TNF-α与过氧化氢通过mTOR非依赖途径激活p85 S6K1
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摘要
哺乳动物雷帕霉素靶蛋白(The mammalian target of rapamycin, mTOR)信号通路整合来自细胞内外的各种信号,对细胞的代谢、生长增殖及存活等生理过程发挥着中心调节作用。许多人类疾病,如癌症和2型糖尿病中都发现mTOR信号通路的调节发生异常。近年来,mTOR信号通路成为基础和临床研究的热点。
mTOR蛋白激酶活性的发挥有赖于其在细胞内与其他分子结合形成复合物：mTOR复合物1 (mTOR complex1, mTORC1)和mTORC2。大环内酯类药物雷帕霉素与细胞内蛋白分子即FK506-结合蛋白12 (FK506-binding protein 12, FKBP12)结合后,对mTORC1发挥特异性的抑制作用。接受生长因子及胰岛素等信号刺激而活化的PI3-K/Akt(蛋白激酶B,PKB),能够磷酸化结节性硬化复合物2(tuberous sclerosis complex 2, TSC2),从而减弱后者对mTORC1的抑制作用。活化的mTORC1可以直接磷酸化其下游底物,40S核糖体蛋白S6激酶(40Sribosomal protein S6 kinase1, S6K1)和真核细胞翻译起始因子4E-结合蛋白1(eukaryotic translation initiation factor 4E binding protein 1,4E-BP1)。S6K1Thr389位点磷酸化后使其激后,并磷酸化核糖体蛋白S6,从而启动5'-Top mRNA (5'-terminal oligopyrimidine tract mRNA)的翻译。被mTORC1磷酸化的4E-BP1则不能再与eIF-4E结合,从而启动5’-帽结构依赖性(5'-cap-dependent)的蛋白翻译。由此二者共同促进蛋白质翻译。
S6Ks属于AGC丝／苏蛋白质激酶家族。哺乳动物细胞主要表达该激酶的两种分子,即S6K1和S6K2(又分别称为S6Kα和S6Kβ),它们由不同的基因编码,但全序列具有很高程度的同源性。S6K1有两种亚型,这两种亚型是由同一个转录子(mRNA)通过选择不同的翻译起始位点而得到的不同产物。p70 S6K1,主要分布于细胞质中；p85 S6K1的N-端较p70 S6K1多了一段由23个氨基酸残基构成的核定位信号(nuclear localization signal, NLS),主要分布于细胞核内。
S6K1是mTORC1最主要的效应分子之一,其调节方式常被作为研究mTORC1信号通路的模型。虽然S6K1的完全及持续活化需要多个生长因子诱导的磷酸化反应,Thr389位点的磷酸化对于S6K1的活化尤其重要,有研究证明,用丙氨酸替代该位点的苏氨酸能阻断S6K1激酶结构域的活化。体外实验证明,mTORC1可以直接磷酸化p70 S6K1的Thr389,而经雷帕霉素处理的细胞,S6K1的Thr389位点发生快速去磷酸化从而使S6K1失活。
最近大量研究报道了S6Ks在调节细胞大小、肿瘤侵袭、迁移以及血管生成、胰岛素抵抗及寿命等生理病理进程中发挥了重要作用。然而,上游信号是如何调节S6Ks以及S6Ks通过何种方式调节细胞的生理过程还没有完全阐明。由于目前还没有能快速抑制S6Ks活性的S6K1或S6K2特异性抑制剂,现有的关于S6Ks活性抑制效应研究的大部分结果,主要通过雷帕霉素抑制mTORC1而间接抑制SK6s的功能而得到。但新近报道,S6K1能通过雷帕霉素抵抗及mTORC1非依赖性的方式发生活化。而且,雷帕霉素及其类似物治疗肿瘤时,常发生雷帕霉素抵抗,其机制还不明确。因此,阐明S6Ks的mTORC1非依赖性活化机制对进一步明确S6Ks功能及其在人类疾病中的作用具有重要的意义。
目前普遍认为S6K1两种亚型,即p70 S6K1和p85 S6K1的调控机制与功能是相同的,所以多数研究主要是针对细胞质亚型的p70 S6K1,而细胞核亚型p85S6K1的功能其及调控机制被忽略,因此对于p85 S6K1的功能及调控机制鲜有报道。
肿瘤坏死因子(tumor necrosis factor-α, TNF-α)和活性氧(reactive oxygenspecies, ROS)在肿瘤形成和肿瘤进展中发挥重要作用。最近研究发现,TNF-α和ROS是mTORC1和p70 S6K1的上游信号分子,TNF-α可通过IκB激酶(IκB Kinase, IKK)磷酸化并抑制TSC1,从而激活mTORC1与p70 S6K1, ROS可通过尚不清楚的机制激活或抑制mTORC1和p70 S6K1,但它们对p85 S6K1的调控及其机制还未见报道。
在本研究中,我们发现p85 S6K1和p70 S6K1具有新的、不同的调节机制与功能。我们首次证明,TNF-α和H2O2磷酸化激活p85 S6K1(T412)是雷帕霉素非依赖的,而对p70 S6K1(T389)的活化却可被雷帕霉素抑制,且这种活化作用不需要mTOR(mTORC1/2),但依赖于IKK。
一、mTOR sh RNA, p70 S6K1和p85 S6K1真核表达载体的构建及谷胱甘肽-S-转移酶(Glutathione-S-Transferase, GST)-S6蛋白的制备
(1)利用PCR技术,将69 bp(23个氨基酸)加到大鼠p70 S6K1 cDNA的N-末端,扩增得到p85 S6K1的cDNA
将p70/p85 S6K1的cDNA克隆至pcDNA3.1-Flag真核表达载体中,成功构建pcDNA3.1-Flag-p70 S6K1和pcDNA3.1-Flag-p85 S6K1真核表达载体。为了防止在p85 S6K1表达载体翻译出p70 S6K1蛋白,我们将p70 S6K1翻译起始位点的ATG (70-72th bp)突变为TTG。
(2)GST-S6蛋白的制备
提取人胚胎肾细胞293 (human embryonic kidney 293, HEK293)的总mRNA作为模板,通过RT-PCR获得人40S核糖体蛋白S6 cDNA,将其克隆至原核表达载体pGEX-6P-1,命名为pGEX-6P-1-S6。GST-S6融合蛋白经异丙基β-D-1-半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)诱导表达后,利用GST-琼脂糖beads进行纯化。纯化的蛋白将作为S6K1体外激酶活性检测的底物。
