scholarly journals Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells

2022 ◽  
Vol 221 (2) ◽  
Author(s):  
Adam N. Keen ◽  
Luke A. Payne ◽  
Vedanta Mehta ◽  
Alistair Rice ◽  
Lisa J. Simpson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Traction Force ◽  
Protein Translation ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Cellular Mechanics ◽  
Mechanical Responses ◽  
Cell Signaling Networks ◽  
Cellular Cytoskeleton

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6–RACK1–ERK1/2–FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.

Cytoskeletal proteins associate with components of the ribosomal maturation and translation apparatus in Xenopus stage I oocytes

Zygote ◽  
2014 ◽  
Vol 23 (5) ◽  
pp. 669-682 ◽  
Author(s):  
Loredana Chierchia ◽  
Margherita Tussellino ◽  
Domenico Guarino ◽  
Rosa Carotenuto ◽  
Nadia DeMarco ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Cytoskeletal Proteins ◽  
Stage I ◽  
Initiation Factor ◽  
Perinuclear Region ◽  
Eukaryotic Initiation Factor ◽  
Translation Apparatus ◽  
Ribosomal Stalk

SummaryActin-based cytoskeleton (CSK) and microtubules may bind to RNAs and related molecules implicated in translation. However, many questions remain to be answered regarding the role of cytoskeletal components in supporting the proteins involved in steps in the maturation and translation processes. Here, we performed co-immunoprecipitation and immunofluorescence to examine the association between spectrins, keratins and tubulin and proteins involved in 60S ribosomal maturation and translation in Xenopus stage I oocytes, including ribosomal rpl10, eukaryotic initiation factor 6 (Eif6), thesaurins A/B, homologs of the eEF1α elongation factor, and P0, the ribosomal stalk protein. We found that rpl10 and eif6 cross-reacted with the actin-based CSK and with tubulin. rpl10 co-localizes with spectrin, particularly in the perinuclear region. eif6 is similarly localized. Given that upon ribosomal maturation, the insertion of rpl10 into the 60S subunit occurs simultaneously with the release of eif6, one can hypothesise that actin-based CSK and microtubules provide the necessary scaffold for the insertion/release of these two molecules and, subsequently, for eif6 transport and binding to the mature 60S subunit. P0 and thesaurins cross-reacted with only spectrin and cytokeratins. Thesaurins aggregated at the oocyte periphery, rendering this a territory favourable site for protein synthesis; the CSK may support the interaction between thesaurins and sites of the translating ribosome. Moreover, given that the assembly of the ribosome stalk, where P0 is located, to the 60S subunit is essential for the release of eif6, it can be hypothesised that the CSK can facilitate the binding of the stalk to the 60S.

scholarly journals Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo

2001 ◽  
Vol 268 (20) ◽  
pp. 5375-5385 ◽  
Author(s):  
Linda McKendrick ◽  
Simon J. Morley ◽  
Virginia M. Pain ◽  
Rosemary Jagus ◽  
Bhavesh Joshi
Keyword(s):  
Protein Synthesis ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation

1994 ◽  
Vol 14 (7) ◽  
pp. 4546-4553
Author(s):  
K V Ramaiah ◽  
M V Davies ◽  
J J Chen ◽  
R J Kaufman
Keyword(s):  
Protein Synthesis ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Guanine Nucleotide Exchange ◽  
Initiation Factor 2 ◽  
Exchange Activity

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.

Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats

2001 ◽  
Vol 281 (1) ◽  
pp. C179-C187 ◽  
Author(s):  
Troy A. Hornberger ◽  
R. Bridge Hunter ◽  
Susan C. Kandarian ◽  
Karyn A. Esser
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Muscle Atrophy ◽  
Protein Translation ◽  
Hindlimb Unloading ◽  
Initiation Factor ◽  
Skeletal Muscle Atrophy ◽  
Α Subunit ◽  
Translation Factors ◽  
Ribosomal S6 Kinase

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 α-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).

