Simon J. Morley

Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability.

The immunosuppressant rapamycin has been shown previously to inhibit the G1/S transition in several cell types by prolonging the G1 phase of the cell cycle. This process appears to be controlled, in part, by the rapamycin-sensitive FK506-binding protein-rapamycin-associated protein-p70 S6 kinase (p70S6k) pathway and the cyclin-dependent kinases (Cdk). We now show that in serum-stimulated NIH 3T3 cells, rapamycin treatment delays the accumulation of cyclin D1 mRNA during progression through G1. Rapamycin also appears to affect stability of the transcript. The combined transcriptional and post-transcriptional effects of the drug ultimately result in decreased levels of cyclin D1 protein. Moreover, degradation of newly synthesized cyclin D1 protein is accelerated by rapamycin, a process prevented by inclusion of the proteasome inhibitor, N-acetyl-Leu-Leu-norleucinal. The overall effect of rapamycin on cyclin D1 leads, in turn, to impaired formation of active complexes with Cdk4, a process which triggers retargeting of the p27Kip1 inhibitor to cyclin E/Cdk2. In view of this novel experimental evidence, we discuss a possible mechanism for the rapamycin-induced cell cycle arrest at the G1/S transition.

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells.

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the α subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2α and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis. Cell Death and Differentiation (2000) 7, 603–615

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N‐terminal (Nt) and C‐terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap‐dependent translation, the cleavage products themselves may directly promote cap‐dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES‐driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap‐independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.

A reevaluation of the cap-binding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate.

The cap-binding eukaryotic initiation factor, eIF4E, is a key target for the regulation of translation in mammalian cells and is widely thought to be present at very low molar concentrations. Here we present observations with the reticulocyte lysate that challenge this view. When reticulocyte ribosomes are harvested by centrifugation, most (75%) of the eIF4E remains in the postribosomal supernatant (PRS). In a reconstituted translation system we find that the ribosome-associated eIF4E alone can sustain much of the overall activity, suggesting that much of the factor in the PRS is functionally redundant. Consistent with this, our estimates of eIF4E in the reticulocyte lysate reveal much higher concentrations than previously reported. The association of a small proportion of eIF4E with the ribosome fraction appears to be functional and dependent on interaction with the factor eIF4G. This fraction of eIF4E is, as expected, more highly phosphorylated than that in the PRS; however, at least half the total phosphorylated eIF4E in reticulocyte lysate translation systems resides in the PRS fraction, suggesting that, while phosphorylation may enhance activity, it is not in itself sufficient to promote utilization of the factor. We also show that the eIF4E-binding factor, eIF4E-BP1 or PHAS-I, which regulates eIF4E activity in insulin-responsive cells, is present in the reticulocyte PRS at an approximately 1:1 molar ratio relative to eIF4E and demonstrate by co-immunoprecipitation studies that the binding of PHAS-I and eIF4G to eIF4E is mutually exclusive. These data are consistent with a potential regulatory role for PHAS-I in the reticulocyte lysate.

Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines

We have investigated the effect of inducing apoptosis in BJAB and Jurkat cells on the cellular content of several polypeptide chain initiation factors. Serum deprivation results in inhibition of protein synthesis and induction of apoptosis in BJAB cells; at early times, there is selective degradation of polypeptide initiation factor eIF4G but no major losses of other key initiation factors. The disappearance of full length eIF4G is accompanied by the appearance of smaller forms of the protein, including a major product of approximately 76 kDa. Apoptosis induced by cycloheximide results in similar effects. Both total cytoplasmic eIF4G and eIF4G associated with eIF4E are degraded with a half-life of 2–4 h under these conditions. Treatment of serum-starved or cycloheximide-treated cells with Z-VAD.FMK or Z-DEVD.FMK, which inhibit caspases required for apoptosis, protects eIF4G from degradation and blocks the appearance of the ca. 76 kDa product. Exposure of BJAB cells to rapamycin rapidly inhibits protein synthesis but does not lead to acute degradation of eIF4G. In both BJAB and Jurkat cells induction of apoptosis with anti-Fas antibody or etoposide also results in the selective loss of eIF4G, which is inhibitable by Z-VAD.FMK. These data suggest that eIF4G is selectively targeted for cleavage as cells undergo apoptosis and is a substrate for proteases activated during this process.