(3)构建重组mTOR sh RNA表达载体(psiLV-H1-mTOR)
成功构建mTOR sh RNA表达载体(psiLV-H1-mTOR),在脂质体的介导下转染人乳腺癌细胞系MCF-7。转染72-96h后可见内源性mTOR的表达明显下调。
二、TNF-α和过氧化氢(hydrogen peroxide, H2O2)以mTOR非依赖,IKK依赖的方式激活p85 S6K1,但不激活p70 S6K1
(1)p70 S6K1和p85 S6K1对TNF-α及H2O2刺激产生不同反应
现有的研究证明,细胞内外各种信号诱导的S6K1(T389/412)和S6(S235/236)的磷酸化反应对雷帕霉素敏感。我们的实验结果显示,在MCF-7细胞中,加入100 nM的雷帕霉素,可以阻断胰岛素及氨基酸诱导的p70 S6K1(T389)、p85 S6K1(T412)和S6(S235/236)的磷酸化。然而,雷帕霉素能抑制TNF-α和HzO2刺激后MCF-7细胞的p70 S6K1的磷酸化,但对p85 S6K1的磷酸化却没有抑制作用。与p85 S6K1(T412)的这种对雷帕霉素不敏感的磷酸化结果一致的是,在有雷帕霉素的作用下,TNF-α和H2O2也能使S6(S235/236)的磷酸化水平增加。而且,H2O2使多种细胞(人骨肉瘤细胞系MG63,小鼠原代成骨细胞OB,乳腺癌细胞系MCF-7和人结肠癌细胞系HCT116的p85 S6K1(T412)发生磷酸化的同时,却抑制p70 S6K1(T389)的磷酸化。以上结果提示,p70 S6K1和p85 S6K1对某些上游信号(如H2O2和TNF-α)存在完全不同的反应。
(2)H2O2以不依赖于雷帕霉素和氨基酸的方式引起p85 S6K1(T412)磷酸化,但不能引起p70 S6K1(T389)的磷酸化
现有研究表明,氨基酸饥饿和雷帕霉素能抑制多种上游信号对mTORC1的活化。我们发现MCF-7, HeLa和HCT116细胞在有雷帕霉素或者氨基酸饥饿的情况下,H2O2能诱导内源性的p85 S6K1(T412)发生磷酸化,但不能诱导p70S6K1(T389)发生磷酸化。而且,H2O2也能使过表达的Flag-p85 S6K1 (T412)发生磷酸化,这种作用对雷帕霉素不敏感。但H2O2不能诱导过表达的Flag-p70S6K1(T389)发生雷帕霉素非依赖磷酸化。
(3)H2O2和TNF-α引起雷帕霉素非依赖的p85 S6K1激活参与雷帕霉素存在时S6(S235/236)的磷酸化激活
S6的S235/236位点的磷酸化对其自身的活化非常关键。我们发现,MCF-7和HCT116细胞中,在有雷帕霉素的情况下,H2O2和TNF-α也能激活S6(S235/236)。过表达的Flag-p85 S6K1而非Flag-p70 S6K1能增强这种雷帕霉素非依赖的S6(S235/236)磷酸化。更重要的是,体外激酶反应结果显示,免疫纯化的Flag-p85 S6K1而不是Flag-p70 S6K1,负责H202引起的S6(S235/236)雷帕霉素非依赖的磷酸化。以上结果提示,是p85 S6K1而非p70 S6K1与雷帕霉素非依赖的S6活化有关。
(4) mTOR (mTORCl和mTORC2)与H2O2引起的p85 S6K1雷帕霉素非依赖的活化无关
利用sh RNA敲低内源性mTOR的表达,使内源性p70/p85 S6K1(T389/412)和S6(S235-36)的磷酸化下降,但不能抑制H2O2诱导的p85 S6K1(T412)和S6(S235/236)的磷酸化。因此,mTOR与H2O2引起的p85 S6K1、S6雷帕霉素非依赖的活化无关。
(5)TSC2不参与H2O2激活p85 S6K1
IKK-β通过磷酸化TSC1作用于mTORC1的上游负性调控分子TSC1/2复合物,激活mTORC1及S6K1。我们选用TSC2野生型及敲除型的小鼠胚胎成纤维细胞(mouse embryo fibroblast, MEF)系TSC2+/+P53-/-MEF和TSC2-/-P53-/-MEF,发现两种细胞在H2O2的作用下,均出现了p85 S6K1(T412)磷酸化水平的增加。而且,有雷帕霉素存在时,两种细胞中均出现剂量效应的p85 S6K1(T412)被H2O2激活,而p70 S6K(T389)被完全抑制,因此,H2O2引起p85 S6K1雷帕霉素非依赖的磷酸化与TSC2无关。
(6)IKK参与H2O2引起的不依赖nTOR的p85 S6K1和S6活化
为进一步研究参与这种不依赖mTOR的p85 S6K1和S6活化的上游信号通路,我们用P13-K和IKK特异性抑制剂进行干预。结果发现,IKK抑制剂能够阻断H2O2诱导的p85 S6K1 (T412)和S6(S235/236)发生的磷酸化,但是PI3-K抑制剂和mTOR抑制剂对它们的磷酸化则没有抑制作用。我们又进一步证明,过表达的myc-IKK-β与Flag-p85 S6K1之间存在相互作用,而且H2O2能增强这种作用。这些结果表明,IKK参与了H2O2诱导的p85 S6K1和S6不依赖nTOR的活化。
综上所述,我们的研究结果揭示了一种新的S6K1调控机制。雷帕霉素存在时,H2O2和TNF-α能激活p85 S6K1但不能激活p70 S6K1,这种激活作用不依赖于mTOR,但是依赖于IKK信号通路,而且这种激活作用负责S6(S235/236)的雷帕霉素及mTOR非依赖性活化。这些发现将有助于我们进一步阐明p85S6K1的功能和雷帕霉素及其类似物治疗肿瘤时形成雷帕霉素抵抗的机制,为发展新的基于mTOR通路的肿瘤靶向治疗方案提供理论基础。
The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth proliferation and survival. The mTOR pathway is deregulated in human diseases such as cancer and type 2 diabetes. Recnet years, mTOR signaling have attracted broad scientific and clinical interest.
