LKB1

LKB1
  • 文章类型: Journal Article
    改变的蛋白质泛素化与癌症有关。据报道,E3泛素连接酶的新型三方基序(TRIM)家族在发育中起着至关重要的作用,增长,和各种肿瘤的转移。TRIM家族成员TRIM27在多种癌症中充当肿瘤发展的潜在启动子。然而,关于胶质母细胞瘤(GBM)中TRIM27的生物学特征和临床相关性知之甚少。这里,我们报告了GBM组织和GBM细胞系中TRIM27表达升高的发现。进一步的功能分析显示,TRIM27缺失在体外和体内均抑制GBM细胞生长。此外,我们发现TRIM27通过增强Warburg效应促进GBM细胞的生长。此外,LKB1/AMPK/mTOR通路的失活对于TRIM27在GBM中的致癌作用至关重要.机械上,TRIM27可以直接与LKB1结合,促进LKB1的泛素化和降解,进而增强Warburg效应和GBM进程。总的来说,这些数据提示TRIM27通过抑制LKB1/AMPK/mTOR轴参与GNM发病机制,可能成为GBM患者潜在的诊断和治疗标志物.
    Altered protein ubiquitination is associated with cancer. The novel tripartite motif (TRIM) family of E3 ubiquitin ligases have been reported to play crucial roles in the development, growth, and metastasis of various tumors. The TRIM family member TRIM27 acts as a potential promoter of tumor development in a wide range of cancers. However, little is known regarding the biological features and clinical relevance of TRIM27 in glioblastoma (GBM). Here, we report findings of elevated TRIM27 expression in GBM tissues and GBM cell lines. Further functional analysis showed that TRIM27 deletion inhibited GBM cell growth both in vitro and in vivo. Furthermore, we found that TRIM27 promoted the growth of GBM cells by enhancing the Warburg effect. Additionally, the inactivation of the LKB1/AMPK/mTOR pathway was critical for the oncogenic effects of TRIM27 in GBM. Mechanistically, TRIM27 could directly bind to LKB1 and promote the ubiquitination and degradation of LKB1, which in turn enhanced the Warburg effect and GBM progression. Collectively, these data suggest that TRIM27 contributes to GNM pathogenesis by inhibiting the LKB1/AMPK/mTOR axis and may be a promising candidate as a potential diagnostic and therapeutic marker for patients with GBM.
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  • 文章类型: Journal Article
    Erianin,在石斛提取物中发现的一种二苄基化合物,具有广泛的抗癌活性。然而,其在胃癌(GC)中的作用机制尚不清楚。LKB1是一种抑癌基因,它的突变是各种癌症的重要驱动因素。然而,一些研究报告了相互矛盾的发现。在这项研究中,我们结合生物信息学和体内外实验来研究Erianin治疗GC的作用和潜在机制。结果表明,LKB1在患者肿瘤组织和GC细胞中高表达,与患者预后不良有关。Erianin能促进GC细胞凋亡,抑制划痕修复,迁移,入侵,和上皮-间质转化(EMT)特征。Erianin剂量依赖性地抑制LKB1,SIK2,SIK3和PARD3的表达,但对SIK1没有显着影响。Erianin还抑制CDX小鼠模型中的肿瘤生长。出乎意料的是,5-FU对LKB1也表现出一定的抑制作用。Erianin和5-FU的组合显著提高了5-FU在GC细胞和异种移植小鼠模型的生长中的抗肿瘤功效。总之,Erianin是一种潜在的抗GC化合物,可以通过靶向LKB1-SIK2/3-PARD3信号轴来抑制GC生长和EMT特性。Erianin和5-FU的协同作用提示了用于GC治疗的有希望的治疗策略。
    Erianin, a bibenzyl compound found in dendrobium extract, has demonstrated broad anticancer activity. However, its mechanism of action in gastric cancer (GC) remains poorly understood. LKB1 is a tumor-suppressor gene, and its mutation is an important driver of various cancers. Yet some studies have reported contradictory findings. In this study, we combined bioinformatics and in vitro and in vivo experiments to investigate the effect and potential mechanism of Erianin in the treatment of GC. The results show that LKB1 was highly expressed in patients\' tumor tissues and GC cells, and it was associated with poor patient prognosis. Erianin could promote GC cell apoptosis and inhibit the scratch repair, migration, invasion, and epithelial-mesenchymal transition (EMT) characteristics. Erianin dose-dependently inhibited the expression of LKB1, SIK2, SIK3, and PARD3 but had no significant effect on SIK1. Erianin also inhibited tumor growth in CDX mice model. Unexpectedly, 5-FU also exhibited a certain inhibitory effect on LKB1. The combination of Erianin and 5-FU significantly improved the anti-tumor efficacy of 5-FU in the growth of GC cells and xenograft mouse models. In summary, Erianin is a potential anti-GC compound that can inhibit GC growth and EMT properties by targeting the LKB1-SIK2/3-PARD3-signaling axis. The synergistic effect of Erianin and 5-FU suggests a promising therapeutic strategy for GC treatment.
