The discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important discoveries in the roles of EPACs greater understanding of their physiological function in cAMP-mediated signaling is impeded by the absence of EPAC-specific antagonist.To overcome this deficit, we have developed a fluorescence-based high throughput assay for screening EPAC specific antagonists. Our assay is highly reproducible and simple to perform using the "mix and measure" format. A pilot screening using the NCI-DTP diversity set library led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity.Our study establishes a robust high throughput screening assay that can be effectively applied for the discovery of EPAC-specific antagonists, which may provide valuable pharmacological tools for elucidating the biological functions of EPAC and for promoting an understanding of disease mechanisms related to EPAC/cAMP signaling.
Abstract Background: Differentiated thyroid cancer (DTC) is commonly diagnosed in women of child-bearing age, but whether pregnancy influences the prognosis of DTC remains controversial. This study aimed to summarize existing evidence regarding the association of pregnancy with recurrence risk in patients previously treated for DTC. Methods: We searched PubMed, Embase, Web of Science, Cochrane, and Scopus based on the prespecified protocol registered at PROSPERO (CRD42022367896). After study selection, two researchers independently extracted data from the included studies. For quantitative data synthesis, we used random-effects meta-analysis models to pool the proportion of recurrence (for pregnant women only) and odds ratio (OR; comparing the risk of recurrence between the pregnancy group and the nonpregnancy group), respectively. Then we conducted subgroup analyses to explore whether risk of recurrence differed by response to therapy status or duration of follow-up time. We also assessed quality of the included studies. Results: A total of ten studies were included. The sample size ranged from 8 to 235, with participants’ age at pregnancy or delivery ranging from 28 to 35 years. The follow-up time varied from 0.1 to 36.0 years. The pooled proportion of recurrence in all pregnant patients was 0.13 (95% confidence intervals [CI]: 0.06–0.25; I 2 : 0.58). Among six included studies reporting response to therapy status before pregnancy, we observed a trend for increasingly higher risk of recurrence from excellent, indeterminate, and biochemically incomplete to structurally incomplete response to therapy ( P trend <0.05). The pooled risk of recurrence in the pregnancy group showed no evidence of a significant difference from that in the nonpregnancy group (OR: 0.75; 95% CI: 0.45–1.23; I 2 : 0). The difference in follow-up time (below/above five years) was not associated with either the proportion of recurrence in all pregnant patients ( P >0.05) or the OR of recurrence in studies with a comparison group ( P >0.05). Two included studies that focused on patients with distant metastasis also did not show a significant difference in disease recurrence between pregnancy and nonpregnancy groups (OR: 0.51 [95% CI: 0.14–1.87; I 2 : 59%]). Conclusion: In general, pregnancy appears to have a minimal association with the disease recurrence of DTC with initial treatment. Clinicians should pay more attention to progression of DTC among pregnant women with biochemical and/or structural persistence. Registration: PROSPERO, https://www.crd.york.ac.uk/PROSPERO/; No. CRD42022367896.
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. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1167) Google Scholar). Epac proteins bind cAMP with high affinity and activate their downstream target Rap1, a Ras superfamily guanine nucleotide-binding protein. Rap1, initially identified as an antagonist for the transforming function of Ras (20.Kitayama H. Suigimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (760) Google Scholar) and on the basis of its high homology to Ras (21.Prizon V. Chardin P. Lerosey I. Olofsson B. Tavitian A. Oncogene. 1998; 3: 201-204Google Scholar), is activated in response to an increase in intracellular cAMP in additional to many other stimuli (22.Altschuler D.L. Peterson S.Sn. Ostrowski M.C. Lapetina E.G. J. Biol. Chem. 1995; 270: 10373-10376Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 23.Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 74-82Abstract Full Text Full Text PDF Scopus (944) Google Scholar, 24.Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). Although PKA can phosphorylate Rap1 at its C terminus (25.Quilliam L.A. Mueller H. Bohl B.P. Prossnitz V. Sklar L.A. Der C.L. Bokoch G.M. J. Immunol. 1991; 147: 1628-1635PubMed Google Scholar), the Rap1S180A mutant that lacks the PKA phosphorylation site 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). Moreover, mutation of the PKA phosphorylation site in Drosophila Rap1 does not impair its biological functions (26.Asha H. deRuiter N.D. Wang M.-G. Hariharan I.K. EMBO J. 1999; 18: 605-615Crossref PubMed Scopus (123) Google Scholar). These observations suggest cAMP-mediated activation of Rap1 may be independent of phosphorylation by PKA. It is most likely that the cAMP-mediated signaling mechanism is much more complex than was believed earlier, and many cAMP-mediated effects that were previously thought to act through PKA are in fact also transduced by Epac. Therefore, it is imperative to reformulate concepts of cAMP-mediated signaling to include the contribution of Epac. The existence of Epac immediately raises many questions regarding the mechanism of cAMP-mediated signaling. Since both PKA and Epac are broadly expressed in many tissues (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. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1167) Google Scholar), an increase in intracellular cAMP levels will lead to the activation of both enzymes, and possibly other potential cAMP target(s) as well. It is conceivable that while PKA acts through a discrete set of signaling pathways, Epac may enlist distinct signaling pathways, and the net cellular effects of cAMP are dictated by the sum of these events. Therefore, the apparent cellular effect of cAMP can vary depending upon the relative cellular abundance and distribution of Epac and PKA. Our hypothesis is that there is a dynamic control of cellular expression and targeting of Epac and PKA which, coupled with the dynamic changes in the concentration of cAMP, are the controlling mechanisms that determine the observed cell type-specific cAMP effects. To test this hypothesis, we examined the specific effects of Epac and PKA on the PI3K/PKB pathway that has been reported to exhibit cell type-specific responses to cAMP. Our study shows for the first time that although Epac and PKA are activated by a common upstream second messenger cAMP, they can exert opposing effects in regulating downstream targets such as PKB. Therefore, the net outcome of cAMP signaling on PKB activation may be dependent upon the dynamic abundance and distribution of intracellular Epac and PKA. Human epaccDNA was a generous gift from Dr. J. L. Bos (University Medical Center Utrecht, The Netherlands). C-terminal FLAG-tagged epac was constructed by PCR using a 5′ primer, 5′-ccatatgctagcATGGTGTTGAGAAGGATGCAC-3′ and a 3′ primer, 5′-ttcggaattcTTATTTGTCGTCGTCTTTGTAGTCTGGCTCCAGCTCTCGGGAGA-3′. The amplified cDNA fragment was subcloned into the NheI-EcoRI sites of pcDNA3.1 mammalian expression vector (Invitrogen). C-terminal, gfp-tagged epac (Epac-EGFP) was constructed by PCR using a 5′ primer 5′-ccatatgctaGCATGGTGTTGAGAAGGATGCAC-3′ and a 3′ primer 5′-tcatatagagctcCTGGCTCCAGCTCTCGGGAGAG-3′. The amplified cDNA fragment was subcloned into the NheI-SacI sites of eukaryotic expression vector pEGFP-N3 (CLONTECH Laboratories), in which the epac gene was fused in-frame and upstream from the gfp gene. By the same method, we constructed the Δ(1–148) epac deletion mutant and Δ(1–148) epac-egfp that lack the first 148 N-terminal Epac amino acid residues. All DNA constructs were confirmed by DNA sequencing. Monoclonal Rap1 antibodies were purchased from Transduction Laboratories. Antibodies that specifically recognize PKB and phosphorylated PKB were from Cell Signaling. Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgGs were from Bio-Rad. The SuperSignal West Pico Chemiluminescent Substrate Reagents kit was obtained from Pierce. Culture media were from Invitrogen. Fetal bovine serum was from Sigma. Forskolin and N6, O2′-dibutyryl adenosine-3′,5′-cyclic monophosphate (dibutyryl cAMP) were fromCalbiochem. Human embryonic kidney (HEK) 293 cells were from American Type Culture Collection. All chemicals were reagent grade. Specific polyclonal antibodies against Epac were generated by Alpha Diagnostic International Inc. (San Antonio, TX) using synthetic Epac peptide spanning residues 41–60, (C)DFSESLEQASTERVLRAGR. The immune sera were initially purified using an ImmunoPure IgG Purification Kit (Pierce) and further affinity purified by immobilized Epac peptide using the SulfoLink Antibody Purification Kit (Pierce). Antibody elution fractions were