Hiroaki Imataka

Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E‐binding proteins (4E‐BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK‐activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C‐terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G‐binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C‐terminal region of the translational inhibitor p97, an eIF4G‐related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.

A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation.

Most eukaryotic mRNAs possess a 5′ cap and a 3′ poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)‐binding protein (PABP) was implicated in poly(A)‐dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5′ rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N‐terminus by 156 amino acids. Co‐immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N‐terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N‐terminal region as the PABP‐binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full‐length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N‐terminal fragment which includes the PABP‐binding site inhibits poly(A)‐dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP‐binding protein, PAIP‐1, eIF4G binds PABP and probably functions in poly(A)‐dependent translation in mammalian cells.

Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene.

The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P‐450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys‐Cys/His‐His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV‐1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus‐1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

A NOVEL FUNCTIONAL HUMAN EUKARYOTIC TRANSLATION INITIATION FACTOR 4G

ABSTRACT Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.

Translational control of hippocampal synaptic plasticity and memory by the eIF2α kinase GCN2

Studies on various forms of synaptic plasticity have shown a link between messenger RNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase that depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins. Activation of postsynaptic targets seems to trigger the transcription of plasticity-related genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. Here we report a unique feature of hippocampal slices from GCN2-/- mice: in CA1, a single 100-Hz train induces a strong and sustained long-term potentiation (late LTP or L-LTP), which is dependent on transcription and translation. In contrast, stimulation that elicits L-LTP in wild-type slices, such as four 100-Hz trains or forskolin, fails to evoke L-LTP in GCN2-/- slices. This aberrant synaptic plasticity is mirrored in the behaviour of GCN2-/- mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, an antagonist of cyclic-AMP-response-element-binding protein (CREB). Thus, in the hippocampus of GCN2-/- mice, the expression of ATF4 is reduced and CREB activity is increased. Our study provides genetic, physiological, behavioural and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory, through modulation of the ATF4/CREB pathway.

Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A.

Mammalian translation initiation factor 4F (eIF4F) consists of three subunits, eIF4A, eIF4E, and eIF4G. eIF4G interacts directly with both eIF4A and eIF4E. The binding site for eIF4E is contained in the amino-terminal third of eIF4G, while the binding site for eIF4A was mapped to the carboxy-terminal third of the molecule. Here we show that human eIF4G possesses two separate eIF4A binding domains in the middle third (amino acids [aa] 478 to 883) and carboxy-terminal third (aa 884 to 1404) of the molecule. The amino acid sequence of the middle portion of eIF4G is well conserved between yeasts and humans. We show that mutations of conserved amino acid stretches in the middle domain abolish or reduce eIF4A binding as well as eIF3 binding. In addition, a separate and nonoverlapping eIF4A binding domain exists in the carboxy-terminal third (aa 1045 to 1404) of eIF4G, which is not present in yeast. The C-terminal two-thirds region (aa 457 to 1404) of eIF4G, containing both eIF4A binding sites, is required for stimulating translation. Neither one of the eIF4A binding domains alone activates translation. In contrast to eIF4G, human p97, a translation inhibitor with homology to eIF4G, binds eIF4A only through the amino-terminal proximal region, which is homologous to the middle domain of eIF4G.

Serum‐stimulated, rapamycin‐sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI

The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two‐dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C‐terminal one‐third of the protein. Mass spectrometry and mutational analyses identified the serum‐stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide‐3‐kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12–rapamycin‐associated protein/mammalian target of rapamycin), prevent the serum‐induced phosphorylation of these residues. Finally, the phosphorylation state of N‐terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C‐terminus to kinases is modulated by this pathway(s).

The major mRNA‐associated protein YB‐1 is a potent 5′ cap‐dependent mRNA stabilizer

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA‐associated protein YB‐1, also termed p50, is a potent cap‐dependent mRNA stabilizer. YB‐1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB‐1 depletion results in accelerated mRNA decay. The cold shock domain of YB‐1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5′ end is required for YB‐1‐mediated stabilization. Significantly, exogenously added YB‐1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB‐1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down‐regulation of eIF4E activity or increasing the YB‐1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.

Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region

ABSTRACT The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with β-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

A new translational regulator with homology to eukaryotic translation initiation factor 4G

Translation initiation in eukaryotes is facilitated by the cap structure, m7GpppN (where N is any nucleotide). Eukaryotic translation initiation factor 4F (eIF4F) is a cap binding protein complex that consists of three subunits: eIF4A, eIF4E and eIF4G. eIF4G interacts directly with eIF4E and eIF4A. The binding site of eIF4E resides in the N‐terminal third of eIF4G, while eIF4A and eIF3 binding sites are present in the C‐terminal two‐thirds. Here, we describe a new eukaryotic translational regulator (hereafter called p97) which exhibits 28% identity to the C‐terminal two‐thirds of eIF4G. p97 mRNA has no initiator AUG and translation starts exclusively at a GUG codon. The GUG‐initiated open reading frame (907 amino acids) has no canonical eIF4E binding site. p97 binds to eIF4A and eIF3, but not to eIF4E. Transient transfection experiments show that p97 suppresses both cap‐dependent and independent translation, while eIF4G supports both translation pathways. Furthermore, inducible expression of p97 reduces overall protein synthesis. These results suggest that p97 functions as a general repressor of translation by forming translationally inactive complexes that include eIF4A and eIF3, but exclude eIF4E.