(A) Induction of DAPK and ZIPK mRNA. U937 cells were treated with IFN-γ for up to 24 h and RT-PCR analysis of total cellular RNA was done using primers targeted against DAPK (top panel), ZIPK (middle panel), and actin (bottom panel).
(B) Temporal expression of DAPK and ZIPK protein. Cell lysates described in (A) were immunoblotted with antibodies against DAPK (top panel), ZIPK (2^nd panel), and actin (3^rd panel). DAPK (△) and ZIPK (▲) expression was quantitated by densitometry and expressed as % of control (mean ± s.e.m.) (bottom panel).
(C) Determination of DAPK and ZIPK activities. Cell lysates were used for in vitro phosphorylation of DAPK- (△) and ZIPK- (▲) specific substrate peptides (mean ± s.e.m.).

(A) siRNA-mediated knock-down of ZIPK or DAPK inhibits L13a phosphorylation. U937 cells were transfected with siRNAs against ZIPK, DAPK, and MK2. Scrambled siRNAs were used as controls. After 24 h, knock-down efficiency was determined by immunoblot analysis of cell lysates (top). Transfected cells were treated with IFN-γ for 16 h, and L13a and phospho-L13a (P-L13a) detected by immunoblot (middle). Immunoblots with anti-actin served as loading controls (bottom).
(B) DAPK is required for ZIPK activation and L13a phosphorylation. U937 cells were transfected with DAPK (or scrambled) siRNA. After recovery, cells were incubated with IFN-γ for 16 h and cell lysates were immunoblotted with anti-DAPK and anti-ZIPK antibodies (left). The same lysates were immunoprecipitated with anti-ZIPK and the immunocomplex was used to phosphorylate recombinant L13a in vitro (right).
(C) DAPK knockdown reduces ZIPK activity. U937 cells were transfected with DAPK (or scrambled) siRNA. After recovery, cells were incubated with IFN-γ for 12 h and metabolically labeled with ³²P-orthophosphoric acid for an additional 4 h. Cytosolic extracts were immunoprecipitated with anti-ZIPK antibody or mouse IgG, and ZIPK phosphorylation visualized by SDS-PAGE and autoradiography.

(A) Multiple sequence alignment of eukaryotic ribosomal protein L13a (and L16A/B paralogs in yeast) using ClustalW. Conserved residues surrounding the conserved R-X-X-S kinase recognition motif are boxed and shaded.
(B) The R-X-X-S/T motif is essential for L13a phosphorylation. Wild-type and mutant peptides (R74A and S77A) containing the L13a kinase recognition motif were subjected to in vitro phosphorylation using lysates from 0- and 16-h, IFN-γ-treated cells.
(C) ZIPK derived from U937 cells phosphorylates L13a. Candidate kinases were immunoprecipitated from lysate of 16-h, IFN-γ-treated cells, and the immunocomplex used to phosphorylate recombinant L13a. Auto-phosphorylated ZIPK (P-ZIPK) is indicated by an open arrow (top). Immunocomplex activity was tested by in vitro phosphorylation using appropriate substrate peptides (Xtide= crosstide, GS= glycogen synthase-derived peptide) (bottom).
(D) Recombinant ZIPK phosphorylates L13a. Active recombinant catalytic domain of DAPK, active full-length ZIPK, and active ERK2 were used to phosphorylate L13a by in vitro phosphorylation.
(E) ZIPK phosphorylates 60S-bound L13a. Recombinant ZIPK was used for in vitro phosphorylation of recombinant L13a (rL13a) and 60S ribosomal subunit isolated from either wild-type or L13a-knockdown (L13a-SHR) U937 cells (top). L13a in 60S subunit was quantitated by western blot and comparison to purified recombinant L13a (not shown). Phosphorylated L13a was quantitated by densitometry and plotted against L13a amount (bottom).

