Protein synthesis in eukaryotic organisms is a complex process that requires cooperation among a large number of polypeptides including ribosomal proteins, modification of enzymes, and ribosome-associated translation factors. The initiation phase of protein synthesis, during which ribosomes select mRNAs to be translated and identify the translational start site, requires a set of eIFs (eukaryotic Translation Initiation Factors), many of which are comprised of multiple polypeptide subunits. eIF2 (Eukaryotic Initiation Factor-2) is a GTP (Guanosine Triphosphate)-binding protein that escorts the initiation-specific form of Met-tRNA (Met-tRNAi) onto the ribosome. It is composed of 3 non-identical subunits, alpha (36 kD), beta (38 kD), and gamma (52 kD). cDNAs for each of the subunits have been cloned and sequenced from a variety of species, and the predicted amino acid sequences of the individual subunits show an exceptional level of conservation among species. The human eIF2alpha, beta, and gamma sequences are respectively, 58%, 7%, and 72%, identical to the corresponding subunits from S. cerevisiae. The conservation among species underscores the important role eIF2 plays in cell viability (Ref.1).
Important functions of eIF2 include delivery of charged initiator methionyl-tRNA to the ribosome, as well as a role in identifying the translational start site. The three-eIF2 subunits are encoded by essential genes (Alpha, SUI2; Beta, SUI3; Gamma, GCD11). A variety of stimuli modulate eIF2 activity, which in turn regulates mRNA translation. Four distinct eIF2-Alpha kinases phosphorylate eIF2-Alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the HRI (Hemin-Regulated Inhibitor), the interferon-inducible dsRNA-dependent kinase PKR (Protein Kinase R), the ER (Endoplasmic Reticulum)-resident kinase PEK (Pancreatic eIF2-Alpha Kinase) and the amino acid deprivation GCN2 (General Control Of Amino Acid Synthesis-2) protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PEK seem to be widely distributed in eukaryotes. The eukaryotic eIF2-Alpha kinases PKR, HRI, and yeast GCN2 share extensive homology within their catalytic domains, but have distinct regulatory domains allowing for different physiologic signals to regulate phosphorylation of eIF2-Alpha (Ref.2). The hemin-regulated phosphorylation of eIF2 is part of a homeostatic mechanism that assures production of equal amounts of heme and globin for the synthesis of hemoglobin. An excess of globin, made through protein synthesis, prevents the accumulation of heme and its oxidation to hemin. HRI kinase activity is activated as a result. The subsequent phosphorylation of eIF2 shuts down globin chain synthesis. Conversely, an excess of heme results in unbound heme, which is oxidized to hemin. Hemin inhibits the HRI kinase, preventing the phosphorylation of eIF2 and allowing further initiation of globin chain synthesis. This homeostatic mechanism is only possible in cells such as reticulocytes that are geared to the production of principally one protein. PKR is another ubiquitously expressed serine-threonine kinase that has been implicated as a signal integrator in translation control pathways. Most of the times, PKR exists at low levels and is inactive in the cell. IFN-Gamma (Interferon-gamma) causes the level to increase, and the same viral dsRNA that induced the synthesis of IFN
is used to convert inactive PKR to the active form. Once the protein binds dsRNA it is phosphorylated by the enzyme PP1 (Protein Phosphatase1) that allows for the dimerization of PKR. This phosphorylated dimer is the active form of PKR. Once activated, PKR has the ability to phosphorylate the protein eIF2-Alpha, which further leads to translation inhibition. PEK is an ER transmembrane protein kinase that phosphorylates eIF2-Alpha in response to ER stress. Phosphorylation of eIF2-Alpha reduces the formation of translation initiation complexes, which leads to reduce recognition of AUG initiation codons and therefore general translational attenuation. Yeast protein kinase GCN2 stimulates the translation of transcriptional activator GCN4 by phosphorylating eIF2-Alpha in response to amino acid starvation (Ref.3).
The guanine nucleotide exchange factor of eIF2, eIF2B
is a heteropentamer, and consists of five different subunits (alpha- epsilon) that have been conserved in eukaryotic evolution. The epsilon-subunit of eIF2B
((Eukaryotic Initiation Factor-2B) is the catalytic one and is phosphorylated in vivo at several different sites. One of these is a target for GSK3
(Glycogen Synthase Kinase-3). Phosphorylation of eIF2B
by GSK3 inhibits its activity. GSK3 is switched off in response to Insulin and growth factors, via a signaling pathway that involves PI3K (Phosphatidylinositol-3 Kinase) and PKB
(Protein Kinase-B). This provides a signaling pathway through which Insulin and other stimuli can activate eIF2B and thus turn on an important step in translation initiation. Recent data suggest that other inputs also regulate eIF2B. For example, signaling via the classical MAPK
(Mitogen Activated Protein Kinase) cascade is involved in the activation of eIF2B
by some growth factors (Ref.4).
eIF2 is an initiation factor for protein synthesis. eIF2 transfers bound initiator met tRNA to the 40S ribosomal subunit-mRNA complex. During initiation, eIF2 forms a complex with GTP and initiator methionyl-tRNAi (met-tRNAi), and this ternary complex subsequently binds to the 40S ribosomal subunit to form the 43S preinitiation complex. Through the action of a translation initiation factor complex referred to as eIF4F (Eukaryotic Initiation Factor-4F), which is comprised of eIF4A
(Eukaryotic Initiation Factor-4A), eIF4E (Eukaryotic Initiation Factor-4E), and eIF4G (Eukaryotic Initiation Factor-4G), mRNA is bound to the 43S preinitiation complex, resulting in formation of the 48S preinitiation complex. During a later step in initiation, the GDP bound to eIF2 is hydrolyzed in an eIF5 (Eukaryotic Initiation Factor5)-mediated process and initiation factors are released fro m the ribosome. Before binding Met-tRNAi and reforming the ternary complex, the GDP bound to eIF2 must be exchanged for GTP, a reaction that is catalyzed by the guanine nucleotide exchange factor, eIF2B, which is present in limiting quantities. eIF2 can be phosphorylated. In the phosphorylated form it is very strongly bound by eIF2B. The binding strength is high enough that the phosphorylated eIF2 is sequestered, tying up much of the available eIF2B. Since eIF2B is present in limiting quantities, phosphorylation of only a small amount of eIF2 can result in total inhibition of translation by sequestration of eIF2B
(Ref.5). The cellular stress response protein GADD34 binds to PP1 and can attenuate translational elongation of key transcriptional factors through dephosphorylation of eIF2-Alpha . Phosphorylation of eIF2 is a key element of the general regulation of nitrogen metabolism in yeast cells. Specific regulation of protein synthesis also occurs. Inhibition of protein translation is a mode of inducing neuronal apoptosis and neurodegeneration in Alzheimers disease. Phosphorylation of eIF2-Alpha terminates global protein translation and induces apoptosis (Ref.6).