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：
8448296
负责人：
Jason A. Chesney
金额：
$ 29.26万
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/8448296
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-2-激酶/果糖-2,6-磷酸酶（PFKFB）磷酸化磷酸胶质果糖-6-磷酸（F6P）至果糖-2,6-磷酸果实（F2,6bp），这是6-磷酸胶质电量的分配率，是一种分配率糖酵解途径。尽管有四种PFKFB酶，但PFKFB3和PFKFB4特别令人感兴趣，因为已经发现这些酶在人类癌症中被激活，并且通过HIF-1？通过HIF-1？的低氧暴露来增加，而对于PFKFB3，对于Ras传输的RAS传输tumors的生长所必需。 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.然后，我们在HeLa细胞中过度表达的PFKFB3，观察到葡萄糖摄取没有变化，而是增殖的增加。真核细胞分裂受细胞周期蛋白依赖性激酶（CDK）的控制，这些激酶（CDKS）与调节性细胞周期蛋白结合并磷酸化数百个控制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，并且在体外纯化了F2,6bp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证实了CDK1对PFKFB3促生殖作用的需求，这反转了PFKFB3过表达引起的增殖增加。重要的是，用PFKFB3特异性siRNA转染HeLa细胞降低了内源性PFKFB3，进而降低CDK1活性，p27表达增加，抑制G1/s转变，诱导凋亡，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凋亡的调节中的独特作用，而不是葡萄糖代谢。我们建议检验以下假设：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中起独特的作用，并且抑制PFKFB3会抑制CDK活性，而不会显着影响正常细胞转化但不影响正常细胞中的葡萄糖代谢。