Identification of Interprotein Interactions between the Subunits of Eukaryotic Initiation Factors eIF2 and eIF2B

Kimball, Scot R.; Heinzinger, Nina K.; Horetsky, Rick L.; Jefferson, Leonard S.

doi:10.1074/jbc.273.5.3039

Cited by 45 publications

(42 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, it remains plausible that complete knockdown has not been entirely obtained (we routinely achieved greater than 95% suppression) and that residual amounts of eIF2B␣ are sufficient to maintain eIF2B activity under normal conditions. Nevertheless, our observations agree with data from previous studies of eIF2B␣ in both yeast and insect cells, where the loss of this subunit did not affect cell growth or normal GEF activity (8,18,28). Additionally, it was reported previously that eIF2B␣ is required for the derepression of GCN4 in yeast following amino acid starvation, an event that is dependent upon eIF2␣ phosphorylation (1,6).…”

Section: Discussionsupporting

confidence: 76%

The Alpha Subunit of Eukaryotic Initiation Factor 2B (eIF2B) Is Required for eIF2-Mediated Translational Suppression of Vesicular Stomatitis Virus

Elsby

Heiber

Reid

et al. 2011

J Virol

Self Cite

View full text Add to dashboard Cite

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2␣), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2␣ phosphorylation. To extend this analysis, we have investigated the importance of eIF2B␣ function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2B␣ harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2␣ homolog SUI2. Significantly, we also demonstrate that the loss of eIF2B␣, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2␣ phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2B␣ in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.The initiation of translation requires the coordinate actions of several protein factors to complete a multiphase process that culminates in the formation of an 80S ribosome on a nascent mRNA molecule (30). The first of these steps is the formation of a stable ternary complex composed of one charged initiator Met-tRNA i molecule, GTP, and eukaryotic initiation factor 2 (eIF2), comprised of its ␣, ␤, and ␥ subunits. Configuration of the ternary complex is required to bring the Met-tRNA i molecule into contact with the 40S ribosomal subunit, which subsequently recognizes mRNA molecules through the eIF4F cap-binding complex (35). These events lead to the recognition of the AUG start codon of the mRNA and the binding of the 60S ribosomal subunit to the 40S subunit (26). At the end of initiation and upon the creation of the 80S ribosome, the accompanying protein factors are released from the ribosome-mRNA molecule to be recycled for future use. In addition to initiation factor release, the GTP bound to eIF2 is hydrolyzed by the action of eIF5, such that the resultant eIF2 is liberated as an inactive binary complex bound to GDP (31, 36). In order for subsequent rounds of initiation to occur, it is necessary to generate new active eIF2-GTP from the existing eIF2-GDP, a reaction catalyzed by the heteropentameric guanine nucleotide exchange factor (GEF) eIF2B (41).eIF2B provides a key regulatory mechanism for cells to decrease protein synthesis rates during periods of stress. Selected cellular stress, such as the accumulation of viral RNA species following infection or misfolded proteins in the endoplasmic...

show abstract

Section: Discussionsupporting

confidence: 76%

The Alpha Subunit of Eukaryotic Initiation Factor 2B (eIF2B) Is Required for eIF2-Mediated Translational Suppression of Vesicular Stomatitis Virus

Elsby

Heiber

Reid

et al. 2011

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…This is consistent with recent reports demonstrating interaction between the C terminus of eIF2␤ and the ␦-and ⑀-subunits of mammalian eIF2B (20) as well as between the N terminus of eIF2␤ and the ⑀-subunit (Gcd6p) of yeast eIF2B (21). The ␤-subunit of eIF2 also appears to interact directly with eIF5 (21)(22)(23)(24).…”

Section: Eukaryotic Translation Initiation Factor Eif2supporting

confidence: 80%

“…Although the ␥-subunit of eIF2 is directly involved in guanine nucleotide binding, no evidence exists for a direct interaction between this eIF2 subunit and the eIF2B catalytic core. However, there is evidence from two studies for direct interaction between eIF2␤ and the ␦-and ⑀-subunits of mammalian eIF2B (20) or the yeast eIF2B⑀ subunit (21), but the region of eIF2␤ required for the interaction remains controversial. Given the effect on K m for the exchange reaction upon removal of eIF2␣, our results indicate that structural interactions between eIF2B and eIF2 that require the presence of eIF2␣ contribute to wild-type rates of catalysis and are consistent with the ability of eIF2B to recognize the presence of the eIF2␣ subunit.…”

Section: H]met-trna Imentioning

confidence: 99%

Biochemical Analysis of the eIF2βγ Complex Reveals a Structural Function for eIF2α in Catalyzed Nucleotide Exchange