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  • 文章类型: Journal Article
    脂质代谢紊乱是几种严重影响公众健康的慢性代谢性疾病的主要原因。Salusin-α,血管活性肽,已被证明可以减轻脂质代谢紊乱,尽管其作用机制尚未报道。为了研究Salusin-α对脂质代谢的影响和潜在机制,使用慢病毒载体过表达或敲低Salusin-α。慢病毒转染HepG2细胞后,游离脂肪酸(FFA)诱导肝细胞脂肪变性。使用油红O染色并通过测量几种生化指标来评估脂质积累的程度。随后,生物信息学用于分析可能参与脂质代谢紊乱的信号通路。最后,半定量PCR和免疫印迹用于验证肝激酶B1(LKB1)/AMPK通路的参与。化合物C,AMPK的抑制剂,被用来进一步证实这种机制的参与。结果表明,Salusin-α显着减弱脂质积累,炎症和氧化应激。此外,Salusin-α增加了LKB1和AMPK的水平,抑制固醇调节元件结合蛋白-1c的表达,脂肪酸合成酶和乙酰辅酶A羧化酶。化合物C的添加消除了Salusin-α介导的AMPK对下游信号分子的调节。总之,Salusin-α的过表达激活了LKB1/AMPK途径,这反过来又抑制了HepG2细胞中的脂质积累。这提供了对Salusin‑α改善脂质代谢紊乱的潜在机制的见解,同时确定了潜在的治疗靶标。
    Lipid metabolism disorders are a major cause of several chronic metabolic diseases which seriously affect public health. Salusin‑α, a vasoactive peptide, has been shown to attenuate lipid metabolism disorders, although its mechanism of action has not been reported. To investigate the effects and potential mechanisms of Salusin‑α on lipid metabolism, Salusin‑α was overexpressed or knocked down using lentiviral vectors. Hepatocyte steatosis was induced by free fatty acid (FFA) after lentiviral transfection into HepG2 cells. The degree of lipid accumulation was assessed using Oil Red O staining and by measuring several biochemical indices. Subsequently, bioinformatics was used to analyze the signaling pathways that may have been involved in lipid metabolism disorders. Finally, semi‑quantitative PCR and western blotting were used to verify the involvement of the liver kinase B1 (LKB1)/AMPK pathway. Compound C, an inhibitor of AMPK, was used to confirm this mechanism\'s involvement further. The results showed that Salusin‑α significantly attenuated lipid accumulation, inflammation and oxidative stress. In addition, Salusin‑α increased the levels of LKB1 and AMPK, which inhibited the expression of sterol regulatory element binding protein‑1c, fatty acid synthase and acetyl‑CoA carboxylase. The addition of Compound C abrogated the Salusin‑α‑mediated regulation of AMPK on downstream signaling molecules. In summary, overexpression of Salusin‑α activated the LKB1/AMPK pathway, which in turn inhibited lipid accumulation in HepG2 cells. This provides insights into the potential mechanism underlying the mechanism by which Salusin‑α ameliorates lipid metabolism disorders while identifying a potential therapeutic target.
