Crystal Structure of the α Subunit of Human Translation Initiation Factor 2B

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the α subunit of human eIF2B at 2.65 Å resolution. The eIF2Bα structure consists of the N-terminal α-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 Å in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 Å in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Bα. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Bα. Interestingly, the yeast eIF2Bα missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Bα missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The β and δ subunits of eIF2B are homologous to eIF2Bα and may have tertiary structures similar to the present eIF2Bα structure. The sulfate-ion-binding residues of eIF2Bα are well conserved in eIF2Bβ/δ. The abovementioned yeast and human missense mutations of eIF2Bβ/δ were also mapped on the eIF2Bα structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.     

Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)

P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a K_d of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5′UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-βs 5′UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3.     

The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the �� Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2α in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2α phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2α phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2α after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2α phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N^G-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2α phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α).² Two eIF2α kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2α in response to UV irradiation (1–4). However, the one or more upstream pathways that activate eIF2α kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO^•) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2α and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO^• and peroxynitrite (ONOO⁻) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6–9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes l-arginine and activates GCN2, which phosphorylates eIF2α (10). UV irradiation also activates NOS and elevates cellular NO^• (11–13). However, the UV-induced NOS activation and NO^• production have never been shown to be related to the activation of eIF2α kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2α.     

Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

The Herpes Simplex Virus US11 Protein Effectively Compensates for the γ134.5 Gene if Present before Activation of Protein Kinase R by Precluding Its Phosphorylation and That of the α Subunit of Eukaryotic Translation Initiation Factor 2