(A) DAPK and ZIPK mRNA 3’UTRs mediate translational inhibition of reporters. Capped, FLuc-DAPK 3’UTR_(1–1225)-A₃₀ RNA and FLuc-ZIPK 3’UTR_(50–645)-A₃₀ RNA was translated in RRL in presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (top). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Fluc expression was normalized by Rluc expression and expressed as % of control reaction without lysate (mean ± s.e.m.); significant differences (p < 0.05, two-tailed t-test) between 24-h and control lysates are indicated (*) (bottom panel).
(B) Secondary structures of GAIT elements. The predicted secondary structures of VEGF-A (3’UTR nt 358–386), Cp (nt 78–106), putative DAPK (nt 1141–1169) and ZIPK (nt 174–206) GAIT elements (by Mfold). Base-pairing between A7:U23 and U8:G22 was disallowed for DAPK RNA, and between A10:U25 for ZIPK RNA.
(C) The GAIT complex binds DAPK and ZIPK GAIT elements. Radiolabeled, GAIT element riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA-protein complexes were resolved by non-denaturing 5% PAGE. RNA-protein complexes were competed with 50- and 100-fold excess of unlabeled Cp GAIT element or mutant Cp GAIT (U10C) element RNA.
(D) EMSA-supershift analysis of GAIT complex binding to DAPK and ZIPK GAIT elements. Cytosolic extracts from IFN-γ-treated U937 cells were incubated with anti-EPRS, anti-L13a and pre-immune IgG and then added to radiolabeled, GAIT element riboprobes. RNA-protein complexes were resolved by non-denaturing 5% PAGE and visualized by autoradiography.

(A) KN-62 inhibits DAPK activity and L13a phosphorylation. U937 cells were treated with IFN-γ for 8 h and then with 5 µM KN-62 (or DMSO) for an additional 8 h. DAPK was immunoprecipitated from cell lysates, and the immunocomplex was used for in vitro phosphorylation of DAPK-specific substrate peptide (left). Lysates were immunoblotted to detect L13a and phospho-L13a (P-L13a) (right).
(B) Inhibition of L13a phosphorylation blocks its release from ribosomes. U937 cells were treated with IFN-γ and DMSO control (above) or KN-62 (below), and polysome (Polys.) and cytosol fractions isolated by ultracentrifugation on a 20% (w/v) sucrose cushion in the presence of cycloheximide. Fractions were analyzed by immunoblot with anti-L13a.
(C) siRNA-mediated knock-down of ZIPK suppresses translational silencing. U937 cells were transfected with siRNA (or scrambled) against ZIPK, and treated with IFN-γ for up to 24 h. Cell lysates were added to in vitro translation reactions of capped, FLuc-Cp-3’ UTR-poly(A) RNA reporter in RRL (top). Capped, RLuc RNA lacking a 3’UTR was co-translated as a control. Fluc expression was normalized by Rluc, both determined by densitometry, and expressed as % of control (mean ± s.e.m., 3 experiments) (bottom). The significant (*) and non-significant (**) differences between 24-h and control lysates are indicated (p < 0.05, two-tailed t-test).
(D) siRNA-mediated knock-down of DAPK inhibits translational silencing. U937 cells were transfected with siRNA (or scrambled control) against DAPK, and in vitro translation determined as in (C).
(E) DAPK and ZIPK knock-down restores VEGF-A expression. U937 cells were transfected with DAPK, ZIPK, or scrambled siRNAs. Cells were treated with IFN-γ for up to 24 h. Cytosolic lysates were immunoblotted with anti-VEGF-A or GAPDH antibodies (top 2 panels). RT-PCR analysis of total cellular RNA was done using primers specific for VEGF-A or β-actin (bottom 2 panels).

