MST1促进细胞凋亡机制方面的研究
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摘要
背景与目的
蛋白激酶MST1(mammalian Sterile 20-like kinase 1)在其氨基端有一个Ste20相关的激酶催化域,羧基端有一个调节区。MST1在细胞增殖、分化、形态和细胞骨架重排方面发挥了重要作用。先前已有研究指出氨基端催化域的缺失会使MST1被半胱氨酸蛋白酶3(caspase-3)在为数众多的细胞凋亡刺激素的作用下所切割,例如由CD95/FasL触发的死亡受体,或被星孢菌素(STS),神经酰胺,热休克和亚砷酸盐处理。被切割后的MST1的氨基末端会转移至核内,然后对于染色质的固缩和随后发生的细胞凋亡产生一定的作用。而且,有实验表明在细胞内过表达MST1的情况下,已经发现了其被切割的情况和因此诱发的细胞凋亡。并且已经有报道证实第326和第349位的天冬氨酸是两个主要的切割位点。突变掉这些切割位点后,MST1的激活,核转位和诱导细胞凋亡的能力都会明显减弱。最近已经明确了在MST1的第八亚结构域的第183位的苏氨酸是其主要的磷酸激活位点并且这个苏氨酸位点的自磷酸化对于MST1激酶的激活是必须的。Hippo是哺乳动物的MST1在果蝇中的同源蛋白,并且已经被大量实验证实其通过抑制转录和(或者)降解细胞周期蛋白E(cyclin E)和DIAPs或者磷酸化并抑制Yorkie来限制细胞生长和增殖。在哺乳动物中,MST1已经被证实可以分别通过促细胞分裂剂激活性蛋白激酶激酶4/促细胞分裂剂激活性蛋白激酶激酶7(MKK4/MKK7)和促细胞分裂剂激活性蛋白激酶激酶3/促细胞分裂剂激活性蛋白激酶激酶6(MKK3/MKK6)激活c-Jun氨基末端激酶(JNK)和p38MAPK激酶信号通路。最近有报道称JNK对于MST1的激活和由MST1介导的通过磷酸化MST1上的第82位丝氨酸引起的细胞凋亡是必须的。此外,JNK的显性失活突变体能抑制MST1诱导的caspase的激活和从而产生的细胞凋亡,而p38的显性失活突变体和p38抑制剂则不能抑制MST1诱导的细胞凋亡。
MST1还能通过磷酸化组蛋白H2B上相对保守的位点(哺乳动物细胞上第14位丝氨酸,酵母菌上第10位丝氨酸)来诱导的细胞凋亡。我们实验室的研究表明MST1能通过磷酸化O亚型叉头框3a(FOXO3a)上的第207位丝氨酸和FOXO1上对应的第212位丝氨酸从而涉及依赖于FOXO的神经元细胞凋亡过程。近期我们还发现磷酸肌醇3(phosphoinositide 3)激酶/Akt能磷酸化第120位苏氨酸从而抑制MST1介导的细胞凋亡前信号通路。
沉默信息调节因子2相关酶1(Sirt1)是NAD+依赖的去乙酰化酶,它有相当数量的底物并且参与了多种细胞进程。去乙酰化这些靶蛋白导致其蛋白活性既有可能被抑制也有可能被激活,从而影响机体生理的许多方面,例如转录沉默,基因水平控制的寿命长短,细胞的新陈代谢,能量的动态平衡,DNA修复和细胞存活。P53作为一个关键肿瘤抑制基因,在反馈细胞所接受的众多压力信号包括DNA损伤,组织缺氧和异常增殖过程中发挥了极其重要的作用。P53维持基因组稳定性的方式主要是依赖于p53诱导的细胞凋亡,包括抑制肿瘤生长或是清除肿瘤。总的来说,p53应对DNA损伤的生物活性是和其转录后的修饰状态密切相关的,特别是特殊位点的磷酸化、乙酰化和泛素化。Sirt1已经被报道能强烈与其底物p53结合,并能去乙酰化p53的第382位赖氨酸。Sirt1介导的去乙酰化能对抗依赖于p53的转录激活并能特定地抑制在DNA损伤或氧化应激下引起的依赖于p53的细胞凋亡。
先前的研究已经指出MST1促进的细胞凋亡有可能是依赖于p53的,但是MST1-p53在细胞凋亡过程中信号通路的分子机制还有很大一部分都是未知。所以本研究旨在明确MST1在遗传毒性因子诱导的依赖于p53的细胞凋亡过程中扮演的角色,并期待着能发现MST1、Sirt1和p53在这个过程中的相互调节机制。
方法
1.1 MST1和Sirt1对p53和p21转录活性的影响
A. H1299细胞在转染14*p53人工荧光素酶报告基因的同时分别共转了p53、MST1、MST1 K59R和Sirt1质粒。细胞裂解液用双荧光素酶报告基因系统检测转录活性。B. H1299细胞在转染p21启动子荧光素酶报告基因的同时分别共转了p53、MST1和Sirt1质粒。细胞裂解液用双荧光素酶报告基因系统检测转录活性。
1.2 MST1和Sirt1对p53介导的细胞凋亡的影响
A. U2OS细胞转染编码MST1和p53 shRNA或者空载体的质粒,然后用Etoposide处理36小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。B. HCT116 p53+/+和HCT116 p53-/-细胞被稳定转染MST1,对照组稳定转染空载体,然后用CDDP处理36小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。C. HCT116 p53+/+和HCT116 p53-/-细胞转染MST1的小干扰RNA,对照组转染随机小干扰RNA,然后用CDDP处理48小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。D. U2OS细胞在转染MST1质粒的同时共转Sirt1质粒或空载体。用Etoposide处理36小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。
2.1 MST1对Sirt1介导的p53去乙酰化作用的影响
A.稳定转染MST1的HCT116 p53+/+细胞和其稳定转染空载体的对照组细胞的裂解液用免疫印迹方法检测,所用的抗体包括抗p53-k382乙酰化抗体和抗p53抗体。上样量通过内参14-3-3β蛋白调整,从而保持一致。B.转染HA-p53或同时转染GFP-MST1的细胞中FLAG-Sirt1的免疫沉淀物用免疫印迹方法检测,所用的抗体包括抗HA抗体和抗FLAG抗体。C.转染p53、p300、Sirt1、MST1质粒的293T细胞的裂解液用免疫印迹方法检测,所用抗体有抗p53-k382乙酰化抗体等。
2.2 MST1如何通过和Sirt1作用增强p53乙酰化
A.体外MST1激酶实验,重组MST1蛋白和其底物GST-Sirt1在32P-ATP存在的情况下进行孵育。反应后用聚丙烯酰胺凝胶电泳分离蛋白质进行荧光放射自显影术测定。B.体外MST1激酶实验,有活性的重组MST1蛋白在32P-ATP存在的情况下和不同的Sirt1蛋白片段(P1、P2、P3)进行孵育。反应后用聚丙烯酰胺凝胶电泳分离蛋白质进行荧光放射自显影术测定。C.体外磷酸化实验,有活性的MST1蛋白和Sirt1蛋白在不带放射性的ATP存在的情况下进行孵育,然后将磷酸化实验产物和乙酰化p53孵育进行去乙酰化实验。反应后用聚丙烯酰胺凝胶电泳分离蛋白质并用免疫印迹方法检测,所用抗体有抗p53-K382乙酰化抗体、抗GST抗体和抗Sirt1抗体。
