Heterotrimeric GAIT complex drives transcript-selective translation inhibition in murine macrophages

Abstract

The gamma interferon (IFN-γ)-activated inhibitor of translation (GAIT) complex in human myeloid cells is heterotetrameric, consisting of glutamyl-prolyl-tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex binds a structural GAIT element in the 3' untranslated region of VEGF-A and other inflammation-related transcripts and inhibits their translation. EPRS is dually phosphorylated by cyclin-dependent kinase 5 (Cdk5) at Ser(886) and then by a Cdk5-dependent-AGC kinase at Ser(999); L13a is phosphorylated at Ser(77) by death-associated protein kinases DAPK and ZIPK. Because profound differences in inflammatory responses between mice and humans are known, we investigated the GAIT system in mouse macrophages. The murine GAIT complex is heterotrimeric, lacking NSAP1. As in humans, IFN-γ activates the mouse macrophage GAIT system via induced phosphorylation of EPRS and L13a. Murine L13a is phosphorylated at Ser(77) by the DAPK-ZIPK cascade, but EPRS is phosphorylated only at Ser(999). Loss of EPRS Ser(886) phosphorylation prevents NSAP1 incorporation into the GAIT complex. However, the triad of Ser(999)-phosphorylated EPRS, Ser(77)-phosphorylated L13a, and GAPDH forms a functional GAIT complex that inhibits translation of GAIT target mRNAs. Thus, translational control by the heterotrimeric GAIT complex in mice exemplifies the distinctive species-specific responses of myeloid cells to inflammatory stimuli.

Fig 1

IFN-γ activates the GAIT system in murine macrophages. (A) RAW 264.7 cells and BMM from wild-type C57BL6/J mice were treated with IFN-γ for 8 and 24 h. Cell lysates were immunoblotted (IB) using anti-VEGF-A and anti-β-actin antibodies. VEGF-A and β-actin mRNA was determined by reverse transcription-PCR (RT-PCR) of total cellular RNA. (B) RAW 264.7 cells were transfected with siRNAs against ZIPK and Cdk5, and control scrambled siRNAs, and incubated with IFN-γ for up to 24 h. Lysates were immunoblotted as shown. (C) Analysis of protein binding to the Cp GAIT element. Cytosolic lysates from IFN-γ-treated U937 and RAW 264.7 cells were incubated with a ³²P-labeled Cp GAIT element RNA probe, and binding was determined by EMSA. (D) In vitro translation. Cells were incubated with IFN-γ and lysates added to rabbit reticulocyte lysates in the presence of capped, polyadenylated luciferase (Luc) reporter RNA bearing the 3′ UTR Cp GAIT element and T7 gene 10 control RNA and [³⁵S]Met. Luc translation was quantified by densitometry, normalized by T7 gene 10, and expressed as a percentage of the control (mean ± SEM; n = 3 experiments).

Fig 2

IFN-γ induces DAPK-ZIPK-mediated L13a phosphorylation at Ser⁷⁷ in mouse macrophages. (A) Multiple-sequence alignment of mammalian L13a protein. The conserved Ser⁷⁷ phosphorylation site is shaded and the kinase recognition motif underlined. (B) Delayed induction of L13a phosphorylation in mouse macrophages. Lysates from IFN-γ-treated RAW 264.7 cells were immunoprecipitated with anti-L13a antibody and phosphorylation determined by immunoblotting with anti-phospho-Ser (P-Ser) antibody. (C) Mouse L13a is phosphorylated on Ser⁷⁷. Human and murine Flag-tagged, wild-type (WT) and Ser⁷⁷-to-Ala (S77A) mutant L13a proteins were transfected in U937 and RAW 264.7 cells and treated with IFN-γ for 16 h. Lysates were immunoprecipitated (IP) with anti-Flag antibody, followed by immunoblotting with anti-P-Ser antibody. (D) DAPK and ZIPK knockdown prevents L13a phosphorylation. RAW 264.7 cells were transfected with siRNAs against DAPK (left) and ZIPK (right) or scrambled control (Cont.) siRNAs. Lysates were immunoprecipitated with anti-L13a antibody, and phosphorylation was determined using anti-P-Ser antibody. (E) Mouse L13a translocates to the GAIT complex. RAW 264.7 and U937 cells were treated with IFN-γ for 24 h in the presence of [³²P]orthophosphate. Lysates were subjected to immunoprecipitation with anti-EPRS antibody and analyzed by immunoblotting with antibodies against EPRS and L13a (top two blots) and by autoradiography (bottom).

