Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells

English version České info

The survival of all living organisms depends on their capacity to adapt their gene expression program to variations in the environment. When subjected to various stresses, eukaryotic cells down-regulate general protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). The yeast Saccharomyces cerevisiae has a single eIF2α kinase, Gcn2, activated by uncharged tRNAs accumulating in amino acid starved cells, which bind to a regulatory domain homologous to histidyl-tRNA synthetase. Gcn2 also contains a degenerate, pseudokinase domain (YKD) of largely unknown function, juxtaposed to the authentic, functional kinase domain (KD). Our study demonstrates that direct interaction between the YKD and KD is essential for activation of Gcn2, and identifies likely KD-contact sites in the YKD that can be altered to either impair or constitutively activate kinase function. Our results provide the first functional insights into the regulatory role of the enigmatic YKD of Gcn2.

Vyšlo v časopise: Enhanced Interaction between Pseudokinase and Kinase Domains in Gcn2 stimulates eIF2α Phosphorylation in Starved Cells. PLoS Genet 10(5): e32767. doi:10.1371/journal.pgen.1004326
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004326

Souhrn

Zdroje

1. LuPD, HardingHP, RonD (2004) Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J Cell Biol 167: 27–33.

2. VattemKM, WekRC (2004) Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci U S A 101: 11269–11274.

3. HinnebuschAG (2005) Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol 59: 407–450.

4. GuoF, CavenerDR (2007) The GCN2 eIF2α kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid. Cell Metab 5: 103–114.

5. HaoS, SharpJW, Ross-IntaCM, McDanielBJ, AnthonyTG, et al. (2005) Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex. Science 307: 1776–1778.

6. Costa-MattioliM, GobertD, HardingH, HerdyB, AzziM, et al. (2005) Translational control of hippocampal synaptic plasticity and memory by the eIF2α kinase GCN2. Nature 436: 1166–1173.

7. MurguiaJR, SerranoR (2012) New functions of protein kinase Gcn2 in yeast and mammals. IUBMB Life 64: 971–974.

8. QiuH, HuC, DongJ, HinnebuschAG (2002) Mutations that bypass tRNA binding activate the intrinsically defective kinase domain in GCN2. Genes Dev 16: 1271–1280.

9. PadyanaAK, QiuH, Roll-MecakA, HinnebuschAG, BurleySK (2005) Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2α protein kinase GCN2. J Biol Chem 280: 29289–29299.

10. GarrizA, QiuH, DeyM, SeoEJ, DeverTE, et al. (2008) A network of hydrophobic residues impeding helix αC rotation maintains latency of eIF2α kinase Gcn2. Mol Cell Biol 29: 1592–1607.

11. WekRC, JacksonBM, HinnebuschAG (1989) Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci USA 86: 4579–4583.

12. WekSA, ZhuS, WekRC (1995) The histidyl-tRNA synthetase-related sequence in the eIF-2α protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids. Mol Cell Biol 15: 4497–4506.

13. ZhuS, SobolevAY, WekRC (1996) Histidyl-tRNA synthetase-related sequences in GCN2 protein kinase regulate in vitro phosphorylation of eIF-2. J Biol Chem 271: 24989–24994.

14. DongJ, QiuH, Garcia-BarrioM, AndersonJ, HinnebuschAG (2000) Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain. Mol Cell 6: 269–279.

15. QiuH, DongJ, HuC, FrancklynCS, HinnebuschAG (2001) The tRNA-binding moiety in GCN2 contains a dimerization domain that interacts with the kinase domain and is required for tRNA binding and kinase activation. EMBO J 20: 1425–1438.

16. RomanoPR, Garcia-BarrioMT, ZhangX, WangQ, TaylorDR, et al. (1998) Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2α kinases PKR and GCN2. Mol Cell Biol 18: 2282–2297.

17. DeyM, CaoC, SicheriF, DeverTE (2007) Conserved intermolecular salt-bridge required for activation of protein kinases PKR, GCN2 and PERK. J Biol Chem 282: 6650–6660.

18. DarAC, DeverTE, SicheriF (2005) Higher-order substrate recognition of eIF2α by the RNA-dependent protein kinase PKR. Cell 122: 887–900.

19. QiuH, Garcia-BarrioMT, HinnebuschAG (1998) Dimerization by translation initiation factor 2 kinase GCN2 is mediated by interactions in the C-terminal ribosome-binding region and the protein kinase domain. Mol Cell Biol 18: 2697–2711.

