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.
Shibata
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S
Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, 569-1094, Japan.
Morino
S
S
Tomoo
K
K
In
Y
Y
Ishida
T
T
D001665:Binding Sites; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D012315:RNA Caps / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012694:Serine; D011493:Protein Kinase C