Xiaodong Cheng

Second messenger cAMP regulates many cellular functions through its effectors, such as cAMP-dependent protein kinase (PKA) and Epac (exchange proteins directly activated by cAMP). Spatial and temporal control of cAMP signaling is crucial to differential regulation of cellular targets involved in various signaling cascades. To investigate the compartmentalized cAMP signaling, we constructed fluorescent indicators that report intracellular cAMP dynamics and Epac activation by sandwiching the full-length Epac1 between cyan and yellow mutants of GFP. Elevations of cAMP decreased FRET and increased the ratio of cyan-to-yellow emissions by 10-30% in living mammalian cells. This response can be reversed by removing cAMP-elevating agents and abolished by mutating the critical residue responsible for cAMP binding. Targeting of the reporter to the plasma membrane, where cAMP is produced in response to the activation of beta-adrenergic receptor, revealed a faster cAMP response at the membrane than in the cytoplasm and mitochondria. Simultaneous imaging with targeted cAMP indicator and PKA activity reporter allowed the detection of a much delayed PKA response in the nucleus after the rapid accumulation of cAMP at the plasma membrane of the same cell, despite the immediate presence of a pool of cAMP in the nucleus. Thus, cAMP dynamics and the activation of its effectors are precisely controlled spatiotemporally in vivo.

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAFE600, but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression. Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAFE600, but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression. Pancreatic ductal adenocarcinoma (PDA) 2The abbreviations used are: PDA, pancreatic ductal adenocarcinoma; FTS, S-trans,transfarnesylthiosalicylic acid; GLI, glioblastoma gene product; Hh, hedgehog; HPDE, human pancreatic ductal epithelium; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; Shh, Sonic hedgehog; SMO, smoothened gene product; siRNA, small interfering RNA. is the fourth leading cause of cancer-related death for both men and women in the United States. It was estimated that in 2006 33,730 new cases would be diagnosed, and 32,300 would die from the disease (American Cancer Society Cancer Facts and Figures 2006). Therefore, PDA is one of the most lethal human diseases, with a 5-year survival rate of less than 4% and a median survival of less than 6 months. PDA is one of the better-characterized neoplasms at the genetic level. There are now sufficient clinical, genetic, and pathological data to support a tumor progression model for PDA in which the pancreatic ductal epithelium progresses from normal to increased grades of pancreatic intraepithelial neoplasia to invasive cancer (1Hruban R.H. Goggins M. Parsons J. Kern S.E. Clin. Cancer Res. 2000; 6: 2969-2972PubMed Google Scholar, 2Klein W.M. Hruban R.H. Klein-Szanto A.J. Wilentz R.E. Mod. Pathol. 2002; 15: 441-447Crossref PubMed Scopus (105) Google Scholar). Accompanying the progressive morphological changes is the sequential accumulation of genetic alterations in the KRAS oncogene and the tumor suppressors INK4A, p53, and SMAD4/DPC4, although these alterations have not been linked to the acquisition of specific histopathological attributes (3Moskaluk C.A. Hruban R.H. Kern S.E. Cancer Res. 1997; 57: 2140-2143PubMed Google Scholar, 4Yamano M. Fujii H. Takagaki T. Kadowaki N. Watanabe H. Shirai T. Am. J. Pathol. 2000; 156: 2123-2133Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 5Luttges J. Galehdari H. Brocker V. Schwarte-Waldhoff I. Henne-Bruns D. Kloppel G. Schmiegel W. Hahn S.A. Am. J. Pathol. 2001; 158: 1677-1683Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). In addition to these frequent genetic abnormalities, mutations in the tumor suppressors BRCA2, TGFBR1, and TGFBR2, the serine-threonine kinases AKT2 and LKB1/STK11, and certain DNA mismatch-repair genes represent other less common genetic events in PDA (6Goggins M. Schutte M. Lu J. Moskaluk C.A. Weinstein C.L. Petersen G.M. Yeo C.J. Jackson C.E. Lynch H.T. Hruban R.H. Kern S.E. Cancer Res. 1996; 56: 5360-5364PubMed Google Scholar, 7Goggins M. Shekher M. Turnacioglu K. Yeo C.J. Hruban R.H. Kern S.E. Cancer Res. 1998; 58: 5329-5332PubMed Google Scholar, 8Friess H. Yamanaka Y. Buchler M. Berger H.G. Kobrin M.S. Baldwin R.L. Korc M. Cancer Res. 1993; 53: 2704-2707PubMed Google Scholar, 9Cheng J.Q. Ruggeri B. Klein W.M. Sonoda G. Altomare D.A. Watson D.K. Testa J.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3636-3641Crossref PubMed Scopus (700) Google Scholar, 10Ruggeri B.A. Huang L. Wood M. Cheng J.Q. Testa J.R. Mol. Carcinog. 1998; 21: 81-86Crossref PubMed Scopus (288) Google Scholar, 11Sahin F. Maitra A. Argani P. Sato N. Maehara N. Montgomery E. Goggins M. Hruban R.H. Su G.H. Mod. Pathol. 2003; 16: 686-691Crossref PubMed Scopus (96) Google Scholar). Aberrant RAS activation plays a critical role in tumorigenesis; activating RAS mutations are found in 30% of all human cancers (12Bos J.L. Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar). Of all human cancers, PDA has the highest incidence of activating KRAS mutations (13Almoguera C. Shibata D. Forrester K. Martin J. Arnheim N. Perucho M. Cell. 1988; 53: 549-554Abstract Full Text PDF PubMed Scopus (1904) Google Scholar). Activating KRAS mutations, representing the earliest genetic changes associated with the transformation of normal ductal epithelium and PDA development, have been detected in pancreatic duct lesions with minimal cytological and architectural atypia and occasionally in histologically normal pancreas (3Moskaluk C.A. Hruban R.H. Kern S.E. Cancer Res. 1997; 57: 2140-2143PubMed Google Scholar, 14Klimstra D.S. Longnecker D.S. Am. J. Pathol. 1994; 145: 1547-1550PubMed Google Scholar, 15Luttges J. Schlehe B. Menke M.A. Vogel I. Henne-Bruns D. Kloppel G. Cancer. 1999; 85: 1703-1710Crossref PubMed Scopus (154) Google Scholar, 16Caldas C. Hahn S.A. Hruban R.H. Redston M.S. Yeo C.J. Kern S.E. Cancer Res. 1994; 54: 3568-3573PubMed Google Scholar, 17Tada M. Ohashi M. Shiratori Y. Okudaira T. Komatsu Y. Kawabe T. Yoshida H. Machinami R. Kishi K. Omata M. Gastroenterology. 1996; 110: 227-231Abstract Full Text PDF PubMed Scopus (285) Google Scholar). The frequency of KRAS mutations correlates with disease progression, reaching almost 100% in pancreatic adenocarcinomas. Targeted endogenous expression of an oncogenic KRAS allele in the mouse pancreas is sufficient to drive the development of pancreatic intraepithelial neoplasia and subsequently at low frequency the progression to both locally invasive adenocarcinoma and metastatic disease with sites of spread exactly as found in human pancreatic cancer (18Aguirre A.J. Bardeesy N. Sinha M. Lopez L. Tuveson D.A. Horner J. Redston M.S. DePinho R.A. Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (806) Google Scholar, 19Hingorani S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1806) Google Scholar, 20Hingorani S.R. Wang L. Multani A.S. Combs C. Deramaudt T.B. Hruban R.H. Rustgi A.K. Chang S. Tuveson D.A. Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1705) Google Scholar). These observations suggest that KRAS plays an essential role in the initiation, development, and maintenance of PDA. Recently, the hedgehog (Hh) signaling pathway has been implicated as playing an important role in the progression and maintenance of PDA (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 22Berman D.M. Karhadkar S.S. Maitra A. Montes D.O. Gerstenblith M.R. Briggs K. Parker A.R. Shimada Y. Eshleman J.R. Watkins D.N. Beachy P.A. Nature. 2003; 425: 846-851Crossref PubMed Scopus (1129) Google Scholar, 23Kayed H. Kleeff J. Keleg S. Guo J. Ketterer K. Berberat N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar). Hh signaling is essential for and cell maintenance in P.W. McMahon Genes Dev. 2001; 15: PubMed Scopus Google Scholar). Hh to of a which in activates downstream that the protein in the GLI L. Beachy P.A. PubMed Scopus Google Scholar). of the Hh signaling including the and the are in human PDA and cell Hh activity a that inhibits Hh signaling through with J. Beachy P.A. Genes Dev. 2002; 16: PubMed Scopus Google in PDA cells with Hh signaling, cell in and tumor in in the and mouse model (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 22Berman D.M. Karhadkar S.S. Maitra A. Montes D.O. Gerstenblith M.R. Briggs K. Parker A.R. Shimada Y. Eshleman J.R. Watkins D.N. Beachy P.A. Nature. 2003; 425: 846-851Crossref PubMed Scopus (1129) Google Scholar, 23Kayed H. Kleeff J. Keleg S. Guo J. Ketterer K. Berberat N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar, G. S. V. D. R. M. C. H. C. A. W. Maitra A. Cancer Res. PubMed Scopus Google Scholar). The of activation of the RAS and Hh pathways in the of PDA that between these two pathways may be a important for the and development of PDA. the effects between KRAS and Hh signaling in pancreatic tumorigenesis are not results from that expression of Hedgehog is sufficient to the signaling pathway by a mutation in the gene (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google and a that activation of the Hedgehog pathway is not sufficient to mutations in the gene to downstream of di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google Scholar). In addition, although expression of endogenous of oncogenic leads to pancreatic intraepithelial neoplasia to all found in the human and PDA in S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1806) Google activation of Hh signaling is not sufficient to pancreatic intraepithelial neoplasia and PDA in a mouse model in which Hh signaling is in the pancreatic epithelium di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google Scholar). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in pancreatic we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh signaling pathway in PDA. that oncogenic transformation of human pancreatic ductal epithelial cells by oncogenic KRAS is by GLI activation and that specific of oncogenic KRAS activity inhibits Hh signaling in PDA cell lines with KRAS These results demonstrate that oncogenic KRAS is involved in activation of the pathway in PDA cells and that between the oncogenic KRAS and Hh pathways may play an important role in cancer development pancreatic PD98059, and from was by J. and from was from kinase and from was from was from and from Jackson and from RNA including and H. C. M. H. 1997; PubMed Google Scholar). for expression of from and by A. 1999; PubMed Google Scholar). was from C. M.S. C. T. C.M. D.M. D.S. Nature. 