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, [7-³²P]-7-[[(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-iodophenyl)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 μM 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.