Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA.
Eurofins Lancaster Laboratories Professional Scientific Services, LLC, Malvern, PA, USA.
Mol Cell Biochem. 2019 Jul;457(1-2):157-168. doi: 10.1007/s11010-019-03520-z. Epub 2019 Mar 16.
Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses. However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on F-actin and ERK2 depending on the assay and Dictyostelium strain used, respectively. Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.
咖啡因在盘基网柄菌中被广泛用于抑制趋化因子 cAMP 的合成，从而防止其干扰对趋化因子诱导反应的研究。然而，咖啡因抑制盘基网柄菌中 cAMP 合成的机制尚未被描述。在这里，我们报告了咖啡因对 cAMP 趋化信号网络的影响。我们发现咖啡因抑制磷酸肌醇 3-激酶（PI3K）和雷帕霉素靶蛋白复合物 2（mTORC2）。PI3K 和 mTORC2 对趋化因子刺激的 cAMP 产生都是必需的，从而为咖啡因介导的 cAMP 合成抑制提供了一种机制。我们的结果还表明，咖啡因处理细胞会导致 cAMP 诱导的 RasG 和 Rap1 激活增加，并抑制 PKA、cGMP、MyoII 和 ERK1 反应。最后，我们观察到咖啡因对 F-肌动蛋白和 ERK2 的影响取决于所使用的测定方法和盘基网柄菌菌株，分别具有相反的作用。总之，我们的研究结果表明，咖啡因会显著影响盘基网柄菌中 cAMP 诱导的趋化信号通路，很可能通过包括 PI3K 和 mTORC2 在内的多个靶点发挥作用。
