Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade
Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade
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DOI：
10.1007/s11010-006-9398-x
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发表时间：
2007-07-01
影响因子：
4.3
通讯作者：
Evans, David R.
中科院分区：
文献类型：
--
作者：
Kotsis, Damian H.;Masko, Elizabeth M.;Evans, David R.
嘧啶从头生物合成途径的通量由多功能蛋白 CAD 控制，该蛋白催化前三个步骤。嘧啶生物合成的细胞周期依赖性调节是 CAD Thr456 和 Ser1406 分别通过 MAP 激酶和 PKA 级联依次磷酸化的结果。该途径的协调调节需要两个磷酸化事件的精确计时。这些研究表明，PKA 对纯化 CAD 的磷酸化会拮抗 MAP 激酶磷酸化，反之亦然。在体内观察到类似的结果。 Forskolin 激活 BHK-21 细胞中的 PKA 导致 Ser1406 磷酸化增加 8.5 倍，并严重抑制 MAP 激酶介导的 CAD Thr456 磷酸化。此外，还发现 MAP 激酶和 PKA 的相对活性决定了 Thr456 磷酸化的程度。表达升高水平的 MAP 激酶的转染子导致 Thr456 磷酸化增加 11 倍，而过表达 PKA 的转染子使 Thr456 磷酸化降低 5 倍。虽然一种激酶对一个位点的磷酸化可能会引起构象变化，从而干扰另一种激酶的磷酸化，但 MAP 激酶和 PKA 与 CAD 形成稳定复合物的观察结果表明，相互拮抗是结合激酶空间干扰的结果。 MAP 激酶和 PKA 对 CAD 磷酸化的相互拮抗作用提供了一种优雅的机制来协调嘧啶生物合成的细胞周期依赖性调节，确保该途径的上调和下调信号不会发生冲突。
The flux through the de novo pyrimidine biosynthetic pathway is controlled by the multifunctional protein CAD, which catalyzes the first three steps. The cell cycle dependent regulation of pyrimidine biosynthesis is a consequence of sequential phosphorylation of CAD Thr456 and Ser1406 by the MAP kinase and PKA cascades, respectively. Coordinated regulation of the pathway requires precise timing of the two phosphorylation events. These studies show that phosphorylation of purified CAD by PKA antagonizes MAP kinase phosphorylation, and vice versa. Similar results were observed in vivo. Forskolin activation of PKA in BHK-21 cells resulted in a 8.5 fold increase in Ser1406 phosphorylation and severely curtailed the MAP kinase mediated phosphorylation of CAD Thr456. Moreover, the relative activity of MAP kinase and PKA was found to determine the extent of Thr456 phosphorylation. Transfectants expressing elevated levels of MAP kinase resulted in a 11-fold increase in Thr456 phosphorylation, whereas transfectants that overexpress PKA reduced Thr456 phosphorylation 5-fold. While phosphorylation of one site by one kinase may induce conformational changes that interfere with phosphorylation by the other, the observation that both MAP kinase and PKA form stable complexes with CAD suggest that the mutual antagonism is the result of steric interference by the bound kinases. The reciprocal antagonism of CAD phosphorylation by MAP kinase and PKA provides an elegant mechanism to coordinate the cell cycle-dependent regulation of pyrimidine biosynthesis ensuring that signals for up- and down-regulation of the pathway do not conflict.