dialyzed in PBS. Specificity of purified antibody was confirmed by Western blot using recombinant Epac protein expressed in insect Sf9 cells. HEK 293 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The cultures were maintained at 37 °C in a humidified chamber supplemented with 5% CO2. The day before the transfection, cells were subcultured into 6-well tissue culture plates, grown to 60–80% confluence overnight, and transfected with purified plasmid DNA at 1 μg/well using LipofectAMINE PlusTM Reagent (Invitrogen). Stable transfectants were selected using G418 at 0.15 mg/ml. Wistar Rat thyroid cells were maintained at 37 °C in 5% CO2 in 3H medium (Coon's modified Ham's F-12 medium containing 1 milliunits/ml of crude bovine TSH, 10 μg/ml insulin, 5 μg/ml transferrin, and 5% calf serum). Cells grown in 6-well plates at 70–90% confluence were serum starved for 20 h before the activation experiments. Cells were treated with forskolin (10 μm), dibutyryl cAMP (1 mm) for various time. Activation was terminated by washing cells twice with PBS. For experiments involving kinase inhibitors, cells were treated with H89 (5 μm), LY294002 (10 μm), or wortmannin (100 nm) 20 min prior to stimulation. Cell lysates were prepared by directly suspending the cells into Laemmli SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.5 mm phenylmethylsulfonyl fluoride). The GTP loading status of Rap1 was assessed using a glutathione S-transferase fusion of the Rap1-binding domain of RalGDS as described earlier (27.de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). Briefly, cells were grown to 75% confluence in 100-mm Corning culture dish, starved in serum-free medium for 48 h, and treated with 10 μm forskolin for 5 min. Following three washes in PBS, the cells were lysed in a buffer containing 50 mm Tris (pH 7.5), 150 mm NaCl, 20 mm MgCl2, 5 mm EGTA, 1% Triton X-100, and Roche EDTA-free protease inhibitors. The cell lysate was mixed with 40 μl of glutathione-Sepharose beads with 30 μg of glutathione S-transferase-RalGDS-Rap1-binding domain bound and incubated at 4 °C for 2 h with gentle agitation. Following three washes in lysis buffer, the beads were suspended in 40 μl of SDS sample buffer. 15 μl of protein samples were loaded onto a 15% SDS-polyacrylamide gel and further analyzed with Western blot using Rap1 specific antibodies. Protein concentration of cell lysates was assayed with the Bio-Rad protein assay reagent, and 30 μg of protein was denatured by heating at 95 °C for 5 min prior to resolution by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) using a Trans-Blot SD transfer cell (Bio-Rad) and a transfer buffer containing 25 mm Tris, 192 mm glycine, 20% methanol, 0.0375% SDS (pH 8.3). Membranes were placed in 5% nonfat milk in Tris-buffered saline/Tween buffer (TTBS) at 4 °C overnight to block nonspecific binding sites, followed by incubation with various antibodies for 1 h at room temperature: anti-PKB antibodies (1:1000), anti-phosphate T308 PKB antibodies (1:1000), anti-phosphate S473 PKB antibodies (1:1000), and affinity purified anti-Epac polyclonal antibody (1 μg/ml). Membranes were then washed with TTBS and incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody for 1 h at room temperature. Following three 10-min washes in TTBS, membranes were treated with substrates for 5 min and exposed to x-ray film at room temperature. At least three independent experiments were performed for each Western blot. PKB activity was measured using an Akt Kinase Assay kit from Cell Signaling following manufacturer's protocol. Mutagenesis was performed using a QuikChangeTM Site-directed Mutagenesis Kit (Stratagene). All DNA constructs were sequenced to confirm the mutation as well as to verify the accuracy of the full-length cDNA sequence. To detect the subcellular localization of Epac-EGFP and Δ(1–148)Epac-EGFP, cells were subcultured after transfection with an appropriate plasmid in 6-well plates with a poly-l-lysine-coated coverslip in each well and grown for 16–24 h. Then the cells were fixed in 2% paraformaldehyde in PBS. The samples were rinsed with PBS, mounted on glass slides, and sealed in 70% glycerol. Fluorescent signals were revealed under the fluorescence microscope (Olympus BX51), using an fluorescein isothiocyanate/GFP filter with maximum excitation at 488 nm and maximum emission at 525 nm. Fluorescence images were recorded using a Hamamatsu digital camera (C4742-95). To elucidate the specific roles of Epac and PKA in modulating the PKB signaling pathway, we examined the effects of cAMP-elevating