TPL2 kinase expression is regulated by the p38γ/p38δ-dependent association of aconitase-1 with TPL2 mRNA

Abstract

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ^-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ^-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.

Keywords: 3′UTR; ACO1; TPL2; mRNA translation; p38γ/p38δ-MAPK.

Fig. 1.

p38γ and p38δ regulate TPL2 protein levels. (A) Lysates of the indicated tissues from WT or p38γ/δ^−/− mice were immunoblotted with anti-TPL2, -p38α, -p38γ, or -p38δ antibodies. p38α expression was used as the loading control. The red asterisks indicate nonspecific protein bands. (B) MEFs and peritoneal macrophage lysates from WT or p38γ/δ^−/− mice were immunoblotted with anti-TPL2 or -p38α. (C) qPCR of TPL2 mRNA from indicated WT or p38γ/δ^−/− tissues. Results were normalized to GAPDH mRNA expression. Data show mean ± SEM (n = 3) from one representative experiment of two with similar results. (D) qPCR of TPL2 mRNA from indicated WT or p38γ/δ^−/− cells. Results were normalized to GAPDH mRNA expression. (E) qPCR of TPL2 mRNA in cytosolic or nuclear RNA from WT or p38γ/δ^−/− MEFs. Results were normalized to GAPDH mRNA expression. Data show mean ± SEM from one representative experiment of two with similar results. ns, not significant; ***P ≤ 0.001 relative to WT.

Fig. 2.

p38γ and p38δ regulate TPL2 protein levels by modulating its stability. (A) HEK293 cells were transfected with plasmids encoding the indicated proteins. After transfection, cells were lysed, pull downs of GST proteins were performed, and pellets were immunoblotted with the indicated antibodies. Total lysates were immunoblotted with the indicated antibodies to examine protein expression. (A, Lower) p38γ and p38δ band intensities in pellets were quantified using the Fiji program. Histogram values are means ± SEM of two independent experiments in duplicate. (B) Metabolic labeling and pulse-chase analysis. WT or p38γ/δ^−/− MEFs transfected with plasmids encoding GST-TPL2 or GST alone as a control were pulse labeled with ³⁵S-Met/Cys and lysed. (B, Left) Pull downs of GST proteins were performed after the times of chase indicated. TPL2 was separated by SDS-PAGE and revealed by autoradiography. (B, Right) TPL2 bands were quantified using the Fiji program, and data show mean ± SEM from two experiments in duplicate. **P ≤ 0.01 relative to WT. (C) CHX-chase analysis. WT or p38γ/δ^−/− MEFs were transfected with a plasmid encoding GST-TPL2 and incubated with 100 μg/mL CHX for the indicated times before lysis. Following cell lysis and SDS-PAGE, immunoblotting was carried out with antitotal TPL2 (SI Appendix, Fig. S2). TPL2 bands were quantified using the Odyssey infrared imaging system, and data show mean ± SEM from two experiments in duplicate. ***P ≤ 0.001 relative to WT. (D) p38γ/δ^−/− MEFs were transfected with plasmids encoding GST-TPL2 alone or with Flag-ABIN2, hemagglutinin (HA)-p38γ, myc-p38δ, or HA-p38γ plus myc-p38δ and treated with CHX as in C. Following cell lysis, SDS-PAGE, and immunoblotting with antitotal TPL2 (SI Appendix, Fig. S2), TPL2 bands were quantified. Data show mean ± SEM from two experiments in duplicate. *P ≤ 0.05 relative to p38γ/δ^−/− MEFs transfected with GST-TPL2 alone; **P ≤ 0.01 relative to p38γ/δ^−/− MEFs transfected with GST-TPL2 alone.

