Figure 7. Ser⁹⁹⁹ Phosphorylation is Required for L13a-mediated Interaction of GAIT Complex with eIF4G.

(A) Ser⁹⁹⁹ phosphorylation is required for GAIT complex interaction with eIF4G during in vitro translation. In vitro translation of GAIT element-bearing reporter in wheat germ extract as in Figure 7B. The reaction mixture was immunoprecipitated with anti-EPRS and eIF4G antibodies after digestion with RNase A/T1, and immunoblotted with antibodies indicated.

(B) Ser⁹⁹⁹ phosphorylation facilitates P-L13a-mediated interaction of GAIT complex with recombinant eIF4G. Individual (left panels) or combined (right panels) GAIT complex proteins were incubated with GST-eIF4G or GST immobilized to glutathione-agarose beads. After washing, the interaction was analyzed by immunoblotting with antibodies against GAIT components and eIF4G.

(C) EPRS Ser⁹⁹⁹ phosphorylation alters protease susceptibility of L13a. P-L13a (150 ng) was preincubated with GAIT components including mutant EPRS linkers and then treated with 10 ng proteinase (Prot.) K for 15 min at 30°C. Untreated and protease-treated P-L13a served as controls. Cleavage was determined by immunoblot with anti-L13a antibody.

(D) Schematic illustrating function of 2-site phosphorylation of EPRS

(i) EPRS resides in the MSC.

(ii) IFN-γ induces phosphorylation of Ser⁸⁸⁶ by a proline-directed kinase.

(iii) IFN-γ induces phosphorylation of Ser⁹⁹⁹ by an AGC kinase group member. Phosphorylation of both Ser⁸⁸⁶ and Ser⁹⁹⁹ (highlighted with radial lines) induces EPRS release from the MSC.

(iv) Diphosphorylated EPRS translocates from the MSC to the cytoplasm.

(v) Phosphorylation of EPRS Ser⁸⁸⁶ is required for NSAP1 binding that blocks binding of EPRS to the GAIT RNA element.

(vi) EPRS Ser⁸⁸⁶ phosphorylation is required for binding of phospho-L13a and GAPDH that induces a conformational shift and permits GAIT RNA element binding to EPRS.

(vii) Phosphorylation of EPRS Ser⁹⁹⁹ is required for translational silencing activity of GAIT complex by permitting interaction of phospho-L13a with eIF4G, and blocking the binding of eIF3 of the 43S ribosomal complex to eIF4G.