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  • 文章类型: Journal Article
    探讨LKB1表达缺失在非小细胞肺癌(NSCLC)中的发生率和预后相关性及协同作用。我们分析了来自188例转移性和60例非转移性可手术I-IIIA期NSCLC患者的手术标本,以评估其与各种过程相关的LKB1和pAMPK蛋白的表达.研究的因素包括抗肿瘤免疫反应调节剂STING和PD-L1;促血管生成,EMT和细胞周期目标,以及与转移相关的(VEGFC,PDGFRα,PDGFRβ,p53,p16,细胞周期蛋白D1,ZEB1,CD24)靶标;和细胞粘附(β-连环蛋白)分子。通过免疫组织化学评估蛋白质表达水平;通过PCR评估LKB1和NEDD9的RNA水平,而KRAS外显子2和BRAFV600E突变通过Sanger测序进行评估。总的来说,在21%(51/248)的患者中观察到LKB1蛋白表达丢失,并且与组织型显着相关(p<0.001),KRAS突变(p<0.001),KC状态(伴随KRAS突变和p16下调)(p<0.001),STING损失(p<0.001),和高CD24表达(p<0.001)。STING丢失也与转移背景下的LKB1表达丢失显著相关(p=0.014)和肺腺癌(LUACs)(p=0.005)。此外,LKB1缺失与仅在LUACs中缺乏或低β-catenin膜表达显著相关,与转移状态(p=0.019)和转移情况(p=0.007)无关。出现LKB1丢失和伴随不存在或低β-catenin膜表达的肿瘤患者的中位总生存期明显低于20.50。52.99个月;p<0.001以及显著更大的死亡风险(HR:3.32,95%c.i.:1.71-6.43;p<0.001)。我们的发现强调了LKB1与STING和β-catenin在NSCLC中的协同作用的影响。在预后方面。
    To investigate the incidence and prognostically significant correlations and cooperations of LKB1 loss of expression in non-small cell lung cancer (NSCLC), surgical specimens from 188 metastatic and 60 non-metastatic operable stage I-IIIA NSCLC patients were analyzed to evaluate their expression of LKB1 and pAMPK proteins in relation to various processes. The investigated factors included antitumor immunity response regulators STING and PD-L1; pro-angiogenic, EMT and cell cycle targets, as well as metastasis-related (VEGFC, PDGFRα, PDGFRβ, p53, p16, Cyclin D1, ZEB1, CD24) targets; and cell adhesion (β-catenin) molecules. The protein expression levels were evaluated via immunohistochemistry; the RNA levels of LKB1 and NEDD9 were evaluated via PCR, while KRAS exon 2 and BRAFV600E mutations were evaluated by Sanger sequencing. Overall, loss of LKB1 protein expression was observed in 21% (51/248) patients and correlated significantly with histotype (p < 0.001), KRAS mutations (p < 0.001), KC status (concomitant KRAS mutation and p16 downregulation) (p < 0.001), STING loss (p < 0.001), and high CD24 expression (p < 0.001). STING loss also correlated significantly with loss of LKB1 expression in the metastatic setting both overall (p = 0.014) and in lung adenocarcinomas (LUACs) (p = 0.005). Additionally, LKB1 loss correlated significantly with a lack of or low β-catenin membranous expression exclusively in LUACs, both independently of the metastatic status (p = 0.019) and in the metastatic setting (p = 0.007). Patients with tumors yielding LKB1 loss and concomitant nonexistent or low β-catenin membrane expression experienced significantly inferior median overall survival of 20.50 vs. 52.99 months; p < 0.001 as well as significantly greater risk of death (HR: 3.32, 95% c.i.: 1.71-6.43; p <0.001). Our findings underscore the impact of the synergy of LKB1 with STING and β-catenin in NSCLC, in prognosis.
