M2型丙酮酸激酶的功能及调节

糖酵解可以为机体迅速提供能量,有氧糖酵解更是肿瘤代谢的主要方式。丙酮酸激酶(pyruvate kinase,PK)是糖酵解途径的限速酶,存在4种类型,其中M2型PK(PKM2)分布最广泛,功能最重要。PKM2具有激酶催化活性,可以异位至线粒体影响细胞生存,进入细胞核后可以调控基因表达,可以作为肿瘤诊断的指标、治疗的靶点和预后的参考。对PKM2功能及其调节机制的了解,可以为疾病的诊断和治疗提供新思路。     

M2型丙酮酸激酶与肿瘤自噬

M2型丙酮酸激酶(M2 isoform pyruvate kinase, PKM2)是糖酵解途径的关键酶。PKM2在多种肿瘤中高表达,且与肿瘤发生发展密切相关。自噬(autophagy)是一个溶酶体依赖的胞内降解过程。自噬通过对肿瘤细胞葡萄糖摄取、糖酵解途径进行调控,从而影响肿瘤的生物学行为。近年来的研究逐步揭示,PKM2与肿瘤自噬之间可相互调控,PKM2可影响肿瘤自噬的发生和结果,自噬可影响PKM2的活性和蛋白量,且二者的相互作用与病人总体生存率密切相关。这些发现将为针对PKM2及自噬的肿瘤临床治疗带来新的思路。     

M2型丙酮酸激酶与宫颈癌关系的研究进展

M2型丙酮酸激酶是近年来发现的一种器官非特异性肿瘤标记物,在多种恶性肿瘤,尤其是在宫颈癌的发生发展中发挥着重要作用.本文综述了M2型丙酮酸激酶的生物学特征、及其在宫颈癌中的表达情况、致癌机制以及应用前景等.     

内皮细胞的M2型丙酮酸激酶有助于维持血管完整性

丙酮酸激酶(pyruvate kinase,PK)是细胞内催化糖酵解的关键酶,其中M2型丙酮酸激酶(PKM2)在增殖能力强的细胞特别是癌细胞中高表达,因此成为临床上肿瘤治疗的一个新靶点。然而,全身应用PKM2抑制剂对于其他细胞如血管内皮细胞是否存在副作用尚不清楚。     

早孕小鼠子宫内膜M2型丙酮酸激酶基因表达的研究

该研究分析了M2型丙酮酸激酶(pyruvate kinase M2,PKM2)基因在早孕小鼠子宫内膜的表达规律。通过建立正常妊娠小鼠模型,收集孕D1、D4、D5、D6、D7小鼠子宫内膜组织及孕D5小鼠着床点及着床旁子宫内膜组织。构建假孕小鼠模型,收集假孕PD1、PD4、PD5、PD6和PD7小鼠子宫内膜组织。用Real-time PCR和Western blot方法检测PKM2 m RNA和蛋白质表达水平;免疫组织化学方法检测PKM2蛋白质在孕D5着床点与着床旁子宫内膜的分布。研究结果显示,在正常妊娠小鼠子宫内膜,PKM2 m RNA表达从孕D5开始出现明显升高,孕D6达高峰,孕D7略有下降,孕D6、孕D7与孕D1相比有明显差异。PKM2蛋白质从孕D6开始出现明显升高,孕D7略有下降,孕D6、孕D7与孕D1相比有明显差异。假孕小鼠子宫内膜PKM2 m RNA水平从PD6开始有明显升高,PD7与PD6水平相当,PD6、PD7与PD1相比有明显差异。PKM2蛋白质水平每两组间无明显差异。孕D5小鼠子宫内膜组织中,PKM2 m RNA及蛋白质水平均呈现着床点明显高于着床旁趋势。该研究初步揭示了PKM2基因在早孕小鼠子宫内膜表达规律,为深入探讨PKM2在维持早孕小鼠子宫内膜正常功能的机制上的作用提供了重要线索。     

M1型丙酮酸激酶在肿瘤中的作用

M型丙酮酸激酶(PKM)包括M1型丙酮酸激酶(PKM1)和M2型丙酮酸激酶(PKM2)。既往的研究认为PKM2与肿瘤存在着密切关系,PKM2通过促进Warburg效应和合成代谢以及发挥蛋白激酶样作用,促进肿瘤细胞增殖,在肿瘤的形成过程中起着关键作用。但近年来的研究表明,PKM1不仅促进线粒体的氧化磷酸化过程,也可促进Warburg效应、生物合成代谢、细胞增殖及肿瘤形成,特别是在肺神经内分泌癌和肝细胞癌的形成和进展中可能发挥着重要作用,有望成为肿瘤治疗的新靶点。     