2005; PubMed Scopus Google Scholar). and an pancreatic ductal epithelial cell was by of and in with and cells by of cells with a and cells from the and in with was to the of to was by cells with the and the and with the cells in the of cells to all the cells RAS of RAS was a of the RAS of as G. J. Cheng Cancer Res. PubMed Scopus Google Scholar). in of cell with and the was on of of at for cell at in a and was between and a The of was from and the was for two was used to the between the two less than was as of cell was with the protein of protein and to in in with for by for detected by cells with the other and the 6 the was with and the cells in at with U0126 other the cells and activity was with the to the the cells in the with of and the was was by at for in a of the was with of and the was of was to the activity The from the was by the from the of to was at with similar and by H. H. C. M. H. 1997; PubMed Google Scholar). RNA was from cells as J. Cheng J. PubMed Scopus Google Scholar). was an and gene expression for of GLI1 and protein in the as by the The of was by to endogenous and to a Oncogenic of by the of oncogene pancreatic we a KRAS human PDA model an cell cell is a human pancreatic duct epithelial cell from normal by genes of human is and of tumor in T. L. J. D.A. M.S. Am. J. Pathol. 1996; Google Scholar, H. L. C. N. J. J. M.S. Am. J. Pathol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). expression of in cells a expression to transformation of the cell all in we used of the of cells to and The cells, increased of RAS activity, and in In addition, expression of in cells to the activation of downstream as and The and in the cells with the cells. These observations are in with results from an KRAS human PDA model the cell J. J. M. M.S. Cancer Res. 2005; PubMed Scopus (105) Google Scholar). of Hh by in the of the activating KRAS oncogene in Hh signaling the oncogenic transformation of HPDE, we the of Hh signaling in and cells. in expression to a increased activation of activity, the expression of the and a mutant that is of GLI not the activity was to the in of activity in an cell These results suggest that activates Hh signaling in cells in a results on the oncogenic KRAS endogenous of GLI1 and protein both of which are Hh in cells as by that the activation of Hh signaling in cells was by activation of the KRAS pathway than an effect associated with the transformation of HPDE, we the oncogenic KRAS activity in cells a specific RAS FTS, which RAS from and M. R. G. D. Y. Y. J. Full Text Full Text PDF PubMed Scopus Google Scholar, B. H. H. E. H. H. M. E. Y. K. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). has been to as a KRAS inhibitor in human pancreatic cell lines that KRAS B. K. M. Y. G. D. P. Y. 1999; PubMed Scopus Google Scholar). in of with to an of and endogenous GLI1 suggest that activates Hh signaling in cells as GLI1 is not a downstream effector but a gene and a of Hh signaling pathway M. G. A.M. F. J. Cancer. Full Text Full Text PDF PubMed Scopus Google Scholar). Oncogenic KRAS for GLI1 in PDA oncogenic KRAS is essential for GLI1 activation in PDA cells, we the expression of the oncogenic KRAS in human PDA cell lines and that mutant RNA that in and cells was used T.B. Hahn J. Cancer Res. 2005; PubMed Scopus Google Scholar). The of KRAS oncogene by the in and cells to RAS with the KRAS the in and cells with cells with the siRNA, the and of of in and cells to and cell death Therefore, to between and cells be knockdown by in a of activity and endogenous of GLI1 in results in cells the that of by was to we used a PDA cell cells not KRAS to the of the activity and GLI1 in cells not These results demonstrate that effects of in and cells are specific to the mutant data that oncogenic KRAS plays an important role in Hh signaling in PDA cells. but the for the of Hh which downstream of the oncogene KRAS the activation of Hh signaling, we the of Hh activity in cells in to specific inhibitors that the RAS downstream MEK and of MEK by U0126 and PD98059 to a of activity and endogenous GLI1 a PI3K-specific These results that the kinase pathway was directly for the activation of Hh signaling in cells. The inhibitory effect of U0126 and PD98059 was in PDA cells activating KRAS and cells with the inhibitors for U0126 and PD98059 inhibited expression as as endogenous GLI1 that the of is not to a of of the we the of and in cells with in U0126 and PD98059 the of the of of which is essential for the but not the is for the activation of Hh signaling, we that a constitutively active but not a constitutively active the inhibitory effect of oncogene KRAS knockdown on Hh we BRAFE600, a constitutively active a constitutively active form of with the and subsequently the in expression of of MEK activity and the inhibitory effect of on Hh signaling the other although the activity inhibited by as by kinase expression of to activity by results cells not These results that the RAF/MEK/MAPK