Gcn2

GCN2 is the only known eukaryotic initiation factor 2α kinase (eIF2α) in Saccharomyces cerevisiae. It inactivates eIF2α by phosphorylation at Serine 51 under conditions of amino acid deprivation, resulting in repression of general protein synthesis whilst allowing selected mRNA, such as the transcription factor GCN4, to be translated due to regions upstream of the coding sequence. Elevated levels of GCN4 stimulate the expression of amino acid biosynthetic genes, which code for enzymes required to synthesize all 20 major amino acids.

Protein kinase GCN2 is a multidomain protein and its C-terminus contains a region homologous to histidyl-tRNA synthetase (HisRS) next to the kinase catalytic moiety.{{cite journal | vauthors = Wek SA, Zhu S, Wek RC | title = The histidyl-tRNA synthetase-related sequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA and is required for activation in response to starvation for different amino acids | journal = Molecular and Cellular Biology | volume = 15 | issue = 8 | pages = 4497–4506 | date = August 1995 | pmid = 7623840 | pmc = 230689 | doi = 10.1128/MCB.15.8.4497 }} This HisRS-like region forms a dimer and dimerization is required for GCN2 function. The crucial contribution to GCN2 function is the promotion of tRNA binding and the stimulation of the kinase domain via physical interaction.{{citation needed|date=February 2019}}

Binding of uncharged tRNA to this synthetase-like domain induces a conformational change in which the GCN2 domains rotate 180° normal to the dimerization surface and thereby transpose from their antiparallel to a parallel orientation. Subsequently, GCN2 is activated.{{cite journal | vauthors = Dey M, Cao C, Sicheri F, Dever TE | title = Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK | journal = The Journal of Biological Chemistry | volume = 282 | issue = 9 | pages = 6653–6660 | date = March 2007 | pmid = 17202131 | doi = 10.1074/jbc.M607897200 | doi-access = free }}

GCN2 activation results from a conformation that facilitates ATP binding, leading to autophosphorylation of an activation loop which leads to maximal GCN2 kinase activity.{{PDB|1ZYC}} {{cite journal | vauthors = Padyana AK, Qiu H, Roll-Mecak A, Hinnebusch AG, Burley SK | title = Structural basis for autoinhibition and mutational activation of eukaryotic initiation factor 2alpha protein kinase GCN2 | journal = The Journal of Biological Chemistry | volume = 280 | issue = 32 | pages = 29289–29299 | date = August 2005 | pmid = 15964839 | doi = 10.1074/jbc.M504096200 | doi-access = free }}

Image:gcn2 function.svg

GCN2 inhibits general translation by phosphorylation of eIF-2α at serine 51 within 15 min of amino acid deprivation, which then subsequently increases the affinity for the guanine exchange factor eIF2B to sequester eIF-2α leading to reduced formation of the ternary complex (TC) consisting of eIF2, GTP and the initiator Met-tRNA required for translation initiation.

eIF2 containing a phosphorylated alpha subunit shows an increased affinity for its only GEF, eIF2B, but

eIF2B is only able to exchange GDP with GTP from unphosphorylated eIF2. So the recycling of eIF2, needed for TC formation, is inhibited by phosphorylation of eIF-2α, which in the end leads to a reduction of global translation rates.

An opposing effect of the reduced availability of TC is the induction of GCN4 expression by translational regulation. Four short ORF's exist in the leader of the GCN4 mRNA. 40S Ribosomal Subunits scanning the mRNA from 5' have TC bound and translate the first upstream open reading frame (uORF). Under non-starving condition there is enough ternary complex that the subunits rebind it before they reach uORF 4. Translation is again initiated, uORF2,3 or 4 translated and the 40S Subunits subsequently dissociate from GCN4 mRNA.

Under starving conditions there is less TC present. Some of the 40S Subunits are not able to rebind TC before they reach uORF 4 but eventually rebind TC before reaching GCN4 coding sequence. Therefore, the reduction in TC formation resulting from GCN2 activation by amino acid starvation leads to the induction of GCN4 translation.