过表达2型丙酮酸激酶通过下调p53稳定性促进线粒体融合

正肿瘤细胞的代谢状态转变是有别于正常细胞的标志。大多数肿瘤细胞在有氧条件下仍表现出活跃的葡萄糖摄取及糖酵解,这种现象被称为Warburg效应。在这一过程中,丙酮酸激酶作为糖酵解的最后一步激酶,可以催化丙酮酸为乳酸并产生ATP。2型丙酮酸激酶(PKM2)高表达于胚胎组织及肿瘤细胞中。     

人转录因子激活蛋白TFAP_2γ促进乳腺癌细胞生长

目的:构建带Flag标签的人转录因子激活蛋白2γ(TFAP_2γ)基因真核表达载体,获得Flag-TFAP_2γ蛋白,检测其对乳腺癌细胞生长的影响。方法:采用PCR技术从乳腺文库中扩增出人TFAP_2γ基因,并将其正确插入Flag载体;将重组质粒与空载体转染人乳腺癌细胞系ZR75-1和MCF-7细胞后,Western印迹检测表达情况,并进行生长曲线实验。结果:双酶切和测序鉴定表明,Flag-TFAP_2γ真核表达质粒构建成功,转染ZR75-1和MCF-7细胞后获得表达;生长曲线实验结果表明,TFAP_2γ可促进乳腺癌细胞的生长。结论:构建了带Flag标签的人TFAP_2γ基因真核表达载体,并能在乳腺癌细胞ZR75-1和MCF-7中表达,且能促进细胞的生长。本实验为进一步研究TFAP_2γ在乳腺癌中的功能奠定了基础。     

Procion Red HE 3B亲和层析纯化人L型丙酮酸激酶

采用国产的红色染料Procion Red HE3B亲和层析和底物ADP洗脱、配合硫酸铵盐析、DEAE-Sephadex A-50和磷酸纤维素层析可将人肝L型丙酮酸激酶提纯410倍,产物电泳纯。其中红色染料一步提纯的倍数平均为7.8倍,回收率平均为59％。     

Reciprocal Regulation of Protein Kinase and Pyruvate Kinase Activities of Pyruvate Kinase M2 by Growth Signals

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598–609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.     

A Novel Pyruvate Kinase M2 Activator Compound that Suppresses Lung Cancer Cell Viability under Hypoxia

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.     

Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is an aggressive tumor, with a high mortality rate due to late symptom presentation and frequent tumor recurrences and metastasis. It is also a rapidly growing tumor supported by different metabolic mechanisms; nevertheless, the biological and molecular mechanisms involved in the metabolic reprogramming in HCC are unclear. In this study, we found that pyruvate kinase M2 (PKM2) was frequently over-expressed in human HCCs and its over-expression was associated with aggressive clinicopathological features and poor prognosis of HCC patients. Furthermore, knockdown of PKM2 suppressed aerobic glycolysis and cell proliferation in HCC cell lines in vitro. Importantly, knockdown of PKM2 hampered HCC growth in both subcutaneous injection and orthotopic liver implantation models, and reduced lung metastasis in vivo. Of significance, PKM2 over-expression in human HCCs was associated with a down-regulation of a liver-specific microRNA, miR-122. We further showed that miR-122 interacted with the 3UTR of the PKM2 gene. Re-expression of miR-122 in HCC cell lines reduced PKM2 expression, decreased glucose uptake in vitro, and suppressed HCC tumor growth in vivo. Our clinical data and functional studies have revealed a novel biological mechanism involved in HCC metabolic reprogramming.     

