SIGNALING CASCADES, ALLOSTERY AND THE PYRIMIDINE PATHWAY

在哺乳动物细胞中，重新合成嘧啶的速率受多功能蛋白CAD的调控。该蛋白催化该途径的前半部分，由6个243 kDa的多肽拷贝组成，这些多肽折叠成不同的结构域，携带谷氨酰胺依赖性氨甲酰磷酸合成酶（CPSase）、天冬氨酸转羧化酶（ATCase）和二氢羟化酶（hoase）活性。该通路的活性受到精确控制，并在细胞被诱导增殖时增加。对纯化蛋白的研究表明，CPSase是该途径中催化初始限速步骤的主要调控位点，并受抑制剂UTP和激活剂PRPP的变构控制。此外，该复合物的活性受蛋白激酶A介导的磷酸化调控，尽管其在调节生长状态和细胞周期变化中对嘧啶生物合成途径活性的作用尚不清楚。最近发现，EGF刺激导致MAP激酶介导的磷酸化和CAD的激活，这使得解决这种模糊性成为可能。本研究的目的是破译相互关系，并评估所有这些控制机制对体内嘧啶生物合成调节的重要性。在培养的哺乳动物细胞和表达一种或多种调节机制失效的CAD突变体的转染物中，将检查de no嘧啶生物合成的速率、变构效应池的大小和蛋白质的磷酸化状态。该方法将推断纯化CAD调控的广泛信息，以开发一个全面的模型，该模型可以解释在不同生长状态、周期阶段和受生长因子刺激的细胞中对新生嘧啶生物合成的体内调控。

In mammalian cells, the rate of de novo pyrimidine biosynthesis is regulated by the multi-functional protein CAD. The protein, which catalyzes the first half of the pathway, consists of six copies of a 243 kDa polypeptide folded into separate structural domains that carry glutamine dependent carbamoyl phosphate synthetase (CPSase), aspartate transcarbomoylase (ATCase) and dihydrooortase (DHOase) activities. The activity of the pathway is precisely controlled and increases when cells are induced to proliferate. Studies of the purified protein have shown that CPSase, which catalyzes the initial, rate limiting step in the pathway is the major locus of regulation and is allosterically controlled by UTP, an inhibitor and PRPP, an activator. In addition, the activity of the complex is regulated by protein kinase A mediated phosphorylation, although its role in regulating the growth state and cell cycle changes in the activity of the pyrimidine biosynthetic pathway is not at all clear. The recent discovery that EGF stimulation results in MAP kinase mediated phosphorylation and activation of CAD makes its possible to resolve this ambiguity. The objective of this research is to decipher the interrelationships and assess the importance of all these control mechanisms on the regulation of pyrimidine biosynthesis in vivo. The rate of de no pyrimidine biosynthesis, the size of the allosteric effector pools and the phosphorylation state of the protein will be examined in mammalian cells grown in culture and in transfectants that express CAD mutants in which one or more of the regulatory mechanisms have been disabled. The approach will be to extrapolate the extensive information developed on the regulation of purified CAD to develop a comprehensive model that can account for the in vivo regulation of de novo pyrimidine biosynthesis in different growth states, phases of the cycle and in cells stimulated by growth factors.