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Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1.
Elia, A; Constantinou, C 1; Clemens, M J
[Article] Oncogene. 27(6):811-822, January 31, 2008.

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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-molecular-weight, 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-molecular-weight 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 m7GTP-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.