GCN4 is the primary regulator in response to amino acid starvation, termed general amino acid control (GAAC). It acts as a transcription factor and activates several genes required for amino acid synthesis.{{cite journal | vauthors = Wek RC | title = eIF-2 kinases: regulators of general and gene-specific translation initiation | journal = Trends in Biochemical Sciences | volume = 19 | issue = 11 | pages = 491–496 | date = November 1994 | pmid = 7855893 | doi = 10.1016/0968-0004(94)90136-8 }}{{cite journal | vauthors = Hinnebusch AG | title = Translational regulation of GCN4 and the general amino acid control of yeast | journal = Annual Review of Microbiology | volume = 59 | pages = 407–450 | year = 2005 | pmid = 16153175 | doi = 10.1146/annurev.micro.59.031805.133833 }}

Recently GCN2 has also been implicated in directing eating behavior in mammals by phosphorylating eIF-2α in the anterior Piriform cortex (APC) of the brain. The molecular mechanisms governing this function are not yet known, but a basic zipper transcription factor called ATF4 is a possible candidate. ATF4 is related to GCN4.{{cite journal | vauthors = Zaborske JM, Narasimhan J, Jiang L, Wek SA, Dittmar KA, Freimoser F, Pan T, Wek RC | display-authors = 6 | title = Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p | journal = The Journal of Biological Chemistry | volume = 284 | issue = 37 | pages = 25254–25267 | date = September 2009 | pmid = 19546227 | pmc = 2757228 | doi = 10.1074/jbc.M109.000877 | doi-access = free }}

GCN2 also regulates the cell cycle by delaying entry into S phase upon ultraviolet (UV) radiation and exposure to methyl methanesulfonate (MMS).{{cite journal | vauthors = Tvegård T, Soltani H, Skjølberg HC, Krohn M, Nilssen EA, Kearsey SE, Grallert B, Boye E | display-authors = 6 | title = A novel checkpoint mechanism regulating the G1/S transition | journal = Genes & Development | volume = 21 | issue = 6 | pages = 649–654 | date = March 2007 | pmid = 17369398 | pmc = 1820939 | doi = 10.1101/gad.421807 }}{{cite journal | vauthors = Krohn M, Skjølberg HC, Soltani H, Grallert B, Boye E | title = The G1-S checkpoint in fission yeast is not a general DNA damage checkpoint | journal = Journal of Cell Science | volume = 121 | issue = Pt 24 | pages = 4047–4054 | date = December 2008 | pmid = 19033384 | doi = 10.1242/jcs.035428 | s2cid = 15903 | doi-access = }} Thereby the cell prevents passing the G1 checkpoint and starting DNA replication when the DNA is damaged. It has been hypothesized, that UV induces nitric oxide synthase activation and NO^. production, which leads to the activation of GCN2 and that the cell cycle regulation by GCN2 is independent of eIF2α phosphorylation.{{cite journal | vauthors = Wang L, Liu Y, Wu S | title = The roles of nitric oxide synthase and eIF2alpha kinases in regulation of cell cycle upon UVB-irradiation | journal = Cell Cycle | volume = 9 | issue = 1 | pages = 38–42 | date = January 2010 | pmid = 20016280 | pmc = 2834424 | doi = 10.4161/cc.9.1.10268 }} Although the causal relationship between GCN2 and cell cycle delay is still under debate, it was suggested that the formation of the pre-replication complex is deferred by GCN2 upon UV-irradiation.

The absence of essential amino acids causes a downregulation of key components of the lipid synthesis such as the fatty acid synthase. Following leucine-deprivation in mammals, GCN2 decreases the expression of lipogenic genes via SREBP-1c.{{cite journal | vauthors = Guo F, Cavener DR | title = The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid | journal = Cell Metabolism | volume = 5 | issue = 2 | pages = 103–114 | date = February 2007 | pmid = 17276353 | doi = 10.1016/j.cmet.2007.01.001 | doi-access = free }} SREBP-1c actions upon genes regulating fatty-acid and triglyceride synthesis and is reduced by leucine deprivation in the liver in a GCN2-dependent manner.