Regulation of eukaryotic initiation factor-2B activity in muscle of diabetic rats

1993 ◽  
Vol 264 (1) ◽  
pp. E101-E108 ◽  
Author(s):  
A. M. Karinch ◽  
S. R. Kimball ◽  
T. C. Vary ◽  
L. S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Cardiac Muscle ◽  
Casein Kinase Ii ◽  
Diabetic Rats ◽  
Casein Kinase ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Fast Twitch

Peptide-chain initiation is inhibited in fast-twitch skeletal muscle, but not heart, of diabetic rats. We have investigated mechanisms that might maintain eukaryotic initiation factor (eIF)-2B activity, preventing loss of efficiency of protein synthesis in heart of diabetic rats but not in fast-twitch skeletal muscle. There was no change in the amount or phosphorylation state of eIF-2 in skeletal or cardiac muscle during diabetes. In contrast, eIF-2B activity was decreased in fast-twitch but not slow-twitch muscle from diabetic animals. NADP+ inhibited partially purified eIF-2B in vitro, but addition of equimolar NADPH reversed the inhibition. The NADPH-to-NADP+ ratio was unchanged in fast-twitch muscle after induction of diabetes but was increased in heart of diabetic rats, suggesting that NADPH also prevents inhibition of eIF-2B in vivo. The activity of casein kinase II, which can phosphorylate and activate eIF-2B in vitro, was significantly lower in extracts of fast-twitch, but not cardiac muscle, of diabetic rats compared with controls. The results presented here demonstrate that changes in eIF-2 alpha phosphorylation are not responsible for the effect of diabetes on eIF-2B activity in fast-twitch skeletal muscle. Modulation of casein kinase II activity may be a factor in the regulation of protein synthesis in muscle during acute diabetes. The activity of eIF-2B in heart might be maintained by the increased NADPH/NADP+.

scholarly journals eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Jennifer Deborah Wuerth ◽  
Matthias Habjan ◽  
Markus Kainulainen ◽  
Besim Berisha ◽  
Damien Bertheloot ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Nonstructural Protein ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mammalian Host ◽  
Eukaryotic Initiation Factor ◽  
Α Subunit ◽  
Immune Factor

ABSTRACT RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α. IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

ANG II activates effectors of mTOR via PI3-K signaling in human coronary smooth muscle cells

2004 ◽  
Vol 287 (3) ◽  
pp. H1232-H1238 ◽  
Author(s):  
Sassan Hafizi ◽  
Xuemin Wang ◽  
Adrian H. Chester ◽  
Magdi H. Yacoub ◽  
Christopher G. Proud
Keyword(s):  
Protein Synthesis ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Signaling Pathway ◽  
Initiation Factor ◽  
Ang Ii ◽  
Eukaryotic Initiation Factor ◽  
Mtor Signaling Pathway ◽  
Phosphatidylinositol 3 Kinase ◽  
Eukaryotic Initiation Factor 4E

We have previously shown that the vasoconstrictive peptide angiotensin II (ANG II) is a hypertrophic agent for human coronary artery smooth muscle cells (cSMCs), which suggests that it plays a role in vascular wall thickening. The present study investigated the intracellular signal transduction pathways involved in the growth response of cSMCs to ANG II. The stimulation of protein synthesis by ANG II in cSMCs was blocked by the immunosuppressant rapamycin, which is an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway that includes the 70-kDa S6 kinase (p70S6k) and plays a key role in cell growth. The inhibitory effect of rapamycin was reversed by a molar excess of FK506; this indicates that both agents act through the common 12-kDa immunophilin FK506-binding protein. ANG II caused a rapid and sustained activation of p70S6k activity that paralleled its phosphorylation, and both processes were blocked by rapamycin. In addition, both of the phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002 abolished the ANG II-induced increase in protein synthesis, and wortmannin also blocked p70S6k phosphorylation. Furthermore, ANG II triggered dissociation of the translation initiation factor, eukaryotic initiation factor-4E, from its regulatory binding protein 4E-BP1, which was also inhibited by rapamycin and wortmannin. In conclusion, we have shown that ANG II activates components of the rapamycin-sensitive mTOR signaling pathway in human cSMCs and involves activation of phosphatidylinositol 3-kinase, p70S6k, and eukaryotic initiation factor-4E, which leads to activation of protein synthesis. These signaling mechanisms may mediate the growth-promoting effect of ANG II in human cSMCs.

Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells

2001 ◽  
Vol 360 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Duraisamy SENTHIL ◽  
Jennifer L. FAULKNER ◽  
Goutam GHOSH CHOUDHURY ◽  
Hanna E. ABBOUD ◽  
Balakuntalam S. KASINATH
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Insulin Signalling ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Tubular Epithelial Cells ◽  
Eukaryotic Initiation Factor 4E ◽  
Proximal Tubular Epithelial Cells ◽  
Proximal Tubular ◽  
Stimulation Of

Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1nM) or insulin (1nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/−2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor β-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/−2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II.

The Translation Initiation Factor eIF4E Is Overexpressed in Multiple Myeloma and Is Critical for Myeloma Cell Proliferation and Survival

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1853-1853 ◽  
Author(s):  
Shirong Li ◽  
MeiHua Jin ◽  
Ailing Liu ◽  
Markus Y. Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Clinical Care ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Abstract 1853 Methods: The translation initiation factor eIF4E is central to protein synthesis in general, and overexpression and/or activation of eIF4E is associated with a malignant phenotype by regulating oncogenic protein translation. Several previous publications indicate that aberrant control of protein synthesis contributes to lymphoma genesis but the exact role of protein translation in multiple myeloma (MM) is less clear. Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. The goal of this study was to determine the role and regulation of eIF4E, as well as the effects of protein translation controlling drugs in MM. Results: By western blot analysis as well as RT-PCR we found that eIF4E protein and mRNA levels are significantly elevated (up to 20 fold) in MM cell lines (H929, RPMI-8226, MM.1S and OPM2) and primary myeloma cells compared to normal plasma cells. Silencing of eIF4E gene expression in RPMI-8226 MM cells by a stable and inducible shRNA system significantly decreased viability of myeloma cells (by ∼ 43%) but not of HEK 293 suggesting a higher dependency of MM cells to protein translation. Next we evaluated different drugs including pomalidomide, rapamycin, pp242, 4EGI-1 and ribavirin, that are known to inhibit protein synthesis for their effects on protein translation in MM. By m7GTP pull down assays we evaluated the effects of the different drugs on eIF4E expression and activity. Rapamycin blocked the phosphorylation of 4EBP1 and eIF4E release, and subsequently inhibited eIF4G binding. The compound 4EGI-1 decreased the interaction between eIF4E and eIF4G. Pomalidomide decreased eIF4E protein expression. All drugs inhibited MM cell DNA synthesis measured by 3H-Thymidine incorporation. Treatment with pomalidomide (10uM), rapamycin (40nM), pp242 (10uM), 4EGI1 (50uM) or ribavirin (50uM) for 48h significantly decreased (p<0.05) proliferation by 43–62% indicating that drugs controlling protein translation inhibit MM growth. We also found that all drugs decreased expression of eIF4E dependent targets such as cyclin D1 and c-myc. Conclusion: Here we show that eIF4E, a key player in translational control, is highly expressed in MM cells and critical for MM growth and survival. Therefore our study helps to understand the function and regulatory mechanism of eIF4E in MM. Further the evaluation of drugs targeting protein translation provides the basis for the optimization of current MM treatment or to open up new strategies such as targeting protein translation in future MM therapy. Disclosures: Lentzsch: Celgene Corp: Consultancy, Research Funding; Onyx: Consultancy; Genzyme: Consultancy; prIME Oncology: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria.

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