title: Upregulation of mTORC2 activation by the selective agonist of EPAC, 8-CPT-2Me-cAMP, in prostate cancer cells: assembly of a multiprotein signaling complex.
abstract: Ligation of cell surface-associated GRP78 by activated α(2) -macroglobulin triggers pro-proliferative cellular responses. In part, this results from activation of adenylyl cyclase leading to an increase in cAMP. We have previously employed the cAMP analog 8-CPT-2Me-cAMP to probe these responses. Here we show in 1-LN prostate cancer cells that 8-CPT-2Me-cAMP causes a dose-dependent increase in Epac1, p-Akt(T308) , p-Akt(S473) , but not p-CREB. By contrast, the PKA activator 6-Benz-cAMP caused a dose-dependent increase in p-CREB, but not Epac1. We measured mTORC2-dependent Akt phosphorylation at S473 in immunoprecipitates of mTOR or Rictor from 1-LN cells. 8-CPT-2Me-cAMP caused a two-threefold increase in p-Akt(S473) and Akt(S473) kinase activity in Rictor immunoprecipitates. By contrast, there was only a negligible effect on p-Akt(T308) in Rictor immunoprecipitates. Silencing Rictor gene expression by RNAi significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of Akt at Ser(473) . These studies represent the first report that Epac1 mediates mTORC2-dependent phosphorylation of Akt(S473) . Pretreatment of these cells with the PI 3-Kinase inhibitor LY294002 significantly suppressed 8-CPT-2Me-cAMP-dependent p-Akt(S473) and p-Akt(S473) kinase activities, and both effects were rapamycin insensitive. This treatment caused a two to threefold increase in S6 Kinase and 4EBP1 phosphorylation, indices of mTORC1 activation. Pretreatment of the cells with LY294002 and rapamycin significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of S6 Kinase and 4EBP1. We further demonstrate that in 8-CPT-2Me-cAMP-treated cells, Epac1 co-immunoprecipitates with AKAP, Raptor, Rictor, PDE3B, and PDE4D suggesting thereby that during Epac1-induced activation of mTORC1 and mTORC2, Epac1 may have an additional function as a "scaffold" protein.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Uma K
authors.initials: UK
authors.lastname: Misra
authors.forename: Salvatore V
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D000091342:Endoplasmic Reticulum Chaperone BiP; D020868:Gene Silencing; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D008297:Male; D000076222:Mechanistic Target of Rapamycin Complex 1; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D013873:Thionucleotides / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: C488718:2'-O-methyl-8-(4-chlorophenylthio)cAMP; D048868:Adaptor Proteins, Signal Transducing; C115163:CREB1 protein, human; D002352:Carrier Proteins; D002867:Chromones; D017362:Cyclic AMP Response Element-Binding Protein; D000091342:Endoplasmic Reticulum Chaperone BiP; D020662:Guanine Nucleotide Exchange Factors; C000717247:HSPA5 protein, human; D009025:Morpholines; D046912:Multiprotein Complexes; C101633:PA2G4 protein, human; D011506:Proteins; C116122:RAPGEF3 protein, human; C492571:RICTOR protein, human; D016601:RNA-Binding Proteins; C463659:RPTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D013873:Thionucleotides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C071595:N(6)-benzyl-cyclic adenosine 5'-monophosphate; D000242:Cyclic AMP; C494918:AKT1 protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517376:PDE4D protein, human; D020123:Sirolimus