Proteomic analysis of mTOR inhibition-mediated phosphorylation changes in ribosomal proteins and eukaryotic translation initiation factors.
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ABSTRACT:
SUBMITTER: Jiang X
PROVIDER: S-EPMC4930769 | biostudies-literature | 2016 Jul
REPOSITORIES: biostudies-literature
Publications
Proteomic analysis of mTOR inhibition-mediated phosphorylation changes in ribosomal proteins and eukaryotic translation initiation factors.
Jiang Xu X Feng Shan S Chen Yuling Y Feng Yun Y Deng Haiteng H
Protein & cell 20160701 7
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Project description:The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.
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Project description:Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon.
| S-EPMC4999511 | biostudies-literature
Project description:Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.
| S-EPMC6066748 | biostudies-literature
Project description:The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the alpha subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2alpha (AteIF2alpha) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2alpha occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2alpha was not phosphorylated.
| S-EPMC2504353 | biostudies-literature
Project description:The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2alpha plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2alpha phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting approximately 90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2alpha, suggesting that this pathway may be broadly utilized by stresses that activate eIF2alpha kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.
| S-EPMC2715810 | biostudies-other
Project description:According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5'-end of mRNA and subsequently migrates along 5'-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5'-UTRs of different lengths and structures in mammalian and plant cell-free systems. For the first time, the relationship between the scanning time and the 5'-UTR length was determined and their linear correlation was experimentally demonstrated. The conclusion is made that the ribosome migration is an unidirectional motion with the rate being virtually independent of a particular mRNA sequence and secondary structure.
| S-EPMC3141257 | biostudies-literature
Project description:Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser/Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G(1) and G(2)/M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5/eIF2/eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation.
| S-EPMC1266118 | biostudies-literature
Project description:Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) inhibits cap-dependent translation in eukaryotes by competing with eIF4G for an interaction with eIF4E. Phosphorylation at Ser-83 of 4E-BP1 occurs during mitosis through the activity of cyclin-dependent kinase 1 (CDK1)/cyclin B rather than through canonical mTOR kinase activity. Here, we investigated the interaction of eIF4E with 4E-BP1 or eIF4G during interphase and mitosis. We observed that 4E-BP1 and eIF4G bind eIF4E at similar levels during interphase and mitosis. The most highly phosphorylated mitotic 4E-BP1 isoform (δ) did not interact with eIF4E, whereas a distinct 4E-BP1 phospho-isoform, EB-γ, phosphorylated at Thr-70, Ser-83, and Ser-101, bound to eIF4E during mitosis. Two-dimensional gel electrophoretic analysis corroborated the identity of the phosphorylation marks on the eIF4E-bound 4E-BP1 isoforms and uncovered a population of phosphorylated 4E-BP1 molecules lacking Thr-37/Thr-46-priming phosphorylation. Moreover, proximity ligation assays for phospho-4E-BP1 and eIF4E revealed different in situ interactions during interphase and mitosis. The eIF4E:eIF4G interaction was not inhibited but rather increased in mitotic cells, consistent with active translation initiation during mitosis. Phosphodefective substitution of 4E-BP1 at Ser-83 did not change global translation or individual mRNA translation profiles as measured by single-cell nascent protein synthesis and eIF4G RNA immunoprecipitation sequencing. Mitotic 5'-terminal oligopyrimidine RNA translation was active and, unlike interphase translation, resistant to mTOR inhibition. Our findings reveal the phosphorylation profiles of 4E-BP1 isoforms and their interactions with eIF4E throughout the cell cycle and indicate that 4E-BP1 does not specifically inhibit translation initiation during mitosis.
| S-EPMC6682726 | biostudies-literature
Project description:Eukaryotic translation initiation factor 6 (eIF6) is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. We here show a novel mode of eIF6 regulation by glycogen synthase kinase 3 (GSK3) that is predominantly active in response to serum starvation. Both GSK3α and GSK3β phosphorylate human eIF6. Multiple residues in the C terminus of eIF6 are phosphorylated by GSK3 in a sequential manner. In response to serum starvation, eIF6 accumulates in the cytoplasm, and this altered localization depends on phosphorylation by GSK3. Disruption of eIF6 phosphorylation exacerbates the translation inhibitory response to serum starvation and stalls cell growth. These results suggest that eIF6 regulation by GSK3 contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress.
| S-EPMC7476728 | biostudies-literature
Project description: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).
| S-EPMC305580 | biostudies-literature