结果与结论
1.1 MST1通过抑制Sirt1活性增强p53转录活性。
在14*p53报告基因中,Sirt1能显著抑制p53的表达,而野生型MST1能明显挽救Sirt1诱导的p53降低,但是ATP结合位点突变的激酶活性失活的MST1(MST1 K59R)不能挽救Sirt1诱导的p53降低。与之相似的,Sirt1抑制了依赖于p53的p21荧光素酶活性,而MST1可以逆转Sirt1诱导的p21表达的降低。综上所述,MST1通过抑制Sirt1来促进p53的转录活性。
1.2 MST1促进细胞凋亡是依赖于p53并且Sirt1能抑制MST1诱导的细胞凋亡。
U2OS细胞在干扰掉p53基因的情况下过表达MST1,用Etoposide处理后,细胞凋亡减少。我们在HCT116 p53-/-细胞中也发现MST1的稳定表达不能增加Cisplatin诱导的细胞凋亡数目,但是在HCT116 p53+/+细胞中MST1的稳定表达却能增加Cisplatin诱导的细胞凋亡数目,并且我们还发现在HCT116 p53+/+细胞中干扰掉MST1后Cisplatin诱导的细胞凋亡明显减少而在HCT116 p53-/-细胞中干扰掉MST1后Cisplatin诱导的细胞凋亡不能被降低。这些实验证据证明MST1在DNA损伤情况下促进细胞凋亡是依赖于p53。同时在Sirt1过表达细胞中,MST1介导的细胞凋亡会减少。
2.1 MST1抑制Sirt1介导的p53去乙酰化。
我们在体外MST1激酶实验中发现MST1不能直接磷酸化p53。而Western blot使用p53特异的乙酰化抗体检测结果表明在MST1过表达细胞中内源p53乙酰化水平有所增高。然后我们用IP实验证明MST1和Sirt1有直接的相互作用。接着,我们观察到MST1的过表达会减弱p53和Sirt1的相互作用。与之一致的,在MST1过表达的细胞中Sirt1介导的p53去乙酰化是被抑制的。综上所述,MST1抑制了Sirt1介导的p53去乙酰化。我们同时发现在体外MST1也能减少Sirt1介导的FOXO3去乙酰化,表明MST1也可能调控Sirt1其他底物的生物活性。
2.2 MST1通过磷酸化Sirt1增加p53乙酰化。
体外MST1激酶实验中用重组Sirt1蛋白作为底物表明Sirt1能被MST1磷酸化。我们接着用重组GST融合蛋白分别编码了3个不重叠的Sirt1区域(肽段P1-P3)并以此指出在Sirt1上的磷酸化区域。在体外激酶实验中显示包含了C末端的第489-747位氨基酸片段的P3是磷酸化的主要区域。在体外磷酸化和去乙酰化实验中,MST1磷酸化Sirt1确实减少了Sirt1诱导的p53去乙酰化。通过以上实验我们阐明了MST1通过负调控Sirt1的去乙酰化活性来调节p53功能的分子机制。
Background and Objectives
The protein kinase mammalian Sterile 20-like kinase 1(MST1) contains a Ste20-related kinase catalytic domain in the amino-terminal segment followed by a regulatory domain at the COOH-terminus. It has been implicated in the diverse biological functions including cell proliferation, differentiation, morphogenesis and cytoskeleton rearrangement. Previous studies have indicated that non-catalytic tail of MST1 is cleaved by caspase-3 upon a numbers of apoptotic stimuli for example death receptor triggering by CD95/FasL and treatments with Staurosporine (STS), Ceramide, as well as heat shock and arsenite. The amino-terminal fragment of cleaved MST1 translocates into the nucleus, where it contributes to chromatin condensation and then apoptosis. Furthermore, under the condition of overexpressing MST1, the cleavage and herein induced apoptosis could also be observed. It has been reported that D326 and D349 are the two major cleavage sites. The kinase activation, nuclear translocation and ability to induce cell death of MST1 areapparently attenuated by mutating these cleavage sites. Recently it has been defined Threonine 183 as a dominating phosphoactivation site in subdomain VIII of MST1 and it is essential for kinase activation with the auto-phosphorylation of Threonine 183 within the MST1 kinase domain. Hippo, a mammalian homolog of MST1/2 in Drosophila, has been extensively shown to restrain cell from growth and proliferation through the inhibition of transcription and/or degradation of cyclin E and DIAPs or phosphorylation and inhibition of Yorkie. In mammals, it has been indicated that MST1 can activate c-Jun n-terminal kinase (JNK) and p38MAPK kinase signaling pathways through MKK4/MKK7 and MKK3/MKK6, respectively. Recently it is suggested that JNK is essential and sufficient for MST1 activation and MST1-mediated apoptosis via phosphorylating the serine 82 on MST1. Furthermore, MST1-induced caspase activation and apoptosis are inhibited by dominant-negative mutant of JNK, not dominant-negative p38 or the p38 inhibitor.
MST1 induces apoptosis by phosphorylating Histone H2B on a relatively conserved site, Ser-14 in mammalian cells and Ser-10 in Saccharomyces cerevisiae, respectively. We reported that MST1 has also been implicated in the control of FOXO-dependent neuronal cell death via phosphorylating FOXO3a at Ser-207 and the corresponding site of FOXO1 at Ser-212. Recently we have shown that phosphorylation of Threonine 120 by phosphoinositide 3-kinase/Akt can inhibit MST1-mediated pro-apoptotic signaling pathway.
Sirt1 is a NAD+-dependent deacetylase with numbers of substrates that participates in various cellular processes. Deacetylation of these target proteins may either inhibit or activate their activities, thus influences many aspects of organism physiology, such as transcriptional silencing, genetic control of aging, cell metabolism, energy homeostasis, DNA repair and cell survival. And the p53 protein functions as a key tumor suppressor, and plays a vital role in invoking cellular responses to numerous stress signals, including DNA damage, hypoxia and aberrant proliferation. The way of p53 in maintaining genome stability is mainly carried out by p53-dependent apoptosis, which is in mediating tumor suppression or tumor clearance. In general, p53 biological activity in responding to DNA damage is tightly regulated by its post-translational modification status, particularly by site-specific phosphorylation, acetylation and ubiquitination. Sirt1 has been reported to strongly bind to its substrate p53 and can deacetylate p53 at Lysine 382. The Sirt1-mediated deacetylation antagonizes p53-dependent transcriptional activation and specifically inhibits p53-dependent apoptosis in response to DNA damage as well as oxidative stress.
Previous studies have shown that MST1 promotes cell death is p53-dependent, but the molecular mechanism underlying MST1-p53 signaling during apoptosis is still largely unknown. In this work, we aim to identify the role of MST1 in promotion of genotoxic agents-induced apoptosis dependent on p53. And we also expect to find out the regulator mechanism among MST1, Sirt1 and p53.
Methods
1.1 Effects of MST1 and Sirt1 on the p53 and p21 transcriptional activity
A. H1299 cells were co-transfected with 14*p53 artificial-luciferase construct with p53, MST1, MST1 K59R or Sirt1. Lysates were assayed for the dual-luciferase activity. B. H1299 cells were co-transfected the p21-luciferase promoter construct with the plasmids encoding p53, MST1 or Sirt1. Lysates were assayed for the dual-luciferase activity as in A.
