Depletion of Ric-8B leads to reduced mTORC2 activity


Autoři: Maíra H. Nagai aff001;  Victor P. S. Xavier aff001;  Luciana M. Gutiyama aff001;  Cleiton F. Machado aff001;  Alice H. Reis aff002;  Elisa R. Donnard aff003;  Pedro A. F. Galante aff003;  Jose G. Abreu aff002;  William T. Festuccia aff004;  Bettina Malnic aff001
Působiště autorů: Department of Biochemistry, University of São Paulo, São Paulo, Brazil aff001;  Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil aff002;  Centro de Oncologia Molecular, Hospital Sírio-Libanês, São Paulo, Brazil aff003;  Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil aff004
Vyšlo v časopise: Depletion of Ric-8B leads to reduced mTORC2 activity. PLoS Genet 16(5): e32767. doi:10.1371/journal.pgen.1008255
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008255

Souhrn

mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.

Klíčová slova:

Apoptosis – Cell signaling – Embryos – Epithelium – G-protein signaling – Gene expression – Phenotypes – Polymerase chain reaction


Zdroje

1. Tall GG, Krumins AM, Gilman AG (2003) Mammalian Ric-8A (synembryn) is a heterotrimeric Gα protein guanine nucleotide exchange factor. J Biol Chem 278: 8356–8362. doi: 10.1074/jbc.M211862200 12509430

2. Von Dannecker L, Mercadante A, Malnic B (2005) Ric-8B, an olfactory putative GTP exchange factor, amplifies signal transduction through the olfactory-specific G-protein Gaolf. J Neurosci 25: 3793–3800. doi: 10.1523/JNEUROSCI.4595-04.2005 15829631

3. Chan P, Gabay M, Wright FA, Tall GG (2011) Ric-8B is a GTP-dependent G protein alphas guanine nucleotide exchange factor. J Biol Chem 286: 19932–19942. doi: 10.1074/jbc.M110.163675 21467038

4. Jones DT, Reed RR (1989) Golf: an olfactory neuron-specific G-protein involved in odorant signal transduction. Science 244: 790–795. doi: 10.1126/science.2499043 2499043

5. Belluscio L, Gold GH, Nemes A, Axel R (1998) Mice deficient in G(olf) are anosmic. Neuron 20: 69–81. doi: 10.1016/s0896-6273(00)80435-3 9459443

6. Zhuang X, Belluscio L, Hen R (2000) Golfα mediates dopamine D1 receptor signaling. J Neurosci 20: RC91. doi: 10.1523/JNEUROSCI.20-16-j0001.2000 10924528

7. Gabay M, Pinter ME, Wright FA, Chan P, Murphy AJ, et al. (2011) Ric-8 proteins are molecular chaperones that direct nascent G protein alpha subunit membrane association. Sci Signal 4: ra79. doi: 10.1126/scisignal.2002223 22114146

8. Nagai Y, Nishimura A, Tago K, Mizuno N, Itoh H (2010) Ric-8B stabilizes the alpha subunit of stimulatory G protein by inhibiting its ubiquitination. J Biol Chem 285: 11114–11120. doi: 10.1074/jbc.M109.063313 20133939

9. Zhuang H, Matsunami H (2007) Synergism of accessory factors in functional expression of mammalian odorant receptors. J Biol Chem 282: 15284–15293. doi: 10.1074/jbc.M700386200 17387175

10. Kerr D, Von Dannecker L, Davalos M, Michaloski J, Malnic B (2008) Ric-8B interacts with Gαolf and Gγ13 and co-localizes with Gαolf, Gβ1 and Gγ13 in the cilia of olfactory sensory neurons. Mol Cell Neurosci 38: 341–348. doi: 10.1016/j.mcn.2008.03.006 18462949

11. Von Dannecker L, Mercadante A, Malnic B (2006) Ric-8B promotes functional expression of odorant receptors. Proc Natl Acad Sci USA 103: 9310–9314. doi: 10.1073/pnas.0600697103 16754875

12. Masuho I, Ostrovskaya O, Kramer GM, Jones CD, Xie K, et al. (2015) Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors. Sci Signal 8: ra123. doi: 10.1126/scisignal.aab4068 26628681

13. Machado CF, Nagai MH, Lyra CS, Reis-Silva TM, Xavier AM, et al. (2017) Conditional deletion of Ric-8b in olfactory sensory neurons leads to olfactory impairment. J Neurosci 37: 12202–12213. doi: 10.1523/JNEUROSCI.0943-17.2017 29118104

14. Hung CM, Garcia-Haro L, Sparks CA, Guertin DA (2012) mTOR-dependent cell survival mechanisms. Cold Spring Harb Perspect Biol 4.

15. Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149: 274–293. doi: 10.1016/j.cell.2012.03.017 22500797

16. Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol 14: 1296–1302. doi: 10.1016/j.cub.2004.06.054 15268862

17. Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, et al. (2004) Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 6: 1122–1128. doi: 10.1038/ncb1183 15467718

18. Cybulski N, Hall MN (2009) TOR complex 2: a signaling pathway of its own. Trends Biochem Sci 34: 620–627. doi: 10.1016/j.tibs.2009.09.004 19875293

19. Xie J, Wang X, Proud CG (2018) Who does TORC2 talk to? Biochem J 475: 1721–1738. doi: 10.1042/BCJ20180130 29794170

20. Oh WJ, Wu CC, Kim SJ, Facchinetti V, Julien LA, et al. (2010) mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide. EMBO J 29: 3939–3951. doi: 10.1038/emboj.2010.271 21045808

21. Zinzalla V, Stracka D, Oppliger W, Hall MN (2011) Activation of mTORC2 by association with the ribosome. Cell 144: 757–768. doi: 10.1016/j.cell.2011.02.014 21376236

22. Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, et al. (2003) BayGenomics: a resource of insertional mutations in mouse embryonic stem cells. Nucleic Acids Res 31: 278–281. doi: 10.1093/nar/gkg064 12520002

23. Stanford WL, Cohn JB, Cordes SP (2001) Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet 2: 756–768. doi: 10.1038/35093548 11584292

24. Munger SD, Leinders-Zufall T, Zufall F (2009) Subsystem organization of the mammalian sense of smell. Annu Rev Physiol 71: 115–140. doi: 10.1146/annurev.physiol.70.113006.100608 18808328

25. Ma M, Grosmaitre X, Iwema CL, Baker H, Greer CA, et al. (2003) Olfactory signal transduction in the mouse septal organ. J Neurosci 23: 317–324. doi: 10.1523/JNEUROSCI.23-01-00317.2003 12514230

26. Roelink H, Porter JA, Chiang C, Tanabe Y, Chang DT, et al. (1995) Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81: 445–455. doi: 10.1016/0092-8674(95)90397-6 7736596

27. Copp AJ (2005) Neurulation in the cranial region—normal and abnormal. J Anat 207: 623–635. doi: 10.1111/j.1469-7580.2005.00476.x 16313396

28. Copp AJ, Greene ND, Murdoch JN (2003) The genetic basis of mammalian neurulation. Nat Rev Genet 4: 784–793. doi: 10.1038/nrg1181 13679871

29. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, et al. (2016) Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Res 44: W90–97. doi: 10.1093/nar/gkw377 27141961

30. Hresko RC, Mueckler M (2005) mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes. J Biol Chem 280: 40406–40416. doi: 10.1074/jbc.M508361200 16221682

31. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM (2005) Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101. doi: 10.1126/science.1106148 15718470

32. Guertin DA, Stevens DM, Thoreen CC, Burds AA, Kalaany NY, et al. (2006) Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCalpha, but not S6K1. Dev Cell 11: 859–871. doi: 10.1016/j.devcel.2006.10.007 17141160

33. Shiota C, Woo JT, Lindner J, Shelton KD, Magnuson MA (2006) Multiallelic disruption of the rictor gene in mice reveals that mTOR complex 2 is essential for fetal growth and viability. Dev Cell 11: 583–589. doi: 10.1016/j.devcel.2006.08.013 16962829

34. Calnan DR, Brunet A (2008) The FoxO code. Oncogene 27: 2276–2288. doi: 10.1038/onc.2008.21 18391970

35. van der Vos KE, Coffer PJ (2011) The extending network of FOXO transcriptional target genes. Antioxid Redox Signal 14: 579–592. doi: 10.1089/ars.2010.3419 20673124

36. Garcia-Martinez JM, Alessi DR (2008) mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1). Biochem J 416: 375–385. doi: 10.1042/BJ20081668 18925875

37. Mei FC, Qiao J, Tsygankova OM, Meinkoth JL, Quilliam LA, et al. (2002) Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation. J Biol Chem 277: 11497–11504. doi: 10.1074/jbc.M110856200 11801596

38. Surve CR, Lehmann D, Smrcka AV (2014) A chemical biology approach demonstrates G protein betagamma subunits are sufficient to mediate directional neutrophil chemotaxis. J Biol Chem 289: 17791–17801. doi: 10.1074/jbc.M114.576827 24808183

39. Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, et al. (2006) SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell 127: 125–137. doi: 10.1016/j.cell.2006.08.033 16962653

40. Okae H, Iwakura Y (2010) Neural tube defects and impaired neural progenitor cell proliferation in Gbeta1-deficient mice. Dev Dyn 239: 1089–1101. doi: 10.1002/dvdy.22256 20186915

41. Murdoch JN, Copp AJ (2010) The relationship between sonic Hedgehog signaling, cilia, and neural tube defects. Birth Defects Res A Clin Mol Teratol 88: 633–652. doi: 10.1002/bdra.20686 20544799

