Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses. However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on F-actin and ERK2 depending on the assay and Dictyostelium strain used, respectively. Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.
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Bode AM, Dong Z (2007) The enigmatic effects of caffeine in cell cycle and cancer. Cancer Lett 247:26–39. https://doi.org/10.1016/j.canlet.2006.03.032
Pohanka M (2015) The perspective of caffeine and caffeine derived compounds in therapy. Bratisl Med J 116:520–530
Reinke A, Chen JCY, Aronova S, Powers T (2006) Caffeine targets TOR complex I and provides evidence for a regulatory link between the FRB and kinase domains of Tor1p. J Biol Chem 281:31616–31626. https://doi.org/10.1074/jbc.M603107200
Brenner M, Thoms SD (1984) Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum. Dev Biol 101:136–146
Reymond CD, Schaap P, Véron M, Williams JG (1995) Dual role of cAMP during Dictyostelium development. Experientia 51:1166–1174. https://doi.org/10.1007/BF01944734
Theibert A, Devreotes PN (1983) Cyclic 3′, 5′-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase. J Cell Biol 97:173–177
Alvarez-Curto E, Weening KE, Schaap P (2007) Pharmacological profiling of the Dictyostelium adenylate cyclases ACA, ACB and ACG. Biochem J 401:309–316. https://doi.org/10.1042/BJ20060880
Pupillo M, Klein P, Vaughan R et al (1988) cAMP receptor and G-protein interactions control development in Dictyostelium. In: Cold spring harbor symposia on quantitative biology. pp 657–665
Kumagai A, Pupillo M, Gundersen R et al (1989) Regulation and function of G alpha protein subunits in Dictyostelium. Cell 57:265–275
Kumagai A, Hadwiger J, Pupillo M, Firtel R (1991) Molecular genetic analysis of two G alpha protein subunits in Dictyostelium. J Biol Chem 266:1220–1228
Devreotes PN, Bhattacharya S, Edwards M et al (2017) Excitable signal transduction networks in directed cell migration. Annu Rev Cell Dev Biol 33:103–125. https://doi.org/10.1146/annurev-cellbio-100616-060739
Lim CJ, Spiegelman GB, Weeks G (2001) RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation. EMBO J 20:4490–4499. https://doi.org/10.1093/emboj/20.16.4490
Lee S, Comer FI, Sasaki A et al (2005) TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium. Mol Biol Cell 16:4572–4583
Chen MY, Long Y, Devreotes PN (1997) A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium. Genes Dev 11:3218–3231
Comer FI, Parent CA (2006) Phosphoinositide 3-kinase activity controls the chemoattractant-mediated activation and adaptation of adenylyl cyclase. Mol Biol Cell 17:357–366. https://doi.org/10.1091/mbc.E05-08-0781
Charest PGG, Shen Z, Lakoduk A et al (2010) A Ras signaling complex controls the RasC-TORC2 pathway and directed cell migration. Dev Cell 18:737–749. https://doi.org/10.1016/j.devcel.2010.03.017
Scavello M, Petlick ARAR, Ramesh R et al (2017) Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium. J Cell Sci 130:1545–1558. https://doi.org/10.1242/jcs.177170
Müller-Taubenberger A, Kortholt A, Eichinger L (2013) Simple system—substantial share: the use of Dictyostelium in cell biology and molecular medicine. Eur J Cell Biol 92:45–53
Bastounis E, Meili R, Alonso-Latorre B et al (2011) The SCAR/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility. Mol Biol Cell 22:3995–4003
Jeon TJ, Lee D-J, Merlot S et al (2007) Rap1 controls cell adhesion and cell motility through the regulation of myosin II. J Cell Biol 176:1021–1033. https://doi.org/10.1083/jcb.200607072
Meili R, Ellsworth C, Lee S et al (1999) Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J 18:2092–2105
Sasaki AT, Chun C, Takeda K, Firtel RA (2004) Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement. J Cell Biol 167:505–518
Fey P, Dodson RJ, Basu S, Chisholm RL (2013) One stop shop for everything Dictyostelium: DictyBase and the Dicty Stock Center in 2012. Methods Mol Biol 983:59–92. https://doi.org/10.1007/978-1-62703-302-2-4
Insall RH, Borleis J, Devreotes PN (1996) The aimless RasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium. Curr Biol 6:719–729
Sasaki AT, Janetopoulos C, Lee S et al (2007) G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. J Cell Biol 178:185–191
Zhang S, Charest PGG, Firtel RAA (2008) Spatiotemporal regulation of Ras activity provides directional sensing. Curr Biol 18:1587–1593. https://doi.org/10.1016/j.cub.2008.08.069
Van Haastert PJ (2006) Analysis of signal transduction: formation of cAMP, cGMP, and Ins(1,4,5)P3 in vivo and in vitro. Methods Mol Biol 346:369–392. https://doi.org/10.1385/1-59745-144-4:369
