Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism

The multisubunit eukaryotic translation initiation factor (eIF) CF recruits 40S ribosomal subunits to the 5' end of mRNA. The eIF4F subunit eIF4E inte racts directly with the mRNA 5' cap structure. Assembly of the eIF4F comple x is inhibited by a family of repressor polypeptides, the eIF4E-binding pro teins (4E-BPs). Binding of the 4E-BPs to eIF4E is regulated by phosphorylat ion: Hypophosphorylated 4E-BP isoforms interact strongly with eIF4E, wherea s hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quie scent cells, but is hyperphosphorylated on multiple sites following exposur e to a variety of extracellular stimuli. The PI3-kinase/Akt pathway and the kinase FRAP/mTOR signal to 4E-BP1. FRAP/mTOR has been reported to phosphor ylate 4E-BP1 directly in vitro. However, it is not known if FRAP/mTOR is re sponsible for the phosphorylation of all 4E-BP1 sites, nor which sites must be phosphorylated to release 4E-BP1 from eIF4E. To address these questions , a recombinant FRAP/mTOR protein and a FRAP/mTOR immunoprecipitate were ut ilized in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide ma pping of the in vitro labeled protein yielded two 4E-BP1 phosphopeptides th at comigrated with phosphopeptides produced in vivo. Mass spectrometry anal ysis indicated that these peptides contain phosphorylated Thr-37 and Thr-46 . Thr-37 and Thr-46 are efficiently phosphorylated in vitro by FRAP/mTOR wh en 4E-BP1 is bound to eIF4E. However, phosphorylation at these sites was no t associated with a loss of eIF4E binding. Phosphorylated Thr-37 and Thr-SB are detected in all phosphorylated in vivo 4E-BPI isoforms, including thos e that interact with eIF4E. Finally, mutational analysis demonstrated that phosphorylation of Thr-37/Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. Taken together, our res ults suggest that 4E-BP1 phosphorylation by FRAP/mTOR on Thr-37 and Thr-46 is a priming event for subsequent phosphorylation of the carboxy-terminal s erum-sensitive sites.