Translation Inhibition by Rocaglates Is Independent of eIF4E Phosphorylation Status

Abstract

Rocaglates are natural products that inhibit protein synthesis in eukaryotes and exhibit antineoplastic activity. In vitro biochemical assays, affinity chromatography experiments coupled with mass spectrometry analysis, and in vivo genetic screens have identified eukaryotic initiation factor (eIF) 4A as a direct molecular target of rocaglates. eIF4A is the RNA helicase subunit of eIF4F, a complex that mediates cap-dependent ribosome recruitment to mRNA templates. The eIF4F complex has been implicated in tumor initiation and maintenance through elevated levels or increased phosphorylation status of its cap-binding subunit, eIF4E, thus furthering the interest toward developing rocaglates as antineoplastic agents. Recent experiments have indicated that rocaglates also interact with prohibitins 1 and 2, proteins implicated in c-Raf-MEK-ERK signaling. Because increased ERK signaling stimulates eIF4E phosphorylation status, rocaglates are also expected to inhibit eIF4E phosphorylation status, a point that has not been thoroughly investigated. It is currently unknown whether the effects on translation observed with rocaglates are solely through eIF4A inhibition or also a feature of blocking eIF4E phosphorylation. Here, we show that rocaglates inhibit translation through an eIF4E phosphorylation-independent mechanism.

Figure 1

A. Proposed mechanism of action of rocaglates on eIF4A recycling through the eIF4F complex. In this model, rocaglates stimulate eIF4A RNA binding, rendering it unavailable to enter into the eIF4F complex. B. Structures of rocaglates used in this study. C. Dose-dependent inhibition of translation by rocaglates in Jurkat and NIH/3T3 cells. Cells were incubated in the presence of compound for a total of 2 hours and protein synthesis rates were determined as described in the Materials and Methods. The relative rates are of translation are calculated by normalizing to DMSO. n = 4; error bars represent the error of the mean.

Figure 2

A. Effects of rocaglates on eIF4E phosphorylation in Jurkat cells. Cells were incubated in the presence of the indicated compounds for 2 hours, lysed, fractionated on a 10% NuPAGE Bis-Tris gel, and transferred to PVDF membranes for western blotting. Blots were probed using antibodies directed to the proteins indicated to the right of the panel. B. Effects of rocaglates on eIF4E phosphorylation status in NIH/3T3 cells. Cells were treated, proteins fractionated, transferred to PVDF membranes, and western blots analyzed as described for Panel A. C. Effects of rocaglates on eIF4E phosphorylation status in RAS-transformed NIH/3T3 cells. Cells were treated, proteins fractionated, transferred to PVDF membranes, and western blots analyzed as described for Panel A. The dashed line indicates that probing for eIF4E and ERK were performed on different membranes. GAPDH levels were used as an internal standard to account for variations in extract levels between lanes. D. Stimulation of eIF4A:RNA binding by rocaglates. Recombinant eIF4A (1.3 μM) was incubated with 35 000 cpm of ³²P-labelled RNA in the presence of 5 μM rocaglate and processed as described in the Materials and Methods. eIF4A:RNA complexes retained on nitrocellulose filters were quantitated by scintillation counting. N = 3; error bars represent error of the mean.

Figure 3

A. Rocaglates inhibit translation in Mnk1^+/+Mnk2^+/+ and Mnk1^−/−Mnk2^−/− MEFs. Protein synthesis rates were determined as described in the Materials and Methods. The relative rates of ³⁵S-Met incorporation are normalized to DMSO. N = 4; error bars represent error of the mean. *, p < 0.001 (Student's t-test). B. Effects of rocaglates on eIF4E phosphorylation in Mnk1^+/+Mnk2^+/+ and Mnk1^−/−Mnk2^−/− cells. Cells were incubated in the presence of the indicated compounds for two hours, lysed, fractionated on a 10% polyacrylamide gel, and transferred to PVDF membranes for western blotting. Blots were probed with antibodies directed to the proteins indicated to the right of the panel. GAPDH levels were used as an internal standard to account for variations in extract levels between lanes. Note that lane 24 is slightly underloaded based on the GAPDH internal standard.