Translational regulation of cyclin D1 by 15-deoxy-delta(12,14)-prostaglandin J(2).

The D-group cyclins play a key role in the progression of cells through the G(1) phase of the cell cycle. Treatment of MCF-7 breast cancer cells with the cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) results in rapid down-regulation of cyclin D1 protein expression and growth arrest in the G(0)/G(1) phase of the cell cycle. 15d-PGJ(2) also down-regulates the expression of cyclin D1 mRNA; however, this effect is delayed relative to the effect on cyclin D1 protein levels, suggesting that the regulation of cyclin D1 occurs at least partly at the level of translation or protein turnover. Treatment of MCF-7 cells with 15d-PGJ(2) leads to a rapid increase in the phosphorylation of protein synthesis initiation factor eukaryotic initiation factor 2alpha (eIF-2alpha) and a shift of cyclin D1 mRNA from the polysome-associated to free mRNA fraction, indicating that 15d-PGJ(2) inhibits the initiation of cyclin D1 mRNA translation. The selective rapid decrease in cyclin D1 protein accumulation is facilitated by its rapid turnover (t(1/2) = 34 min) after inhibition of cyclin D1 protein synthesis. The half-life of cyclin D1 protein is not significantly altered in cells treated with 15d-PGJ(2). Treatment of cells with 15d-PGJ(2) results in strong induction of heat shock protein 70 (HSP70) gene expression, suggesting that 15d-PGJ(2) might activate protein kinase R (PKR), an eIF-2alpha kinase shown previously to be responsive to agents that induce stress. 15d-PGJ(2) strongly stimulates eIF-2alpha phosphorylation and down-regulates cyclin D1 expression in a cell line derived from wild-type mouse embryo fibroblasts but has an attenuated effect in PKR-null cells, providing evidence that PKR is involved in mediating the effect of 15d-PGJ(2) on eIF-2alpha phosphorylation and cyclin D1 expression. In summary, treatment of MCF-7 cells with 15d-PGJ(2) results in increased phosphorylation of eIF-2alpha and inhibition of cyclin D1 mRNA translation initiation. At later time points, repression of cyclin D1 mRNA expression may also contribute to the decrease in cyclin D1 protein.     

Cell cycle-dependent phosphorylation of the translational repressor eIF-4E binding protein-1 (4E-BP1).

A fundamental control point in the regulation of the initiation of protein synthesis is the formation of the eukaryotic initiation factor 4F (eIF-4F) complex. The formation of this complex depends upon the availability of the mRNA cap binding protein, eIF-4E, which is sequestered away from the translational machinery by the tight association of eIF-4E binding proteins (4E-BPs). Phosphorylation of 4E-BP1 is critical in causing its dissociation from eIF-4E, leaving 4E available to form translationally active eIF-4F complexes, switching on mRNA translation. In this report, we provide the first evidence that the phosphorylation of 4E-BP1 increases during mitosis and identify Ser-65 and Thr-70 as phosphorylated sites. Phosphorylation of Thr-70 has been implicated in the regulation of 4E-BP1 function, but the kinase phosphorylating this site was unknown. We show that the cyclin-dependent kinase, cdc2, phosphorylates 4E-BP1 at Thr-70 and that phosphorylation of this site is permissive for Ser-65 phosphorylation. Crucially, the increased phosphorylation of 4E-BP1 during mitosis results in its complete dissociation from eIF-4E.     

Translation initiation factors induce DNA synthesis and transform NIH 3T3 cells

Several polypeptide factors that are essential for the initiation of protein synthesis bind to eukaryotic mRNAs and facilitate the formation of ribosome initiation complexes. Purified mRNA-binding translation initiation factors were microinjected into quiescent NIH 3T3 cells to study the possible growth-promoting role of these factors in living cells. We report that recombinant eIF-4E and rabbit reticulocyte eIF-4F induce a dose-dependent increase of DNA synthesis and morphologically transform NIH 3T3 cells. These results suggest that polypeptides involved in activating the rate-limiting step of protein synthesis (initiation complex formation) can be mitogenic and oncogenic when overexpressed in a cell by direct injection. Thus, eIF-4E and eIF-4F represent a class of proto-oncogenic proteins that is cytoplasmic, is involved in protein synthesis initiation, and is distinct from the proto-oncogenes that have been identified previously.     

Simultaneous cytoplasmic redistribution of ribosomal protein L32 mRNA and phosphorylation of eukaryotic initiation factor 4E after mitogenic stimulation of Swiss 3T3 cells

Ribosomal protein L32 mRNA moved from messenger ribonucleoprotein particles into polysomes following serum activation of quiescent Swiss 3T3 cells. This redistribution of the mRNA into a translationally active state began by 1 h and was complete by 3 h after activation. In contrast, actin mRNA showed no translational control, being found predominantly in polysomes in both quiescent and activated cultures. The phosphorylation state of eukaryotic initiation factor (eIF) 4E, which binds mRNA caps, was examined in parallel. eIF-4E phosphorylation was elevated by 1 h following serum activation and reached a peak by 3-5 h. Treatment of resting cells with phorbol ester also simultaneously stimulated eIF-4E phosphorylation and the movement of L32 mRNA into polysomes. These results are consistent with a model in which mitogen-induced phosphorylation increases the pool of active eIF-4E molecules, which in turn cause the recruitment of translationally controlled mRNAs to actively synthesizing ribosomes.     

Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus infected L cells

Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.     