In herpes simplex virus-infected cells, viral γ₁34.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1α to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). The amino acid sequence of the γ₁34.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the α47 promoter next to U_S11, a γ₂ (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the γ₁34.5 gene. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. We report the following: (i) in cells infected with these mutants, U_S11 protein was made early in infection; (ii) U_S11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, U_S11 blocked the phosphorylation of eIF-2α by PKR activated by poly(I-C); and (iv) U_S11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, U_S11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2α, whereas U_S11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The γ₁34.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. U_S11 was retained as a γ₂ gene because, like many viral proteins, it has multiple functions.The herpes simplex virus 1 (HSV-1) genome encodes two sets of functions. The first and paramount are functions related to viral gene expression, replication of viral DNA, synthesis of virion proteins, assembly, packaging, and egress of the virus from the infected cell. The second set of functions, no less important in the survival of the virus in the human population, is creation of the environment necessary to maximize the yield and spread of virus from cell to cell and from infected to uninfected individuals (reviewed in reference 38). Of these known genes, several play a significant role in abating or delaying a host response to infection. The earliest to be expressed is the U_L41 gene which encodes a protein that is introduced into the cell in virions during infection (26, 27). This protein reduces the synthesis of host proteins by causing the destruction of mRNA in a rather nonspecific manner and therefore could be expected to reduce the synthesis of cellular proteins deleterious to viral replication (26, 27, 44).A second and very different approach to blocking host defense mechanisms is exemplified by infected cell protein 47 (ICP47). Proteosomal degradation of viral proteins could be expected to produce antigenic peptides which, if presented on the cell surface, could provoke a cytotoxic cell response early in infection and thus reduce viral yield. ICP47, an α protein made immediately after infection, blocks the presentation of antigenic peptides on the surface of the infected cells (20).The focus of this laboratory has been on a third viral pathway designed to block cellular response to infection. In cells infected with most viruses, the synthesis of complementary mRNA leads to activation of double-stranded RNA-dependent protein kinase R (PKR). This enzyme phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) (23). A consequence of this phosphorylation is total shutoff of protein synthesis. This would be an example of a noble sacrifice of the infected cell for the sake of survival of the organism were it not for the fact that viruses, while activating the PKR kinase pathway by making double-stranded RNA, also express functions which block this host defense system (2–4, 6, 7, 10, 28, 30, 34). In the case of HSV-1, more than 50% of the viral DNA is represented late in infection in the form of cRNA (21, 25), and the gene whose product blocks the consequences of activation of PKR is γ₁34.5 (7). In the absence of the gene, eIF-2α is phosphorylated and protein synthesis is impaired beginning approximately 5 h after infection (7, 9). In its presence, protein synthesis continues unabated even though PKR is activated (9). Recent studies have shown that the carboxyl terminus of the γ₁34.5 gene binds to the protein phosphatase 1α (PP1) and redirects it to dephosphorylate eIF-2α (19). The effectiveness of the γ₁34.5-PP1 complex is apparent from the observation that the rate of dephosphorylation of eIF-2α in cells infected with wild-type virus is more than 1000 times that of uninfected cells or cells infected with the γ₁34.5⁻ virus (5, 19).The studies described in this report concern another aspect of virus-induced block of the consequence of activation of PKR. Briefly, Mohr and Gluzman reported that serial passage of a γ₁34.5⁻ mutant resulted in the selection of a compensatory mutation capable of sustained protein synthesis (35). A characteristic of the compensatory mutants isolated by Mohr and Gluzman is a deletion in the α47 gene resulting in the juxtaposition of the promoter of the α47 gene next to the 5′ end of U_S11, a late (γ₂) viral gene. Preliminary studies of those mutants revealed that PKR was activated in cells infected with either the wild-type parent or the γ₁34.5⁻ virus, but protein synthesis was unaffected in cells infected with wild-type virus or the mutant carrying the compensatory mutations (5, 18).In an attempt to define the phenotype of the virus carrying the compensatory mutation, we constructed a mutant lacking the γ₁34.5 and the U_S8 to -12 genes. This mutant, designated R5103, activated PKR and caused a shutoff of protein synthesis (5). We then inserted into the R5103 genome a DNA fragment consisting of the intact U_S10 gene and the U_S11 open reading frame fused to the α47 promoter. This virus, designated R5104, activated PKR but did not induce the shutoff of protein synthesis. Consistent with the conclusion of Mohr and Gluzman (35), the mutation maps in the domain inserted into the R5104 virus (5). Further studies yielded two significant observations. First, in stark contrast to lysates of cells infected with R5103 and other γ₁34.5⁻ mutants, the lysates of R5104 virus failed to phosphorylate the α subunit of eIF-2 (5). Second, in striking contrast to lysates of wild-type virus-infected cells, the phosphatase activity of lysates of R5104 virus-infected cells specific for eIF-2α could not be differentiated from that of mock-infected cells or those of cells infected with other γ₁34.5⁻ mutants (5). These results indicated that the compensatory mutation blocks PKR from phosphorylating eIF-2α.The studies summarized in this report focused on U_S11 protein. We report that in cells infected with the R5104 recombinant the U_S11 protein is made early in infection, that U_S11 protein interacts with PKR and blocks the phosphorylation of eIF-2α by activated PKR in in vitro assays, and that the effectiveness of the U_S11 protein is greater if the protein is present in the reaction before activation of PKR than if it is after PKR has been activated by the addition of poly(I-C). We also found that U_S11 is phosphorylated in the presence of activated PKR but not in its absence. We conclude that U_S11 may have been an ancient mechanism for blocking the effects of activated PKR and that it has been supplanted by acquisition of the carboxyl-terminal domain of the γ₁34.5 protein from a cellular gene. We also note that U_S11 protein made late in infection, after PKR has been activated, is ineffective.Relevant to this report are some of the properties of the U_S11 protein. U_S11 is one of the most abundant viral proteins expressed at late times in viral infection (22, 31). It binds mRNA in a sequence- and conformation-specific fashion (39–41). In HSV-1-infected cells, U_S11 suppresses the synthesis of a truncated RNA colinear with the 5′ domain of the U_L34 mRNA (40). The protein accumulates in nucleoli, in the cytoplasm in association with the 60S ribosomal subunit, and it is also packaged in virions (31, 37, 41). In newly infected cells, the U_S11 protein has been found associated with ribosomes (41).Recently a plethora of reports suggested that U_S11 may have novel functions not readily apparent from its localization in the infected cell. Thus, U_S11 protein has been reported to have functions similar to those of human immunodeficiency Tat and Rev proteins and has also been reported to complement Rev function in a Rev⁻ human immunodeficiency virus mutant (11). The U_S11 protein has been reported to confer thermotolerance and help restore protein synthesis in HeLa cells subjected to thermal injury (12).     