Gcn2 is held in its inactive state via several auto-inhibitory molecular interactions until exposed to an activating signal. Binding of uncharged tRNA to the synthetase-like domain results in allosteric re-arrangements. This leads to Gcn2 auto-phosphorylation at specific sites in the activation loop of the kinase domain. This phosphorylation then allows Gcn2 to efficiently phosphorylate eIF2α.{{cite journal | vauthors = Castilho BA, Shanmugam R, Silva RC, Ramesh R, Himme BM, Sattlegger E | title = Keeping the eIF2 alpha kinase Gcn2 in check | journal = Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | volume = 1843 | issue = 9 | pages = 1948–1968 | date = September 2014 | pmid = 24732012 | doi = 10.1016/j.bbamcr.2014.04.006 | doi-access = free }}

In yeast cells, GCN2 is kept inactive via phosphorylation at serine 577, which is thought to depend on the activity of TORC1. Inactivation of TORC1 by Rapamycin affects GCN2 and at least partly by dephosphorylation of serine 577. This leads to activation of GCN2 even in amino acid replete cells, probably by increasing the affinity of GCN2 for uncharged tRNA, so that even basal levels permit tRNA binding.{{cite journal | vauthors = Yuan W, Guo S, Gao J, Zhong M, Yan G, Wu W, Chao Y, Jiang Y | display-authors = 6 | title = General Control Nonderepressible 2 (GCN2) Kinase Inhibits Target of Rapamycin Complex 1 in Response to Amino Acid Starvation in Saccharomyces cerevisiae | journal = The Journal of Biological Chemistry | volume = 292 | issue = 7 | pages = 2660–2669 | date = February 2017 | pmid = 28057755 | pmc = 5314164 | doi = 10.1074/jbc.M116.772194 | doi-access = free }} However, this phosphorylation site in Gcn2 is not conserved in fission yeast or in mammalian cells.{{cite journal | vauthors = Rødland GE, Tvegård T, Boye E, Grallert B | title = Crosstalk between the Tor and Gcn2 pathways in response to different stresses | journal = Cell Cycle | volume = 13 | issue = 3 | pages = 453–461 | date = 2014-02-01 | pmid = 24280780 | pmc = 3956541 | doi = 10.4161/cc.27270 }}

Another stimulatory input to GCN2 is exerted by a complex of GCN1/GCN20. GCN1/GCN20 shows structural similarity to eEF3, a factor important in the binding of tRNA to ribosomes. The GCN1/GCN20 complex physically interacts with GCN2 by binding to its N-terminus.{{cite journal | vauthors = Garcia-Barrio M, Dong J, Ufano S, Hinnebusch AG | title = Association of GCN1-GCN20 regulatory complex with the N-terminus of eIF2alpha kinase GCN2 is required for GCN2 activation | journal = The EMBO Journal | volume = 19 | issue = 8 | pages = 1887–1899 | date = April 2000 | pmid = 10775272 | pmc = 302013 | doi = 10.1093/emboj/19.8.1887 }} It is thought that GCN1/GCN20 facilitates the transfer of tRNA from the ribosomal A site to the HisRS-like domain of GCN2. An additional mechanism of regulation of this protein is through the conserved protein IMPACT, that acts both in yeast, nematodes and mammals as an inhibitor of GCN2.{{cite journal | vauthors = Sattlegger E, Swanson MJ, Ashcraft EA, Jennings JL, Fekete RA, Link AJ, Hinnebusch AG | title = YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed | journal = The Journal of Biological Chemistry | volume = 279 | issue = 29 | pages = 29952–29962 | date = July 2004 | pmid = 15126500 | doi = 10.1074/jbc.M404009200 | doi-access = free }}{{cite journal | vauthors = Pereira CM, Sattlegger E, Jiang HY, Longo BM, Jaqueta CB, Hinnebusch AG, Wek RC, Mello LE, Castilho BA | display-authors = 6 | title = IMPACT, a protein preferentially expressed in the mouse brain, binds GCN1 and inhibits GCN2 activation | journal = The Journal of Biological Chemistry | volume = 280 | issue = 31 | pages = 28316–28323 | date = August 2005 | pmid = 15937339 | doi = 10.1074/jbc.M408571200 | doi-access = free}}{{cite journal | vauthors = Ferraz RC, Camara H, De-Souza EA, Pinto S, Pinca AP, Silva RC, Sato VN, Castilho BA, Mori MA | display-authors = 6 | title = IMPACT is a GCN2 inhibitor that limits lifespan in Caenorhabditis elegans | journal = BMC Biology | volume = 14 | issue = 1 | pages = 87 | date = October 2016 | pmid = 27717342 | pmc = 5054600 | doi = 10.1186/s12915-016-0301-2 | doi-access = free }}

