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

• 批准号: 8250362
• 负责人: Jason A. Chesney
• 金额: $ 31.07万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 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/8250362
• 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; Screening procedure; 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; small molecule; tumor; tumor growth; tumor progression; virtual

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. PUBLIC HEALTH RELEVANCE: The research proposed in this application may enable the identification of a novel molecular target - the CDK1 F2,6BP-binding domain - that is required for the regulation of the cell cycle and cancer progression. This binding pocket then can be used for receptor-based virtual screening to identify novel anti-neoplastic agents.

描述(申请人提供)：6-phosphofructo-2-kinase/fructose-2，6-bisphosphatases(全氟化钾)将果糖-6-磷酸(F6P)磷酸化为果糖-2，6-二磷酸(F2，6BP)，这是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。虽然有四种PFKFB酶，但PFKFB3和PFKFB4尤其令人感兴趣，因为已经发现这些酶在人类癌症中被激活，通过HIF-1？在低氧暴露下增加，并且在PFKFB3的情况下，是RAS转化的肿瘤生长所必需的。为了更好地了解PFKFB2-4对糖酵解的相对贡献，我们研究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(糖酵解的部位)，而PFKFB3定位于细胞核。然后，我们在HeLa细胞中过表达了PFKFB3，并观察到葡萄糖摄取没有变化，而是增殖增加。真核细胞分裂是由细胞周期蛋白依赖性蛋白激酶(CDK)控制的，它与调控细胞周期蛋白结合，并磷酸化数百种控制DNA复制、转录和有丝分裂的底物。我们发现过表达的PFKFB3能刺激HeLa细胞中CDK1的活性，纯化的F2，6BP在体外能刺激重组单体CDK1的活性。然后，我们通过证明CDK1 siRNA而不是CDK2、CDK4或CDK6 siRNA逆转了由PFKFB3过表达引起的增殖增加，从而证实了CDK1对PFKFB3促增殖作用的需求。重要的是，转染PFKFB3特异性siRNA的HeLa细胞内源性PFKFB3减少，进而降低CDK1活性，增加p27表达，抑制G1/S转换，诱导细胞凋亡，但对葡萄糖摄取没有影响。这些数据支持PFKFB3在细胞周期进程和细胞凋亡中的调节作用，而不是在葡萄糖代谢中的作用。我们建议检验由PFKFB3产生的核F2，6BP激活CDK并促进细胞周期进展的假设。我们预计，核F2，6BP可能在调节CDK1和其他CDK方面发挥独特的作用，抑制PFKFB3将抑制CDK的活性，而不会显著影响转化细胞中的糖代谢，而不是在正常细胞中。 公共卫生相关性：本申请中提出的研究可能使识别一种新的分子靶点-CDK1 F2，6BP结合域-对细胞周期和癌症进展的调节所需。然后，这种结合口袋可以用于基于受体的虚拟筛选，以识别新的抗肿瘤药物。