Nika

Rippel

Hannig

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Eukaryotic translation initiation factor eIF2 is a heterotrimer that binds and delivers Met-tRNA iMet to the 40 S ribosomal subunit in a GTP-dependent manner. Initiation requires hydrolysis of eIF2-bound GTP, which releases an eIF2⅐GDP complex that is recycled to the GTP form by the nucleotide exchange factor eIF2B. The ␣-subunit of eIF2 plays a critical role in regulating nucleotide exchange via phosphorylation at serine 51, which converts eIF2 into a competitive inhibitor of the eIF2B-catalyzed exchange reaction. We purified a form of eIF2 (eIF2␤␥) completely devoid of the ␣-subunit to further study the role of eIF2␣ in eIF2 function. These studies utilized a yeast strain genetically altered to bypass a deletion of the normally essential eIF2␣ structural gene (SUI2). Removal of the ␣-subunit did not appear to significantly alter binding of guanine nucleotide or Met-tRNA i Met ligands by eIF2 in vitro. Qualitative assays to detect 43 S initiation complex formation and eIF5-dependent GTP hydrolysis revealed no differences between eIF2␤␥ and the wild-type eIF2 heterotrimer. However, steady-state kinetic analysis of eIF2B-catalyzed nucleotide exchange revealed that the absence of the ␣-subunit increased K m for eIF2␤␥⅐GDP by an order of magnitude, with a smaller increase in V max . These data indicate that eIF2␣ is required for structural interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange. We suggest that this function contributes to the ability of the ␣-subunit to control the rate of nucleotide exchange through reversible phosphorylation. Eukaryotic translation initiation factor eIF21 is a heterotrimeric GTP-binding protein that in yeast is encoded by the essential genes SUI2, SUI3, and GCD11. eIF2 binds charged initiator tRNA (Met-tRNA i Met ) in a GTP-dependent manner to form a stable ternary complex that interacts with the small ribosomal subunit and additional initiation factors (including eIF3 and eIF1A) to form a 43 S initiation complex (reviewed in Refs. 1 and 2-5). This complex binds at or near the 5Ј-end of capped mRNAs and "scans" the mRNA in a 5Ј to 3Ј direction until an AUG start codon in proper context is encountered. Genetic analyses in yeast have demonstrated a function for eIF2 in the selection of the translational start site (6, 7). Recognition of the start codon is accompanied by eIF5-mediated hydrolysis of eIF2-bound GTP, which facilitates release of an inactive eIF2⅐GDP binary complex as well as several other initiation factors. The 40 S/mRNA/Met-tRNA iMet complex interacts with a 60 S ribosomal subunit to form an 80 S initiation complex that can then enter the elongation phase of protein synthesis. Conversion of the inactive eIF2⅐GDP binary complex to eIF2⅐GTP, which is capable of rebinding initiator tRNA, is catalyzed by the nucleotide exchange factor eIF2B.To execute its functions in translation initiation, eIF2 must interact with multiple ligands as well as other components of the translational apparatus. The former include guanine nucleotides and initi...

show abstract

“…11). However, data primarily from assays using human recombinant proteins suggest that phosphorylation of eIF2␣ increases the interaction between eIF2␤ (not eIF2␣) and eIF2B␦ and eIF2B⑀ (14,34).…”

Section: Expression Of a Distinct Isoform Of Eif2b␦ Interferes With Tmentioning

confidence: 99%

Regulation of the Unfolded Protein Response by eIF2Bδ Isoforms

Martin

Kimball

Gardner

2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2␣. eIF2␣ phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2␣ phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2B␦ variant 1 (V1). Replacement of total eIF2B␦ with V1 renders cells insensitive to eIF2␣ phosphorylation; specifically, they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2B␦ in ER stress response-deficient cells restores the stress response. Our data suggest that V1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2␣ phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases that result in ER stress.

show abstract

Identification of Interprotein Interactions between the Subunits of Eukaryotic Initiation Factors eIF2 and eIF2B

Cited by 45 publications

References 18 publications

The Alpha Subunit of Eukaryotic Initiation Factor 2B (eIF2B) Is Required for eIF2-Mediated Translational Suppression of Vesicular Stomatitis Virus

The Alpha Subunit of Eukaryotic Initiation Factor 2B (eIF2B) Is Required for eIF2-Mediated Translational Suppression of Vesicular Stomatitis Virus

Biochemical Analysis of the eIF2βγ Complex Reveals a Structural Function for eIF2α in Catalyzed Nucleotide Exchange

Regulation of the Unfolded Protein Response by eIF2Bδ Isoforms

Contact Info

Product

Resources

About