signaling, but not the is critical for GLI activation in PDA. KRAS, through the RAF/MEK/MAPK GLI1 the by which oncogene KRAS activation of Hh signaling, we oncogenic KRAS GLI1 at the protein level. Because of the was specific to the endogenous GLI protein we an GLI1 expression T. S. J. J. Full Text Full Text PDF PubMed Scopus Google and and cells and subsequently the expression of GLI1 in the expression of GLI1 in both and cells with oncogenic KRAS activity by of new protein by we that of MEK by U0126 to a of GLI1 in cells The of GLI1 protein in cells was the of GLI1 protein in cells was estimated to be less than GLI1 protein was by the inhibitor in the of these results suggest the oncogene KRAS, through the blocks GLI1 and leads to the activation of Hh signaling in pancreatic cancer cells. of GLI the of KRAS the and of Hh signaling activation in pancreatic cancer development, we GLI1 expression an P. A.M. B. A.J. A.M. A. M. S. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of GLI1 has been P. A.M. B. A.J. A.M. A. M. S. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the of the GLI1 siRNA, we a GLI1 with GLI1 and cells. GLI1 GLI1 protein expression in cells GLI1 not of and cells GLI1 gene to a attenuation of of cells as by the results cells not we a in and cells. specific has been to effects on Hh signaling both in and in A. 1999; PubMed Google Scholar). of to Hh signaling as by the of activity in cells expression of of in a similar to that of GLI1 similar results in cells not In GLI1 gene effect on of cells, which KRAS mutation these data suggest that activation of Hh signaling is important for KRAS oncogenic transformation in pancreatic cancer cells. active KRAS mutations are one of the earliest and most common genetic alterations in pancreatic (13Almoguera C. Shibata D. Forrester K. Martin J. Arnheim N. Perucho M. Cell. 1988; 53: 549-554Abstract Full Text PDF PubMed Scopus (1904) Google Scholar). between KRAS and other oncogenic pathways in PDA have not been In we the between oncogenic KRAS and the Hh signaling which has been to be in human PDA and cell lines (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 22Berman D.M. Karhadkar S.S. Maitra A. Montes D.O. Gerstenblith M.R. Briggs K. Parker A.R. Shimada Y. Eshleman J.R. Watkins D.N. Beachy P.A. Nature. 2003; 425: 846-851Crossref PubMed Scopus (1129) Google Scholar, 23Kayed H. Kleeff J. Keleg S. Guo J. Ketterer K. Berberat N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar). In the human pancreatic ductal epithelial cell we that to increased endogenous GLI1 and transcriptional activity, suppressing oncogenic KRAS expression by inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS we found that Hh activation was by inhibitors specific for MEK, but not PI3K, in and mutant KRAS PDA cell of active form of BRAFE600, but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. suggest that oncogenic KRAS, through the RAF/MEK/MAPK GLI1 protein and plays an important role in activating the Hh signaling pathway in the of Hh pancreatic and genetic suggest that GLI and play as as in Hh development E. Clin. 2005; PubMed Scopus Google the of and in pancreatic tumorigenesis are not of with other Hh signaling as Sonic hedgehog SMO, and have been in human pancreatic cancer and pancreatic cancer cell lines (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar, 22Berman D.M. Karhadkar S.S. Maitra A. Montes D.O. Gerstenblith M.R. Briggs K. Parker A.R. Shimada Y. Eshleman J.R. Watkins D.N. Beachy P.A. Nature. 2003; 425: 846-851Crossref PubMed Scopus (1129) Google Scholar, 23Kayed H. Kleeff J. Keleg S. Guo J. Ketterer K. Berberat N. I. T. Buchler H. J. Cancer. 110: PubMed Scopus Google Scholar). the other the of in human pancreatic cancer has not been of a on a active form of the suggest that although activation is sufficient to drive pancreatic not human pancreatic di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google Scholar). the and between GLI1 and is that these are by signaling pathway in a similar suggest that expression of is inhibited by KRAS knockdown and MEK GLI1 expression in both and cells that oncogenic KRAS activates Hh signaling through of GLI in PDA. of signaling in in the pancreatic epithelium has been to expression S.R. Wang L. Multani A.S. Combs C. Deramaudt T.B. Hruban R.H. Rustgi A.K. Chang S. Tuveson D.A. Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1705) Google we that activation of the Hh pathway represents important by which oncogenic KRAS promotes tumor The of oncogenic KRAS to Hh signaling in the of Hh an for than of PDA cells lines with Hh signaling activity are to (21Thayer S.P. di Magliano M.P. Heiser P.W. Nielsen C.M. Roberts D.J. Lauwers G.Y. Qi Y.P. Gysin S. Fernandez-del Castillo C. Yajnik V. Antoniu B. McMahon M. Warshaw A.L. Hebrok M. Nature. 2003; 425: 851-856Crossref PubMed Scopus (1317) Google Scholar). The with an that Hh activation activates and the RAS pathway J. M. C. E. F. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google suggest that RAS and Hh signaling pathways form a to tumorigenesis in pancreatic and are essential for signaling in the of in and of cells, activation of the pathway is not sufficient to drive activation Lu K. G.