The mTOR target 4E‐BP1 contributes to differential protein expression during normoxia and hypoxia through changes in mRNA translation efficiency

Abstract

Hypoxia causes a rapid and sustained inhibition in mRNA translation that is characterized by both a transient phosphorylation of eukaryotic initiation factor 2‐alpha (eIF2α) and by inhibition of the mRNA cap binding protein eIF4E via activation of two distinct inhibitory proteins, the mammalian target of rapamycin (mTOR) target 4E‐BP1 and the eIF4E transporter 4E‐T. Although the importance of eIF2α phosphorylation during hypoxia has been clearly demonstrated, there is little information on the potential relevance of eIF4E regulation. We generated HeLa cells stably expressing a short hairpin interfering RNA (shRNA) against 4E‐BP1 and found that despite efficient knockdown, no significant changes occurred in the overall inhibition of mRNA translation during hypoxia. However, using a proteomics approach we identified seven proteins that were exclusively expressed in the 4E‐BP1 knockdown cells during both normoxic and hypoxic conditions. Further investigation of the transcriptional and translational regulation of these genes by quantitative RT‐PCR indicated that the loss of 4E‐BP1 causes a significant increase in the rate of protein synthesis of S100 calcium‐binding protein A4 (S100A4) and transgelin 2. These 4E‐BP1 regulated proteins have previously been associated with tumor cell motility, invasion and metastasis and may thus contribute to an adverse tumor phenotype.