Modulation of the mitogenic activity of eukaryotic translation initiation factor-4E by protein kinase C

Eukaryotic initiation factor-4E (eIF-4E) binds to the cap structure of eukaryotic mRNAs and is a component of the cap-binding protein complex eIF-4F. eIF-4E is present in cells in limiting concentrations and is phosphorylated both in vivo and in vitro by protein kinase C (PKC). Recently, eIF-4E has been implicated as an intracellular transducer of extracellular growth signals; microinjection of recombinant eIF-4E into quiescent NIH 3T3 cells induced DNA synthesis. In the present report, the mitogenic activity of eIF-4E was examined after coinjection with PKC. Recombinant eIF-4E was phosphorylated by PKC at the same amino acid that is phosphorylated in cultured cells and reticulocytes in response to phorbol ester. At limiting concentrations of eIF-4E, coinjection with PKC induced a fivefold increase in the mitogenic activity of eIF-4E. Injection of PKC alone or coinjection of eIF-4E with cAMP-dependent protein kinase (PKA) or the Raf protein had no effect. These results suggest that the mitogenic activity of eIF-4E is enhanced by PKC-specific phosphorylation and that phosphate addition is a rate-limiting step in eIF-4E activity.     

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.     

Ras transformation of cloned rat embryo fibroblasts results in increased rates of protein synthesis and phosphorylation of eukaryotic initiation factor 4E.

Eukaryotic initiation factor 4E (eIF-4E) is a 25-kDa phosphoprotein that binds to the 7-methylguanosine cap of mRNA and acts, along with other eIF-4 polypeptides, to unwind mRNA secondary structure at the 5' terminus. Recent studies have indicated that eIF-4E acts as a protooncogene, but only in its phosphorylated state. In order to determine the role of eIF-4E in oncogenesis, we examined its regulation and expression in cloned rat embryo fibroblasts transformed with the Harvey ras (Ha-ras) oncogene. The expression of Ha-ras increased the rate of protein synthesis but did not increase the levels of eIF-4E mRNA or protein. However, a dramatic increase (7-fold) in phosphate incorporation into eIF-4E was observed. The percentage of eIF-4E in the phosphorylated state was the same in transfected and control cells, indicating that both phosphorylation and dephosphorylation of eIF-4E were increased. Phosphopeptide mapping of eIF-4E from transformed cells indicated a single site of phosphorylation at Ser-53, which is the same as that identified previously in eIF-4E from reticulocytes and HeLa cells. These results indicate that p21ras is part of the signal transduction pathway leading to phosphorylation of eIF-4E. These findings also provide a potential mechanism for cell transformation by p21ras which involves the preferential stimulation of translation of certain mRNAs.     

Preferential translation of heat shock mRNAs in HeLa cells deficient in protein synthesis initiation factors eIF-4E and eIF-4 gamma.

Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HeLa cells causes a reduction in the levels of both eIF-4E and eIF-4 gamma (p220) and a concomitant decrease in the rates of both cell growth and protein synthesis (De Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. Cell Biol. 11, 5435-5445). The synthesis of most proteins in the antisense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to be synthesized. In the present study, we identified many of these as stress-inducible or heat shock proteins (HSPs). By mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to be HSP 90, HSP 70, HSP 65, and HSP 27. The steady-state levels of HSP 90, 70, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprecipitation indicated that HSP 90 and HSP 70 were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of HSPs in the AS cells was not due, however, to increased mRNA levels; the levels of HSP 90 and 70 mRNAs either remained the same or decreased after induction of antisense RNA expression. Actin mRNA, a typical cellular mRNA, was found on high polysomes in control cells but shifted to smaller polysomes in AS cells, as expected from the general decrease in translational initiation caused by eIF-4E and eIF-4 gamma depletion. HSP 90 and 70 mRNAs showed the opposite behavior; they were associated with small polysomes in control cells but shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in vivo for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of translational initiation.     

Interactions of the eIF-4F subunits in the yeast Saccharomyces cerevisiae.

Recognition of the cap structure at the 5' end of mRNA is one of the first events in initiation of eukaryotic translation. This step is mediated by the translation initiation factor 4F (eIF-4F). In mammalian cells this factor is composed of the cap-binding protein eIF-4E, eIF-4A, and a 220-kDa polypeptide. In yeast Saccharomyces cerevisiae, eIF-4E is found associated with a 150-kDa protein (p150) and a 20-kDa protein (p20). The resulting protein complex is proposed to represent yeast eIF-4F. To study the functions of p150 and p20 and their interaction with eIF-4E, we disrupted the genes encoding p150 and p20 and analyzed the effects on protein complex formation and cell viability. Yeast cells with single and double disruptions of the genes encoding p150 and p20 are viable, but p150 single and p150/p20 double disruptions show a slow growth phenotype. Gel chromatography and immunoadsorption experiments with a monoclonal anti-eIF-4E antibody coupled to protein G-Sepharose show that both p150 and p20 bind independently of each other to eIF-4E.     

Expression of antisense RNA against initiation factor eIF-4E mRNA in HeLa cells results in lengthened cell division times, diminished translation rates, and reduced levels of both eIF-4E and the p220 component of eIF-4F.

HeLa cells were transformed to express antisense RNA against initiation factor eIF-4E mRNA from an inducible promoter. In the absence of inducer, these cells (AS cells) were morphologically similar to control cells but grew four- to sevenfold more slowly. Induction of antisense RNA production was lethal. Both eIF-4E mRNA and protein levels were reduced in proportion to the degree of antisense RNA expression, as were the rates of protein synthesis in vivo and in vitro. Polysomes were disaggregated with a concomitant increase in ribosomal subunits. Translation in vitro was restored by addition of the initiation factor complex eIF-4F but not by eIF-4E alone. Immunological analysis revealed that the p220 component of eIF-4F was decreased in extracts of AS cells and undetectable in AS cells treated with inducer, suggesting that p220 and eIF-4E levels are coordinately regulated. eIF-4A, another component of eIF-4F, was unaltered.