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  • 文章类型: Journal Article
    线粒体电子传递链(ETC)在调节铁死亡中的作用尚未完全阐明。这里,我们发现,ETC复合物I的药理学抑制降低泛醇水平,同时降低ATP水平并激活AMP激活的蛋白激酶(AMPK),这两种效应以其在促进和抑制铁性凋亡中的作用而闻名,分别。因此,复合物I抑制剂对谷胱甘肽过氧化物酶4(GPX4)抑制诱导的铁凋亡的影响有限。复合物I在LKB1-AMPK灭活细胞中的药理学抑制作用,或复合物I的遗传消融(不会触发明显的AMPK激活),消除AMPK介导的铁凋亡抑制作用,并使癌细胞对GPX4失活诱导的铁凋亡敏感。此外,复合物I抑制与放射疗法(RT)协同作用,通过在小鼠模型中诱导铁凋亡来选择性抑制LKB1缺陷型肿瘤的生长。我们的数据证明了复合物I在调节铁凋亡中的多方面作用,并提出了针对LKB1缺陷型癌症的铁凋亡诱导治疗策略。
    The role of the mitochondrial electron transport chain (ETC) in regulating ferroptosis is not fully elucidated. Here, we reveal that pharmacological inhibition of the ETC complex I reduces ubiquinol levels while decreasing ATP levels and activating AMP-activated protein kinase (AMPK), the two effects known for their roles in promoting and suppressing ferroptosis, respectively. Consequently, the impact of complex I inhibitors on ferroptosis induced by glutathione peroxidase 4 (GPX4) inhibition is limited. The pharmacological inhibition of complex I in LKB1-AMPK-inactivated cells, or genetic ablation of complex I (which does not trigger apparent AMPK activation), abrogates the AMPK-mediated ferroptosis-suppressive effect and sensitizes cancer cells to GPX4-inactivation-induced ferroptosis. Furthermore, complex I inhibition synergizes with radiotherapy (RT) to selectively suppress the growth of LKB1-deficient tumors by inducing ferroptosis in mouse models. Our data demonstrate a multifaceted role of complex I in regulating ferroptosis and propose a ferroptosis-inducing therapeutic strategy for LKB1-deficient cancers.
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  • 文章类型: Journal Article
    肿瘤抑制因子LKB1是一种丝氨酸/苏氨酸蛋白激酶,在人肺腺癌(LUAD)中经常发生突变。LKB1调节已知控制细胞极性和代谢的复杂信号网络;然而,介导LKB1肿瘤抑制活性的途径尚未完全确定.为了确定LKB1介导的生长抑制的机制,我们开发了一种基于球体的细胞培养试验来研究LKB1依赖性生长。然后,我们在球形培养物中进行了全基因组CRISPR筛选,发现LKB1抑制生长,在某种程度上,通过激活PIKFYVE脂质激酶。最后,我们使用化学抑制剂和pH敏感报告子确定LKB1通过以PIKFYVE依赖性方式促进野生型EGFR的内在化而损害生长.
    The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.
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  • 文章类型: Journal Article
    社会记忆是区分熟悉和未知物种的能力。它是社会认知的重要组成部分,因此对于建立社会关系至关重要。尽管社会记忆编码背后的神经回路机制已经得到了很好的研究,很少关注社会记忆处理的调节机制。多巴胺能系统,起源于中脑腹侧被盖区(VTA),是认知功能的关键调节剂。本研究旨在阐明其在调节社会记忆中的作用,并探讨其可能的分子机制。这里,我们表明,VTA多巴胺(DA)神经元的激活是形成所必需的,但不是检索,社会记忆。在社交互动之前抑制VTADA神经元,但不是在社交互动后24小时,第二天严重损害了社会歧视。此外,我们表明,VTADA神经元的激活受丝氨酸/苏氨酸蛋白激酶肝激酶B1(Lkb1)的调节。VTADA神经元中Lkb1的缺失降低了多巴胺能神经元爆发的频率。此外,Lkb1在调节社会行为中起着重要作用。成年小鼠VTA中Lkb1的遗传和病毒介导的缺失均损害了社会记忆,随后减弱了社会熟悉。总之,我们的研究结果提供了将社会记忆形成与小鼠VTADA神经元激活联系起来的直接证据,并说明了Lkb1在调节VTADA神经元功能中的关键作用。
