The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchan

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• 作者：Uma K. Misra ; Steven Kaczowka ; Salvatore V. Pizzo
• 关键词：Cyclic AMP generation in macrophages ; 8-CPT-2-O-Me-cAMP and cyclic AMP-dependent regulation in macrophages ; Akt protein kinase activation ; Epac1 and Akt protein kinase activation
• 刊名：Cellular Signalling
• 出版年：2008
• 期刊代码：42_08986568
• 类别：bio
• 出版时间：August 2008
• 卷：20
• 期：8
• 页码：1459-1470
• 文件大小：1285 K

摘要

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt^Thr-308, and p-Akt^Ser-473 in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1•GTP, p-ERK1/2, p-B-Raf, p110rc="http://www.sciencedirect.com/scidirimg/entities/204e.gif" alt="greek small letter alpha" title="greek small letter alpha" border="0"> catalytic subunit of PI 3-kinase, p-PDK, and p-p^70s6k. Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt^Thr-308 protein kinase activity and p-Akt^Thr-308 levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt^Ser-473 kinase activity and p-Akt^Ser-473 levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.