agents on activation of PKB in parental HEK293 and HEK293 cells that have been stably transfected with Epac. As shown in Fig. 1A, HEK293 cells expressed normal levels of PKA but undetectable amounts of Epac when cell lysates from HEK293 and HEK293/Epac cells were probed by affinity purified PKA catalytic subunit antibodies and affinity purified Epac antibodies, respectively. A protein with the apparent molecular weight of 100,000 corresponding to the calculated size of Epac was readily detectable in the Epac-transfected HEK293 cells. Furthermore, overexpression of Epac in HEK293 cells did not affect the protein levels of PKA (Fig. 1A). When treated with forskolin, endogenous PKB activity in HEK293/Epac cells was increased about 3-fold (Fig. 1B), as monitored by phosphorylation of Ser473 and Thr308, whose phosphorylation status is critical for the kinase activity of PKB (28.Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar, 29.Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (909) Google Scholar). This observation was in marked contrast to that of the parental non-transfected HEK293 cells where forskolin caused an apparent inhibition in phosphorylation of the endogenous PKB at both Ser473 and Thr308(Fig. 1C). Inhibition of PKB phosphorylation by cAMP was blocked following treatment with a specific PKA inhibitor H89 (Fig. 1C), suggesting that the inhibitory effect of cAMP on PKB is likely mediated by PKA. Moreover, H89 also augmented the cAMP-mediated PKB activation in HEK293/Epac cells (Fig. 1B), confirming the inhibitory effect of PKA on PKB activation. The basal levels of PKB activation was slightly elevated (∼30%) in 293/Epac cells as compared with that of the parental 293 cells when the PKB phosphorylation levels of 293 and 293/Epac cells were monitored on the same blot (Fig. 1D). In addition to monitoring the phosphorylation status of PKB Ser473 and Thr308, we also measured the endogenous cellular PKB kinase activity directly. Results from these kinase activity experiments confirmed the Western blotting data (Table I). To our knowledge, this is the first demonstration that Epac and PKA regulate the PKB signaling cascade in opposite directions. To monitor the temporal relationship between the roles of Epac and PKA on modulating PKB activation, we examined the effects of forskolin on PKB phosphorylation in 293 and 293/Epac cells as a function of time. As shown in Fig. 1E, PKB activation by the Epac/Rap1 pathway in the 293/Epac cells was fast and sustained: reaching maximal within 5 min while the inhibitory effect of PKA on PKB peaked around 20 min and slowly attenuated after 30 min.Table IEndogenous PKB activity in HEK293 and HEK293/Epac cells−Forskolin+Forskolin+Forskolin + H89HEK2931.0aThe basal PKB activity in the absence of forskolin treatment was set to 1.0 and PKB activities in the presence of treatments were normalized to the basal PKB level.0.3 ± 0.21.5 ± 0.3HEK293/Epac1.0aThe basal PKB activity in the absence of forskolin treatment was set to 1.0 and PKB activities in the presence of treatments were normalized to the basal PKB level.bThe basal PKB activity in HEK293/Epac cells is about 40% higher than that in HEK293 cells.3.4 ± 0.95.9 ± 1.0Endogenous PKB activities in HEK293 and HEK293/Epac with and without forskolin treatments were measured using Akt Kinase Assay kit from Cell Signaling following the manufacturer's protocol.a The basal PKB activity in the absence of forskolin treatment was set to 1.0 and PKB activities in the presence of treatments were normalized to the basal PKB level.b The basal PKB activity in HEK293/Epac cells is about 40% higher than that in HEK293 cells. Open table in a new tab Endogenous PKB activities in HEK293 and HEK293/Epac with and without forskolin treatments were measured using Akt Kinase Assay kit from Cell Signaling following the manufacturer's protocol. We used dibutyryl cAMP, a classical cell membrane-permeable cAMP analog (30.Posternak T. Weimmann G. Methods Enzymol. 