Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors

  • A. F. M. Tariqul Islam
  • Margarethakay Scavello
  • Pouya Lotfi
  • Dustin Daniel
  • Pearce Haldeman
  • Pascale G. CharestEmail author


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.


Dictyostelium Chemotaxis cAMP Caffeine PI3K mTORC2 



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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11010_2019_3520_MOESM1_ESM.pdf (1.7 mb)
Supplementary material 1 (PDF 1721 KB)


  1. 1.
    Bode AM, Dong Z (2007) The enigmatic effects of caffeine in cell cycle and cancer. Cancer Lett 247:26–39. CrossRefGoogle Scholar
  2. 2.
    Pohanka M (2015) The perspective of caffeine and caffeine derived compounds in therapy. Bratisl Med J 116:520–530CrossRefGoogle Scholar
  3. 3.
    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. CrossRefGoogle Scholar
  4. 4.
    Brenner M, Thoms SD (1984) Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum. Dev Biol 101:136–146CrossRefGoogle Scholar
  5. 5.
    Reymond CD, Schaap P, Véron M, Williams JG (1995) Dual role of cAMP during Dictyostelium development. Experientia 51:1166–1174. CrossRefGoogle Scholar
  6. 6.
    Theibert A, Devreotes PN (1983) Cyclic 3′, 5′-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase. J Cell Biol 97:173–177CrossRefGoogle Scholar
  7. 7.
    Alvarez-Curto E, Weening KE, Schaap P (2007) Pharmacological profiling of the Dictyostelium adenylate cyclases ACA, ACB and ACG. Biochem J 401:309–316. CrossRefGoogle Scholar
  8. 8.
    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 657–665Google Scholar
  9. 9.
    Kumagai A, Pupillo M, Gundersen R et al (1989) Regulation and function of G alpha protein subunits in Dictyostelium. Cell 57:265–275CrossRefGoogle Scholar
  10. 10.
    Kumagai A, Hadwiger J, Pupillo M, Firtel R (1991) Molecular genetic analysis of two G alpha protein subunits in Dictyostelium. J Biol Chem 266:1220–1228Google Scholar
  11. 11.
    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. CrossRefGoogle Scholar
  12. 12.
    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. CrossRefGoogle Scholar
  13. 13.
    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 16:4572–4583CrossRefGoogle Scholar
  14. 14.
    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 11:3218–3231CrossRefGoogle Scholar
  15. 15.
    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. CrossRefGoogle Scholar
  16. 16.
    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. CrossRefGoogle Scholar
  17. 17.
    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. CrossRefGoogle Scholar
  18. 18.
    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 92:45–53CrossRefGoogle Scholar
  19. 19.
    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 22:3995–4003CrossRefGoogle Scholar
  20. 20.
    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. CrossRefGoogle Scholar
  21. 21.
    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 18:2092–2105CrossRefGoogle Scholar
  22. 22.
    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 167:505–518CrossRefGoogle Scholar
  23. 23.
    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. CrossRefGoogle Scholar
  24. 24.
    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 6:719–729CrossRefGoogle Scholar
  25. 25.
    Sasaki AT, Janetopoulos C, Lee S et al (2007) G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility. J Cell Biol 178:185–191CrossRefGoogle Scholar
  26. 26.
    Zhang S, Charest PGG, Firtel RAA (2008) Spatiotemporal regulation of Ras activity provides directional sensing. Curr Biol 18:1587–1593. CrossRefGoogle Scholar
  27. 27.
    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. Google Scholar
  28. 28.
    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 147:559–576CrossRefGoogle Scholar
  29. 29.
    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. CrossRefGoogle Scholar
  30. 30.
    Dormann D, Weijer G, Parent CA et al (2002) Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development. Curr Biol 12:1178–1188CrossRefGoogle Scholar
  31. 31.
    Parent CA, Blacklock BJ, Froehlich WM et al (1998) G protein signaling events are activated at the leading edge of chemotactic cells. Cell 95:81–91CrossRefGoogle Scholar
  32. 32.
    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. CrossRefGoogle Scholar
  33. 33.
    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. CrossRefGoogle Scholar
  34. 34.
    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. CrossRefGoogle Scholar
  35. 35.
    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. CrossRefGoogle Scholar
  36. 36.
    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. CrossRefGoogle Scholar
  37. 37.
    Schwebs DJ, Hadwiger JA (2015) The Dictyostelium MAPK ERK1 is phosphorylated in a secondary response to early developmental signaling. Cell Signal 27:147–155. CrossRefGoogle Scholar
  38. 38.
    van Haastert PJ, Kuwayama H (1997) cGMP as second messenger during Dictyostelium chemotaxis. FEBS Lett 410:25–28CrossRefGoogle Scholar
  39. 39.
    Veltman D, Van Haastert PJM (2003) Regulation of Dictyostelium guanylyl cyclases. Protist 154:33–42. CrossRefGoogle Scholar
  40. 40.
    Riedl J, Crevenna AH, Kessenbrock K et al (2008) Lifeact: a versatile marker to visualize F-actin. Nat Methods 5:605–607. CrossRefGoogle Scholar
  41. 41.
    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. CrossRefGoogle Scholar
  42. 42.
    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:.
  43. 43.
    Charest PGG, Firtel RAA (2006) Feedback signaling controls leading-edge formation during chemotaxis. Curr Opin Genet Dev 16:339–347. CrossRefGoogle Scholar
  44. 44.
    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 15:3361–3368CrossRefGoogle Scholar
  45. 45.
    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 17:4220–4227CrossRefGoogle Scholar
  46. 46.
    Takeda K, Sasaki AT, Ha H et al (2007) Role of PI3 kinases in chemotaxis in Dictyostelium. J Biol Chem 282:11874–11884CrossRefGoogle Scholar
  47. 47.
    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. CrossRefGoogle Scholar
  48. 48.
    Gonzalez C, Klein G, Satre M (1990) Caffeine, an inhibitor of endocytosis in Dictyostelium discoideum amoebae. J Cell Physiol 144:408–415. CrossRefGoogle Scholar
  49. 49.
    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):283–294Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of ArizonaTucsonUSA
  2. 2.Eurofins Lancaster Laboratories Professional Scientific Services, LLCMalvernUSA
  3. 3.Department of Basic Medical SciencesUniversity of ArizonaPhoenixUSA
  4. 4.Division of Biology and Biological Engineering, Joint Center for Transitional MedicineCalifornia Institute of TechnologyPasadenaUSA

Personalised recommendations