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. Author manuscript; available in PMC: 2010 Jul 31.
Published in final edited form as: Mol Cell. 2009 Jul 31;35(2):164–180. doi: 10.1016/j.molcel.2009.05.028

Figure 4. EPRS Phosphorylation is Required for Release from MSC.

Figure 4

(A) Inhibition of phosphorylation blocks EPRS release from MSC. U937 cells were treated with IFN-γ and roscovitine or DSMO as in Fig. 3D. Lysates were immunoblotted with antibodies indicated. Same lysates were immunoprecipitated with anti-EPRS antibody and immunoblotted with antibodies against MSC components, KRS and AIMP3/p18, or against the pre-GAIT complex protein, NSAP1.

(B) Both Ser phosphorylation events are required for EPRS release from MSC. Flag-tagged, full-length, wild-type (WT) or phosphorylation-defective (Ser-to-Ala, S-A) EPRS mutants were cloned into pCMV6-XL5 vector and expressed in U937 cells by transient transfection. After 18 h, cycloheximide (50 μg/ml, 30 min) was added to block new synthesis, and then incubated with IFN-γ for 4 h. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with antibodies indicated.

(C) Incorporation of EPRS phospho-mimetics into MSC. Flag-tagged, full-length, WT and phospho-mimetic (Ser-to-Asp, S-D) mutant EPRS was expressed in U937 cells by transient transfection as in (B) and measured by immunoblot with anti-Flag antibody. Endogenous EPRS was detected by anti-EPRS antibody. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with antibodies indicated.

(D) Release of S886D phospho-mimetic establishes dual-site phosphorylation-dependent release of EPRS from MSC. Cells expressing Flag-tagged, full-length EPRS were incubated with IFN-γ for 4 h. Lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with antibodies indicated.