Graves L M, Guy H I, Kozlowski P, Huang M, Lazarowski E, Pope R M, Collins M A, Dahlstrand E N, Earp H S, Evans D R
Department of Pharmacology, Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599-7365, USA.
Nature. 2000 Jan 20;403(6767):328-32. doi: 10.1038/35002111.
The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.
嘧啶核苷酸的从头合成是哺乳动物细胞增殖所必需的。该途径中的限速步骤由氨甲酰磷酸合成酶（CPS II）催化，CPS II是多功能酶CAD的一部分。在此，我们描述了丝裂原活化蛋白（MAP）激酶级联对CAD的调控。当在体外被MAP激酶磷酸化或在体内被表皮生长因子激活时，CAD失去其反馈抑制作用（这依赖于三磷酸尿苷），并对激活作用（这依赖于磷酸核糖焦磷酸）变得更加敏感。这两种别构调节变化均有利于嘧啶的生物合成以促进生长。它们伴随着体内表皮生长因子依赖性CAD磷酸化的增加，并可通过抑制MAP激酶来阻止。一个共有MAP激酶磷酸化位点的突变消除了生长因子刺激引起的CAD别构调节变化。最后，与MAP激酶信号传导对CPS II活性的影响一致，表皮生长因子增加了细胞内三磷酸尿苷，并且这种增加可通过抑制MAP激酶而逆转。因此，这些研究可能表明MAP激酶级联的激活与嘧啶核苷酸的从头生物合成之间存在直接联系。