Kinase acitivity of mTOR depends on the complexes formation of mTOR by combining with other molecules in cells, mTOR complex 1 (mTORC1) and mTORC2. Macrolides rapamycin specifically inhibits the activity of mTORC1 by combining with intracellular protein FK506-binding-protein 12 (FKBP12). Activated phosphoinositide 3-kinase (PI3-K)/Akt (protein kinase B, PKB) induced by growth factor and insulin stimulation may phosphorylate tuberous sclerosis complex 2 (TSC2), which will attenuate inhibitive effect on mTORCl. Activated mTORC1 will directly phosphorylate its downstream substrates,40S ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Phosphorylation at Thr389 site of S6K1 results in its activation and may phosphorylate and activate 40S ribosomal protein S6, which will initiate translation of 5'-terminal oligopyrimidine tract mRNA (5'-Top mRNAs). Phosphorylated 4E-BP1 by mTOR can no longer bind eIF4E, thus triggers a 5'-cap dependent protein translation. Consequently, protein synthesis begins.
S6Ks belongs to the AGC family of Ser/Thr kinases. Mammalian cells express two forms of the kinase, S6K1 and S6K2 (also known as S6Kαand S6Kβrespectively), which are encoded by two different genes and share a high level of overall sequence homology. S6K1 has two isoforms produced from the same transcript by alternative translational start sites. The shorter form, which is largely cytoplasmic, was initially termed p70 S6K1, whereas the larger form, which appears to be exclusively nuclear, was referred to as p85 S6K1. In the case of p85 S6K1, the additional 23-amino-acid sequence residing at its amino terminus has been shown to function as a nuclear localization signal (NLS).
S6K1 is the best characterized effector of mTOR, and its regulation serves as a model for mTOR signaling. A full and sustained S6K1 activation requires multiple growth factor-induced phosphorylation events. The phosphorylation of Thr389 by mTORC1 is particularly important because subsititution of the residue with alanine blocks the activation of the kinase domain. There is also evidence indicating that p70 S6K1 (T389) is directly phoshorylated by mTORC1 in vitro and rapamycin treatment of cells triggers the rapid dephosphorylation of Thr389 and inactivation of S6K1.
Recent studies have addressed many important roles of S6Ks in cell size control, tumor invasiveness, motility and angiogenesis, insulin resistance and lifespan. Regulation of S6Ks by upstream signals and mechanisms through which S6Ks regulate cellular process, however, remains to be clarified. For lack of S6K1-or S6K-2-specific inhibitors which is useful in defining the effects of acute inhibition of S6Ks, most results for S6Ks functions come from inhibition of mTORC1 by rapamycin. But recent studies have addressed activation of S6K1 in rapamycin-resistant and mTORC1-independent manner. Moreover, mechanisms involved in rapamycin-resistance in rapamycin-based cancer therapy remain unclear. Defining mTORC1-independent mechanisms that regulate S6Ks is important for understanding the functions of S6Ks and its roles in human diseases.
Findings to date would suggest that two isoforms of S6K1, p70 S6Kland p85 S6K1 are controlled in a similar manner. Most studies have been focused on cytoplasmic isoform p70 S6K1. Functions and regulation of nuclear isoform p85 S6K1 have been neglected and are poorly understood.
TNF-αand reactive oxygen species (ROS) play important role in carcinogenesis and tumor progression. Previous studies have demonstrated that TNF-αand ROS are important upstream signals of mTORC1 and p70 S6K1. But their roles in regulation of p85 S6K1 have not been reported.
In this study, we found novel and differential regulation and function of p70 and p85 S6K1. We demonstrated for the first time that p85 but not p70 S6K1 was activated by TNF-αand H2O2 through mTOR-independent, I (?)B-kinase (IKK)-dependent mechanism.