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). that activation of the RAS pathway is sufficient to drive Hh signaling activation in PDA cells through of GLI expression the addition of Hh and the effect of RAS is mediated by the RAF/MEK/MAPK pathway of of GLI1 expression in oncogenic PDA cells leads to a attenuation of in that activation of the Hh signaling pathway may represent an important for pancreatic is with a report by Hebrok and di Magliano M. S. A. J. Hebrok M. Genes Dev. PubMed Scopus Google that although Hh signaling not human pancreatic of Hh and signaling the of in active and with results are in with an in which through protein kinase and the signal-regulated kinase transcriptional activity in cells in a G.M. Cancer Res. PubMed Scopus Google Scholar). these observations and suggest that activation of the by and at the of GLI is an of the Hh signaling. The of between and Hh signaling pathways in pancreatic that the RAS and Hh pathways may represent a new for PDA. of for cells, of for J. for FTS, A. for the Cancer for Cheng for of and of for critical of the with

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. Cyclic adenosine 3′,5′-monophosphate (cAMP) is produced as an intracellular second messenger in response to a variety of extracellular signals, including hormones, growth factors, and neurotransmitters. cAMP regulates a wide range of important biological processes, which, alongside cell metabolism, include cell division, growth, differentiation, secretion, memory, and neoplastic transformation. For many years, major intracellular effects of cAMP in mammalian cells were believed to be mediated by cAMP-dependent protein kinase (PKA). 1The abbreviations used are: PKAcAMP-dependent protein kinaseCcAMP-dependent protein kinase catalytic subunitRcAMP-dependent protein kinase regulatory subunitEpacexchange protein directly activated by cAMPGEFguanine nucleotide exchange factorPI3Kphosphatidylinositol 3-kinasedibutyryl cAMPN6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphatePBSphosphate-buffered salinePDKphosphoinositide-dependent kinaseHEKhuman embryonic kidneyEGFPepidermal growth factor proteinWRTWistar rat thyroidTSHthyrotropin 1The abbreviations used are: PKAcAMP-dependent protein kinaseCcAMP-dependent protein kinase catalytic subunitRcAMP-dependent protein kinase regulatory subunitEpacexchange protein directly activated by cAMPGEFguanine nucleotide exchange factorPI3Kphosphatidylinositol 3-kinasedibutyryl cAMPN6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphatePBSphosphate-buffered salinePDKphosphoinositide-dependent kinaseHEKhuman embryonic kidneyEGFPepidermal growth factor proteinWRTWistar rat thyroidTSHthyrotropin The regulation of PKA is achieved via a unique three-component signaling system in which PKA is composed of two separate subunits, the catalytic (C) and regulatory (R) subunits that interact to form an inactive holoenzyme complex (1.Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar). Although phosphorylation of Thr197 in the activation loop of the C subunit is necessary for the maturation and optimal catalytic activity of PKA (2.Steinberg R.A. Cauthron R.D. Symcox M.M. Shunton H. Mol. Cell. Biol. 1993; 13: 2332-2341Crossref PubMed Google Scholar, 3.Cheng X. Ma Y. Moore M. Hemmings B.A. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9849-9854Crossref PubMed Scopus (191) Google Scholar), unlike most other kinases whose activity is regulated by dynamic phosphorylation/dephosphorylation of the activation loop this phosphorylation step does not seem to be a regulatory mechanism for PKA in vivo. Once phosphorylated, PKA is fully active in its catalytic potential and the Thr197 phosphate does not turn over readily (4.Adams J.A. McGlone M.L. Gibson R. Taylor S.S. Biochemistry. 1995; 34: 2447-2454Crossref PubMed Scopus (133) Google Scholar). The activation of PKA is achieved by binding of the second messenger cAMP to the R subunit, which consequently induces a conformational change in the R subunit and leads to the dissociation of the holoenzyme into its constituent subunits (1.Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (955) Google Scholar). The free active C subunit can then affect a range of diverse cellular events by phosphorylating an array of cytoplasmic and nuclear protein substrates, including enzymes and transcription factors (5.Zetterqvist Ö. Z. Ragnarsson U. Engstrom L. Kemp B.E. Peptides and Protein Phosphorylation. CRC Press Inc., Boca Raton, FL1990: 1-41Google Scholar). cAMP-dependent protein kinase cAMP-dependent protein kinase catalytic subunit cAMP-dependent protein kinase regulatory subunit exchange protein directly activated by cAMP guanine nucleotide exchange factor phosphatidylinositol 3-kinase N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate phosphate-buffered saline phosphoinositide-dependent kinase human embryonic kidney epidermal growth factor protein Wistar rat thyroid thyrotropin cAMP-dependent protein kinase cAMP-dependent protein kinase catalytic subunit cAMP-dependent protein kinase regulatory subunit exchange protein directly activated by cAMP guanine nucleotide exchange factor phosphatidylinositol 3-kinase N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate phosphate-buffered saline phosphoinositide-dependent kinase human embryonic kidney epidermal growth factor protein Wistar rat thyroid thyrotropin The effect of cAMP on certain cellular functions has been shown to be dependent on cell-type and biological responses (6.Grave L.M. Lawrance Jr., J.C. Trends Endocrinol. Metab. 