1.2 Effects of MST1 and Sirt1 on the p53-mediated cell death
A. U2OS cells were transfected with the plasmids encoding MST1 together with p53 shRNA or control vector, then treated with Etoposide (36h) before apoptosis analysis and the apoptosis was analyzed by Annexin-V staining followed by flow cytometry. B. Both HCT116 p53+/+ and p53-/- cells stably transfected with MST1 or control vectors were treated with Cisplatin (CDDP) and the cell death analysis was performed as in A. C. U2OS cells were transfected with the plasmids encoding MST1 together with Sirt1 or control empty vector and the cell death analysis was performed as in A.
2.1 Effects of MST1 on Sirt1-mediated deacetylation of p53
A. Lysates of HCT116 p53+/+ cells stably transfected with MST1 or control vector were immunoblotted with anti-p53-K382Ac or anti-p53 antibody and the loading was normalized by using the protein 14-3-3β. B. FLAG-Sirt1 immunoprecipitates from cells transfected with HA-p53 or GFP-MST1 were immunoblotted with anti-HA or anti-FLAG antibody. C. Lysates of 293T cells transfected with the plasmids encoding p53, p300, Sirt1, MST1 were immunoblotted with anti-p53-K382Ac antibody as well as the other antibodies.
2.2 How MST1 enhances p53 acetylation through its interaction with Sirt1
A. In vitro MST1 kinase assay was performed by incubating the recombinant active MST1 with GST-Sirt1 as substrate in the presence of 32P-ATP. The reaction was analyzed by SDS-PAGE followed by autoradiography. B. In vitro MST1 kinase assay was performed by incubating the recombinant active MST1 with different Sirt1 fragments (P1, P2, and P3) in the presence of 32P-ATP. The reaction was analyzed by SDS-PAGE followed by autoradiography. C. In vitro phosphorylation was performed by incubating active MST1 and Sirt1 in the presence of cold ATP. Then the deacetylation reaction was performed by incubating the products of in vitro phosphorylation reaction with the acetylated p53. The reaction was analyzed by SDS-PAGE followed by immunoblotting with anti-p53-K382Ac, anti-GST or Sirt1 antibody.
Results and Conclusions
1.1 MST1 promotes p53 transcriptional activities by inhibiting Sirt1 activity.
Sirt1 dramatically inhibits p53-mediated expression of the 14*p53 reporter. Wild type MST1, significantly rescues Sirt1-induced p53 repression, but the kinase dead MST1 in which the ATP binding site was mutated (MST1 K59R) failed to do so. Similarly, Sirt1 inhibits p53-dependent p21 luciferase’s activity and MST1 could reverse Sirt1-induced p21 repression. Taken together, MST1 promotes p53 biological activities by inhibiting Sirt1.
1.2 MST1 promotes cell death is p53-dependent and Sirt1 can inhibition of MST1-induced apoptosis.
P53 knockdown in U2OS cells reduces MST1 overexpression-induced cell death upon Etoposide treatment. We also found that MST1 cannot induce Cisplatin-triggered cell death in HCT116 p53-/- cells. However, MST1 increases Cisplatin-induced cell death in HCT116 p53+/+ cells. These experiments together strikingly support our conclusion that MST1 induces cell death under DNA damage is p53-dependent. And Sirt1 overexpression decreases MST1 mediated apoptosis.
2.1 MST1 inhibits Sirt1-mediated deacetylation of p53.
In vitro MST1 kinase assay using recombinant p53 or Histone H2B as the substrate, we found MST1 failed to phosphorylate p53 in vitro. Western blot analysis by using p53 specific acetylation antibody revealed that the acetylation of endogenous p53 was upregulated in the MST1 overexpressing cells. Then first, we examined the physical interaction between MST1 and Sirt1, indicating they might interact functionally. Secondly, we observed that MST1 overexpression reduced the interaction between p53 and Sirt1. Consistently, the Sirt1 mediated deacetylation of p53 is inhibited in the presence of MST1 in cells. Collectively, MST1 inhibits Sirt1 mediated p53 deacetylation. We also found that MST1 reduces Sirt1-mediated FOXO3 deacetylation in vitro, indicating that MST1 might regulate the biological function of other Sirt1’s substrates.
2.2 MST1 enhances p53 acetylation through phosphorylating Sirt1.
In vitro MST1 kinase assay using the recombinant Sirt1 as the substrate indicates Sirt1 could be phosphorylated by MST1. We then delineated the phosphorylated region within Sirt1 using recombinant GST fusion proteins encoding three non-overlapping Sirt1 domains (peptides P1-P3). In vitro kinase assay shows the P3 fragment containing C-terminal 489-747 amino acids is the major region of phosphorylation. And MST1 phosphorylation of Sirt1 indeed decreases Sirt1-induced p53 deacetylation by using in vitro phosphorylation followed by in vitro deacetylation reaction. Here we elucidate the mechanism of MST1 regulating p53 through negative regulation of Sirt1’s deacetylation activity.