Coordination of Eukaryotic Translation Elongation Factor 1A (eEF1A) Function in Actin Organization and Translation Elongation by the Guanine Nucleotide Exchange Factor eEF1B��

Eukaryotic translation elongation factor 1A (eEF1A) both shuttles aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide exchange factor eEF1Bα. We show that eEF1Bα has the ability to disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα F163, which binds in this domain, demonstrates effects on growth, eEF1A binding, nucleotide exchange activity, and cell morphology. These phenotypes can be partially restored by an intragenic W130A mutation. Furthermore, the combination of F163A with the lethal K205A mutation restores viability by drastically reducing eEF1Bα affinity for eEF1A. This also results in a consistent increase in actin bundling and partially corrected morphology. The consequences of the overlapping functions in this eEF1A domain and its unique differences from the bacterial homologs provide a novel function for eEF1Bα to balance the dual roles in actin bundling and protein synthesis.The final step of gene expression takes place at the ribosome as mRNA is translated into protein. In the yeast Saccharomyces cerevisiae, elongation of the polypeptide chain requires the orchestrated action of three soluble factors. The eukaryotic elongation factor 1 (eEF1)² complex delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating ribosome (1). The eEF1A subunit is a classic G-protein that acts as a “molecular switch” for the active and inactive states based on whether GTP or GDP is bound, respectively (2). Once an anticodon-codon match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely slow (3, 4), a guanine nucleotide exchange factor (GEF) complex, eEF1B, is required (5, 6). The yeast S. cerevisiae eEF1B complex contains two subunits, the essential catalytic subunit eEF1Bα (5) and the non-essential subunit eEF1Bγ (7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP: Mg²⁺ and eEF1A:eEF1Bα C terminus:GDPNP (8, 9) demonstrated a surprising structural divergence from the bacterial EF-Tu-EF-Ts (10) and mammalian mitochondrial EF-Tu_mt-EF-Ts_mt (11). While the G-proteins have a similar topology and consist of three well-defined domains, a striking difference was observed in binding sites for their GEFs. The C terminus of eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A, creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts and mammalian mitochondrial EF-Ts_mt, make extensive contacts with domain I and III of EF-Tu and EF-Tu_mt, respectively. The altered binding interface of eEF1Bα to domain II of eEF1A is particularly unexpected given the functions associated with domain II of eEF1A and EF-Tu. The crystal structure of the EF-Tu:GDPNP:Phe-tRNA^Phe complex reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II and tRNA to sustain stable contacts (12). That eEF1A employs the same aa-tRNA binding site is supported by genetic and biochemical data (13-15). Interestingly, eEF1Bα contacts many domain II eEF1A residues in the region hypothesized to be involved in the binding of the aa-tRNA CCA end (8). Because, the shared binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is significantly different between the eukaryotic and bacterial/mitochondrial systems, eEF1Bα may play a unique function aside from guanine nucleotide release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This noncanonical function of eEF1A was initially observed in Dictyostelium amoebae (16). It is estimated that greater than 60% of Dictyostelium eEF1A is associated with the actin cytoskeleton (17). The eEF1A-actin interaction is conserved among species from yeast to mammals, suggesting the importance of eEF1A for cytoskeleton integrity. Using a unique genetic approach, multiple eEF1A mutations were identified that altered cell growth and morphology, and are deficient in bundling actin in vitro (18, 19). Intriguingly, most mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding site. Previous studies have demonstrated that actin bundling by eEF1A is significantly reduced in the presence of aa-tRNA while eEF1A bound to actin filaments is not in complex with aa-tRNA (20). Therefore, actin and aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of yeast eEF1A or actin-bundling deficient mutants do not affect translation elongation (18, 19, 21), suggesting eEF1A-dependent cytoskeletal organization is independent of its translation elongation function (18, 20). Thus, while aa-tRNA binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling function associated with eEF1A domain II places greater importance on its relationship with the “novel” binding interface between eEF1A domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates the equilibrium between actin and translation functions of eEF1A and is perhaps the result of evolutionary selective pressure to balance the eukaryotic-specific role of eEF1A in actin organization. Here, we present kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the importance of the interactions between domain II of eEF1A and eEF1Bα to prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A support that this complex formation is a determining factor for eEF1A-induced actin organization. Interestingly, the F163A that reduces eEF1A affinity is an intragenic suppressor of the lethal K205A eEF1Bα mutant that displays increased affinity for eEF1A. This, along with a consistent change in the actin bundling correlated with the affinity of eEF1Bα for eEF1A, indicates that eEF1Bα is a balancer, directing eEF1A to translation elongation and away from actin, and alterations in this balance result in detrimental effects on cell growth and eEF1A function.     