Fig. 3.

p38γ and p38δ regulate TPL2 translation. (A) Polysome profiling (10 to 50% sucrose gradient) of total WT (dark blue) and p38γ/δ^−/− (red) MEF lysates. The figure shows one representative profile (n = 3). (B) qPCR of TPL2, ABIN2, and β-actin mRNA from polysome profile fractions. Results were normalized to GAPDH mRNA expression. Data show mean ± SEM from one representative experiment in triplicate. *P ≤ 0.05 relative to WT MEFs. (C, Upper and D, Upper) Schematic representations of mouse TPL2 mRNA (C) and ABIN2 mRNA (D). TPL2 is expressed as two isoforms due to the alternative translational initiation at methionine 1 (M1) or methionine 30 (M30) (27). Representations show 5′UTR, coding regions (M1-Tpl2, M30-Tpl2, and ABIN2), and 3′UTR. Cloning sites for psiCHECK2 plasmids are shown on the 3′UTRs, with forward (Fw) and reverse (Rv) primers indicated. Some binding sites for the RBPs (ACO1, NonO, PABPC1, and ZFP36) at the TPL2 3′UTR are indicated (C, Upper). (C, Lower) Schematic representation of the psiCHECK2-Renilla-TPL2 3′UTR (TPL2 3′UTR) and psiCHECK2-Renilla-ABIN2 3′UTR (ABIN2 3′UTR). (E) WT and p38γ/δ^−/− MEFs were transfected with luciferase plasmids psiCHEK2 as empty vector (EV) or containing TPL2 3′UTR or ABIN2 3′UTR. Renilla values were normalized against Firefly levels, and repression fold was calculated for the TPL2 3′UTR or the ABIN2 3′UTR reporter relative to EV for each condition. Data are mean ± SEM from one representative experiment in triplicate. ns, not significant. ***P ≤ 0.001 relative to WT.

Fig. 4.

Analysis of the proteins bound to the 3′UTR of TPL2 mRNA. (A) Schematic representation of the streptavidin aptamer (1sm)-tagged RNA–protein capture methodology and identification of TPL2 3′UTR-bound proteins. LC-MS/MS, liquid chromatography–tandem mass spectrometry. (B) SDS-PAGE and silver staining of proteins bound to 3′UTR of TPL2 mRNA from WT and p38γ/δ^−/− macrophage extracts. Lanes 1 and 2 are biological replicas. (C) Venn diagrams (https://bioinfogp.cnb.csic.es/tools/venny/) showing the number of common and specific proteins bound to 3′UTR of TPL2 mRNA from WT and p38γ/δ^−/− cell extracts. (D and E) Enrichment analysis of KEGG pathways (D) and GO biological processes (E) of the proteins bound to 3′UTR of TPL2 mRNA from WT and p38γ/δ^−/− cell extracts. (F) Venn diagrams showing the number of common and specific proteins bound to 3′UTR of TPL2 mRNA from WT and p38γ/δ^−/− cells and RBPs containing binding sites in the TPL2 mRNA. Th. TPL2 mRNA-RBP, proteins containing predicted binding sites in TPL2 mRNA 3′UTR. (G) Endogenous ACO1 was immunoprecipitated from WT and p38γ/δ^−/− MEFs lysates under Ribonuclease (RNase)-free conditions. RNA was isolated from pellets and retrotranscribed to complementary DNA (cDNA) for qPCR analysis. TPL2 3′UTR RNA levels were quantified and normalized to 18S RNA levels and to each input control. Data show mean ± SEM from two experiments in duplicate. *P ≤ 0.05 relative to p38γ/δ^−/− MEFs. (H) p38γ/δ^−/− MEFs were transfected with empty HA vector (p38γ/δ^−/−) or with plasmids encoding HA-p38γ (p38γ/δ^−/− + p38γ), HA-p38δ (p38γ/δ^−/− + p38δ), or HA-p38γ plus HA-p38δ (p38γ/δ^−/− + p38γ/p38δ), and endogenous ACO1 was immunoprecipitated as described in G. TPL2 3′UTR RNA levels were quantified and normalized to 18S RNA levels and to each input control. Data show mean ± SEM from two experiments in duplicate. ns, not significant. *P ≤ 0.05 relative to p38γ/δ^−/− MEFs.

Fig. 5.