    Social memory is the ability to discriminate between familiar and unknown conspecifics. It is an important component of social cognition and is therefore essential for the establishment of social relationships. Although the neural circuit mechanisms underlying social memory encoding have been well investigated, little focus has been placed on the regulatory mechanisms of social memory processing. The dopaminergic system, originating from the midbrain ventral tegmental area (VTA), is a key modulator of cognitive function. This study aimed to illustrate its role in modulating social memory and explore the possible molecular mechanisms. Here, we show that the activation of VTA dopamine (DA) neurons is required for the formation, but not the retrieval, of social memory. Inhibition of VTA DA neurons before social interaction, but not 24 h after social interaction, significantly impaired social discrimination the following day. In addition, we showed that the activation of VTA DA neurons was regulated by the serine/threonine protein kinase liver kinase B1 (Lkb1). Deletion of Lkb1 in VTA DA neurons reduced the frequency of burst firing of dopaminergic neurons. Furthermore, Lkb1 plays an important role in regulating social behaviors. Both genetic and virus-mediated deletions of Lkb1 in the VTA of adult mice impaired social memory and subsequently attenuated social familiarization. Altogether, our results provide direct evidence linking social memory formation to the activation of VTA DA neurons in mice and illustrate the crucial role of Lkb1 in regulating VTA DA neuron function.
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  • 文章类型: Journal Article
    背景:肥胖与给患者和社会带来巨大负担的多种代谢紊乱有关。白色脂肪组织(WAT)中的“褐变”现象已成为对抗代谢紊乱的有希望的治疗策略。然而,尽管抗糖尿病药物达格列净(DAPA)被认为可以促进“褐变”,“这种具体机制以前还不清楚。
    方法:在本研究中,用C57BL/6J雄性小鼠通过高脂饮食建立肥胖模型,和3T3-L1细胞用于诱导成熟脂肪细胞,并通过体内外实验结合探讨DAPA在“褐变”中的作用和机制。
    结果:结果表明,DAPA促进了WAT\“褐变\”并改善了代谢紊乱。此外,我们发现DAPA通过成纤维细胞生长因子受体1-肝激酶B1-磷酸腺苷活化蛋白激酶信号通路激活“褐变”。
    结论:这些发现为DAPA通过促进白色脂肪组织褐变治疗肥胖提供了合理的依据。
    BACKGROUND: Obesity is associated with a wide variety of metabolic disorders that impose significant burdens on patients and society. The \"browning\" phenomenon in white adipose tissue (WAT) has emerged as a promising therapeutic strategy to combat metabolic disturbances. However, though the anti-diabetic drug dapagliflozin (DAPA) is thought to promote \"browning,\" the specific mechanism of this was previously unclear.
    METHODS: In this study, C57BL/6 J male mice were used to establish an obesity model by high-fat diet feeding, and 3T3-L1 cells were used to induce mature adipocytes and to explore the role and mechanism of DAPA in \"browning\" through a combination of in vitro and in vivo experiments.
    RESULTS: The results show that DAPA promotes WAT \"browning\" and improves metabolic disorders. Furthermore, we discovered that DAPA activated \"browning\" through the fibroblast growth factor receptors 1-liver kinase B1-adenosine monophosphate-activated protein kinase signaling pathway.
    CONCLUSIONS: These findings provide a rational basis for the use of DAPA in treating obesity by promoting the browning of white adipose tissue.
