时Cdk1的磷酸化水平明显下降。为了进一步验证Artemis通过影响cyclin B而实施对Cdk1磷酸化的调控，本课题分别应用cyclin B、phospho-S126-Cyclin B、Artemis和Gamma-tubulin抗体进行Immunostaining染色，结果显示：细胞经IR处理后18小时，在表达野生型Artemis和表达S516-645D的HEK293细胞系中，cyclin B的激活及核内转运均为正常，而在表达S516-645A的HEK293细胞系中phospho-S126-cyclin B则主要存在于中心体(centrosome)；在表达野生型Artemis的HEK293细胞系中，只有极少数细胞的中心体中存在Artemis蛋白的表达(约为1％)，而在表达S516-645A的HEK293细胞系中，在中心体中观察到Artemis蛋白表达的细胞数则明显增加(约为10％)；此外，相比于表达野生型Artemis的细胞系，在表达S516-645A的细胞系中，Artemis蛋白和phospho-S126-cyclin B的细胞内共定位(colocalization)显著增加。结论：1．当细胞受到IR处理后，Artemis蛋白中的S516、S534、S538和S645四个位点发生磷酸化。2．当细胞受到小剂量IR处理后，ATM是负责S516、S534、S538和S645四个位点磷酸化的主要激酶；当细胞受到大剂量IR处理后，DNA-PK也参与了这四个位点的磷酸化。3．IR处理后，这四个位点的磷酸化动力学是不同的。S534和S538在短时间内快速磷酸化，又在短时间内被快速去磷酸化；S516和S645在短时间内发生磷酸化，并在长时间内维持较高的磷酸化水平。因此，S534和S538可能具有相同或相似的功能，而S516和S645可能具有相同或相似的功能。4．S516和S645的磷酸化参与了DNA损伤后的G2／M checkpoint recovery，使得细胞在G2／M checkpoint被关闭后恢复正常的细胞周期，促进细胞由G2期向M期转换。5．DNA损伤后，Artemis是Cdk1磷酸化的调节蛋白。6．Artemis通过影响cyclin B实施对Cdk1磷酸化的调控。7．Artemis对Cdk1磷酸化状态的调节独立于Cdc25通路和Plk1通路。更多还原

【Abstract】 Cell proliferation is governed by the cell cycle that is strictly regulated by cyclin-dependent kinases (Cdks). In mammalian cells, the cell cycle is divided into four discrete phases: G1, S, G2 and M. During the cell cycle DNA damage is a relatively common event occurred which induces several cellular responses including cell cycle checkpoint, DNA repair and apoptosis. In eukaryotic cells these responses are mediated to a large degree by a family of PIKKs that include ATM, ATR and DNA-PK. Numerous downstream targets of ATM and/or ATR have been identified including Artemis, p53, Brca1, Nbs1, Smc1, Chk1, Chk2, 53BP1, Mdc1 and Rad17. These proteins are involved in transducing or mediating checkpoint signals, and may in some cases also play a direct role in the repair of DNA damage.Artemis is a member of the SNM1/PSO2 gene family. Members of this family,which in humans also include SNM1, SNM1B, ELAC2, and CPSF73, share a region of homology termed the SNM1 domain, which contains a metallo-β -lactamase fold that it possesses nucleolytic activity and an appended β -CASP domain that is a predicted nucleic acid binding motif, but are otherwise distinct between the yeast and human. In addition to the SNM1 domain, there is a SCD (S/TQ cluster domain) domain located at the carboxyl-terminal half of Artemis protein and has been shown those (S/T)Q motifs in this domain are preferred sites of phosphorylation by ATM, ATR, and DNA-PK.Artemis which located on the short arm of chromosome 10 and encoding 692 amino acid residues, was cloned by linkage analysis. Its original characterization was based on the finding that mutations in Artemis lead to a human severe combined immunodeficiency syndrome (SCID) due to a defect in V(D)J recombination which resulting in premature arrest of both B- and T-lymphocytes maturation. In addition, Artemis-deficient cells were shown to be radiosensitive leading to the designation of the complete phenotype as RS-SCID. Biochemical studies of Artemis have shown that it possesses a 5’-3’ exonucleolytic activity on a single-stranded DNA, and when complexed with and phosphorylated by DNA-PKcs, its exonucleolytic activity is changed to endonucleolytic activity on 5’ and 3’ overhangs and the ability to open DNA hairpins. During the V(D)J recombination, a process by which immunoglobulin and T cell receptor variable domain exons are assembled from V, D, and J elements, Artemis forms the complex with DNA-PKcs and opens the hairpins produced by the RAG complex which endonucleolytically nicks the DNA 5’ of the heptamer, where it borders each V, D, or J element. Artemis has also been reported to be required in nonhomologous DNA end-joining pathway (NHEJ) for the repair ofdouble-stranded breaks (DSBs) that possess damaged termini and this function has been shown to require activation by either ATM and/or DNA-PK. In addition, Zhang et al. reported that Artemis plays a role in the cell cycle since a defect was observed in the maintenance of the G2/M checkpoint upon DNA damage in human cancer cell lines depleted of Artemis.Artemis has been shown to be a phosphorylation target of DNA-PK, ATM, and ATR in vitro and in vivo. This modification is dependent upon the nature of the DNA damage agent as IR induces phosphorylation of Artemis by both DNA-PK and ATM, and UV induces phosphorylation by ATR. However, the details of Artemis phosphorylations induced by DNA damage and their functions on cell response to DNA damage