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

Abstract

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.

Phosphorylation of Eukaryotic Initiation Factor 4E Markedly Reduces Its Affinity for Capped mRNA

“…Finally, some stress conditions will lead to a severe inhibition of protein synthesis although the phosphorylation of eIF4E increased under those conditions (8,9,31). An explanation for these apparent contradicting results might come from recent data, which suggest that the intact eIF4F complex may be required for de novo initiation events but not for reinitiation (32).…”

“…Phosphorylation of eIF4E is increased by mitogenic stimuli that activate translation (reviewed in Ref. 7) and by cytokines (8,9). Recently, two kinases were identified that phosphorylate Ser 209 in eIF4E and are targets for the mitogen-activated extracellular signal-regulated kinase and stress/cytokine-activated p38 mitogen-activated protein kinase pathways (9 -13).…”

Phosphorylation of Eukaryotic Initiation Factor (eIF) 4E Is Not Required for de Novo Protein Synthesis following Recovery from Hypertonic Stress in Human Kidney Cells

“…SDS-PAGE, Isoelectric Focusing, and Immunoblotting-Samples containing equal amounts of protein were resolved by SDS-PAGE and processed as described previously (21,22). Antiserum specific for the C-terminal domain of eIF4GI and those specific to eIF4E, eIF2␣, and phospho-eIF2␣ were as described previously (22); total levels of eIF4G, eIF4E, eIF2␣, PABP, eIF4A, and 4E-BP1 were visualized with alkaline phosphatase-conjugated secondary antibodies, while the determinations using commercial phosphospecific antisera and those for p38 MAP kinase used enhanced chemiluminescence for detection.…”

“…In all cases, care was taken to ensure that detection was within the linear range of the response. One-dimensional vertical isoelectric focusing gels used for the analysis of eIF4E phosphorylation were performed as described previously (21,22).…”

“…First, increased phosphorylation of eIF4E observed during cell stress is sometimes associated with a global inhibition of protein synthesis (5,21,22,25). In addition, phosphorylation of eIF4E is not required for restoration of translation in an eIF4E-dependent system in vitro, and both wild-type and phosphorylation site variants are able to rescue the lethal phenotype of eIF4E deletion in Saccharomyces cerevisiae (35).…”