Chung CY, Firtel RA (1999) PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis. J Cell Biol 147:559–576
Takeda K, Shao D, Adler M et al (2012) Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway. Sci Signal 5:ra2. https://doi.org/10.1126/scisignal.2002413
Dormann D, Weijer G, Parent CA et al (2002) Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development. Curr Biol 12:1178–1188
Parent CA, Blacklock BJ, Froehlich WM et al (1998) G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95:81–91
Kamimura Y, Devreotes PN (2010) Phosphoinositide-dependent protein kinase (PDK) activity regulates phosphatidylinositol 3,4,5-trisphosphate-dependent and -independent protein kinase B activation and chemotaxis. J Biol Chem 285:7938–7946. https://doi.org/10.1074/jbc.M109.089235
Liao X-HH, Buggey J, Kimmel AR et al (2010) Chemotactic activation of Dictyostelium AGC-family kinases AKT and PKBR1 requires separate but coordinated functions of PDK1 and TORC2. J Cell Sci 123:983–992. https://doi.org/10.1242/jcs.064022
Kamimura Y, Xiong Y, Iglesias PA et al (2008) PIP3-independent activation of TorC2 and PKB at the cell’s leading edge mediates chemotaxis. Curr Biol 18:1034–1043. https://doi.org/10.1016/j.cub.2008.06.068
Liu Q, Wang J, Kang S et al (2011) Discovery of 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl) benzo[h][1,6]naphthyridin-2(1H)-one (Torin2) as a potent, selective, and orally available mammalian target of rapamycin (mTOR) inhibitor for treatment of cancer. J Med Chem 54:1473–1480. https://doi.org/10.1021/jm101520v
Gomer RHRH, Armstrong DD, Leichtling BHBH, Firtel RARA (1986) cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor. Proc Natl Acad Sci USA 83:8624–8628. https://doi.org/10.1073/pnas.83.22.8624
Schwebs DJ, Hadwiger JA (2015) The Dictyostelium MAPK ERK1 is phosphorylated in a secondary response to early developmental signaling. Cell Signal 27:147–155. https://doi.org/10.1016/j.cellsig.2014.10.009
van Haastert PJ, Kuwayama H (1997) cGMP as second messenger during Dictyostelium chemotaxis. FEBS Lett 410:25–28
Veltman D, Van Haastert PJM (2003) Regulation of Dictyostelium guanylyl cyclases. Protist 154:33–42. https://doi.org/10.1078/143446103764928477
Riedl J, Crevenna AH, Kessenbrock K et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5:605–607. https://doi.org/10.1038/nmeth.1220
Hall AL, Warren V, Dharmawardhane S, Condeelis J (1989) Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation. J Cell Biol 109:2207–2213. https://doi.org/10.1083/jcb.109.5.2207
Sasaki AT, Janetopoulos C, Lee S et al (2007) G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. J Cell Biol 178:. https://doi.org/10.1083/jcb.200611138
Charest PGG, Firtel RAA (2006) Feedback signaling controls leading-edge formation during chemotaxis. Curr Opin Genet Dev 16:339–347. https://doi.org/10.1016/j.gde.2006.06.016
Knetsch MLW, Epskamp SJP, Schenk PW et al (1996) Dual role of cAMP and involvement of both G-proteins and ras in regulation of ERK2 in Dictyostelium discoideum. EMBO J 15:3361–3368
Brzostowski JA, Kimmel AR (2006) Nonadaptive regulation of ERK2 in Dictyostelium: implications for mechanisms of cAMP Relay10.1091/mbc.E06-05-0376. Mol Biol Cell 17:4220–4227
Takeda K, Sasaki AT, Ha H et al (2007) Role of PI3 kinases in chemotaxis in Dictyostelium. J Biol Chem 282:11874–11884
Tariqul Islam AFM, Yue H, Scavello M et al (2018) The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a negative effect of caffeine and potential role of microtubules. Cell Signal 48:25–37. https://doi.org/10.1016/j.cellsig.2018.04.005
Gonzalez C, Klein G, Satre M (1990) Caffeine, an inhibitor of endocytosis in Dictyostelium discoideum amoebae. J Cell Physiol 144:408–415. https://doi.org/10.1002/jcp.1041440307
Zhou K, Pandol S, Bokoch G, Traynor-Kaplan AE (1998) Disruption of Dictyostelium PI3K genes reduces [32P]phosphatidylinositol 3,4 bisphosphate and [32P]phosphatidylinositol trisphosphate levels, alters F-actin distribution and impairs pinocytosis. J Cell Sci 111(Pt 2):283–294
We are grateful to the Dicty Stock Center and its material depositors for providing cells and DNA constructs.
This study was funded by a Research Scholar Grant 127940-RSG-15-024-01-CSM from the American Cancer Society to P.G.C. M.S. was supported by NIH T32 Grant GM008804 and P.L. was supported by a U.S. Public Health Service Grant GM037830.
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Tariqul Islam, A.F.M., Scavello, M., Lotfi, P. et al. Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors. Mol Cell Biochem 457, 157–168 (2019). https://doi.org/10.1007/s11010-019-03520-z