have not been elucidated. We therefore try to answer these questions and results are reported as follows:Part I. Identification of Sites of Phosphorylation in Artemis and the Responsible Kinases.To identify specific phosphorylated residues in Artemis, we initiated an analysis by mass spectroscopy. GST-Artemis was stably expressed in HEK293 cells and GST fusion proteins were purified with or without exposure to IR. This analysis indicated with a high probability that the S516 residue was phosphorylated upon exposure of cells to IR. S516 which located in the SCD is a SQ motif and there are other six SQ motifs in this domain, including SQ534, SQ538, SQ548, SQ553, SQ562 and SQ645. So we used gel mobility shift assay to identify additional six sites. For this purpose we independently mutated each of the six SQ motifs to AQ, and expressed GST fusion protein in HEK293 cells. This analysis indicated a slightly increase in the mobility for the S645A mutantand S534AS538A mutant compared to the wild-type protein after IR treatment. Taken together, these results suggested that S516, S534, S538 and S645 were candidate sites for phosphorylation in Artemis in vivo after DNA damage. In order to further analyze these four candidate sites, we prepared affinity purified phospho-specific peptide antibodies for each site. These antibodies were then used for Western blotting against either GST-Artemis wild-type protein or the appropriate S to A mutant. The experiments indicated that all four antibodies were specific for phosphorylation at the target site, and that the reactivity of the antibodies increased after exposure of cells to IR. This suggested that S516, S534, S538 and S645 in Artemis were phosphorylated after DNA damage.To determine the kinase(s) responsible for IR-induced phosphorylation at each of the four identified sites, we used the PIKKs inhibitors caffeine and wortmannin to treat the HEK293 cells. Caffeine is an inhibitor of ATM and ATR, but not DNA-PK while wortmannin inhibits all three enzymes. For these experiments we examined the phosphorylation of endogenous Artemis. Interestingly, at a low dose of IR (3Gy) both caffeine and wortmannin inhibited phosphorylation at each site to approximately the same degree, while at a high dose of IR (10Gy) wortmannin was the more potent inhibitor. These results suggest that at low dose of IR the phosphorylation is primarily carried out by ATM/ATR while at high dose IR DNA-PK may play a significant role. To further examine this issue, we used siRNA transfection to deplete ATM, ATR, or DNA-PKcs, and then examined phosphorylation at each site. At low dose of IR siRNA-mediated knockdown of ATM significantly reduced the level of phosphorylation at each site. Knockdown of DNA-PKcs at low dose IR had little or no affect on the phosphorylation at any of the four sites, while at high dose ofIR a significant decrease in signal was observed at all four sites. Taken together, these findings indicate that ATM is the principal kinase that modifies Artemis at these sites after the lower and more physiological relevant induction of DSBs, while at high level of DNA damage Artemis also becomes a substrate for DNA-PK.All of the following experiments were carried out using 3Gy of IR to treat the cells.Part II. Role of DNA Damage-Induced Phosphorylation of Artemis in Cell Cycle Regulation.We examined the kinetics of phosphorylation at each of the four SQ sites after IR treatment. Phosphorylation at S534 and S538 showed rapid phosphorylation that was readily apparent by 30min; however, this phosphorylation was highly transient and had disappeared by 2hrs. On the other hand, phosphorylation at S516 and S645 was observed at 30min, remained strong until 6hrs, and then slowly declined out to 24hrs.We independently mutated each of the four SQ motifs to AQ, and stably expressed in HEK293 cells and then treated with IR, cells expressing these single mutants had no effect on the cell cycle. Since either S516 and S645 residues or S534 and S538 residues displayed similar kinetics of phosphorylation and dephosphorylation after DNA damage. We prepared two double mutants in which both residues were changed to alanine (refer to these two double mutants as S516-645A and S534-538A, respectively.), and then prepared two HEK293 cell lines that stably expressed these two double mutant proteins, respectively, and a control cell line that over-expressed wild-type Artemis. Interestingly, cells