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  • 文章类型: Journal Article
    γδT细胞在免疫监视中起着至关重要的作用,并且是先天免疫和适应性免疫之间的桥梁。然而,γδT细胞发育和功能的代谢需求和调节仍然知之甚少。在这项研究中,我们研究了肝激酶B1(Lkb1)的作用,丝氨酸/苏氨酸激酶,将细胞代谢与细胞生长和增殖联系起来,在γδT细胞生物学中。我们的发现表明,Lkb1不仅参与调节γδT谱系承诺,而且在γδT细胞效应子功能中起关键作用。具体来说,Lkb1的T细胞特异性缺失导致胸腺细胞发育受损,胸腺和外周淋巴组织中γδT细胞亚群发生明显变化。值得注意的是,Lkb1的缺失抑制了Vγ1和Vγ4γδT细胞的表达,促进产生IL-17的Vγ6γδT细胞的成熟,并导致致命的自身免疫性肝炎(AIH)的发生。值得注意的是,清除γδT细胞或阻断IL-17可显着减弱AIH。机械上,Lkb1缺乏破坏了代谢稳态和AMPK活性,伴随着mTORC1激活的增加,从而引起γδT细胞的过度活化并增强凋亡。有趣的是,在Lkb1缺陷小鼠中,AMPK的激活或mTORC1信号传导的抑制可有效抑制IL-17水平并减弱AIH.我们的发现强调了Lkb1在维持γδT细胞稳态和预防IL-17介导的自身免疫性疾病中的关键作用。为控制胸腺γδT细胞亚群确定和功能分化的代谢程序提供新的见解。
    γδ T cells play a crucial role in immune surveillance and serve as a bridge between innate and adaptive immunity. However, the metabolic requirements and regulation of γδ T-cell development and function remain poorly understood. In this study, we investigated the role of liver kinase B1 (Lkb1), a serine/threonine kinase that links cellular metabolism with cell growth and proliferation, in γδ T-cell biology. Our findings demonstrate that Lkb1 is not only involved in regulating γδ T lineage commitment but also plays a critical role in γδ T-cell effector function. Specifically, T-cell-specific deletion of Lkb1 resulted in impaired thymocyte development and distinct alterations in γδ T-cell subsets in both the thymus and peripheral lymphoid tissues. Notably, loss of Lkb1 inhibited the commitment of Vγ1 and Vγ4 γδ T cells, promoted the maturation of IL-17-producing Vγ6 γδ T cells, and led to the occurrence of fatal autoimmune hepatitis (AIH). Notably, clearance of γδ T cells or blockade of IL-17 significantly attenuated AIH. Mechanistically, Lkb1 deficiency disrupted metabolic homeostasis and AMPK activity, accompanied by increased mTORC1 activation, thereby causing overactivation of γδ T cells and enhanced apoptosis. Interestingly, activation of AMPK or suppression of mTORC1 signaling effectively inhibited IL-17 levels and attenuated AIH in Lkb1-deficient mice. Our findings highlight the pivotal role of Lkb1 in maintaining the homeostasis of γδ T cells and preventing IL-17-mediated autoimmune diseases, providing new insights into the metabolic programs governing the subset determination and functional differentiation of thymic γδ T cells.
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  • 文章类型: Journal Article
    AMP激活的蛋白激酶(AMPK)是细胞能量状态的传感器。当ADP:ATP和/或AMP:ATP比率(信号能量不足)增加激活时,AMPK用于恢复能量平衡。AMP与AMPK-γ亚基上的三个CBS重复序列(CBS1,CBS3,CBS4)中的一个或多个的结合通过三种互补机制激活激酶复合物:(i)通过上游激酶LKB1促进α-亚基Thr172磷酸化;(ii)保护免受Thr172去磷酸化;(iii)变构激活。令人惊讶的是,据报道,ADP的结合模拟了前两种效应,但不是第三个。我们现在表明,在生理相关浓度的Mg。ATP2-(高于标准测定中使用的那些)ADP结合确实引起变构激活。然而,ADP仅引起适度的激活,因为(与AMP不同),浓度略高于激活变得明显的浓度,ADP开始在催化位点引起竞争性抑制。我们的结果对ADP作用的生理相关性表示怀疑,并表明AMP是体内的主要激活剂。我们还在人α2β2γ1复合物的三个γ亚基CBS重复序列中的每一个上对与腺嘌呤核苷酸结合的疏水残基进行了突变,并检查了它们对AMP和ADP调节的影响。CBS3位点的突变对AMP激活的所有三种机制具有最大的影响,特别是在较低的ATP浓度下,而CBS4的突变降低了对AMP的敏感性。ADP的变构激活似乎需要所有三个位点。
    The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.
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