| Serine/threonine-protein kinase GCN2 | |||||||
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 Crystal structure of GCN2 | |||||||
| Identifiers | |||||||
| Symbol | GCN2 | ||||||
| Alt. symbols | AAS1 | ||||||
| NCBI gene | 851877 | ||||||
| PDB | 1zyc | ||||||
| UniProt | P15442 | ||||||
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GCN2 (general control nonderepressible 2) is a serine/threonine-protein kinase that senses amino acid deficiency through binding to uncharged transfer RNA (tRNA). It plays a key role in modulating amino acid metabolism as a response to nutrient deprivation.
GCN2 is the only known eukaryotic initiation factor 2α kinase (eIF2α) in Saccharomyces cerevisiae . [1] 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 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. [2] 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 ]
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. [3]
GCN2 activation results from a conformation that facilitates ATP binding, leading to autophosphorylation of an activation loop which leads to maximal GCN2 kinase activity. [4]
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. [5] [6] [7]
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. [5] ATF4 is related to GCN4. [1]
GCN2 also regulates the cell cycle by delaying entry into S phase upon ultraviolet (UV) radiation and exposure to methyl methanesulfonate (MMS). [8] [9] 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. [10] 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. [8]
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. [11] 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. [12]
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. [13] However, this phosphorylation site in Gcn2 is not conserved in fission yeast or in mammalian cells. [14]
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. [15] It is thought that GCN1/GCN20 facilitates the transfer of tRNA from the ribosomal A site to the HisRS-like domain of GCN2. [7] 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. [16] [17] [18]
There are also GCN2 homologues in Neurospora crassa , [19] C. elegans , [18] Drosophila melanogaster [20] [21] and mice. [22] Thus, GCN2 may be the most widespread and founding member of the eIF-2α kinase subfamily. [23]
The mammalian target of rapamycin (mTOR), also referred to as the mechanistic target of rapamycin, and sometimes called FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), is a kinase that in humans is encoded by the MTOR gene. mTOR is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases.
Eukaryotic translation is the biological process by which messenger RNA is translated into proteins in eukaryotes. It consists of four phases: initiation, elongation, termination, and recapping.
eIF-2-alpha kinase is a kinase enzyme that phosphorylates eIF2α. There are four forms in mammals:
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Eukaryotic initiation factors (eIFs) are proteins or protein complexes involved in the initiation phase of eukaryotic translation. These proteins help stabilize the formation of ribosomal preinitiation complexes around the start codon and are an important input for post-transcription gene regulation. Several initiation factors form a complex with the small 40S ribosomal subunit and Met-tRNAiMet called the 43S preinitiation complex. Additional factors of the eIF4F complex recruit the 43S PIC to the five-prime cap structure of the mRNA, from which the 43S particle scans 5'-->3' along the mRNA to reach an AUG start codon. Recognition of the start codon by the Met-tRNAiMet promotes gated phosphate and eIF1 release to form the 48S preinitiation complex, followed by large 60S ribosomal subunit recruitment to form the 80S ribosome. There exist many more eukaryotic initiation factors than prokaryotic initiation factors, reflecting the greater biological complexity of eukaryotic translation. There are at least twelve eukaryotic initiation factors, composed of many more polypeptides, and these are described below.
Eukaryotic translation initiation factor 4E-binding protein 1 is a protein that in humans is encoded by the EIF4EBP1 gene. inhibits cap-dependent translation by binding to translation initiation factor eIF4E. Phosphorylation of 4E-BP1 results in its release from eIF4E, thereby allows cap-dependent translation to continue thereby increasing the rate of protein synthesis.
DNA replication licensing factor MCM3 is a protein that in humans is encoded by the MCM3 gene.
DNA damage-inducible transcript 3, also known as C/EBP homologous protein (CHOP), is a pro-apoptotic transcription factor that is encoded by the DDIT3 gene. It is a member of the CCAAT/enhancer-binding protein (C/EBP) family of DNA-binding transcription factors. The protein functions as a dominant-negative inhibitor by forming heterodimers with other C/EBP members, preventing their DNA binding activity. The protein is implicated in adipogenesis and erythropoiesis and has an important role in the cell's stress response.
Eukaryotic translation initiation factor 2 subunit 1 (eIF2α) is a protein that in humans is encoded by the EIF2S1 gene.
Eukaryotic translation initiation factor 2-alpha kinase 3, also known as protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), is an enzyme that in humans is encoded by the EIF2AK3 gene.
Eukaryotic translation initiation factor 2 subunit 2 (eIF2β) is a protein that in humans is encoded by the EIF2S2 gene.
MAP kinase-interacting serine/threonine-protein kinase 1 is an enzyme that in humans is encoded by the MKNK1 gene.
Eukaryotic translation initiation factor 2A (eIF2A) is a protein that in humans is encoded by the EIF2A gene. The eIF2A protein is not to be confused with eIF2α, a subunit of the heterotrimeric eIF2 complex. Instead, eIF2A functions by a separate mechanism in eukaryotic translation.
Eukaryotic translation initiation factor 3 subunit D (eIF3d) is a protein that in humans is encoded by the EIF3D gene.
Translational activator GCN1 is a protein that in humans is encoded by the GCN1L1 gene.
Eukaryotic Initiation Factor 2 (eIF2) is an eukaryotic initiation factor. It is required for most forms of eukaryotic translation initiation. eIF2 mediates the binding of tRNAiMet to the ribosome in a GTP-dependent manner. eIF2 is a heterotrimer consisting of an alpha, a beta, and a gamma subunit.
Salubrinal is a drug which acts as a specific inhibitor of eIF2α phosphatase enzymes and is primarily used experimentally, to study stress responses in eukaryotic cells associated with the action of eIF2. Salubrinal indirectly inhibits eIF2 as a result of reduced dephosphorylation of its α-subunit, resulting in activation of stress response pathways usually triggered by events such as oxidative stress or buildup of unfolded protein in the endoplasmic reticulum. Salubrinal has putative therapeutic value due to its function, but is as yet only used experimentally. Salubrinal is being studied at Indiana University for its potential to fight osteoporosis and accelerate bone healing.
Amino acid response is the mechanism triggered in mammalian cells by amino acid starvation.
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The transactivation domain or trans-activating domain (TAD) is a transcription factor scaffold domain which contains binding sites for other proteins such as transcription coregulators. These binding sites are frequently referred to as activation functions (AFs). TADs are named after their amino acid composition. These amino acids are either essential for the activity or simply the most abundant in the TAD. Transactivation by the Gal4 transcription factor is mediated by acidic amino acids, whereas hydrophobic residues in Gcn4 play a similar role. Hence, the TADs in Gal4 and Gcn4 are referred to as acidic or hydrophobic, respectively.