1990; 7: 43-50Abstract Full Text PDF Scopus (44) Google Scholar). For example, in PC12 cells, Swiss 3T3 cells, and thyrocytes, cAMP activates MAP kinases, potentiates the effects of growth factors on differentiation and gene expression, and/or stimulates cell growth and promotes the G1 to S phase transition in the cell cycle (7.Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6217Abstract Full Text PDF PubMed Google Scholar, 8.Vaillancourt R.R. Gardner A.M. Johnson G.L. Mol. Cell. Biol. 1994; 14: 6522-6530Crossref PubMed Scopus (149) Google Scholar, 9.Withers D.J. Bloom S.R. Rozengurt E. J. Biol. Chem. 1995; 270: 21411-21419Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 10.Medina D.L. Santisteban P. Eur. J. Endocrinol. 2000; 143: 161-178Crossref PubMed Scopus (92) Google Scholar). In contrast, cAMP inhibits the proliferation of many cells, including fibroblasts (Rat1 and NIH 3T3), smooth muscle cells, and cells transformed by oncogenes such as ras (11.Burgering B.M.T. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (316) Google Scholar, 12.Wu J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (823) Google Scholar, 13.Graves L.M. Bornfeldt K.E. Raines E.W. Potts B.C. Macdonald S.G. Ross R. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10300-10304Crossref PubMed Scopus (404) Google Scholar, 14.Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (865) Google Scholar). Despite extensive studies, the molecular mechanism underlying the cell type-specific effects of cAMP remains elusive. The growth inhibitory effect of cAMP is believed to be mediated partly through activation of PKA, which interferes with Ras/MAPK signaling pathways (15.Qiu W. Zhuang S. von Lintig F.C. Boss G.R. Pilz R.B. J. Biol. Chem. 2000; 274: 31921-31929Abstract Full Text Full Text PDF Scopus (92) Google Scholar). Recent studies suggested that PI3K activity may be required for cAMP-stimulated cell proliferation in thyroid cells (16.Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar, 17.Ciullo I. Diez-Roux G. Di Domenico M. Migliaccio A. Avvedimento E.V. Oncogene. 2001; 20: 1186-1192Crossref PubMed Scopus (88) Google Scholar). Interestingly, the effects of cAMP on PI3K/PKB pathways are also cell type-specific and correlate well with the mitogenic effects of cAMP (16.Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar). In cells in which cAMP is mitogenic, cAMP stimulates PKB phosphorylation and membrane ruffling. Furthermore, cAMP effects on PKB and membrane ruffling are PKA independent (16.Cass L.A. Summers S.A. Prendergast G.V. Backer J.M. Birnbaum M.J. Meinkoth J.L. Mol. Cell. Biol. 1999; 19: 5882-5891Crossref PubMed Scopus (168) Google Scholar). These findings indicate that multiple cAMP-mediated pathways exist and only some are PKA dependent. Therefore, the recently discovered cAMP receptor Epac (exchange protein directly activated by cAMP) or cAMP-GEF (cAMP-regulated guanine nucleotide exchange factor) may represent an important piece of the puzzle that is critical to our understanding of cAMP-mediated cell signaling. Epac contains a cAMP-binding domain that is homologous to the R subunit of PKA and a guanine exchange factor (GEF) domain (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar, 19.Kawasaki H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). Epac cAMP with and a guanine as an for the of H. Y. T. Y. M. Cell. Full Text PDF PubMed Scopus Google and on the of its to Chardin P. I. A. Oncogene. 1998; Scholar), is activated in response to an in intracellular cAMP in additional to many other D.L. E.G. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, H. R.D. Cell. Full Text Full Text PDF Scopus Google Scholar, J.L. Rooij J. Rev. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). Although PKA can its C L.A. H. L.A. G.M. J. Google Scholar), the that the PKA phosphorylation can be activated by Epac in response to cAMP (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar). of the PKA phosphorylation in does not its biological functions H. EMBO J. 1999; PubMed Scopus Google Scholar). These cAMP-mediated activation of may be independent of phosphorylation by PKA. It is most that the cAMP-mediated signaling mechanism is complex believed and many cAMP-mediated effects that were previously to through PKA are in fact also by Epac. Therefore, is to of cAMP-mediated signaling to include the of Epac. The of Epac many the mechanism of cAMP-mediated signaling. PKA and Epac are in many (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar, 19.Kawasaki H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar), an in intracellular cAMP to the activation of and other potential cAMP as It is conceivable that while PKA through a of signaling Epac may signaling and the net cellular effects of cAMP are by the of