Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1.
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 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.
Elia
A
A
Translational Control Group, Division of Basic Medical Sciences, Centre for Molecular and Metabolic Signalling, St George's, University of London, London, UK.
Constantinou
C
C
Clemens
M J
MJ
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects; D014176:Protein Biosynthesis; D025801:Ubiquitin / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D015853:Cysteine Proteinase Inhibitors; C087000:EIF4EBP1 protein, human; D039561:Eukaryotic Initiation Factor-4E; D007976:Leupeptins; D010750:Phosphoproteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde