title: 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 exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages.
abstract: 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, p110alpha 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.
authors.affiliation: Department of Pathology, M301 Davison Building, Duke University Medical Center, Durham, North Carolina 27710, United States.
authors.forename: Uma K
authors.initials: UK
authors.lastname: Misra
authors.forename: Steven
authors.initials: S
authors.lastname: Kaczowka
authors.forename: Salvatore V
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D020935:MAP Kinase Signaling System; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000378:metabolism*; D034622:RNA Interference; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015854:Up-Regulation; D020827:rap1 GTP-Binding Proteins / Q000032:analysis
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000242:Cyclic AMP; D004247:DNA; D020662:Guanine Nucleotide Exchange Factors; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020827:rap1 GTP-Binding Proteins; C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; C482601:Epac protein, mouse