蛋白激酶MST1(mammalian Sterile 20-like kinase 1)在其氨基端有一个Ste20相关的激酶催化域,羧基端有一个调节区。MST1在细胞增殖、分化、形态和细胞骨架重排方面发挥了重要作用。先前已有研究指出氨基端催化域的缺失会使MST1被半胱氨酸蛋白酶3(caspase-3)在为数众多的细胞凋亡刺激素的作用下所切割,例如由CD95/FasL触发的死亡受体,或被星孢菌素(STS),神经酰胺,热休克和亚砷酸盐处理。被切割后的MST1的氨基末端会转移至核内,然后对于染色质的固缩和随后发生的细胞凋亡产生一定的作用。而且,有实验表明在细胞内过表达MST1的情况下,已经发现了其被切割的情况和因此诱发的细胞凋亡。并且已经有报道证实第326和第349位的天冬氨酸是两个主要的切割位点。突变掉这些切割位点后,MST1的激活,核转位和诱导细胞凋亡的能力都会明显减弱。最近已经明确了在MST1的第八亚结构域的第183位的苏氨酸是其主要的磷酸激活位点并且这个苏氨酸位点的自磷酸化对于MST1激酶的激活是必须的。Hippo是哺乳动物的MST1在果蝇中的同源蛋白,并且已经被大量实验证实其通过抑制转录和(或者)降解细胞周期蛋白E(cyclin E)和DIAPs或者磷酸化并抑制Yorkie来限制细胞生长和增殖。在哺乳动物中,MST1已经被证实可以分别通过促细胞分裂剂激活性蛋白激酶激酶4/促细胞分裂剂激活性蛋白激酶激酶7(MKK4/MKK7)和促细胞分裂剂激活性蛋白激酶激酶3/促细胞分裂剂激活性蛋白激酶激酶6(MKK3/MKK6)激活c-Jun氨基末端激酶(JNK)和p38MAPK激酶信号通路。最近有报道称JNK对于MST1的激活和由MST1介导的通过磷酸化MST1上的第82位丝氨酸引起的细胞凋亡是必须的。此外,JNK的显性失活突变体能抑制MST1诱导的caspase的激活和从而产生的细胞凋亡,而p38的显性失活突变体和p38抑制剂则不能抑制MST1诱导的细胞凋亡。
MST1还能通过磷酸化组蛋白H2B上相对保守的位点(哺乳动物细胞上第14位丝氨酸,酵母菌上第10位丝氨酸)来诱导的细胞凋亡。我们实验室的研究表明MST1能通过磷酸化O亚型叉头框3a(FOXO3a)上的第207位丝氨酸和FOXO1上对应的第212位丝氨酸从而涉及依赖于FOXO的神经元细胞凋亡过程。近期我们还发现磷酸肌醇3(phosphoinositide 3)激酶/Akt能磷酸化第120位苏氨酸从而抑制MST1介导的细胞凋亡前信号通路。
沉默信息调节因子2相关酶1(Sirt1)是NAD+依赖的去乙酰化酶,它有相当数量的底物并且参与了多种细胞进程。去乙酰化这些靶蛋白导致其蛋白活性既有可能被抑制也有可能被激活,从而影响机体生理的许多方面,例如转录沉默,基因水平控制的寿命长短,细胞的新陈代谢,能量的动态平衡,DNA修复和细胞存活。P53作为一个关键肿瘤抑制基因,在反馈细胞所接受的众多压力信号包括DNA损伤,组织缺氧和异常增殖过程中发挥了极其重要的作用。P53维持基因组稳定性的方式主要是依赖于p53诱导的细胞凋亡,包括抑制肿瘤生长或是清除肿瘤。总的来说,p53应对DNA损伤的生物活性是和其转录后的修饰状态密切相关的,特别是特殊位点的磷酸化、乙酰化和泛素化。Sirt1已经被报道能强烈与其底物p53结合,并能去乙酰化p53的第382位赖氨酸。Sirt1介导的去乙酰化能对抗依赖于p53的转录激活并能特定地抑制在DNA损伤或氧化应激下引起的依赖于p53的细胞凋亡。
先前的研究已经指出MST1促进的细胞凋亡有可能是依赖于p53的,但是MST1-p53在细胞凋亡过程中信号通路的分子机制还有很大一部分都是未知。所以本研究旨在明确MST1在遗传毒性因子诱导的依赖于p53的细胞凋亡过程中扮演的角色,并期待着能发现MST1、Sirt1和p53在这个过程中的相互调节机制。
方法
1.1 MST1和Sirt1对p53和p21转录活性的影响
A. H1299细胞在转染14*p53人工荧光素酶报告基因的同时分别共转了p53、MST1、MST1 K59R和Sirt1质粒。细胞裂解液用双荧光素酶报告基因系统检测转录活性。B. H1299细胞在转染p21启动子荧光素酶报告基因的同时分别共转了p53、MST1和Sirt1质粒。细胞裂解液用双荧光素酶报告基因系统检测转录活性。
1.2 MST1和Sirt1对p53介导的细胞凋亡的影响
A. U2OS细胞转染编码MST1和p53 shRNA或者空载体的质粒,然后用Etoposide处理36小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。B. HCT116 p53+/+和HCT116 p53-/-细胞被稳定转染MST1,对照组稳定转染空载体,然后用CDDP处理36小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。C. HCT116 p53+/+和HCT116 p53-/-细胞转染MST1的小干扰RNA,对照组转染随机小干扰RNA,然后用CDDP处理48小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。D. U2OS细胞在转染MST1质粒的同时共转Sirt1质粒或空载体。用Etoposide处理36小时后用流式细胞仪检测Annexin-V染色的细胞凋亡情况。
2.1 MST1对Sirt1介导的p53去乙酰化作用的影响
A.稳定转染MST1的HCT116 p53+/+细胞和其稳定转染空载体的对照组细胞的裂解液用免疫印迹方法检测,所用的抗体包括抗p53-k382乙酰化抗体和抗p53抗体。上样量通过内参14-3-3β蛋白调整,从而保持一致。B.转染HA-p53或同时转染GFP-MST1的细胞中FLAG-Sirt1的免疫沉淀物用免疫印迹方法检测,所用的抗体包括抗HA抗体和抗FLAG抗体。C.转染p53、p300、Sirt1、MST1质粒的293T细胞的裂解液用免疫印迹方法检测,所用抗体有抗p53-k382乙酰化抗体等。
2.2 MST1如何通过和Sirt1作用增强p53乙酰化
A.体外MST1激酶实验,重组MST1蛋白和其底物GST-Sirt1在32P-ATP存在的情况下进行孵育。反应后用聚丙烯酰胺凝胶电泳分离蛋白质进行荧光放射自显影术测定。B.体外MST1激酶实验,有活性的重组MST1蛋白在32P-ATP存在的情况下和不同的Sirt1蛋白片段(P1、P2、P3)进行孵育。反应后用聚丙烯酰胺凝胶电泳分离蛋白质进行荧光放射自显影术测定。C.体外磷酸化实验,有活性的MST1蛋白和Sirt1蛋白在不带放射性的ATP存在的情况下进行孵育,然后将磷酸化实验产物和乙酰化p53孵育进行去乙酰化实验。反应后用聚丙烯酰胺凝胶电泳分离蛋白质并用免疫印迹方法检测,所用抗体有抗p53-K382乙酰化抗体、抗GST抗体和抗Sirt1抗体。
结果与结论
1.1 MST1通过抑制Sirt1活性增强p53转录活性。
在14*p53报告基因中,Sirt1能显著抑制p53的表达,而野生型MST1能明显挽救Sirt1诱导的p53降低,但是ATP结合位点突变的激酶活性失活的MST1(MST1 K59R)不能挽救Sirt1诱导的p53降低。与之相似的,Sirt1抑制了依赖于p53的p21荧光素酶活性,而MST1可以逆转Sirt1诱导的p21表达的降低。综上所述,MST1通过抑制Sirt1来促进p53的转录活性。
1.2 MST1促进细胞凋亡是依赖于p53并且Sirt1能抑制MST1诱导的细胞凋亡。
U2OS细胞在干扰掉p53基因的情况下过表达MST1,用Etoposide处理后,细胞凋亡减少。我们在HCT116 p53-/-细胞中也发现MST1的稳定表达不能增加Cisplatin诱导的细胞凋亡数目,但是在HCT116 p53+/+细胞中MST1的稳定表达却能增加Cisplatin诱导的细胞凋亡数目,并且我们还发现在HCT116 p53+/+细胞中干扰掉MST1后Cisplatin诱导的细胞凋亡明显减少而在HCT116 p53-/-细胞中干扰掉MST1后Cisplatin诱导的细胞凋亡不能被降低。这些实验证据证明MST1在DNA损伤情况下促进细胞凋亡是依赖于p53。同时在Sirt1过表达细胞中,MST1介导的细胞凋亡会减少。
2.1 MST1抑制Sirt1介导的p53去乙酰化。
我们在体外MST1激酶实验中发现MST1不能直接磷酸化p53。而Western blot使用p53特异的乙酰化抗体检测结果表明在MST1过表达细胞中内源p53乙酰化水平有所增高。然后我们用IP实验证明MST1和Sirt1有直接的相互作用。接着,我们观察到MST1的过表达会减弱p53和Sirt1的相互作用。与之一致的,在MST1过表达的细胞中Sirt1介导的p53去乙酰化是被抑制的。综上所述,MST1抑制了Sirt1介导的p53去乙酰化。我们同时发现在体外MST1也能减少Sirt1介导的FOXO3去乙酰化,表明MST1也可能调控Sirt1其他底物的生物活性。
2.2 MST1通过磷酸化Sirt1增加p53乙酰化。
体外MST1激酶实验中用重组Sirt1蛋白作为底物表明Sirt1能被MST1磷酸化。我们接着用重组GST融合蛋白分别编码了3个不重叠的Sirt1区域(肽段P1-P3)并以此指出在Sirt1上的磷酸化区域。在体外激酶实验中显示包含了C末端的第489-747位氨基酸片段的P3是磷酸化的主要区域。在体外磷酸化和去乙酰化实验中,MST1磷酸化Sirt1确实减少了Sirt1诱导的p53去乙酰化。通过以上实验我们阐明了MST1通过负调控Sirt1的去乙酰化活性来调节p53功能的分子机制。
Background and Objectives
The protein kinase mammalian Sterile 20-like kinase 1(MST1) contains a Ste20-related kinase catalytic domain in the amino-terminal segment followed by a regulatory domain at the COOH-terminus. It has been implicated in the diverse biological functions including cell proliferation, differentiation, morphogenesis and cytoskeleton rearrangement. Previous studies have indicated that non-catalytic tail of MST1 is cleaved by caspase-3 upon a numbers of apoptotic stimuli for example death receptor triggering by CD95/FasL and treatments with Staurosporine (STS), Ceramide, as well as heat shock and arsenite. The amino-terminal fragment of cleaved MST1 translocates into the nucleus, where it contributes to chromatin condensation and then apoptosis. Furthermore, under the condition of overexpressing MST1, the cleavage and herein induced apoptosis could also be observed. It has been reported that D326 and D349 are the two major cleavage sites. The kinase activation, nuclear translocation and ability to induce cell death of MST1 areapparently attenuated by mutating these cleavage sites. Recently it has been defined Threonine 183 as a dominating phosphoactivation site in subdomain VIII of MST1 and it is essential for kinase activation with the auto-phosphorylation of Threonine 183 within the MST1 kinase domain. Hippo, a mammalian homolog of MST1/2 in Drosophila, has been extensively shown to restrain cell from growth and proliferation through the inhibition of transcription and/or degradation of cyclin E and DIAPs or phosphorylation and inhibition of Yorkie. In mammals, it has been indicated that MST1 can activate c-Jun n-terminal kinase (JNK) and p38MAPK kinase signaling pathways through MKK4/MKK7 and MKK3/MKK6, respectively. Recently it is suggested that JNK is essential and sufficient for MST1 activation and MST1-mediated apoptosis via phosphorylating the serine 82 on MST1. Furthermore, MST1-induced caspase activation and apoptosis are inhibited by dominant-negative mutant of JNK, not dominant-negative p38 or the p38 inhibitor.
