Fig. 4.

Cdk5-mediated EPRS phosphorylation is required for the release of EPRS from the MSC and for binding to GAIT element RNA. (A) EPRS linker phosphorylated at Ser⁸⁸⁶ by Cdk5 reconstitutes the GAIT complex assembly and binding to GAIT element RNA. His-tagged, WT, and mutant (S886A and S999A) EPRS linker proteins were phosphorylated by incubation with lysate from IFN-γ–treated U937 cells or with recombinant Cdk5/p35, and then repurified using Ni-affinity resin (Top, Left). Specific phosphorylation was confirmed using anti-phosphospecific antibodies. Unmodified (Top, Right), lysate-phosphorylated (Middle, Left), or Cdk5-phosphorylated WT (Middle, Right), S999A (Bottom, Left), and S886A (Bottom, Right) EPRS linkers were preincubated with NSAP1 or with NSAP1, GAPDH, and phospho-L13a (P-L13a), and binding to biotinylated Cp GAIT element RNA immobilized on a streptavidin sensor chip was determined by SPR and expressed as resonance units (RU). (B) Cdk5-mediated phosphorylation induces EPRS release from MSC. U937 cells were transfected with siRNA targeting Cdk5 (and control siRNA) and then treated with IFN-γ for 4 h. Lysates were immunoprecipitated with antibodies against EPRS, LysRS (KRS, MSC constituent), and NSAP1 (pre-GAIT complex constituent), and evaluated by immunoblot analysis.