Fig 3

IFN-γ induces single-site EPRS phosphorylation at Ser⁹⁹⁹. (A) Multiple-sequence alignment of mammalian EPRS protein. Conserved Ser⁸⁸⁶ and Ser⁹⁹⁹ phosphorylation sites are shaded, and the kinase recognition motif around human Ser⁸⁸⁶ is underlined. (B) EPRS is phosphorylated at Ser⁹⁹⁹ in RAW 264.7 cells. Lysates from IFN-γ-treated cells were probed with phospho-specific antibodies and with anti-EPRS antibody as a loading control. (C) Ser⁹⁹⁹ is the only EPRS linker site phosphorylated in murine cells. Human and mouse pcDNA3-Flag-tagged, wild-type as well as single and double Ser-to-Ala mutant EPRS linkers were transfected into U937 and RAW 264.7 cells. Cells were treated with IFN-γ for 4 h, and lysates were immunoprecipitated with anti-Flag antibody and probed with anti-Flag and anti-P-Ser antibodies. (D) IFN-γ-activated mouse macrophages phosphorylate human EPRS at both Ser⁸⁸⁶ and Ser⁹⁹⁹. Lysates from IFN-γ-treated cells were used to in vitro phosphorylate recombinant EPRS linkers bearing Ser⁹⁹⁹-to-Ala and Ser⁸⁸⁶-to-Ala mutations in the presence of [γ-³²P]ATP. (E) IFN-γ induces Cdk5 activity in mouse macrophages. U937 and RAW 264.7 cells were treated with IFN-γ for 4 h, and the lysates were immunoprecipitated with anti-Cdk5 antibody and used for in vitro phosphorylation of a Ser⁸⁸⁶-containing EPRS peptide (QRRDR⁸⁸⁶SPTRNREPA) substrate (mean ± SEM; n = 4 experiments). (F) Cdk5 knockdown in mouse macrophages blocks EPRS phosphorylation. RAW 264.7 cells were transfected with siRNA against Cdk5 or scrambled control siRNA. Lysates were subjected to immunoblot analysis with antibody against P-EPRS-Ser⁹⁹⁹. (G) A Cdk5-dependent AGC group kinase phosphorylates Ser⁹⁹⁹. RAW 264.7 cells were incubated with IFN-γ for 0.5 h and then with specific kinase inhibitors for an additional 3.5 h. Pro-directed kinase group inhibitors were roscovitine (10 μM) and olomoucine (50 μM); AGC kinase group inhibitors were H-7 (10 μM), H-8 (10 μM), staurosporine (20 nM), rottlerin (100 μM), LY294002 (2.5 μM), Akt inhibitor VIII (2.5 μM), and rapamycin (10 nM). Lysates were analyzed by immunoblotting as indicated. (H) Schematic of IFN-γ-induced EPRS phosphorylation in human and mouse macrophages.

Fig 4

Ser⁹⁹⁹ phosphorylation induces EPRS translocation from MSC to GAIT complex. Lysates from IFN-γ-treated RAW 264.7 cells were immunoprecipitated with anti-EPRS antibody and subjected to immunoblot analysis with anti-AIMP3/p18 and anti-L13a antibodies to determine association with MSC and GAIT complex, respectively. Phospho-specific antibodies were used to determine EPRS phosphorylation.

Fig 5

Mouse GAIT complex lacks NSAP1. (A) RNA EMSA supershift investigation of binding of mouse GAIT complex proteins to GAIT element. Lysates from IFN-γ-treated RAW 264.7 cells were incubated with antibodies against GAIT complex constituents and then with a ³²P-labeled Cp GAIT element RNA probe. (B) Lysates from IFN-γ-treated U937 and RAW 264.7 cells were immunoblotted with antibodies against human GAIT complex constituents (left). Lysates were immunoprecipitated with anti-EPRS (middle) and anti-NSAP1 (right) antibodies and subjected to immunoblot analysis as indicated.

Fig 6

NSAP1 is not essential for reconstitution of GAIT complex function. (A) Schematic of human heterotetrameric (top) and mouse heterotrimeric (bottom) GAIT RNP; (B) reconstitution of GAIT function with human EPRS linker. His-tagged, wild-type (WT), and phospho-mimetic (Ser-to-Asp, S-to-D) human EPRS linker proteins were generated. Phosphorylated WT (P-WT) linker was generated by incubation with IFN-γ-activated cell lysate and repurified by Ni affinity chromatography. Linker proteins were incubated with other GAIT constituents as indicated and added to wheat germ extract for in vitro translation of luciferase (Luc)-Cp-GAIT element and T7 gene 10 reporters. (C) Reconstitution of GAIT function with mouse EPRS linker. His-tagged WT, P-WT, phospho-defective (S999A), phosphorylated S999A (P-S999A; generated by incubation with IFN-γ-activated cell lysate and repurified) and phospho-mimetic (S999D) mouse EPRS linker proteins were incubated other GAIT constituents and used in the in vitro experiment.

Fig 7

Comparison of human and mouse GAIT systems. Human heterotetrameric (top) and mouse heterotrimeric (bottom) GAIT complexes are shown.