1 Construction of mTOR shRNA and p70 S6K1, p85 S6K1 expression vectors and preparation of Glutathione-S-Transferase (GST)-S6 protein
(1) N-terminal (69 bp,23 amino acids) of p85 S6K1 was added to rat p70-S6K1 cDNA (pRK7-p70 S6K1, from Addgene) by PCR, and both cDNAs of p70S6K1 and p85 S6K1 were subcloned into pcDNA3.1-Flag eukaryotic expression vectors, namely pcDNA3.1-Flag-p70 S6K1, pcDNA3.1-Flag-p85 S6K1. Notably, to avoid expression of p70 S6K1 in p85 S6K1 expression vector, the p70 translation initiate site (70-72th bp, ATG) was changed into TTG.
(2) Human 40S ribosomal protein S6 cDNA was obtained from HEK293 cells by RT-PCR and was subcloned into prokaryotic expression vector pGEX-6P-1 (pGEX-6P-1-S6). GST-S6 protein was induced by isopropylβ-D-1-thiogalactopyranoside (IPTG) and purified using GST-agarose beads. Purified GST-S6 protein will be used as substrate for S6K1 in vitro kinase assay.
(3) Recombinant mTOR shRNA expression vector (psiLV-H1-mTOR) was constructed and transfected into human breast cancer cell line MCF-7. Endogenous mTOR expression was significantly down-regulated after 72-96 h.
2 mTOR-independent, IKK-dependent activation of p85 but not p70 S6K1 by tumor necrosis factor-α(TNF-α) and hydrogen peroxide (H2O2).
(1) Different reaction of p70 S6K1 and p85 S6K1 to H2O2 and TNF-α.
It has been demonstrated that phosphorylation of S6K1 (T389/412) and S6 (S235/236) stimulated by extracellular and intracellular signals is rapamycin-sensitive. We found that phosphorylation of p70 S6K1 (T389), p85 S6K1 (T412) and S6 (S235/236) stimulated by insulin and amino acid were blocked completely by 100 nM rapamycin in MCF-7 cells. When stimulated by H2O2 and TNF-α, however, p70 S6K1 (T389) but not p85 S6K1 (T412) was inhibited by rapamycin. Coordinate with the rapamycin-insensitive activation of p85 S6K1 (T412), phoshorylation of S6 (S235/236) was also elevated by H2O2 and TNF-αin the presence of rapamycin. Moreover, H2O2 stimulated phosphorylation of p85 S6K1 (T412) but inhibited p70 S6K1 (T389) in a variety of cells (MG63, osteoblasts, MCF-7 and HCT116). Taken together, it is suggested that p70 S6K1 and p85 S6K1 may react differently to some signals such as H2O2 and TNF-α.
(2) Rapamycin and amino acid-independent phosphorylation of p85 S6K1 (T412) but not p70 S6K1 (T389) by H2O2.
Previous studies suggest that amino acid-deprivation and rapamycin prevent mTORC1 activation by vairous upstream signals. In this study, we found that in MCF-7, HeLa and HCT116 cells, H2O2 could induce endogenous p85 S6K1 (T412) but not p70 S6K1 (T389) phosphorylation in the presence rapamycin or deprivation of amino acid. Moreover, overexpressed p85 S6K1 but not p70 S6K1 was also phosphorylated on Thr412 by H2O2 in rapamycin-insensitive manner.
(3) Rapamycin-insensitive activation of p85 S6K1 by H2O2 and TNF-αis responsible for rapamycin-independent S6 (S235/236) phosphorylation.
Phosphorylation of S6 on S235/236 is critical for its activation. We found that H2O2 and TNF-αstimulated phosphorylation of S6 (S235/236) in the presence of rapamycin in MCF-7 and HCT116 cells. Furthermore, overexpression of p85 S6K1 but not p70 S6K1 enhanced rapamycin-independent phosphorylation S6 (S235/236). Most importantly, in vitro kinase assay clearly showed that immuno-purified Flag-p85 S6K1 but not Flag-p70 S6K1 was responsible for H2O2 stimulated rapamycin-independent phosphorylation of S6 (S235/236). It is suggested that p85 S6K1 but not p70 S6K1 is responsible for rapamycin-independent activation of S6.
(4) mTOR (mTORC1 and mTORC2) is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.
Knockdown of endogenous mTOR expression by shRNA deceased constitutive phosphorylation of p70/p85 S6K1 (T389/412) and S6 (S235/236), but was unable to prevent H2O2 induced p85 S6K1 (T412) and S6 (S235/236) phosphorylation.
(5) TSC2 is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.
It has been shown that IKK-βactivated mTORC1 and S6K1 by phosphorylation and inhibition mTORC1 negative regulator TSC1/TSC2. It was found in this study that H2O2 induced p85 S6K1 (T412) but not p70 S6K1 (T389) phosphorylation both in TSC2+/+P53-/- mouse embryonic fibroblast (MEF) and in TSC2-/- P53-/-MEFs either in the presence or absence of rapamycin. This suggests that TSC2 is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.
(6) IKK is required for mTOR-independent activation of p85 S6K1 and S6 by H2O2.
To detect upstream signals involved in mTOR-independent activation of p85 S6K1 and S6, PI3-K and IKK specific inhibitors were used. It is revealed that IKK inhibitor, but not PI3-K and mTOR inhibitor blocks H2O2-induced p85 S6K1 (T412) and S6 (S235/236) phosphorylation. Further studies demonstrated that overexpressed myc-IKK-βassociated with Flag-p85 S6K1 and this association was enhanced by H2O2 treatment. These results demonstrate that IKK is required for mTOR-independent activation of p85 S6K1 and S6 by H2O2.
