Caspase Cleavage of Initiation Factor 4E-Binding Protein 1 Yields a Dominant Inhibitor of Cap-Dependent Translation and Reveals a Novel Regulatory Motif

Abstract

Eukaryotic initiation factor 4E (eIF4E) binding proteins (4E-BPs) regulate the assembly of initiation complexes required for cap-dependent mRNA translation. 4E-BP1 undergoes insulin-stimulated phosphorylation, resulting in its release from eIF4E, allowing initiation complex assembly. 4E-BP1 undergoes caspase-dependent cleavage in cells undergoing apoptosis. Here we show that cleavage occurs after Asp24, giving rise to the N-terminally truncated polypeptide Δ4E-BP1, which possesses the eIF4E-binding site and all the known phosphorylation sites. Δ4E-BP1 binds to eIF4E and fails to become sufficiently phosphorylated upon insulin stimulation to bring about its release from eIF4E. Therefore, Δ4E-BP1 acts as a potent inhibitor of cap-dependent translation. Using a mutagenesis approach, we identify a novel regulatory motif of four amino acids (RAIP) which lies within the first 24 residues of 4E-BP1 and which is necessary for efficient phosphorylation of 4E-BP1. This motif is conserved among sequences of 4E-BP1 and 4E-BP2 but is absent from 4E-BP3. Insulin increased the phosphorylation of 4E-BP3 but not sufficiently to cause its release from eIF4E. However, a chimeric protein that was generated by replacing the N terminus of 4E-BP3 with the N-terminal sequence of 4E-BP1 (containing this RAIP motif) underwent a higher degree of phosphorylation and was released from eIF4E. This suggests that the N-terminal sequence of 4E-BP1 is required for optimal regulation of 4E-BPs by insulin.

We thank A. A. M. Thomas (University of Utrecht) and R. M. Denton (University of Bristol) for supplying antibodies. The cDNA encoding human 4E-BP3 was a kind gift from F. Poulin and N. Sonenberg (McGill University, Montreal). We thank N. Morrice (University of Dundee) for his help in sequencing Δ4E-BP1. We also thank J. E. Harthill (University of Dundee) for technical assistance in isoelectric focusing, P. R. Shepherd (University College, London) for providing the FLAG-tagged mTOR construct, P. H. Scott (University of Glasgow) for supplying the recombinant FKBP12 protein, and both G. C. Scheper and T. P. Herbert (University of Dundee) for critical reading of the manuscript.

This work was supported by the Wellcome Trust through the award of a Prize Studentship to A. R. Tee.