Effect of mRNA cap structure on eIF-4E phosphorylation and cap binding analyses using Ser209-mutated eIF-4Es.

The in vitro phosphorylation of human recombinant eIF-4E by protein kinase C was most effective in the absence of m7GTP, supporting a 'performed complex model' as the mRNA binding step of initiation, i. e., eIF-4E first forms an initiation complex eIF-4F and is phosphorylated before interacting with mRNA. On the other hand, the comparison of m7GTP-binding ability of wild-type eIF-4E with those of four Ser209-mutated ones (S209A, S209D, S209E and S209K) showed that the addition of anionic charge on Ser209 increases the cap affinity of eIF-4E by repressing the release of the cap from the complex, not by increasing the complex formation, suggesting the importance of a retractable ionic bridge between Ser209 and Lys159 in controlling the cap binding by eIF-4E phosphorylation.