Therefore, the cellular effect of cAMP can upon the cellular abundance and distribution of Epac and PKA. Our is that is a dynamic of cellular and targeting of Epac and PKA which, with the dynamic in the of are the that the cell type-specific cAMP this we examined the specific effects of Epac and PKA on the PI3K/PKB that has been to cell type-specific responses to cAMP. Our study for the that Epac and PKA are activated by a second messenger can opposing effects in regulating such as Therefore, the net outcome of cAMP signaling on PKB activation may be dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. a J. L. Bos The by a and a The into the of mammalian by a and a The into the of in which the gene and the the we the and that the Epac were by were that PKB and PKB were and were The were and N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate cAMP) were embryonic kidney cells were were Epac were by Epac The were an and further by Epac the were in of by Epac protein in cells were in with The were in a with The the cells were into to and with were Wistar thyroid cells were in in of and in were for the activation were with cAMP for Activation by cells with For kinase cells were with or to were by directly the cells into The of a of the domain of as Rooij J. Bos J.L. Oncogene. 14: PubMed Scopus Google Scholar). cells were to in in for and with for in the cells were in a and The cell with of with of domain and for with in the were in of of protein were a and further with specific Protein of cell with the protein and of protein by for to by were to membrane a cell and a were in in to binding by with for PKB PKB and were then with and with or for in were with for and to independent were for PKB activity an a were to the as well as to the of the the subcellular of and cells were with an in with a in well and for the cells were in in The were with on and in were the an with and were a the specific roles of Epac and PKA in the PKB signaling we examined the effects of on activation of PKB in and cells that been with Epac. shown in cells of PKA of Epac cell and cells were probed by PKA catalytic subunit and Epac protein with the molecular of to the of Epac readily in the Furthermore, of Epac in cells not affect the protein of PKA with PKB activity in cells as by phosphorylation of and whose phosphorylation is critical for the kinase activity of PKB S.R. P. Biol. 7: Full Text Full Text PDF PubMed Google Scholar, L. H. McCormick F. P. J. Science. 1998; PubMed Scopus Google Scholar). in to that of the cells an in phosphorylation of the PKB and of PKB phosphorylation by cAMP with a specific PKA that the inhibitory effect of cAMP on PKB is mediated by PKA. also the cAMP-mediated PKB activation in cells the inhibitory effect of PKA on PKB The of PKB activation in cells as with that of the cells the PKB phosphorylation of and cells were on the In to monitoring the phosphorylation of PKB and we also the cellular PKB kinase activity kinase activity the our this is the that Epac and PKA the PKB signaling in the between the roles of Epac and PKA on PKB activation, we examined the effects of on PKB phosphorylation in and cells as a of shown in PKB activation by the in the cells and while the inhibitory effect of PKA on PKB and PKB activity in and PKB activity in the of to and PKB in the of were to the PKB PKB activity in the of to and PKB in the of were to the PKB PKB activity in cells is that in PKB in and with and were the The PKB activity in the of to and PKB in the of were to the PKB The PKB activity in cells is that in in a new PKB in and with and were the used a cell cAMP T. G. PubMed Scopus Google as a for Epac and PKA to the effect of in our shown in cAMP the PKB activation in cells while the activation of PKB in cells in a to that of our that of cAMP to or activation of PKB in cells and cells, that the PKB activation in cells in the of is the of activation of Epac by a of a critical in the domain of Epac is in J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Taylor S.S. J. Biol. Chem. Full Text PDF PubMed Google and directly with the phosphate of cAMP Y. L. Taylor S.S. Science. 1995; 269: PubMed Scopus Google Scholar). this leads to the of nucleotide binding activity and biological activity of Epac H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). cells the Epac, cells in a to the cells in response to in of PKB activity to that the cAMP-mediated PKB activation the of Epac activation and required a is the only for Epac activation to PKB activation, we the of in and and shown in the of in cells the of the and not the of in well with our that the of Epac in cells is the of Epac In contrast, cells of with is with the that Epac is to in the of and Epac is of further in response to cAMP (18.de Rooij J. Zwartkruis F.J.T. Verheijen M.H.G. Cool R.H. Nijman S.M.B. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1607) Google Scholar, 19.Kawasaki H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). with further the of in These that activation is in PKB activation of is required for PKB activation, we a in cells to the in activation of of the of Epac to PKB in cells in response to intracellular cAMP. activated PKB the fact that in cells in our and not in cells, the effect of may be that is of Epac and that activation is required for PKB activation in proper subcellular targeting of Epac is required for cAMP-mediated PKB activation and to further that the effect of cAMP on PKB phosphorylation in cells to the of a Epac, we into the cells a whose been Epac is inactive be to the subcellular Epac exerts its shown in the protein, protein the cell while the Epac a the of the Epac is for proper cellular targeting of Epac. cells the were with of PKB as shown in cells, cAMP the phosphorylation of PKB in with the inhibitory effect of as we in the cells domain also that of in the and of stimulation cAMP binding activity and its activity can be regulated by cAMP as the Epac in with A. Cool R.H. Rooij J. Bos J.L. Wittinghofer A. J. Mol. Biol. 2001; PubMed Scopus Google Scholar), with our of the targeting domain of the Epac to its proper subcellular activates the that is necessary for PKB activation in response to intracellular cAMP These further that Epac is for the effects of cAMP on PKB in cells and proper targeting of Epac is for the cAMP-mediated activation of PKB in vivo. a of the activation of PKB is dependent upon the of PI3K in many S.R. P. Biol. 7: Full Text Full Text PDF PubMed Google Scholar, L. H. McCormick F. P. J. Science. 1998; PubMed Scopus Google Scholar), stimulation of PKB can also be achieved in a Hemmings Van Obberghen E. PubMed Scopus Google Scholar, Hemmings Van Obberghen E. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). PKB activation is dependent on PI3K we examined the effect of a specific PI3K on cAMP-mediated activation of PKB in can be in and phosphorylation of of PKB in cells Epac by of the cells with were with These indicate that PKB activation by by a that of Epac is the thyroid as has been shown by that the of Epac is that human H. Springett G.M. S. M. M. A.M. Science. 1998; PubMed Scopus Google Scholar). cell Wistar rat thyroid cells by Epac a protein Epac is in In the of Epac in with a to that of the cell Interestingly, unlike the cells, cells to the including thyrotropin in a to that of cAMP activated the PKB Furthermore, a active in cells to a in PKB activation in response to In to directly regulating many important cellular processes, cAMP an array of intracellular signaling pathways such the MAP kinase pathways (7.Frodin M. Peraldi P. Van Obberghen E. J. Biol. Chem. 1994; 269: 6207-6217Abstract Full Text PDF PubMed Google Scholar, B.M.T. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. 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Chem. 2001; Full Text Full Text PDF Scopus Google Scholar). In to the inhibitory effects of cAMP on PKB in cells, our study that cAMP activated PKB in Epac These that the cAMP activates Epac and PKA, can effects on PKB The cellular effects of Epac on PKB activation can be mediated by a or Although we the that Epac activates PKB through a our suggests that activation of is necessary for PKB activation as of a form of the effect of Epac in of Epac leads to a activation of in activation of to Epac and activity of M. 1993; Google Scholar), is most to the PKB activation the of PKB activity is only in cells that only a specific of is important for PKB a specific Epac further this of the of Epac the to be to proper subcellular is to PKB through activation of specific cellular activation of is an important mechanism for in regulation. It has been previously that guanine nucleotide exchange factor that a domain with Epac, can the membrane of while the the cellular X. T. Y. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). on our a for cAMP-mediated PKB regulation is shown in the two intracellular cAMP Epac and PKA, mediate the opposing effects of cAMP on PKB the through PKA, can PKB recent study by S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google that of PKB by PKA is It further suggested that PKA inhibits PKB activity by PI3K kinase activity and consequently PKB its the membrane S. H. J. J. Biol. Chem. 2001; Full Text Full Text PDF Scopus Google Scholar). The mechanism of PI3K is not and It is that PKA directly PI3K and consequently its kinase activity. It has also been proposed that PKA regulates activation through phosphorylation A. L.A. Meinkoth J.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). the other our study that cAMP can also PKB through an signaling in a of activation of PKB our understanding of the cAMP-mediated signaling pathways. Our of opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is conceivable that the net of the cellular effects of cAMP may be dependent upon the dynamic of Epac and PKA and specific subcellular distribution in a Our that activates PKB in cells that of Epac our that Epac activates while PKA inhibits PKB in response to cAMP and the cellular effects of cAMP are of the Epac and PKA signaling pathways. Furthermore, our that the effects of Epac and PKA activation on a specific may provide an additional regulatory mechanism for intracellular cAMP signaling. the complex and important that cAMP in regulating cell differentiation, and is that other cAMP-mediated signaling pathways may also L. Bos The for the Epac and for in