1974; 38: 399-409Crossref PubMed Scopus (43) Google Scholar) as a direct activating agent for Epac and PKA to verify the effect of forskolin in our study. As shown in Fig. 2, dibutyryl cAMP inhibited the PKB activation in parental 293 cells while promoted the activation of PKB in 293/Epac cells in a manner similar to that of forskolin. Taken together, our results strongly suggest that elevation of cAMP lead to suppression or activation of PKB in 293 cells and 293/Epac cells, respectively. To unambiguously demonstrate that the apparent PKB activation observed in Epac-expressing HEK293 cells in the presence of cAMP-elevating agents is indeed the result of direct activation of Epac by cAMP, a single point mutation of a critical residue, Arg279, in the cyclic nucleotide-binding domain of Epac was created. This Arg residue is conserved in all known cyclic nucleotide-binding proteins (31.Shabb J.B. Corbin J.D. J. Biol. Chem. 1992; 267: 5723-5726Abstract Full Text PDF PubMed Google Scholar, 32.Bubis J. Neitzel J.J. Saraswat L.D. Taylor S.S. J. Biol. Chem. 1988; 263: 9668-9673Abstract Full Text PDF PubMed Google Scholar) and interacts directly with the phosphate of cAMP (33.Su Y. Dostmann R.G. Herberg F.W. Durick K. Xuong N-h. Ten Eyck L. Taylor S.S. Varughese K.I. Science. 1995; 269: 807-813Crossref PubMed Scopus (343) Google Scholar). Mutation at this Arg residue leads to the loss of cyclic nucleotide binding activity and biological activity of Epac (19.Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1167) Google Scholar). Unlike HEK293 cells expressing the wild-type Epac, EpacR279E-expressing HEK293 cells behaved in a manner similar to the parental HEK293 cells in response to forskolin stimulation, resulting in inhibition of PKB activity (Fig. 3). This led us to conclude that the observed cAMP-mediated PKB activation was the direct consequence of Epac activation and required a functional cyclic nucleotide-binding domain. Rap1 is the only known downstream effector for Epac described so far. To test if Rap1 activation contributes to Epac-mediated PKB activation, we compared the levels of GTP-bound Rap1 in serum-starved and forskolin-treated HEK293/Epac and parental HEK293 cells. As shown in Fig. 4A, the amount of GTP-bound Rap1 in parental HEK293 cells was below the detection limit of the pull-down assay and forskolin did not significantly increase the GTP loading of Rap1 in these cells. This fits very well with our observation that the expression of Epac in HEK293 cells is very low, under the detection limit of Western blot using affinity purified Epac antibodies. In contrast, Epac-expressing HEK293 cells exhibited basally elevated levels of GTP-bound Rap1 compared with parental HEK293 cells. This observation is consistent with the original report that Epac overexpression is sufficient to partially activate Rap1 even in the absence of cAMP-elevating agent and Epac is capable of further activating Rap1 specifically 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. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1167) Google Scholar). Treatment with forskolin further increased the proportion of GTP-bound Rap1 in HEK293/Epac cells. These results suggest that Rap1 activation is potentially involved in Epac-mediated PKB activation. To examine if activation of Rap1 is required for Epac-mediated PKB activ
β-Arrestin 1 and 2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic β cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating β cell function and whole-body glucose homeostasis. Initially, we inactivated the Barr1 gene in β cells of adult mice (β-barr1-KO mice). β-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, 2 widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and coimmunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in β cells may prove useful for the development of efficacious antidiabetic drugs.
This paper describes the development of single-domain recombinant antibodies against human telomerase core protein. A His-tagged hTERT spanning main reverse-transcriptase domain of hTERT was purified from host E. coli and used to immunize BALB/c mice. The VHs (heavy chain variable region genes) were amplified by PCR from total RNA of splenocytes and further induced random mutagenesis by DNA shuffling to enrich the repertoire of VH library. All VHs were cloned into phagemid vectors and displayed to generate 4 × 1010 phage libraries. The candidates carrying VH domains against hTERT were primarily screened through three times of panning procedure on His-tagged hTERT coated microplates, and specific antibodies were further selected by West-Western blot. Two clones, designated as a3 and b8, were confirmed to interact with the target in the solid-phase assay. DNA sequencing proved their mouse VH origin. The purified single-domain antibody of b8 could not only recognize native hTERT, but also neutralize human telomerase activity on inhibitory assay and b8 showed the stronger suppressive efficacy compared with a3. The data demonstrated that the developed single-domain recombinant antibodies were hTERT-specific with high potential of binding and activity inhibition.