In a summary, our study reveals a novel regulatory mechanism about S6K1. H2O2 and TNF-αactivate p85 but not p70 S6K1 by mTOR-independent, IKK-dependent pathway, which is responsible for rapamycin-insnesitive and mTORC1-independnet phosphorylation of S6 (S235/236). The finding will be helpful for us to understand the functions of p85 S6K1 and mechanisms involved in rapamycin-resistance in rapamycin-based cancer therapy.
mTOR蛋白激酶活性的发挥有赖于其在细胞内与其他分子结合形成复合物：mTOR复合物1 (mTOR complex1, mTORC1)和mTORC2。大环内酯类药物雷帕霉素与细胞内蛋白分子即FK506-结合蛋白12 (FK506-binding protein 12, FKBP12)结合后,对mTORC1发挥特异性的抑制作用。接受生长因子及胰岛素等信号刺激而活化的PI3-K/Akt(蛋白激酶B,PKB),能够磷酸化结节性硬化复合物2(tuberous sclerosis complex 2, TSC2),从而减弱后者对mTORC1的抑制作用。活化的mTORC1可以直接磷酸化其下游底物,40S核糖体蛋白S6激酶(40Sribosomal protein S6 kinase1, S6K1)和真核细胞翻译起始因子4E-结合蛋白1(eukaryotic translation initiation factor 4E binding protein 1,4E-BP1)。S6K1Thr389位点磷酸化后使其激后,并磷酸化核糖体蛋白S6,从而启动5'-Top mRNA (5'-terminal oligopyrimidine tract mRNA)的翻译。被mTORC1磷酸化的4E-BP1则不能再与eIF-4E结合,从而启动5’-帽结构依赖性(5'-cap-dependent)的蛋白翻译。由此二者共同促进蛋白质翻译。
S6Ks属于AGC丝／苏蛋白质激酶家族。哺乳动物细胞主要表达该激酶的两种分子,即S6K1和S6K2(又分别称为S6Kα和S6Kβ),它们由不同的基因编码,但全序列具有很高程度的同源性。S6K1有两种亚型,这两种亚型是由同一个转录子(mRNA)通过选择不同的翻译起始位点而得到的不同产物。p70 S6K1,主要分布于细胞质中；p85 S6K1的N-端较p70 S6K1多了一段由23个氨基酸残基构成的核定位信号(nuclear localization signal, NLS),主要分布于细胞核内。
S6K1是mTORC1最主要的效应分子之一,其调节方式常被作为研究mTORC1信号通路的模型。虽然S6K1的完全及持续活化需要多个生长因子诱导的磷酸化反应,Thr389位点的磷酸化对于S6K1的活化尤其重要,有研究证明,用丙氨酸替代该位点的苏氨酸能阻断S6K1激酶结构域的活化。体外实验证明,mTORC1可以直接磷酸化p70 S6K1的Thr389,而经雷帕霉素处理的细胞,S6K1的Thr389位点发生快速去磷酸化从而使S6K1失活。
最近大量研究报道了S6Ks在调节细胞大小、肿瘤侵袭、迁移以及血管生成、胰岛素抵抗及寿命等生理病理进程中发挥了重要作用。然而,上游信号是如何调节S6Ks以及S6Ks通过何种方式调节细胞的生理过程还没有完全阐明。由于目前还没有能快速抑制S6Ks活性的S6K1或S6K2特异性抑制剂,现有的关于S6Ks活性抑制效应研究的大部分结果,主要通过雷帕霉素抑制mTORC1而间接抑制SK6s的功能而得到。但新近报道,S6K1能通过雷帕霉素抵抗及mTORC1非依赖性的方式发生活化。而且,雷帕霉素及其类似物治疗肿瘤时,常发生雷帕霉素抵抗,其机制还不明确。因此,阐明S6Ks的mTORC1非依赖性活化机制对进一步明确S6Ks功能及其在人类疾病中的作用具有重要的意义。
目前普遍认为S6K1两种亚型,即p70 S6K1和p85 S6K1的调控机制与功能是相同的,所以多数研究主要是针对细胞质亚型的p70 S6K1,而细胞核亚型p85S6K1的功能其及调控机制被忽略,因此对于p85 S6K1的功能及调控机制鲜有报道。
肿瘤坏死因子(tumor necrosis factor-α, TNF-α)和活性氧(reactive oxygenspecies, ROS)在肿瘤形成和肿瘤进展中发挥重要作用。最近研究发现,TNF-α和ROS是mTORC1和p70 S6K1的上游信号分子,TNF-α可通过IκB激酶(IκB Kinase, IKK)磷酸化并抑制TSC1,从而激活mTORC1与p70 S6K1, ROS可通过尚不清楚的机制激活或抑制mTORC1和p70 S6K1,但它们对p85 S6K1的调控及其机制还未见报道。
在本研究中,我们发现p85 S6K1和p70 S6K1具有新的、不同的调节机制与功能。我们首次证明,TNF-α和H2O2磷酸化激活p85 S6K1(T412)是雷帕霉素非依赖的,而对p70 S6K1(T389)的活化却可被雷帕霉素抑制,且这种活化作用不需要mTOR(mTORC1/2),但依赖于IKK。
一、mTOR sh RNA, p70 S6K1和p85 S6K1真核表达载体的构建及谷胱甘肽-S-转移酶(Glutathione-S-Transferase, GST)-S6蛋白的制备
(1)利用PCR技术,将69 bp(23个氨基酸)加到大鼠p70 S6K1 cDNA的N-末端,扩增得到p85 S6K1的cDNA
将p70/p85 S6K1的cDNA克隆至pcDNA3.1-Flag真核表达载体中,成功构建pcDNA3.1-Flag-p70 S6K1和pcDNA3.1-Flag-p85 S6K1真核表达载体。为了防止在p85 S6K1表达载体翻译出p70 S6K1蛋白,我们将p70 S6K1翻译起始位点的ATG (70-72th bp)突变为TTG。
(2)GST-S6蛋白的制备