42. Mukhopadhyay S, Wen X, Ratti N, Loktev A, Rangell L, et al. (2013) The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 152: 210–223. doi: 10.1016/j.cell.2012.12.026 23332756

43. Regard JB, Malhotra D, Gvozdenovic-Jeremic J, Josey M, Chen M, et al. (2013) Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification. Nat Med 19: 1505–1512. doi: 10.1038/nm.3314 24076664

44. Rodgers BD, Levine MA, Bernier M, Montrose-Rafizadeh C (2001) Insulin regulation of a novel WD-40 repeat protein in adipocytes. J Endocrinol 168: 325–332. doi: 10.1677/joe.0.1680325 11182770

45. Oh WJ, Jacinto E (2011) mTOR complex 2 signaling and functions. Cell Cycle 10: 2305–2316. doi: 10.4161/cc.10.14.16586 21670596

46. Liu L, Luo Y, Chen L, Shen T, Xu B, et al. (2010) Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity. J Biol Chem 285: 38362–38373. doi: 10.1074/jbc.M110.141168 20937815

47. Son G, Hines IN, Lindquist J, Schrum LW, Rippe RA (2009) Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis. Hepatology 50: 1512–1523. doi: 10.1002/hep.23186 19790269

48. Shimobayashi M, Hall MN (2014) Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 15: 155–162. doi: 10.1038/nrm3757 24556838

49. Saxton RA, Sabatini DM (2017) mTOR Signaling in Growth, Metabolism, and Disease. Cell 168: 960–976. doi: 10.1016/j.cell.2017.02.004 28283069

50. Albert V, Svensson K, Shimobayashi M, Colombi M, Munoz S, et al. (2016) mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue. EMBO Mol Med 8: 232–246. doi: 10.15252/emmm.201505610 26772600

51. Mukaida S, Evans BA, Bengtsson T, Hutchinson DS, Sato M (2017) Adrenoceptors promote glucose uptake into adipocytes and muscle by an insulin-independent signaling pathway involving mechanistic target of rapamycin complex 2. Pharmacol Res 116: 87–92. doi: 10.1016/j.phrs.2016.12.022 28025104

52. Sato M, Evans BA, Sandstrom AL, Chia LY, Mukaida S, et al. (2018) alpha1A-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes. Biochem Pharmacol 148: 27–40. doi: 10.1016/j.bcp.2017.11.016 29175420

53. Dbouk HA, Vadas O, Shymanets A, Burke JE, Salamon RS, et al. (2012) G protein-coupled receptor-mediated activation of p110beta by Gbetagamma is required for cellular transformation and invasiveness. Sci Signal 5: ra89. doi: 10.1126/scisignal.2003264 23211529

54. Leopoldt D, Hanck T, Exner T, Maier U, Wetzker R, et al. (1998) Gbetagamma stimulates phosphoinositide 3-kinase-gamma by direct interaction with two domains of the catalytic p110 subunit. J Biol Chem 273: 7024–7029. doi: 10.1074/jbc.273.12.7024 9507010

55. O'Hayre M, Degese MS, Gutkind JS (2014) Novel insights into G protein and G protein-coupled receptor signaling in cancer. Curr Opin Cell Biol 27: 126–135. doi: 10.1016/j.ceb.2014.01.005 24508914

56. Verma R, Marchese A (2015) The endosomal sorting complex required for transport pathway mediates chemokine receptor CXCR4-promoted lysosomal degradation of the mammalian target of rapamycin antagonist DEPTOR. J Biol Chem 290: 6810–6824. doi: 10.1074/jbc.M114.606699 25605718

57. Mombaerts P, Wang F, Dulac C, Chao S, Nemes A, et al. (1996) Visualizing an olfactory sensory map. Cell 87: 675–686. doi: 10.1016/s0092-8674(00)81387-2 8929536

58. Liberles S, Buck L (2006) A second class of chemosensory receptors in the olfactory epithelium. Nature 442: 645–650. doi: 10.1038/nature05066 16878137

59. Michaloski J, Galante P, Malnic B (2006) Identification of potential regulatory motifs in odorant receptor genes by analysis of promoter sequences. Genome Research 16: 1091–1098. doi: 10.1101/gr.5185406 16902085

60. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402–408. doi: 10.1006/meth.2001.1262 11846609

61. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25: 1105–1111. doi: 10.1093/bioinformatics/btp120 19289445

62. Yates A, Akanni W, Amode MR, Barrell D, Billis K, et al. (2016) Ensembl 2016. Nucleic Acids Res 44: D710–716. doi: 10.1093/nar/gkv1157 26687719

63. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079. doi: 10.1093/bioinformatics/btp352 19505943

64. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, et al. (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7: 562–578. doi: 10.1038/nprot.2012.016 22383036

65. Wang B, Fallon JF, Beachy PA (2000) Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100: 423–434. doi: 10.1016/s0092-8674(00)80678-9 10693759

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