There are also GCN2 homologues in Neurospora crassa,{{cite journal | vauthors = Sattlegger E, Hinnebusch AG, Barthelmess IB | title = cpc-3, the Neurospora crassa homologue of yeast GCN2, encodes a polypeptide with juxtaposed eIF2alpha kinase and histidyl-tRNA synthetase-related domains required for general amino acid control | journal = The Journal of Biological Chemistry | volume = 273 | issue = 32 | pages = 20404–20416 | date = August 1998 | pmid = 9685394 | doi = 10.1074/jbc.273.32.20404 | doi-access = free }} C. elegans, Drosophila melanogaster{{cite journal | vauthors = Santoyo J, Alcalde J, Méndez R, Pulido D, de Haro C | title = Cloning and characterization of a cDNA encoding a protein synthesis initiation factor-2alpha (eIF-2alpha) kinase from Drosophila melanogaster. Homology To yeast GCN2 protein kinase | journal = The Journal of Biological Chemistry | volume = 272 | issue = 19 | pages = 12544–12550 | date = May 1997 | pmid = 9139706 | doi = 10.1074/jbc.272.19.12544 | doi-access = free | hdl = 20.500.11820/6f055e71-f712-4052-89d3-3160e7b2eaad | hdl-access = free }}{{cite journal | vauthors = Olsen DS, Jordan B, Chen D, Wek RC, Cavener DR | title = Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase | journal = Genetics | volume = 149 | issue = 3 | pages = 1495–1509 | date = July 1998 | pmid = 9649537 | pmc = 1460234 | doi = 10.1093/genetics/149.3.1495 }} and mice.{{cite journal | vauthors = Sood R, Porter AC, Olsen DA, Cavener DR, Wek RC | title = A mammalian homologue of GCN2 protein kinase important for translational control by phosphorylation of eukaryotic initiation factor-2alpha | journal = Genetics | volume = 154 | issue = 2 | pages = 787–801 | date = February 2000 | pmid = 10655230 | pmc = 1460965 | doi = 10.1093/genetics/154.2.787 }} Thus, GCN2 may be the most widespread and founding member of the eIF-2α kinase subfamily.{{cite journal | vauthors = Dever TE | title = Translation initiation: adept at adapting | journal = Trends in Biochemical Sciences | volume = 24 | issue = 10 | pages = 398–403 | date = October 1999 | pmid = 10500305 | doi = 10.1016/s0968-0004(99)01457-7 }}

• Eukaryotic initiation factors
• The three subunits of eIF2:
• α – EIF2S1
• β – EIF2S2
• γ – EIF2S3
• Kinases of eIF2
• HRI (Heme-regulated inhibitor kinase) or EIF2AK1
• PKR (Protein kinase R)
• PERK (PKR-like ER-localized eIF2α kinase)
• EIF2AK4

{{Reflist|3}}

Category:Proteins

Category:Saccharomyces cerevisiae genes