Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form.
Eukaryotic translation initiation factor eIF-4E plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its participation in protein synthesis. However, the biochemical basis for the functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocytes by chromatography on rRNA-Sepharose. Using the resultant purified forms, we have studied the protein's interaction with the cap analogs m7GTP and m7GpppG and with the cap of globin mRNA by fluorescence quenching of tryptophan residues. It was found that phosphorylated eIF-4E had 3- to 4-fold greater affinity for cap analogs and mRNA than nonphosphorylated eIF-4E. The equilibrium binding constants (x 10(5), expressed as M-1) for the interaction of phosphorylated eIF-4E with m7GTP, m7GpppG, and globin mRNA were 20.0 +/- 0.1, 16.4 +/- 0.1, and 31.0 +/- 0.1, respectively, whereas those for the nonphosphorylated form were 5.5 +/- 0.4, 4.3 +/- 0.4, and 10.0 +/- 0.1, respectively. Treatment with potato acid phosphatase converted the phosphorylated form to the nonphosphorylated form and decreased the binding constant for m7GTP by a factor of 3. The increased affinity for mRNA caps may account for the in vivo and in vitro correlations between eIF-4E phosphorylation and accelerated protein synthesis and cell growth.
Minich
W B
WB
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130.
Balasta
M L
ML
Goss
D J
DJ
Rhoads
R E
RE
D000818:Animals; D039561:Eukaryotic Initiation Factor-4E; D005914:Globins / Q000235:genetics; D008956:Models, Chemical; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012314:RNA Cap Analogs / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012315:RNA Caps; D014364:Tryptophan; D005914:Globins