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3′-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the “on” rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52–90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3′ BTE with higher affinity than for either m⁷G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3′ BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation.     

The B56γ3 Regulatory Subunit of Protein Phosphatase 2A (PP2A) Regulates S Phase-specific Nuclear Accumulation of PP2A and the G1 to S Transition

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56γ3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56γ3 becomes enriched in the nucleus at the G₁/S border and in S phase. The S phase-specific nuclear enrichment of B56γ3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56γ3 promotes nuclear localization of the A and C subunits, whereas silencing both B56γ2 and B56γ3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56γ3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G₁ to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56γ3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.     

Cannabinoid Modulation of Eukaryotic Initiation Factors (eIF2α and eIF2B1) and Behavioral Cross-Sensitization to Cocaine in Adolescent Rats

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Hypoxia-inducible Factor-1α (HIF-1α) Promotes Cap-dependent Translation of Selective mRNAs through Up-regulating Initiation Factor eIF4E1 in Breast Cancer Cells under Hypoxia Conditions

Hypoxia promotes tumor evolution and metastasis, and hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxia-related cellular processes in cancer. The eIF4E translation initiation factors, eIF4E1, eIF4E2, and eIF4E3, are essential for translation initiation. However, whether and how HIF-1α affects cap-dependent translation through eIF4Es in hypoxic cancer cells has been unknown. Here, we report that HIF-1α promoted cap-dependent translation of selective mRNAs through up-regulation of eIF4E1 in hypoxic breast cancer cells. Hypoxia-promoted breast cancer tumorsphere growth was HIF-1α-dependent. We found that eIF4E1, not eIF4E2 or eIF4E3, is the dominant eIF4E family member in breast cancer cells under both normoxia and hypoxia conditions. eIF4E3 expression was largely sequestered in breast cancer cells at normoxia and hypoxia. Hypoxia up-regulated the expression of eIF4E1 and eIF4E2, but only eIF4E1 expression was HIF-1α-dependent. In hypoxic cancer cells, HIF-1α-up-regulated eIF4E1 enhanced cap-dependent translation of a subset of mRNAs encoding proteins important for breast cancer cell mammosphere growth. In searching for correlations, we discovered that human eIF4E1 promoter harbors multiple potential hypoxia response elements. Furthermore, using chromatin immunoprecipitation (ChIP) and luciferase and point mutation assays, we found that HIF-1α utilized hypoxia response elements in the human eIF4E1 proximal promoter region to activate eIF4E1 expression. Our study suggests that HIF-1α promotes cap-dependent translation of selective mRNAs through up-regulating eIF4E1, which contributes to tumorsphere growth of breast cancer cells at hypoxia. The data shown provide new insights into protein synthesis mechanisms in cancer cells at low oxygen levels.     

Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.     

α6 Integrin Transactivates Insulin-like Growth Factor Receptor-1 (IGF-1R) to Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation

The canonical mitochondrial death pathway was first discovered for its role in signaling apoptosis. It has since been found to have a requisite function in differentiation initiation in many cell types including the lens through low level activation of the caspase-3 protease. The ability of this pathway to function as a molecular switch in lens differentiation depends on the concurrent induction of survival molecules in the Bcl-2 and IAP families, induced downstream of an IGF-1R/NFκB coordinate survival signal, to regulate caspase-3 activity. Here we investigated whether α6 integrin signals upstream to this IGF-1R-mediated survival-linked differentiation signal. Our findings show that IGF-1R is recruited to and activated specifically in α6 integrin receptor signaling complexes in the lens equatorial region, where lens epithelial cells initiate their differentiation program. In studies with both α6 integrin knock-out mice lenses and primary lens cell cultures following α6 integrin siRNA knockdown, we show that IGF-1R activation is dependent on α6 integrin and that this transactivation requires Src kinase activity. In addition, without α6 integrin, activation and expression of NFκB was diminished, and expression of Bcl-2 and IAP family members were down-regulated, resulting in high levels of caspase-3 activation. As a result, a number of hallmarks of lens differentiation failed to be induced; including nuclear translocation of Prox1 in the differentiation initiation zone and apoptosis was promoted. We conclude that α6 integrin is an essential upstream regulator of the IGF-1R survival pathway that regulates the activity level of caspase-3 for it to signal differentiation initiation of lens epithelial cells.