Involvement of stress-activated protein kinase and p38/RK mitogen activated protein kinase signaling pathways in the enhanced phosphorylation of initiation factor 4E in NIH 3T3 cells

The initiation factor (eIF) 4E is regulated by modulating both the phosphorylation and the availability of the protein to participate in the initiation process. Here we show that either serum treatment or activation of the stress-activated protein kinase (JNK/SAPK) led to enhanced phosphorylation of eIF4E in quiescent NIH 3T3 cells. Although the immunosuppressant, rapamycin, was found to stabilize the association of eIF4E with its negative regulator, 4E-BP1, this drug did not prevent the early effects of serum stimulation on the overall rate of translation, polysome formation, the phosphorylation status of eIF4E, or the recruitment of eIF4E into the eIF4F complex. However, the rapid enhancement of eIF4E phosphorylation in response to serum was largely prevented by the inhibitor of mitogen-activated protein (MAP) kinase activation, PD98059. Activation of the JNK/SAPK signaling pathway with anisomycin resulted in enhanced phosphorylation of eIF4E, which was prevented by either rapamycin or the highly specific p38 MAP kinase inhibitor, SB203580. These data illustrate that multiple signaling pathways, including those of distinct members of the MAP kinase family, mediate the phosphorylation of eIF4E and that the association of eIF4E with 4E-BP1 does not necessarily prevent phosphorylation of eIF4E in vivo.

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m7 guanosine cap

ABSTRACT In eukaryotes the majority of mRNAs have an m7G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3′ end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.

Activity of the Hepatitis A Virus IRES Requires Association between the Cap-Binding Translation Initiation Factor (eIF4E) and eIF4G

ABSTRACT The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m7GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.

Phosphorylation of eukaryotic initiation factor (eIF) 4E is not required for de novo protein synthesis following recovery from hypertonic stress in human kidney cells.

Previous work has suggested that increased phosphorylation of eukaryotic initiation factor (eIF) 4E at Ser-209 in the C-terminal loop of the protein often correlates with increased translation rates. However, the functional consequences of phosphorylation have remained contentious with our understanding of the role of eIF4E phosphorylation in translational control far from complete. To investigate the role for eIF4E phosphorylation in de novo translation, we studied the recovery of human kidney cells from hypertonic stress. Results show that hypertonic shock caused a rapid inhibition of protein synthesis and the disaggregation of polysomes. These changes were associated with the dephosphorylation of eIF4G, eIF4E, 4E-binding protein 1 (4E-BP1), and ribosomal protein S6. In addition, decreased levels of the eIF4F complex and increased association of 4E-BP1 with eIF4E were observed over a similar time course. The return of cells to isotonic medium rapidly promoted the phosphorylation of these initiation factors, increased levels of eIF4F complexes, promoted polysome assembly, and increased rates of translation. However, by using a cell-permeable, specific inhibitor of eIF4E kinase, Mnk1 (CGP57380), we show that de novoinitiation of translation and eIF4F complex assembly during this recovery phase did not require eIF4E phosphorylation.

Disruption of the interaction of mammalian protein synthesis eukaryotic initiation factor 4B with the poly(A)-binding protein by caspase- and viral protease-mediated cleavages

Eukaryotic initiation factor (eIF) 4B interacts with several components of the initiation pathway and is targeted for cleavage during apoptosis. In a cell-free system, cleavage of eIF4B by caspase-3 coincides with a general inhibition of protein synthetic activity. Affinity chromatography demonstrates that mammalian eIF4B interacts with the poly(A)-binding protein and that a region consisting of the N-terminal 80 amino acids of eIF4B is both necessary and sufficient for such binding. This interaction is lost when eIF4B is cleaved by caspase-3, which removes the N-terminal 45 amino acids. Similarly, the association of eIF4B with the poly(A)-binding proteinin vivo is reduced when cells are induced to undergo apoptosis. Cleavage of the poly(A)-binding protein itself, using human rhinovirus 3C protease, also eliminates the interaction with eIF4B. Thus, disruption of the association between mammalian eIF4B and the poly(A)-binding protein can occur during both apoptosis and picornaviral infection and is likely to contribute to the inhibition of translation observed under these conditions.