提取人胚胎肾细胞293 (human embryonic kidney 293, HEK293)的总mRNA作为模板,通过RT-PCR获得人40S核糖体蛋白S6 cDNA,将其克隆至原核表达载体pGEX-6P-1,命名为pGEX-6P-1-S6。GST-S6融合蛋白经异丙基β-D-1-半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)诱导表达后,利用GST-琼脂糖beads进行纯化。纯化的蛋白将作为S6K1体外激酶活性检测的底物。
(3)构建重组mTOR sh RNA表达载体(psiLV-H1-mTOR)
成功构建mTOR sh RNA表达载体(psiLV-H1-mTOR),在脂质体的介导下转染人乳腺癌细胞系MCF-7。转染72-96h后可见内源性mTOR的表达明显下调。
二、TNF-α和过氧化氢(hydrogen peroxide, H2O2)以mTOR非依赖,IKK依赖的方式激活p85 S6K1,但不激活p70 S6K1
(1)p70 S6K1和p85 S6K1对TNF-α及H2O2刺激产生不同反应
现有的研究证明,细胞内外各种信号诱导的S6K1(T389/412)和S6(S235/236)的磷酸化反应对雷帕霉素敏感。我们的实验结果显示,在MCF-7细胞中,加入100 nM的雷帕霉素,可以阻断胰岛素及氨基酸诱导的p70 S6K1(T389)、p85 S6K1(T412)和S6(S235/236)的磷酸化。然而,雷帕霉素能抑制TNF-α和HzO2刺激后MCF-7细胞的p70 S6K1的磷酸化,但对p85 S6K1的磷酸化却没有抑制作用。与p85 S6K1(T412)的这种对雷帕霉素不敏感的磷酸化结果一致的是,在有雷帕霉素的作用下,TNF-α和H2O2也能使S6(S235/236)的磷酸化水平增加。而且,H2O2使多种细胞(人骨肉瘤细胞系MG63,小鼠原代成骨细胞OB,乳腺癌细胞系MCF-7和人结肠癌细胞系HCT116的p85 S6K1(T412)发生磷酸化的同时,却抑制p70 S6K1(T389)的磷酸化。以上结果提示,p70 S6K1和p85 S6K1对某些上游信号(如H2O2和TNF-α)存在完全不同的反应。
(2)H2O2以不依赖于雷帕霉素和氨基酸的方式引起p85 S6K1(T412)磷酸化,但不能引起p70 S6K1(T389)的磷酸化
现有研究表明,氨基酸饥饿和雷帕霉素能抑制多种上游信号对mTORC1的活化。我们发现MCF-7, HeLa和HCT116细胞在有雷帕霉素或者氨基酸饥饿的情况下,H2O2能诱导内源性的p85 S6K1(T412)发生磷酸化,但不能诱导p70S6K1(T389)发生磷酸化。而且,H2O2也能使过表达的Flag-p85 S6K1 (T412)发生磷酸化,这种作用对雷帕霉素不敏感。但H2O2不能诱导过表达的Flag-p70S6K1(T389)发生雷帕霉素非依赖磷酸化。
(3)H2O2和TNF-α引起雷帕霉素非依赖的p85 S6K1激活参与雷帕霉素存在时S6(S235/236)的磷酸化激活
S6的S235/236位点的磷酸化对其自身的活化非常关键。我们发现,MCF-7和HCT116细胞中,在有雷帕霉素的情况下,H2O2和TNF-α也能激活S6(S235/236)。过表达的Flag-p85 S6K1而非Flag-p70 S6K1能增强这种雷帕霉素非依赖的S6(S235/236)磷酸化。更重要的是,体外激酶反应结果显示,免疫纯化的Flag-p85 S6K1而不是Flag-p70 S6K1,负责H202引起的S6(S235/236)雷帕霉素非依赖的磷酸化。以上结果提示,是p85 S6K1而非p70 S6K1与雷帕霉素非依赖的S6活化有关。
(4) mTOR (mTORCl和mTORC2)与H2O2引起的p85 S6K1雷帕霉素非依赖的活化无关
利用sh RNA敲低内源性mTOR的表达,使内源性p70/p85 S6K1(T389/412)和S6(S235-36)的磷酸化下降,但不能抑制H2O2诱导的p85 S6K1(T412)和S6(S235/236)的磷酸化。因此,mTOR与H2O2引起的p85 S6K1、S6雷帕霉素非依赖的活化无关。
(5)TSC2不参与H2O2激活p85 S6K1
IKK-β通过磷酸化TSC1作用于mTORC1的上游负性调控分子TSC1/2复合物,激活mTORC1及S6K1。我们选用TSC2野生型及敲除型的小鼠胚胎成纤维细胞(mouse embryo fibroblast, MEF)系TSC2+/+P53-/-MEF和TSC2-/-P53-/-MEF,发现两种细胞在H2O2的作用下,均出现了p85 S6K1(T412)磷酸化水平的增加。而且,有雷帕霉素存在时,两种细胞中均出现剂量效应的p85 S6K1(T412)被H2O2激活,而p70 S6K(T389)被完全抑制,因此,H2O2引起p85 S6K1雷帕霉素非依赖的磷酸化与TSC2无关。
(6)IKK参与H2O2引起的不依赖nTOR的p85 S6K1和S6活化
为进一步研究参与这种不依赖mTOR的p85 S6K1和S6活化的上游信号通路,我们用P13-K和IKK特异性抑制剂进行干预。结果发现,IKK抑制剂能够阻断H2O2诱导的p85 S6K1 (T412)和S6(S235/236)发生的磷酸化,但是PI3-K抑制剂和mTOR抑制剂对它们的磷酸化则没有抑制作用。我们又进一步证明,过表达的myc-IKK-β与Flag-p85 S6K1之间存在相互作用,而且H2O2能增强这种作用。这些结果表明,IKK参与了H2O2诱导的p85 S6K1和S6不依赖nTOR的活化。
综上所述,我们的研究结果揭示了一种新的S6K1调控机制。雷帕霉素存在时,H2O2和TNF-α能激活p85 S6K1但不能激活p70 S6K1,这种激活作用不依赖于mTOR,但是依赖于IKK信号通路,而且这种激活作用负责S6(S235/236)的雷帕霉素及mTOR非依赖性活化。这些发现将有助于我们进一步阐明p85S6K1的功能和雷帕霉素及其类似物治疗肿瘤时形成雷帕霉素抵抗的机制,为发展新的基于mTOR通路的肿瘤靶向治疗方案提供理论基础。
The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth proliferation and survival. The mTOR pathway is deregulated in human diseases such as cancer and type 2 diabetes. Recnet years, mTOR signaling have attracted broad scientific and clinical interest.