20. NarasimhanJ, StaschkeKA, WekRC (2004) Dimerization is required for activation of eIF2 kinase Gcn2 in response to diverse environmental stress conditions. J Biol Chem 279: 22820–22832.

21. RamirezM, WekRC, HinnebuschAG (1991) Ribosome-association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisae. Mol Cell Biol 11: 3027–3036.

22. ZhuS, WekRC (1998) Ribosome-binding domain of eukaryotic initiation factor-2 kinase GCN2 facilitates translation control. J Biol Chem 273: 1808–1814.

23. VisweswaraiahJ, LageixS, CastilhoBA, IzotovaL, KinzyTG, et al. (2011) Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity. J Biol Chem 286: 36568–36579.

24. MartonMJ, Vasquez de AldanaCR, QiuH, CharkraburttyK, HinnebuschAG (1997) Evidence that GCN1 and GCN20, translational regulators of GCN4, function on enlongating ribosomes in activation of the eIF2a kinase GCN2. Mol Cell Biol 17: 4474–4489.

25. Vazquez de AldanaCR, MartonMJ, HinnebuschAG (1995) GCN20, a novel ATP binding cassette protein, and GCN1 reside in a complex that mediates activation of the eIF-2α kinase GCN2 in amino acid-starved cells. EMBO J 14: 3184–3199.

26. Garcia-BarrioM, DongJ, UfanoS, HinnebuschAG (2000) Association of GCN1/GCN20 regulatory complex with the conserved N-terminal domain of eIF2a kinase GCN2 is required for GCN2 activation in vivo. EMBO J 19: 1887–1899.

27. SattleggerE, HinnebuschAG (2005) Polyribosome binding by GCN1 is required for full activation of eukaryotic translation initiation factor 2α kinase GCN2 during amino acid starvation. J Biol Chem 280: 16514–16521.

28. SattleggerE, HinnebuschAG (2000) Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid-starved cells. EMBO J 19: 6622–6633.

29. VisweswaraiahJ, LeeSJ, HinnebuschAG, SattleggerE (2012) Overexpression of eukaryotic translation elongation factor 3 impairs Gcn2 protein activation. J Biol Chem 287: 37757–37768.

30. MurphyJM, ZhangQ, YoungSN, ReeseML, BaileyFP, et al. (2013) A robust methodology to subclassify pseudokinases based on their nucleotide binding properties. Biochem J 457: 323–34.

31. BoudeauJ, Miranda-SaavedraD, BartonGJ, AlessiDR (2006) Emerging roles of pseudokinases. Trends Cell Biol 16: 443–452.

32. WekRC, RamirezM, JacksonBM, HinnebuschAG (1990) Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression. Mol Cell Biol 10: 2820–2831.

33. RamirezM, WekRC, Vazquez de AldanaCR, JacksonBM, FreemanB, et al. (1992) Mutations activating the yeast eIF-2α kinase GCN2: Isolation of alleles altering the domain related to histidyl-tRNA synthetases. Mol Cell Biol 12: 5801–5815.

34. Garcia-BarrioM, DongJ, CherkasovaVA, ZhangX, ZhangF, et al. (2002) Serine-577 is phosphorylated and inhibits the tRNA binding and eIF2α kinase activities of GCN2. J Biol Chem 277: 30675–30683.

35. ZeqirajE, FilippiBM, DeakM, AlessiDR, van AaltenDM (2009) Structure of the LKB1-STRAD-MO25 complex reveals an allosteric mechanism of kinase activation. Science 326: 1707–1711.

36. LandauM, MayroseI, RosenbergY, GlaserF, MartzE, et al. (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res 33: W299–302.

37. DeLano WL (2002) The PyMOL Molecular Graphics System. Palo Alto, CA.

38. MoehleCM, HinnebuschAG (1991) Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae. Mol Cell Biol 11: 2723–2735.

39. ReidGA, SchatzG (1982) Import of proteins into mitochondria. Yeast cells grown in the presence of carbonyl cyanide m-chlorophenylhydrazone accumulate massive amounts of some mitochondrial precursor polypeptides. J Biol Chem 257: 13056–13061.

40. CiganAM, PabichEK, FengL, DonahueTF (1989) Yeast translation initiation suppressor sui2 encodes the α subunit of eukaryotic initiation factor 2 and shares identity with the human a subunit. Proc Natl Acad Sci USA 86: 2784–2788.

41. BardwellL, CooperAJ, FriedbergEC (1992) Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro. Mol Cell Biol 12: 3041–3049.