MST1 induces apoptosis by phosphorylating Histone H2B on a relatively conserved site, Ser-14 in mammalian cells and Ser-10 in Saccharomyces cerevisiae, respectively. We reported that MST1 has also been implicated in the control of FOXO-dependent neuronal cell death via phosphorylating FOXO3a at Ser-207 and the corresponding site of FOXO1 at Ser-212. Recently we have shown that phosphorylation of Threonine 120 by phosphoinositide 3-kinase/Akt can inhibit MST1-mediated pro-apoptotic signaling pathway.
Sirt1 is a NAD+-dependent deacetylase with numbers of substrates that participates in various cellular processes. Deacetylation of these target proteins may either inhibit or activate their activities, thus influences many aspects of organism physiology, such as transcriptional silencing, genetic control of aging, cell metabolism, energy homeostasis, DNA repair and cell survival. And the p53 protein functions as a key tumor suppressor, and plays a vital role in invoking cellular responses to numerous stress signals, including DNA damage, hypoxia and aberrant proliferation. The way of p53 in maintaining genome stability is mainly carried out by p53-dependent apoptosis, which is in mediating tumor suppression or tumor clearance. In general, p53 biological activity in responding to DNA damage is tightly regulated by its post-translational modification status, particularly by site-specific phosphorylation, acetylation and ubiquitination. Sirt1 has been reported to strongly bind to its substrate p53 and can deacetylate p53 at Lysine 382. The Sirt1-mediated deacetylation antagonizes p53-dependent transcriptional activation and specifically inhibits p53-dependent apoptosis in response to DNA damage as well as oxidative stress.
Previous studies have shown that MST1 promotes cell death is p53-dependent, but the molecular mechanism underlying MST1-p53 signaling during apoptosis is still largely unknown. In this work, we aim to identify the role of MST1 in promotion of genotoxic agents-induced apoptosis dependent on p53. And we also expect to find out the regulator mechanism among MST1, Sirt1 and p53.
Methods
1.1 Effects of MST1 and Sirt1 on the p53 and p21 transcriptional activity
A. H1299 cells were co-transfected with 14*p53 artificial-luciferase construct with p53, MST1, MST1 K59R or Sirt1. Lysates were assayed for the dual-luciferase activity. B. H1299 cells were co-transfected the p21-luciferase promoter construct with the plasmids encoding p53, MST1 or Sirt1. Lysates were assayed for the dual-luciferase activity as in A.