(A) Knockdown of ZIPK prevents translational silencing of DAPK and vice versa. U937 cells were transfected with siRNA directed against ZIPK (or scrambled). After recovery, cells were treated with IFN-γ up to 24 h and lysates immunoblotted with anti-DAPK and anti-GAPDH antibodies (left, top 2 panels). Similarly, cells were transfected with siRNA against DAPK, and lysates were probed with anti-ZIPK and anti-GAPDH antibodies (right, top 2 panel). Significant (*) and non-significant (**) differences between 24-h and control lysates are indicated (p < 0.001, two-tailed t-test). RT-PCR analysis of total cellular RNA was performed with primers targeted against DAPK and ZIPK (bottom).
(B) Time course of L13a phosphorylation and VEGF-A translation after recurrent IFN-γ treatment. U937 cells were treated with IFN-γ initially for up to 24 h (0–24 h). Cells were then washed and resuspended in fresh medium without IFN-γ for another 8 or 16 h (t = 32, 40 h). At that time, cells were washed again and incubated with IFN-γ or medium for an additional 8 or 16 h (t = 48, 56 h). Lysates were immunoblotted with anti-L13a (top), anti-VEGF-A (middle), and anti-GAPDH (bottom) antibodies.
(C) RNA oligomers complementary to DAPK and ZIPK GAIT elements disrupt negative-feedback regulation. U937 cells were transfected with DAPK and ZIPK (or control) morpholino antisense oligomers (10 µM). Cells were treated with IFN-γ for up to 24 h, then washed and resuspended in medium without IFN-γ for another 16 h (40 h). Lysates were immunoblotted with anti-DAPK, anti-ZIPK, anti-L13a and anti-GAPDH antibodies (panels 1–4, respectively).
(D) Schematic illustrating negative-feedback regulation in the GAIT system. IFN-γ causes transcriptional induction of inflammatory genes including Cp, VEGF, DAPK, and ZIPK. The kinases then activate the GAIT complex that ultimately silences translation of GAIT element-bearing genes, including both kinases, thereby inactivating the GAIT system.

(A) Delayed phosphorylation of L13a. Lysates from U937 cells treated with IFN-γ for up to 24 h were subjected to SDS-PAGE (12%) followed by immunoblot with anti-L13a antibody. The positions of phosphorylated (P-L13a) and non-phosphorylated L13a are indicated.
(B) Induction of L13a kinase activity in IFN-γ-treated cells. Lysates were used for in vitro phosphorylation assay using [γ-³²P]ATP and recombinant L13a as substrate. Proteins were resolved by SDS-PAGE and subjected to autoradiography (top). Band intensities from three independent experiments were quantitated by densitometry and expressed as fold-induction compared to lysates from untreated cells (mean ± s.e.m., 3 independent experiments) (bottom).
(C) L13a is phosphorylated at Ser. Lysates from 24-h, IFN-γ-treated cells were immuno-precipitated with anti-phospho-Ser, phospho-Thr (Biodesign, Saco, ME) and phospho-Tyr (Santa Cruz) antibodies, followed by immunoblot analysis with anti-L13a antibody.
(D) Mass spectrometric analysis of phosphorylation site in L13a. Cell lysates from Hi-5 insect cells expressing His-tagged human L13a cDNA cloned into pFASTBAC1 vector, or empty vector, were resolved on SDS-PAGE and stained with Coomassie blue (inset). The phospho-L13a band was excised and the in-gel tryptic digest analyzed by mass spectrometry. The CID spectrum of the phosphopeptide APSRIFWR containing phosphorylated Ser⁷⁷ revealed facile loss of H₃PO₄ to produce the doubly-charged base peak in the spectrum (m/z 508.2). The peptide sequence and site of phosphorylation were established by interpretation of low-abundance fragment ions, including labeled ions at m/z 473.1 and 424.1, doubly-charged ions corresponding to y7-H₃PO₄ and y6-H₃PO₄ ions, respectively.
(E) L13a is phosphorylated on Ser⁷⁷ in vitro. His-tagged, wild-type (WT) and mutant (S77A) L13a were expressed in E. coli, purified by Ni-affinity chromatography, and used as substrate for in vitro phosphorylation with 0- or 16-h IFN-γ-treated cell lysates. Phosphorylation was visualized by SDS-PAGE and autoradiography.
(F) L13a is phosphorylated on Ser⁷⁷ in vivo. Flag-tagged, wild-type and mutant (S77A) L13a were expressed in U937 cells. Expression was determined by immunoblot with anti-Flag antibody (top). Transfected cells were treated with IFN-γ for 20 h followed by a 4-h pulse of ³²P-orthophosphate. Lysates were immunoprecipitated with anti-Flag antibody, and L13a phosphorylation determined by autoradiography (middle). Lysates from transfected cells were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-phospho-Ser antibody (bottom).