Phenyl azide substituted and benzophenone-substituted phosphonamides of 7-methylguanosine 5'-triphosphate as photoaffinity probes for protein synthesis initiation factor eIF-4E and a proteolytic fragment containing the cap-binding site

Three photoactive derivatives of the 7-methylguanosine-containing cap of eukaryotic mRNA were used to investigate protein synthesis initiation factor eIF-4E from human erythrocytes and rabbit reticulocytes. Sensitive and specific labeling of eIF-4E was observed with the previously described probe, gamma-32P]-gamma-(4-benzoylphenyl)methyl]amido]-7-methyl-GTP Blaas et al. (1982) Virology 116, 339; abbreviated 32P]BPM]. A second probe was synthesized that was an azidophenyltyrosine derivative of m7GTP ( 125I]APTM), the monoanhydride of m7GDP with 125I]-N-(4-azidophenyl)-2-(phosphoramido)-3-(4-hydroxy-3-iodop hen yl) propionamide. This probe allowed rapid and quantitative introduction of radioactivity in the last rather than the first step of synthesis and placed the radioactive label on the protein-proximal side of the weak P-N bond. A dissociation constant of 6.9 microM was determined for 125I]APTM, which is comparable to the published values for m7GTP. m7GTP and APTM were equally effective as competitive inhibitors of eIF-4E labeling with 125I]APTM. Like 32P]BPM, 125I]APTM labeled both the full-length (25 kDa) polypeptide and a 16-kDa degradation product, designated eIF-4E*, with labeling occurring in proportion to the amounts of each polypeptide present. A third probe, an azidophenylglycine derivative of m7GTP ( 32P]APGM), the monoanhydride of m7GDP with 32P]-N-(4-azidophenyl)-2-(phosphoramido)acetamide, was also synthesized and shown to label eIF-4E specifically. Unlike 32P]BPM and 125I]APTM, however, 32P]APGM labeled eIF-4E* approximately 4-fold more readily than intact eIF-4E. Tryptic and CNBr cleavage suggested that eIF-4E* consists of a protease-resistant core of eIF-4E that retains the cap-binding site and consists of approximately residues 47-182.