expressing the S516-645A mutant showed an accumulation of cells in G2/M thatwas readily detected between 12 and 24 hrs after IR treatment compared to cells expressing wild-type Artemis. In addition, expression of a mutant Artemis (S5106-645D), in which the serines at S516 and S645 were mutated to aspartic acid residues in order to mimic phosphorylation, resulted in a wild-type phenotype. To determine if the accumulation observed with the S516-645A mutant was due to an affect on the G2/M checkpoint, we examined phospho-H3 staining in each of the three cell lines, S516-645A, S516-645D, and wild-type. The results showed that the transition from G2 to M phase is delayed in S516-645A mutant as opposed to cells expressing wild-type Artemis or the S516-645D mutant. To insure that the accumulation in G2 observed after IR treatment was not the result of affects in earlier stages of the cell cycle, we pulse labeled cells with BrdU, and then treated cells with IR. These experiments indicated that labeled cells expressing the S516-645D mutant were found to transit into G1 as early as 9 hrs after IR treatment, while G1 phase cells were not observed in the S516-645A expressing cells even at 12 hrs after IR.We also performed same experiments in cells expressing S534-538A as in cells expressing S516-645A, However, S534-538A did not exhibit any effect on cell cycle.Taken together, these results suggest that combined mutations of both S516 and S645 are required for the observed delay in the transition from G2 to M phase after IR treatment.There are two explanations for the observed prolonged delay in the G2 phase in cells expressing the S516-645A Artemis mutant. Such cells may have a reduced ability to repair DSBs leading to a prolonged G2/M checkpoint, or may be defective in recovery from the G2/M checkpoint. First, we examinedgamma-H2AX levels after IR treatment. Cells expressing the S516-645A mutant did not show any difference of gamma-H2AX levels and sustaining time as compared to cells expressing Artemis wild-type or the S516-645D mutant. Second, depletion of Artemis by siRNA actually gave rise to an accelerated transition from G2 to M phase and a more rapid entry into the G1 phase compared to control cells after IR treatment as determined by cell cycle analysis, phopho-H3 staining and BrdU labeling, which is contrary to the result obtained with cells expressing the S516-645A mutant. On the other hand, levels of gamma-H2AX were not sustained at higher levels after IR in cells depleted of Artemis as compared to control cells. These results indicate that the S516-645A mutant induces a prolonged G2 arrest, the cause is not due to a failure to repair DSBs, but due to a defective in recovery from the G2/M checkpoint. Part III: Molecular Mechanisms of Phosphorylations of Both S516 and S645 in G2/M Checkpoint Recovery.Since G2-M transition is driven by activation of cyclin-dependent kinase1 (Cdk1)-cyclin B complex. The activity of Cdk1-Cyclin B complex is largely determined by the phosphorylation state of Cdk1. We examined the phosphorylation status of Cdk1 after IR in cells expressing the S516-645A mutant and observed an extended duration of phosphorylation of this kinase compared to cells expressing wild-type Artemis or the S516-645D mutant, by 18 hrs after IR, the phosphorylation of Cdk1 in cells expressing wild-type or S516-645D mutant has disappeared while in cells expressing S516-645A mutant it still was sustained at high level. Thus, this result validates the cell cycle data of S516-645 A expressing cells shown above, and indicates that Artemis is a regulator of Cdk1 after exposure of cells to DNA damage.It is clear that the balance between the inhibiting kinases Myt1/Wee1 and the activating Cdc25C tyrosine phosphotase is a critical regulator of phosphorylation of Cdk1. We examined Wee1 and Cdc25C in cells expressing the S516-645A mutant and found either that no changes in the levels occurred, or the changes that did occur were not in the direction predicted by an extended G2 delay. On the other hand, we examined the distribution of Cdc25C in cells expressing S516-645A mutant and found no changes occurred. These results suggest that the effect of Artemis on Cdk1 phosphorylation is independent of Wee1 and Cdc25C pathways.Previous findings have shown that Plk1 is invoved in G2/M checkpoint recovey by dephosphorylating Wee1 and thus promoting its degradation. We examined Plk1 in cells expressing the S516-645A mutant and found the changes in the levels occurred were not in the direction predicted by an extended G2 delay. This suggests that Artemis regulating G2/M checkpoint recovery is not involved in Plk1 pathway.We examined the Cyclin B and found that Artemis status had a significant impact on the levels of this protein. In cells expressing the S516-645A mutant cyclin B was stabilized, even by 24 hrs after IR it was still sustained at high level. Next we examined the localization and phosphorylation status of cyclin B after IR. Compared to cells expressing wild-type Artemis, expression of the S516-645A mutant resulted in a significant increase in cyctoplasmic levels of Cyclin B and a decreased level of phosphorylation. In addition, to determine if Artemis status has an effect on the cyclin B-Cdk1 interaction, we performed reciprocal co-immunoprecipitation experiments. The results indicated that the interaction between cyclin B and Cdk1 was dramatically increased in cellsexpressing S516-645A mutant compared to cells expressing the wild-type protein. Also noted that cyclin B appeared to interact with the phosphorylated form of Cdk1 in cells expressing the S516-645A mutant compared to cells expressing the wild-type Artemis. In addition, the interaction between cyclin B and Wee1 was enhanced by expression of the S516-645A mutant. Finally, immunoprecipitation of Artemis after exposure of cells to IR showed that cyclin B weakly interacts with wild-type Artemis, but that this interaction was significantly stronger in the presence of the S516-645A mutant. Taken together, these results suggest that Artemis regulates cyclin B by affecting its stabilization, dephosphorylation and cytoplasmic retention, and that the S516-645A mutant enhances the accumulation of inactive cyclin B-Cdk1 complexes.We also examined Cdk1 phosphorylation status and cyclin protein level after knockdown of Artemis by siRNA and found that the phosphorylation forms of Cdk1 and the cyclin B levels were greatly reduced compared to control cells.To further confirm the results shown above that Artemis affects the phosphorylation of Cdk1 via regulating cyclin B. Immunofluorescence was used to examine the localization of cyclin B in different cell lines after exposure to IR for 18hr. For these experiments we used an antibody to Artemis, an antibody to cyclin B, and a phospho-specific antibody that recognizes phospho-S126 of cyclin B, which is one of the sites required for activation of cyclin B. The results showed that activation and nuclear import of cyclin B occurred normally in cells expressing Artemis wild-type and the S516-645D mutant, whereas, the phospho-S126 form of cyclin B appeared to be preferentially localized to the centrosome in cells expressing the S516-645A mutant. Gamma-tubulin staining was used to confirm the centrosomal localization of phospho-S126-cyclin B. Inwild-type expressing cells the occurrence of Artemis in the centrosome was rare (approximately 1%), while significantly more cells exhibited this localization in cells expressing the S516-645A mutant (approximately 10%). In addition, the colocalization of Artemis and phospho-S126-cyclin B was significantly enhanced in the S516-645A mutant expressing cells compared to cells expressing wild-type Artemis.Conclusions:1. Artemis is phosphorylated at S516, S534, S538 and S645 after IR treatment.2. ATM is the principal kinase that modifies Artemis at S516, S534, S538 and S645 after the lower and more physiological relevant induction of DSBs, while at high levels of DNA damage Artemis also becomes a substrate for DNA-PK.3. The S516 and S645 residues share a similar kinetics of phosphorylation anddephosphorylation after DNA damage, which were phosphorylated immediately after DNA damage and the phosphorylation were sustained at high level for long time. The residues S534 and S538 have a different kinetic signature of IR-induced phosphorylation compared to the S516 and S645 residues. Both S534 and S538 were rapidly phosphorylated after DNA damage and dephosphorylated in short time.4. The phosphorylations at S516 and S645 regulate recovery from the G2/Mcheckpoint and drive the cells from G2 phase into M phase upon the G2/M checkpoint silenced.5. Artemis is a regulator of the phosphorylation of Cdk1 after DNA damage.6. Artemis affects the phosphorylation of Cdk1 via regulation of Cyclin B.7. The effect of Artemis on Cdk1 phosphorylation is independent of Plk1 and Cdc25C pathways.更多还原