Alternative p38MAPKs interact with ACO1, and p38δ expression increases TPL2 protein levels. (A) WT and p38γ/δ^−/− MEFs cells were transfected with plasmid encoding FLAG-ACO1 or with empty vector (EV). After transfection, cell lysates were immunoblotted with anti-TPL2 or -p38α antibodies (A, Lower). The intensity of TPL2 bands was quantified using Fiji program. Histogram values are means ± SEM of two independent experiments in duplicate. (B) WT and p38γ/δ^−/− MEFs were transfected with plasmids encoding either FLAG-ACO1 or with EV (Flag-EV), and luciferase plasmid psiCHEK2-TPL2 3′UTR or luciferase plasmid psiCHEK2-EV was used as a control. Renilla luciferase luminescence values were measured and normalized against Firefly luciferase levels. Repression fold was calculated as described in Fig. 3. Data are mean ± SEM from one representative experiment in triplicate. *P ≤ 0.05 relative to WT; **P ≤ 0.01 relative to WT. (C) WT and p38γ/δ^−/− MEFs were stimulated with 0.5 M sorbitol for 20 min and lysed. Lysates were run in a Phos-tag gel and immunoblotted with the indicated antibodies. White and red circles indicate unphosphorylated and phosphorylated proteins, respectively. (D) WT MEFs were transfected with luciferase plasmids psiCHEK2 as EV or containing TPL2 3′UTR. Cells were incubated with the indicated p38MAPK inhibitor (or Dimethyl sulfoxide (DMSO) for control) for 6 h before lysis. Repression fold was determined as in B. Data are mean ± SEM from one representative experiment in triplicate. ns, not significant; ***P ≤ 0.001(E) HEK293 cells were transfected with plasmids encoding the indicated proteins. After transfection, cells were lysed, and immunoprecipitation of ACO1 with anti-Flag antibody was performed. Pellets were immunoblotted with the indicated antibodies (panels immunoprecipitation (IP): ACO1). Total lysates were immunoblotted with the indicated antibodies to examine protein expression (panels Lysate). (E, Lower) p38γ, p38δ, and ACO1 band intensities in pellets were quantified using the Fiji program. p38γ and p38δ intensity bands were normalized to immunoprecipitated ACO1. Histogram values are means ± SEM of two independent experiments in duplicate. (F) WT MEFs or p38γ/δ^−/− MEFs stably expressing p38γ, p38δ, or EV as control were lysed, and 50 µg of total lysate protein was immunoblotted with the indicated antibodies. Representative blots are shown. In F, Lower, the intensity of TPL2 bands was quantified using the Fiji program. Histogram values are means ± SEM of three independent experiments. (G) MEFs were transfected with luciferase plasmids psiCHEK2-EV or with psiCHEK2-TPL2 3′UTR or psiCHEK2-ABIN2 3′UTR, and with plasmids encoding HA-p38γ, or HA-p38δ or GFP-p38γ or HA-p38δKD as indicated. Renilla luciferase values were normalized against Firefly luciferase levels, and repression fold was calculated for the TPL2 3′UTR or the ABIN2 3′UTR reporter relative to EV for each condition. Data are mean ± SEM from one representative experiment in triplicate. ns, not significant; ***P ≤ 0.001. (H) Proposed model for the regulation of TPL2 protein levels by p38γ and p38δ. In WT cells, p38γ and p38δ associate with ACO1 (Left panel (a)) and also, with the TPL2/ABIN2/p105 complex (Left panel (b)). In WT cells, the p38γ/p38δ/ACO1 complex prevents ACO1 from binding to TPL2 3′UTR, and TPL2 mRNA is translated (Left panel (a)). In p38γ/δ^−/− cells, free ACO1 binds to TPL2 3′UTR and impairs TPL2 mRNA translation by a yet unknown mechanism (Right panel (a)). In WT cells, the p38γ/p38δ/TPL2/ABIN2/p105 complex stabilizes TPL2 protein (Left panel (b)), whereas p38γ/p38δ absence decreases TPL2 stability and increases its degradation (Right panel (b)). ns, not significant; *P ≤ 0.05.