Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1

Abstract

The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecularweight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecularweight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m 7 GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.

Association of Maternal mRNA and Phosphorylated EIF4EBP1 Variants With the Spindle in Mouse Oocytes: Localized Translational Control Supporting Female Meiosis in Mammals

“…A dual effect of phosphorylation of EIF4EBP1 was reported elsewhere, either reducing affinity of EIF4EBP1 for EIF4E, or promoting polyubiquitination and decreased EIF4EBP1 stability (Elia et al 2008). Our data are consistent with EIF4EBP1 degradation subsequent to phosphorylation.…”

Association between LRRK2 and 4E-BP1 protein levels in normal and malignant cells

“…As it is shown in Fig. 5, 4E-BP1 phosphorylation may promote degradation of a fraction of 4E-BP1, mediated by the proteasome; this agrees with previous studies in which a role for the proteasome is proposed in 4E-BP1 degradation (27,29). Furthermore, our data suggest that degradation is sensitive to the phosphorylation status of 4E-BP1.…”

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

“…It is also feasible that only pre-phosphorylated forms of 4E-BP1 can then undergo further modifications, analogous to the observation that only phosphorylated 4E-BP1 is subjected to ubiquitination. 40,41 Our investigations of the eIF4G paralogs were successful in identifying 2 sites of phosphorylation on eIF4GII at Ser 384 and Ser 392, through a combination of deletion analysis and unphosphorylatable serine-alanine mutations. While we have not conclusively identified the kinase responsible for this modification, it does appear likely to be Cdk1.…”