Kinase acitivity of mTOR depends on the complexes formation of mTOR by combining with other molecules in cells, mTOR complex 1 (mTORC1) and mTORC2. Macrolides rapamycin specifically inhibits the activity of mTORC1 by combining with intracellular protein FK506-binding-protein 12 (FKBP12). Activated phosphoinositide 3-kinase (PI3-K)/Akt (protein kinase B, PKB) induced by growth factor and insulin stimulation may phosphorylate tuberous sclerosis complex 2 (TSC2), which will attenuate inhibitive effect on mTORCl. Activated mTORC1 will directly phosphorylate its downstream substrates,40S ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Phosphorylation at Thr389 site of S6K1 results in its activation and may phosphorylate and activate 40S ribosomal protein S6, which will initiate translation of 5'-terminal oligopyrimidine tract mRNA (5'-Top mRNAs). Phosphorylated 4E-BP1 by mTOR can no longer bind eIF4E, thus triggers a 5'-cap dependent protein translation. Consequently, protein synthesis begins.
S6Ks belongs to the AGC family of Ser/Thr kinases. Mammalian cells express two forms of the kinase, S6K1 and S6K2 (also known as S6Kαand S6Kβrespectively), which are encoded by two different genes and share a high level of overall sequence homology. S6K1 has two isoforms produced from the same transcript by alternative translational start sites. The shorter form, which is largely cytoplasmic, was initially termed p70 S6K1, whereas the larger form, which appears to be exclusively nuclear, was referred to as p85 S6K1. In the case of p85 S6K1, the additional 23-amino-acid sequence residing at its amino terminus has been shown to function as a nuclear localization signal (NLS).
S6K1 is the best characterized effector of mTOR, and its regulation serves as a model for mTOR signaling. A full and sustained S6K1 activation requires multiple growth factor-induced phosphorylation events. The phosphorylation of Thr389 by mTORC1 is particularly important because subsititution of the residue with alanine blocks the activation of the kinase domain. There is also evidence indicating that p70 S6K1 (T389) is directly phoshorylated by mTORC1 in vitro and rapamycin treatment of cells triggers the rapid dephosphorylation of Thr389 and inactivation of S6K1.
Recent studies have addressed many important roles of S6Ks in cell size control, tumor invasiveness, motility and angiogenesis, insulin resistance and lifespan. Regulation of S6Ks by upstream signals and mechanisms through which S6Ks regulate cellular process, however, remains to be clarified. For lack of S6K1-or S6K-2-specific inhibitors which is useful in defining the effects of acute inhibition of S6Ks, most results for S6Ks functions come from inhibition of mTORC1 by rapamycin. But recent studies have addressed activation of S6K1 in rapamycin-resistant and mTORC1-independent manner. Moreover, mechanisms involved in rapamycin-resistance in rapamycin-based cancer therapy remain unclear. Defining mTORC1-independent mechanisms that regulate S6Ks is important for understanding the functions of S6Ks and its roles in human diseases.
Findings to date would suggest that two isoforms of S6K1, p70 S6Kland p85 S6K1 are controlled in a similar manner. Most studies have been focused on cytoplasmic isoform p70 S6K1. Functions and regulation of nuclear isoform p85 S6K1 have been neglected and are poorly understood.
TNF-αand reactive oxygen species (ROS) play important role in carcinogenesis and tumor progression. Previous studies have demonstrated that TNF-αand ROS are important upstream signals of mTORC1 and p70 S6K1. But their roles in regulation of p85 S6K1 have not been reported.
In this study, we found novel and differential regulation and function of p70 and p85 S6K1. We demonstrated for the first time that p85 but not p70 S6K1 was activated by TNF-αand H2O2 through mTOR-independent, I (?)B-kinase (IKK)-dependent mechanism.
1 Construction of mTOR shRNA and p70 S6K1, p85 S6K1 expression vectors and preparation of Glutathione-S-Transferase (GST)-S6 protein
(1) N-terminal (69 bp,23 amino acids) of p85 S6K1 was added to rat p70-S6K1 cDNA (pRK7-p70 S6K1, from Addgene) by PCR, and both cDNAs of p70S6K1 and p85 S6K1 were subcloned into pcDNA3.1-Flag eukaryotic expression vectors, namely pcDNA3.1-Flag-p70 S6K1, pcDNA3.1-Flag-p85 S6K1. Notably, to avoid expression of p70 S6K1 in p85 S6K1 expression vector, the p70 translation initiate site (70-72th bp, ATG) was changed into TTG.
