SIGNALING CASCADES, ALLOSTERY AND THE PYRIMIDINE PATHWAY

信号级联、变构和嘧啶途径

• 批准号: 6913797
• 负责人: HEDEEL I. GUY EVANS
• 金额: $ 24.6万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6913797
• 关键词: allosteric site; amidohydrolases; antibody; aspartate carbamoyltransferase; biological signal transduction; biosynthesis; carbamoylphosphate synthase; enzyme activity; enzyme mechanism; fluorescence microscopy; gene expression; gene mutation; genetic regulation; immunofluorescence technique; laboratory rabbit; mitochondria; mitogen activated protein kinase; molecular dynamics; phosphorylation; protein kinase A; protein kinase C; protein localization; protein transport; pyrimidines

DESCRIPTION (provided by applicant): The rate of de novo pyrimidine biosynthesis in mammalian cells is regulated by CAD, a large multifunctional protein that catalyzes the first three steps in the pathway. The flux through the pathway is precisely controlled and increases in tumors and in cells induced to proliferate. Carbamoyl phosphate synthetase, the CAD component that catalyzes the initial, rate limiting step, the locus of regulation, is controlled by UTP inhibition, PRPP activation, by protein kinase A (PKA) mediated phosphorylation and by phosphorylation of CAD via the MAP kinase cascade in response to EGF stimulation. The pathway is up-regulated just prior to S phase by MAP kinase mediated phosphorylation and subsequently down-regulated as the cells exit the S phase by dephosphorylation of the MAP kinase site and phosphorylation by PKA. The first Specific Aim is to elucidate the mechanism and physiological significance of three newly discovered, putative regulatory mechanisms; 1) autophosphorylation of CAD that results in large changes in catalytic activity and regulation, 2) protein kinase C that acts in concert with MAP kinase to activate CAD and 3) the regulated signaling complexes of CAD, MAP kinase, PKA and PP1 that may regulate the timing of the sequential phosphorylations. The synergistic and antagonist interactions between the individual control mechanisms that allow them to work in concert to respond to varying demands for pyrimidine nucleotides are of special interest. The second Specific Aim is to assess the physiological significance of the growth state dependent changes in CAD intracellular dynamics. Microscopic and biochemical approaches have shown that an appreciable fraction of the CAD phosphorylated by MAP kinase is localized in the nucleus and that a fraction of the CAD in the cytosol is associated with the microtubules. The postulated direct transfer of dihydroorotate to the mitochondrial dehydrogenase, catalyzing the next step in the pathway, will be investigated in vivo using a histochemical assay. Fluorescence microscopy of live cells in conjunction with mutants and constructs that target CAD to different cellular compartments will be used to elucidate the functional significance of CAD nucleocytoplasmic dynamics. The overall objective is to develop a comprehensive model for the regulation of the pathway in normal and neoplastic cells taking into consideration the integration of signals and the interplay of these diverse regulatory mechanisms.

描述（由申请人提供）：哺乳动物细胞中从头嘧啶生物合成的速率受CAD调节，CAD是一种催化途径中前三步的大型多功能蛋白质。通过该途径的流量受到精确控制，并且在肿瘤和诱导增殖的细胞中增加。氨甲酰磷酸合成酶，催化初始限速步骤的CAD组分，调节位点，受UTP抑制、PRPP激活、蛋白激酶A（PKA）介导的磷酸化和CAD磷酸化控制，通过MAP激酶级联反应响应EGF刺激。该途径在S期之前通过MAP激酶介导的磷酸化上调，随后在细胞退出S期时通过MAP激酶位点的去磷酸化和PKA的磷酸化下调。第一个具体目标是阐明三个新发现的，假定的调节机制的机制和生理意义; 1）CAD的自身磷酸化，其导致催化活性和调节的巨大变化，2）与MAP激酶协同作用以激活CAD的蛋白激酶C，和3）CAD，MAP激酶，PKA和PP 1可能调节顺序磷酸化的时间。个体控制机制之间的协同和拮抗相互作用使它们能够协同工作以响应对嘧啶核苷酸的不同需求，这是特别感兴趣的。第二个具体目标是评估CAD细胞内动力学中生长状态依赖性变化的生理意义。显微镜和生物化学的方法已经表明，相当一部分的CAD磷酸化的MAP激酶是本地化的细胞核和一小部分的CAD在胞质溶胶是与微管。将使用组织化学测定法在体内研究假定的二氢乳清酸直接转移到线粒体脱氢酶，催化途径中的下一步。荧光显微镜的活细胞与突变体和结构，目标CAD到不同的细胞隔室将被用来阐明CAD核质动力学的功能意义。总体目标是开发一个全面的模型，在正常和肿瘤细胞中的信号的整合和这些不同的调节机制的相互作用的途径的调节。