Secreted Pyruvate Kinase M2 Promotes Lung Cancer Metastasis through Activating the Integrin Beta1/FAK Signaling Pathway

1. Download : Download high-res image (196KB)
2. Download : Download full-size image

     

NSun2 Promotes Cell Growth via Elevating Cyclin-Dependent Kinase 1 Translation

The tRNA methytransferase NSun2 promotes cell proliferation, but the molecular mechanism has not been elucidated. Here, we report that NSun2 regulates cyclin-dependent kinase 1 (CDK1) expression in a cell cycle-dependent manner. Knockdown of NSun2 decreased the CDK1 protein level, while overexpression of NSun2 elevated it without altering CDK1 mRNA levels. Further studies revealed that NSun2 methylated CDK1 mRNA in vitro and in cells and that methylation by NSun2 enhanced CDK1 translation. Importantly, NSun2-mediated regulation of CDK1 expression had an impact on the cell division cycle. These results provide new insight into the regulation of CDK1 during the cell division cycle.     

Pyruvate Kinase M2 Expression,but Not Pyruvate Kinase Activity,Is Up-Regulated in a Grade-Specific Manner in Human Glioma

Normal tissues express the M1 isoform of pyruvate kinase (PK) that helps generate and funnel pyruvate into the mitochondria for ATP production. Tumors, in contrast, express the less active PKM2 isoform, which limits pyruvate production and spares glycolytic intermediates for the generation of macromolecules needed for proliferation. Although high PKM2 expression and low PK activity are considered defining features of tumors, very little is known about how PKM expression and PK activity change along the continuum from low grade to high grade tumors, and how these changes relate to tumor growth. To address this issue, we measured PKM isoform expression and PK activity in normal brain, neural progenitor cells, and in a series of over 100 astrocytomas ranging from benign grade I pilocytic astrocytomas to highly aggressive grade IV glioblastoma multiforme (GBM). All glioma exhibited comparably reduced levels of PKM1 expression and PK activity relative to normal brain. In contrast, while grade I-III gliomas all had modestly increased levels of PKM2 RNA and protein expression relative to normal brain, GBM, regardless of whether they arose de novo or progressed from lower grade tumors, showed a 3–5 fold further increase in PKM2 RNA and protein expression. Low levels of PKM1 expression and PK activity were important for cell growth as PKM1 over-expression and the accompanying increases in PK activity slowed the growth of GBM cells. The increased expression of PKM2, however, was also important, because shRNA-mediated PKM2 knockdown decreased total PKM2 and the already low levels of PK activity, but paradoxically also limited cell growth in vitro and in vivo. These results show that pyruvate kinase M expression, but not pyruvate kinase activity, is regulated in a grade-specific manner in glioma, but that changes in both PK activity and PKM2 expression contribute to growth of GBM.     

Dominant Negative Mutations Affect Oligomerization of Human Pyruvate Kinase M2 Isozyme and Promote Cellular Growth and Polyploidy

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM₂) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM₂ in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM₂ by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM₂ showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM₂ by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.     

Pyruvate Kinase M2 in Blood Circulation Facilitates Tumor Growth by Promoting Angiogenesis

It is long known that pyruvate kinase isoform M2 (PKM2) is released into the circulation of cancer patients. The PKM2 levels in patients have been suggested as a diagnostic marker for many types of cancers. However, it is not known how PKM2 is released in the blood, and whether the circulating PKM2 has any physiological function(s) in tumor progression. In this report, we demonstrate that PKM2 in the blood facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes tumor angiogenesis by increasing endothelial cell proliferation, migration, and cell-ECM adhesion. Only the dimeric PKM2 possess the activity in promoting tumor angiogenesis, which is consistent with the observations that PKM2 in circulation of cancer patients is a dimer form.     

In Vitro Effects of Some Flavones on Human Pyruvate Kinase Isoenzyme M2

PKM2 is an important target for designing anticancer drug. Inhibitors and activators of this enzyme are suitable molecules for use in treating cancer. The aim of the present study was to investigate the effect of certain flavones on PKM2. Apigenin, wogonin, flavone, 3‐hydroxyflavone, 5‐hydroxyflavone, 6‐hydroxyflavone, and 7‐hydroxyflavone effectively inhibited PKM2, with IC₅₀ in the range of 0.99–2.120 μM. The kinetic study indicated that these compounds acted as noncompetitive with K_i values of 3.53–5.67 μM toward phosphoenolpyruvate. Scutellarin and tangeritin demonstrated strong activation effect with AC₅₀ values < 2 μM. Diosmetin, baicalin, baicalein, and luteolin showed an intermediate‐level activator effect. These results demonstrate that flavone and their analogs could serve as leading compounds to develop new potent and selective inhibitor and activator for PKM2.