Nuclear eukaryotic initiation factor 4E (eIF4E) colocalizes with splicing factors in speckles.

The eukaryotic initiation factor 4E (eIF4E) plays a pivotal role in the control of protein synthesis. eIF4E binds to the mRNA 5' cap structure, m(7)GpppN (where N is any nucleotide) and promotes ribosome binding to the mRNA. It was previously shown that a fraction of eIF4E localizes to the nucleus (Lejbkowicz, F., C. Goyer, A. Darveau, S. Neron, R. Lemieux, and N. Sonenberg. 1992. Proc. Natl. Acad. Sci. USA. 89:9612-9616). Here, we show that the nuclear eIF4E is present throughout the nucleoplasm, but is concentrated in speckled regions. Double label immunofluorescence confocal microscopy shows that eIF4E colocalizes with Sm and U1snRNP. We also demonstrate that eIF4E is specifically released from the speckles by the cap analogue m(7)GpppG in a cell permeabilization assay. However, eIF4E is not released from the speckles by RNase A treatment, suggesting that retention of eIF4E in the speckles is not RNA-mediated. 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) treatment of cells causes the condensation of eIF4E nuclear speckles. In addition, overexpression of the dual specificity kinase, Clk/Sty, but not of the catalytically inactive form, results in the dispersion of eIF4E nuclear speckles.

Suppression of cap-dependent translation in mitosis.

Cap-dependent translation is mediated by eIF4F, a protein complex composed of three subunits as follows: eIF4E, which recognizes the mRNA 5' cap structure; eIF4A, an RNA-helicase; and eIF4G, a scaffolding protein that binds eIF4E, eIF4A, and the eIF4E-kinase Mnk1 simultaneously. eIF4E is hypophosphorylated and cap-dependent translation is reduced at mitosis. Here, we show that 4E-BP1, a suppressor of eIF4E function, is also hypophosphorylated in mitosis, resulting in disruption of the eIF4F complex. Consequently, eIF4E is sequestered from the eIF4G/Mnk1 complex. These results explain the specific inhibition of cap-dependent translation in mitosis and also explain how eIF4E is rendered hypophosphorylated during mitosis. Furthermore, eIF4E interaction with eIF4GII is strongly decreased coincident with hyperphosphorylation of eIF4GII. Thus, inhibition of cap-dependent translation in mitosis results from a combination of phosphorylation modifications leading to eIF4F complex disruption.

Regulatable expression of the interferon-induced double-stranded RNA dependent protein kinase PKR induces apoptosis and fas receptor expression.

PKR is an interferon-induced dsRNA-dependent protein kinase involved in the antiviral response as well as in cell growth and differentiation. Studies using a transdominant negative mutant of PKR also have implicated the kinase in tumor suppression and apoptosis. However, functional studies of PKR have been hampered by the lack of a suitable expression system. In this study, we used a tetracycline-regulated inducible system in NIH3T3 cells to investigate the involvement of PKR in programmed cell death (apoptosis). We show that expression of wild-type PKR causes apoptosis and correlates with increased mRNA levels for the Fas receptor, a member of the tumor necrosis family of proteins. Expression of an inactive form of PKR (K296R) or the vector alone did not induce apoptosis or elevate Fas mRNA levels. Our results clearly demonstrate that expression of an active form of PKR triggers apoptosis, possibly through upregulation of the Fas receptor.

A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E.

The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in the control of cell growth. eIF4E binds to the mRNA 5' cap structure m(7)GpppN (where N is any nucleotide), and promotes ribosome binding to the mRNA in the cytoplasm. However, a fraction of eIF4E localizes to the nucleus. Here we describe the cloning and functional characterization of a new eIF4E-binding protein, referred to as 4E-T (eIF4E-Transporter). We demonstrate that 4E-T is a nucleocytoplasmic shuttling protein that contains an eIF4E-binding site, one bipartite nuclear localization signal and two leucine-rich nuclear export signals. eIF4E forms a complex with the importin alphabeta heterodimer only in the presence of 4E-T. Overexpression of wild-type 4E-T, but not of a mutant defective for eIF4E binding, causes the nuclear accumulation of HA-eIF4E in cells treated with leptomycin B. Taken together, these results demonstrate that the novel nucleocytoplasmic shuttling protein 4E-T mediates the nuclear import of eIF4E via the importin alphabeta pathway by a piggy-back mechanism.

Specific cyclic nucleotide phosphodiesterase inhibitors differently modulate contractile kinetics in airway smooth muscle.

The involvement of various phosphodiesterases (PDEs) in controlling the time-dependent mechanical properties of guinea pig trachealis smooth muscles was determined by using different classes of PDE inhibitors as pharmacological tools. These drugs produced low amplitude and long-lasting dose-dependent relaxations on the resting tone with the following EC50 values: rolipram, 3 nM; indolidan, 0.11 microM; and zaprinast, 0.5 nM and 1 microM. These PDE inhibitors were 50% less active than 1 microM norepinephrine. The effects of the drugs were also tested on carbachol-induced contractions and norepinephrine-evoked relaxations. Zaprinast, but not rolipram nor indolidan, decreased the rate of rise of contraction, thus prolonging the time to reach the plateau by 75% without modifying the magnitude of the responses. Zaprinast and rolipram significantly increased the total length of the norepinephrine effect by 25 and 35%, respectively. Similar results were obtained in a dose-dependent manner on isoproterenol-induced relaxations. In contrast, a higher concentration of indolidan was required to affect the amplitude, duration, and time to peak of isoproterenol- or norepinephrine-induced relaxations. These results indicate that PDE IV (rolipram sensitive) and PDE I, and less likely PDE V (both zaprinast sensitive), are involved in the control of guinea pig airway contractile kinetics, whereas PDE III (indolidan sensitive) is essentially involved in the modulation of the resting tone. Four cytosolic isozymes were identified in bovine airway smooth muscles (ASMs); PDE I (calmodulin-dependent PDE), PDE II (cGMP-stimulated PDE), PDE IV (cAMP-specific and rolipram-sensitive PDE), and PDE V (cGMP-specific and zaprinast-sensitive PDE). Characterization of PDE isoforms present in the microsomal fraction by HPLC showed the presence of PDE IV, PDE V, and to a lesser extent PDE III. However, PDE III was not detected in ASM cytosol. Using newly synthesized radioligands, binding studies confirmed the low level of expression of PDE III and the presence of PDE IV. We conclude that PDE I controls the rate of contraction, whereas PDE V and PDE IV prolong the time of relaxation induced by NE. PDE V would control the ASM responsiveness by regulating the intracellular cGMP concentration, which in turn would both activate PKG and stimulate PDE II (cGS-PDE). Since the various isozymes of PDE are differently involved in the kinetic control of the mechanical events in ASM, they represent physiologically relevant and important pharmacological targets.