Posttranslational Modification of 6-phosphofructo-1-kinase as an Important Feature of Cancer Metabolism

BackgroundHuman cancers consume larger amounts of glucose compared to normal tissues with most being converted and excreted as lactate despite abundant oxygen availability (Warburg effect). The underlying higher rate of glycolysis is therefore at the root of tumor formation and growth. Normal control of glycolytic allosteric enzymes appears impaired in tumors; however, the phenomenon has not been fully resolved. Methodology/Principal FindingsIn the present paper, we show evidence that the native 85-kDa 6-phosphofructo-1-kinase (PFK1), a key regulatory enzyme of glycolysis that is normally under the control of feedback inhibition, undergoes posttranslational modification. After proteolytic cleavage of the C-terminal portion of the enzyme, an active, shorter 47-kDa fragment was formed that was insensitive to citrate and ATP inhibition. In tumorigenic cell lines, only the short fragments but not the native 85-kDa PFK1 were detected by immunoblotting. Similar fragments were detected also in a tumor tissue that developed in mice after the subcutaneous infection with tumorigenic B16-F10 cells. Based on limited proteolytic digestion of the rabbit muscle PFK-M, an active citrate inhibition-resistant shorter form was obtained, indicating that a single posttranslational modification step was possible. The exact molecular masses of the active shorter PFK1 fragments were determined by inserting the truncated genes constructed from human muscle PFK1 cDNA into a pfk null E. coli strain. Two E. coli transformants encoding for the modified PFK1s of 45,551 Da and 47,835 Da grew in glucose medium. The insertion of modified truncated human pfkM genes also stimulated glucose consumption and lactate excretion in stable transfectants of non-tumorigenic human HEK cell, suggesting the important role of shorter PFK1 fragments in enhancing glycolytic flux. Conclusions/SignificancePosttranslational modification of PFK1 enzyme might be the pivotal factor of deregulated glycolytic flux in tumors that in combination with altered signaling mechanisms essentially supports fast proliferation of cancer cells.背景：与正常组织相比，人类癌症会消耗更多葡萄糖，即便氧气供应充足，大部分葡萄糖仍会被转化并以乳酸形式排出，这一现象即沃伯格效应（Warburg effect）。糖酵解速率异常升高是肿瘤发生与生长的核心诱因。肿瘤中糖酵解变构酶的正常调控似乎受损，但该现象的具体机制尚未完全阐明。 材料与方法/主要结果：本研究证实，天然85 kDa的6-磷酸果糖-1-激酶（6-phosphofructo-1-kinase, PFK1）——糖酵解的关键调控酶，正常情况下受反馈抑制调控——可发生翻译后修饰。对该酶的C端区域进行蛋白水解切割后，可形成一段具有活性的47 kDa截短片段，该片段对柠檬酸与ATP的抑制作用不敏感。在致瘤细胞系中，免疫印迹法仅能检测到该截短片段，而无法检测到天然85 kDa的PFK1。在皮下接种致瘤性B16-F10细胞后形成的小鼠肿瘤组织中，同样可检测到类似的截短片段。对兔肌肉PFK-M进行有限蛋白水解消化后，可获得具有活性且抗柠檬酸抑制的截短形式，提示仅需单次翻译后修饰即可产生该截短片段。通过将由人肌肉PFK1 cDNA构建的截短基因转入pfk基因敲除大肠杆菌（E. coli）菌株，确定了活性截短PFK1片段的精确分子量。两株编码分子量分别为45551 Da与47835 Da的修饰型PFK1的大肠杆菌转化子，可在葡萄糖培养基中存活增殖。将截短修饰后的人pfkM基因转入非致瘤性人HEK细胞的稳定转染株后，同样可促进葡萄糖消耗与乳酸分泌，提示截短型PFK1片段在增强糖酵解通量中发挥关键作用。 结论与意义：PFK1酶的翻译后修饰可能是肿瘤中糖酵解通量失调的核心因素，该修饰与异常信号通路共同作用，可显著促进癌细胞的快速增殖。