RhoE Inhibits 4E-BP1 Phosphorylation and eIF4E Function Impairing Cap-dependent Translation

Abstract

The Rho GTPase family member RhoE inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have previously reported that RhoE also inhibits cell cycle progression and Ras-induced transformation, specifically preventing cyclin D1 translation. Here we investigate the molecular mechanisms underlying those observations. RhoE inhibits the phosphorylation of the translational repressor 4E-BP1 in response to extracellular stimuli. However, RhoE does not affect the activation of mTOR, the major kinase regulating 4E-BP1 phosphorylation, as indicated by the phosphorylation levels of the mTOR substrate S6K, the dynamics of mTOR/Raptor association, and the observation that RhoE, as opposed to rapamycin, does not impair cellular growth. Interestingly, RhoE prevents the release of the eukaryotic initiation factor eIF4E from 4E-BP1, inhibiting cap-dependent translation. Accordingly, RhoE also inhibits the expression and the transcriptional activity of the eIF4E target c-Myc. Consistent with its crucial role in cell proliferation, we show that eIF4E can rescue both cell cycle progression and Ras-induced transformation in RhoE-expressing cells, indicating that the inhibition of eIF4E function is critical to mediate the anti-proliferative effects of RhoE.Rnd proteins (Rnd1, Rnd2, and RhoE/Rnd3) constitute a separate subfamily within the Rho family of small GTPases that have attracted recent attention because of their atypical features (1). Despite their high level of sequence similarity with RhoA, -B, and -C, Rnd proteins are remarkably different at both biochemical and functional levels. For instance, Rnd proteins do not cycle between an active form and an inactive form. Instead, they are thought to be constitutively GTP bound because of their extremely low GTPase activity and their increased affinity for GTP over GDP. Moreover, both Rnd1 and RhoE/Rnd3 induce opposite effects to those induced by RhoA on the actin cytoskeleton, promoting the disassembly of actin stress fibers and the loss of focal adhesions (2, 3).

Rnd proteins: Multifunctional regulators of the cytoskeleton and cell cycle progression

“…First, RhoA/ROCK inhibition does not account for the cell cycle arrest induced by Rnd3, and second, the two effects show remarkably different kinetics: loss of stress fibres is a transient response to Rnd3, whereas cell cycle inhibition is sustained over several days [11]. Instead, Rnd3 appears to inhibit cell cycle progression by reducing the phosphorylation of the translation regulator 4EBP1 [56] (Fig. 3).…”

Mammalian Target of Rapamycin (mTOR): Conducting the Cellular Signaling Symphony

“…As raptor knockdown phenocopies the effect of mTOR catalytic inhibitors by ablating 4EBP1 phosphorylation (15), these data unexpectedly reveal that depending on substrate, rapamycin does not inhibit all mTOR/raptor-dependent functions and can signal in a rapamycin-resistant manner. Recently, RhoE overexpression was found to suppress 4EBP1 but not S6K1 phosphorylation (31). Perhaps RhoE functions as an inhibitor of rapamycin-insensitive mTORC1?…”

The GTPase-deficient Rnd Proteins Are Stabilized by Their Effectors

“…Transient expression of Rnd1 or Rnd3 disassembles actin stress fibers and stimulates cell migration (15). Overexpression of Rnd3 can inhibit cell cycle progression and Ras-induced transformation, but this does not appear to involve RhoA, ERK, or PI3K/Akt pathways (16). Interestingly the micro-RNA associated with epithelial-mesenchymal transition, mir-200b, interacts with predicted target sites in the 3Ј-untranslated region of Rnd3 mRNA.…”