(2) Human 40S ribosomal protein S6 cDNA was obtained from HEK293 cells by RT-PCR and was subcloned into prokaryotic expression vector pGEX-6P-1 (pGEX-6P-1-S6). GST-S6 protein was induced by isopropylβ-D-1-thiogalactopyranoside (IPTG) and purified using GST-agarose beads. Purified GST-S6 protein will be used as substrate for S6K1 in vitro kinase assay.
(3) Recombinant mTOR shRNA expression vector (psiLV-H1-mTOR) was constructed and transfected into human breast cancer cell line MCF-7. Endogenous mTOR expression was significantly down-regulated after 72-96 h.
2 mTOR-independent, IKK-dependent activation of p85 but not p70 S6K1 by tumor necrosis factor-α(TNF-α) and hydrogen peroxide (H2O2).
(1) Different reaction of p70 S6K1 and p85 S6K1 to H2O2 and TNF-α.
It has been demonstrated that phosphorylation of S6K1 (T389/412) and S6 (S235/236) stimulated by extracellular and intracellular signals is rapamycin-sensitive. We found that phosphorylation of p70 S6K1 (T389), p85 S6K1 (T412) and S6 (S235/236) stimulated by insulin and amino acid were blocked completely by 100 nM rapamycin in MCF-7 cells. When stimulated by H2O2 and TNF-α, however, p70 S6K1 (T389) but not p85 S6K1 (T412) was inhibited by rapamycin. Coordinate with the rapamycin-insensitive activation of p85 S6K1 (T412), phoshorylation of S6 (S235/236) was also elevated by H2O2 and TNF-αin the presence of rapamycin. Moreover, H2O2 stimulated phosphorylation of p85 S6K1 (T412) but inhibited p70 S6K1 (T389) in a variety of cells (MG63, osteoblasts, MCF-7 and HCT116). Taken together, it is suggested that p70 S6K1 and p85 S6K1 may react differently to some signals such as H2O2 and TNF-α.
(2) Rapamycin and amino acid-independent phosphorylation of p85 S6K1 (T412) but not p70 S6K1 (T389) by H2O2.
Previous studies suggest that amino acid-deprivation and rapamycin prevent mTORC1 activation by vairous upstream signals. In this study, we found that in MCF-7, HeLa and HCT116 cells, H2O2 could induce endogenous p85 S6K1 (T412) but not p70 S6K1 (T389) phosphorylation in the presence rapamycin or deprivation of amino acid. Moreover, overexpressed p85 S6K1 but not p70 S6K1 was also phosphorylated on Thr412 by H2O2 in rapamycin-insensitive manner.
(3) Rapamycin-insensitive activation of p85 S6K1 by H2O2 and TNF-αis responsible for rapamycin-independent S6 (S235/236) phosphorylation.
Phosphorylation of S6 on S235/236 is critical for its activation. We found that H2O2 and TNF-αstimulated phosphorylation of S6 (S235/236) in the presence of rapamycin in MCF-7 and HCT116 cells. Furthermore, overexpression of p85 S6K1 but not p70 S6K1 enhanced rapamycin-independent phosphorylation S6 (S235/236). Most importantly, in vitro kinase assay clearly showed that immuno-purified Flag-p85 S6K1 but not Flag-p70 S6K1 was responsible for H2O2 stimulated rapamycin-independent phosphorylation of S6 (S235/236). It is suggested that p85 S6K1 but not p70 S6K1 is responsible for rapamycin-independent activation of S6.
(4) mTOR (mTORC1 and mTORC2) is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.
Knockdown of endogenous mTOR expression by shRNA deceased constitutive phosphorylation of p70/p85 S6K1 (T389/412) and S6 (S235/236), but was unable to prevent H2O2 induced p85 S6K1 (T412) and S6 (S235/236) phosphorylation.
(5) TSC2 is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.
It has been shown that IKK-βactivated mTORC1 and S6K1 by phosphorylation and inhibition mTORC1 negative regulator TSC1/TSC2. It was found in this study that H2O2 induced p85 S6K1 (T412) but not p70 S6K1 (T389) phosphorylation both in TSC2+/+P53-/- mouse embryonic fibroblast (MEF) and in TSC2-/- P53-/-MEFs either in the presence or absence of rapamycin. This suggests that TSC2 is not required for rapamycin-insensitive activation of p85 S6K1 by H2O2.
(6) IKK is required for mTOR-independent activation of p85 S6K1 and S6 by H2O2.
To detect upstream signals involved in mTOR-independent activation of p85 S6K1 and S6, PI3-K and IKK specific inhibitors were used. It is revealed that IKK inhibitor, but not PI3-K and mTOR inhibitor blocks H2O2-induced p85 S6K1 (T412) and S6 (S235/236) phosphorylation. Further studies demonstrated that overexpressed myc-IKK-βassociated with Flag-p85 S6K1 and this association was enhanced by H2O2 treatment. These results demonstrate that IKK is required for mTOR-independent activation of p85 S6K1 and S6 by H2O2.
In a summary, our study reveals a novel regulatory mechanism about S6K1. H2O2 and TNF-αactivate p85 but not p70 S6K1 by mTOR-independent, IKK-dependent pathway, which is responsible for rapamycin-insnesitive and mTORC1-independnet phosphorylation of S6 (S235/236). The finding will be helpful for us to understand the functions of p85 S6K1 and mechanisms involved in rapamycin-resistance in rapamycin-based cancer therapy.
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