Objective:To study the expression of p53?bcl 2 and the relationship among p53?bcl 2 and cell cycle in acute leukemia(AL).Methods:49 patients with AL were selected as a study group and 14 patients without hematologic malignancies in the bone marrow sample were selected as a control group. The patient′s bone marrow was withdrawn and marked with monoclonal antibody. The expression of mutant type p53 protein and bcl 2 protein were detected with flow cytometry.26 patients was detected with flow cytometry for cell cycle at the same time.Results:The expression rate of p53 and bcl 2 proteins in AL group is 44.9 % and 49.0 % respectively,which is significantly higher than those in the control group.There were no difference between ALL group and ANLL group, but was significantly lower in M 3 group than in the other non M 3 ANLL group. Mutant type p53 protein was positively related to the expression of bcl 2 protein, and the two proteins might cooperate in the pathogenesis of leukemia. The numbers of cell in the cycle of S+G 2/M in patients with leukemia were significantly lower than those in control. There were no relationship between cell cycle and the expression of p53?bcl 2.Conclusion:p53 mutation and bcl 2 may be related to leukemia.p53 mutation and bcl 2 expression might cooperate in the progression of leukemia.Combined detection of p53 mutation and bcl 2 expression may be helpful for forcasting the prognosis of leukemia.
Abstract Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, precisely how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation remains elusive. Herein, we report that cAMP, a major stress-response second messenger, acts through Epac1 as a regulator of cellular SUMOylation. The Epac1-associated proteome is highly enriched with components of the SUMOylation pathway. Activation of Epac1 by intracellular cAMP triggers phase separation and the formation of nuclear condensates containing Epac1 and general components of the SUMOylation machinery to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low-density lipoprotein induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. These results provide a direct nexus connecting two major cellular stress responses to define a molecular mechanism in which cAMP regulates the dynamics of cellular condensates to modulate protein SUMOylation.
Abstract IMPORTANCE Differentiated thyroid cancer (DTC) is commonly diagnosed in women of child-bearing age, but whether pregnancy influences the prognosis of DTC remained controversial. OBJECTIVE This systematic review and meta-analysis aimed to summarize and appraise the existing evidence of the impact of pregnancy on the prognosis of patients previously treated for DTC. DATA SOURCES We searched PubMed, Embase, Web of Science, Cochrane, and Scopus until February 2023. STUDY SELECTION Studies of patients diagnosed and treated for DTC before pregnancy reporting the recurrence/progression condition of DTC were included. Case reports and studies failing to identify the time of diagnosis or initial treatment were excluded. DATA EXTRACTION AND SYNTHESIS Meta-analyses were conducted according to MOOSE guideline. Data extraction was conducted by two independent investigators with a standard form. Pooled effect estimates were calculated in a random-effects model. MAIN OUTCOMES AND MEASURES DTC recurrence/progression and the type of recurrence/progression (structural or biochemical). RESULTS Among the 10 included studies (n = 625), 4 (n = 143) of them compared the pregnancy group with the non-pregnancy group while the remaining 6 (n = 482) only included the pregnant patients. The pooled proportion of recurrence/progression in all pregnant patients was 13% (95% CI, 6%, 25%). Compared with the non-pregnancy group, the pooled odds ratio of recurrence/progression in the pregnancy group was 0.75 (95% CI, 0.45, 1.23). Two included studies focused on patients with distant metastasis and also did not observe difference in disease recurrence/progression between the pregnancy group and the non-pregnancy group [OR, 0.51 (95% CI, 0.14-1.87)]. Six included studies also reported response to therapy status prior to pregnancy, and the pooled proportion for recurrence/progression in pregnant DTC patients with excellent response (n=287), indeterminate response (n=44), biochemical incomplete response (n=41) and structural incomplete response (n=70) was 0.00 (95% CI, 0.00-0.86), 0.09 (95% CI, 0.00-0.99), 0.20 (95% CI, 0.06-0.46) and 0.45 (95% CI, 0.17-0.76), respectively. There was a trend for an increasingly higher risk of recurrence/progression from excellent, indeterminate, biochemical incomplete to structural incomplete response to therapy ( P <0.05). CONCLUSIONS AND RELEVANCE Pregnancy appears to have a minimal impact on the prognosis of DTC with initial treatment. Clinicians may pay more attention to the progression of DTC among pregnant women with biochemical and/or structural persistence. Key Points Question Does subsequent pregnancy has an impact on the prognosis of patients previously treated for differentiated thyroid cancer (DTC)? Findings In this systematic review and meta-analysis of 10 studies including 625 patients previously treated for DTC and underwent pregnancy subsequently, pregnancy might have a minimal impact on DTC recurrence/progression. Patients with biochemical and/or structural incomplete response to DTC treatment prior to pregnancy appears to have a higher risk of DTC recurrence/progression compared to those with excellent or indeterminate response. Meaning Though pregnancy appears to have little influence on the prognosis of patients previously treated for DTC, patients with biochemical and/or structural persistence should be more carefully monitored during pregnancy.
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