Activation of Cyclin-Dependent Kinases by Fructose-2,6-Bisphosphate

2,6-二磷酸果糖激活细胞周期蛋白依赖性激酶

基本信息

批准号：
8636908
负责人：
Jason A. Chesney
金额：
$ 30.19万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8636908
关键词：
6-Phosphofructo-2-kinase 6-Phosphofructokinase Affect Amino Acids Apoptosis Binding CDC2 Protein Kinase CDK2 gene CDK4 gene Cancer cell line Cell Cycle Cell Cycle Progression Cell Cycle Regulation Cell Nucleus Cell division Cells Cyclin-Dependent Kinases Cyclins Cytoplasm Cytostatics DNA biosynthesis Data Enzymes Epithelial Epithelial Cells Eukaryotic Cell Family Family member Fructose G1/S Transition Genes Genetic Transcription Genomics Glycolysis Growth Hela Cells Hematopoietic Human Human Development Hypoxia In Vitro Liver Malignant Neoplasms Mitosis Molecular Target Mus Mutation Normal Cell Normal tissue morphology Nuclear Oncogenes Pathway interactions Phosphoric Monoester Hydrolases Phosphotransferases Protein p53 Recombinants Regulation Relative (related person)Research Role Signal Pathway Site Small Interfering RNA Testing Tissues Transfection Transformed Cell Line Transgenic Organisms base cancer cell chemotherapeutic agent clinically significant fructose-6-phosphate glucose metabolism glucose uptake in vitro activity interest mutant neoplastic novel public health relevance receptor screening small molecule tumor tumor growth tumor progression virtual

项目摘要

DESCRIPTION (provided by applicant): The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB) phosphorylate fructose-6-phosphate (F6P) to fructose-2,6-bisphosphate (F2,6BP), which is an allosteric activator of 6-phosphofructo-1-kinase, a rate-limiting enzyme in the glycolytic pathway. Although there are four PFKFB enzymes, PFKFB3 and PFKFB4 are of particular interest since these enzymes have been found to be activated in human cancers, to be increased by hypoxic exposure via HIF-1?, and, in the case of PFKFB3, to be required for the growth of Ras-transformed tumors. In order to better understand the relative contributions of PFKFB2-4 to glycolysis, we examined the subcellular localization of these enzymes and were surprised to find that whereas PFKFB2 and PFKFB4 localized to the cytoplasm (the site of glycolysis), PFKFB3 localized to the nucleus. We then over-expressed PFKFB3 in HeLa cells and observed no change in glucose uptake but rather an increase in proliferation. Eukaryotic cell division is controlled by cyclin dependent kinases (CDKs) that bind to regulatory cyclins and phosphorylate hundreds of substrates that control DNA replication, transcription and mitosis. We found that over-expression of PFKFB3 stimulated CDK1 activity in HeLa cells and that purified F2,6BP stimulated recombinant monomeric CDK1 in vitro. We then confirmed the requirement of CDK1 for the pro-proliferative effects of PFKFB3 by demonstrating that CDK1 siRNA but not CDK2, CDK4 or CDK6 siRNA reversed the increased proliferation caused by over-expression of PFKFB3. Importantly, transfection of HeLa cells with PFKFB3-specific siRNA decreased endogenous PFKFB3 which in turn reduced CDK1 activity, increased p27 expression, suppressed G1/S transition, induced apoptosis but had no impact on glucose uptake. These data support a distinct role for PFKFB3 in the regulation of cell cycle progression and apoptosis, and not glucose metabolism. We propose to test the hypothesis that nuclear F2,6BP generated by PFKFB3 activates CDKs and promotes cell cycle progression. We anticipate that nuclear F2,6BP may serve unique roles in regulating CDK1, and other CDKs, and that inhibition of PFKFB3 will suppress CDK activities without significantly affecting glucose metabolism in transformed but not in normal cells.

描述（由申请人提供）：6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶（PFKFB）将果糖-6-磷酸（F6P）磷酸化为果糖-2,6-二磷酸（F2,6BP），其是 6-磷酸果糖-1-激酶的变构激活剂，6-磷酸果糖-1-激酶是糖酵解途径中的限速酶。尽管有四种 PFKFB 酶，但 PFKFB3 和 PFKFB4 特别令人感兴趣，因为已发现这些酶在人类癌症中被激活，并通过 HIF-1? 暴露于缺氧环境中而增加，并且，就 PFKFB3 而言，需要用于 Ras 转化肿瘤的生长。为了更好地了解 PFKFB2-4 对糖酵解的相对贡献，我们检查了这些酶的亚细胞定位，并惊讶地发现 PFKFB2 和 PFKFB4 定位于细胞质（糖酵解位点），而 PFKFB3 定位于细胞核。然后，我们在 HeLa 细胞中过度表达 PFKFB3，观察到葡萄糖摄取没有变化，但增殖增加。真核细胞分裂由细胞周期蛋白依赖性激酶 (CDK) 控制，CDK 与调节细胞周期蛋白结合并磷酸化数百种控制 DNA 复制、转录和有丝分裂的底物。我们发现PFKFB3的过表达刺激了HeLa细胞中的CDK1活性，并且纯化的F2,6BP在体外刺激了重组单体CDK1。然后，我们通过证明 CDK1 siRNA 而不是 CDK2、CDK4 或 CDK6 siRNA 逆转了 PFKFB3 过度表达引起的增殖增加，证实了 CDK1 对 PFKFB3 促增殖作用的需要。重要的是，用 PFKFB3 特异性 siRNA 转染 HeLa 细胞会减少内源性 PFKFB3，进而降低 CDK1 活性，增加 p27 表达，抑制 G1/S 转变，诱导细胞凋亡，但对葡萄糖摄取没有影响。这些数据支持 PFKFB3 在调节细胞周期进程和细胞凋亡（而不是葡萄糖代谢）中发挥独特作用。我们建议测试以下假设：PFKFB3 产生的核 F2,6BP 激活 CDK 并促进细胞周期进程。我们预计核 F2,6BP 可能在调节 CDK1 和其他 CDK 中发挥独特的作用，并且抑制 PFKFB3 将抑制 CDK 活性，而不显着影响转化细胞中的葡萄糖代谢，但不会显着影响正常细胞中的葡萄糖代谢。