cAMP analog mapping of Epac1 and cAMP kinase - Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension
cAMP analog mapping of Epac1 and cAMP kinase - Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension
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DOI：
10.1074/jbc.m302179200
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发表时间：
2003-09-12
影响因子：
4.8
通讯作者：
Doskeland, SO
中科院分区：
文献类型：
--
作者：
Christensen, AE;Selheim, F;Doskeland, SO
关于 cAMP 依赖性蛋白激酶 (cAPK) 和由 cAMP (Epac) 直接激活的鸟嘌呤交换因子作为 cAMP 作用介质的相对作用知之甚少。我们测试了 cAMP 类似物选择性激活 Epac1 或 cAPK 以及区分 Epac 与 cAPKI 和 cAPKII 结合位点的能力。我们发现常用的 cAMP 类似物，如 8-Br-cAMP 和 8-pCPT-cAMP，与 cAMP 一样能同等地激活 Epac 和 cAPK，即是完全激动剂。相比之下，6-修饰的 cAMP 类似物（如 N-6-苯甲酰基-cAMP）是低效的 Epac 激活剂和完整的 cAPK 激活剂。核糖 2' 位修饰的类似物比 cAMP 诱导更强的 Epac1 激活，但仅是 cAPK 的部分激动剂。 cAMP 的 2'-O-烷基取代将 Epac/cAPK 结合选择性提高了 10-100 倍。 8 位的苯硫基取代基，特别是对位的 MeO- 或 Cl-，进一步增强了 Epac/cAPK 的选择性。 8-pCPT- 和 2'-O-甲基取代的组合将 Epac/cAPK 结合选择性提高了约三个数量级。 6-取代的 cAMP 类似物的 cAPK 选择性、中等浓度的 Rp-cAMPS 类似物对 cAPK 的优先抑制以及 8-pCPT-2'-O-甲基-cAMP 的 Epac 选择性也在完整细胞中得到证实。使用这些化合物选择性调节 PC-12 细胞中的 Epac 和 cAPK，我们观察到选择性激活 Epac 的类似物与 cAPK 特异性类似物强烈协同作用，诱导神经突生长。因此，我们得出结论，cAMP 诱导的神经突生长是由 Epac 和 cAPK 介导的。
Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP ( Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAPKII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N-6-benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2'-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2'-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10-100-fold. Phenylthio substituents in position 8, particularly with MeO- or Cl- in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT- and 2'-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2'-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.