1.2 Effects of MST1 and Sirt1 on the p53-mediated cell death
A. U2OS cells were transfected with the plasmids encoding MST1 together with p53 shRNA or control vector, then treated with Etoposide (36h) before apoptosis analysis and the apoptosis was analyzed by Annexin-V staining followed by flow cytometry. B. Both HCT116 p53+/+ and p53-/- cells stably transfected with MST1 or control vectors were treated with Cisplatin (CDDP) and the cell death analysis was performed as in A. C. U2OS cells were transfected with the plasmids encoding MST1 together with Sirt1 or control empty vector and the cell death analysis was performed as in A.
2.1 Effects of MST1 on Sirt1-mediated deacetylation of p53
A. Lysates of HCT116 p53+/+ cells stably transfected with MST1 or control vector were immunoblotted with anti-p53-K382Ac or anti-p53 antibody and the loading was normalized by using the protein 14-3-3β. B. FLAG-Sirt1 immunoprecipitates from cells transfected with HA-p53 or GFP-MST1 were immunoblotted with anti-HA or anti-FLAG antibody. C. Lysates of 293T cells transfected with the plasmids encoding p53, p300, Sirt1, MST1 were immunoblotted with anti-p53-K382Ac antibody as well as the other antibodies.
2.2 How MST1 enhances p53 acetylation through its interaction with Sirt1
A. In vitro MST1 kinase assay was performed by incubating the recombinant active MST1 with GST-Sirt1 as substrate in the presence of 32P-ATP. The reaction was analyzed by SDS-PAGE followed by autoradiography. B. In vitro MST1 kinase assay was performed by incubating the recombinant active MST1 with different Sirt1 fragments (P1, P2, and P3) in the presence of 32P-ATP. The reaction was analyzed by SDS-PAGE followed by autoradiography. C. In vitro phosphorylation was performed by incubating active MST1 and Sirt1 in the presence of cold ATP. Then the deacetylation reaction was performed by incubating the products of in vitro phosphorylation reaction with the acetylated p53. The reaction was analyzed by SDS-PAGE followed by immunoblotting with anti-p53-K382Ac, anti-GST or Sirt1 antibody.
Results and Conclusions
1.1 MST1 promotes p53 transcriptional activities by inhibiting Sirt1 activity.
Sirt1 dramatically inhibits p53-mediated expression of the 14*p53 reporter. Wild type MST1, significantly rescues Sirt1-induced p53 repression, but the kinase dead MST1 in which the ATP binding site was mutated (MST1 K59R) failed to do so. Similarly, Sirt1 inhibits p53-dependent p21 luciferase’s activity and MST1 could reverse Sirt1-induced p21 repression. Taken together, MST1 promotes p53 biological activities by inhibiting Sirt1.
1.2 MST1 promotes cell death is p53-dependent and Sirt1 can inhibition of MST1-induced apoptosis.
P53 knockdown in U2OS cells reduces MST1 overexpression-induced cell death upon Etoposide treatment. We also found that MST1 cannot induce Cisplatin-triggered cell death in HCT116 p53-/- cells. However, MST1 increases Cisplatin-induced cell death in HCT116 p53+/+ cells. These experiments together strikingly support our conclusion that MST1 induces cell death under DNA damage is p53-dependent. And Sirt1 overexpression decreases MST1 mediated apoptosis.
2.1 MST1 inhibits Sirt1-mediated deacetylation of p53.
In vitro MST1 kinase assay using recombinant p53 or Histone H2B as the substrate, we found MST1 failed to phosphorylate p53 in vitro. Western blot analysis by using p53 specific acetylation antibody revealed that the acetylation of endogenous p53 was upregulated in the MST1 overexpressing cells. Then first, we examined the physical interaction between MST1 and Sirt1, indicating they might interact functionally. Secondly, we observed that MST1 overexpression reduced the interaction between p53 and Sirt1. Consistently, the Sirt1 mediated deacetylation of p53 is inhibited in the presence of MST1 in cells. Collectively, MST1 inhibits Sirt1 mediated p53 deacetylation. We also found that MST1 reduces Sirt1-mediated FOXO3 deacetylation in vitro, indicating that MST1 might regulate the biological function of other Sirt1’s substrates.
2.2 MST1 enhances p53 acetylation through phosphorylating Sirt1.
In vitro MST1 kinase assay using the recombinant Sirt1 as the substrate indicates Sirt1 could be phosphorylated by MST1. We then delineated the phosphorylated region within Sirt1 using recombinant GST fusion proteins encoding three non-overlapping Sirt1 domains (peptides P1-P3). In vitro kinase assay shows the P3 fragment containing C-terminal 489-747 amino acids is the major region of phosphorylation. And MST1 phosphorylation of Sirt1 indeed decreases Sirt1-induced p53 deacetylation by using in vitro phosphorylation followed by in vitro deacetylation reaction. Here we elucidate the mechanism of MST1 regulating p53 through negative regulation of Sirt1’s deacetylation activity.
引文
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