Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage

Abstract

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤30min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment.

Fig. 1

Western blot of p38, ERK and JNK in SILAC-labeled RAW 264.7 with total and phospho-specific antibodies. with GAPDH as internal control. The activation of MAPKs was comparable to that in unlabeled RAW 264.7 ensuring the SILAC-labeled behave similarly to their unlabeled counterparts. Results shown are representative of 3 replicate experiments.

Fig. 2

Experimental design for SILAC-based quantitative phosphoproteomic analysis of DON-induced RSR. RAW 264.7 cultured in media supplemented with normal lysine and arginine (Lys0, Arg0), medium-labeled lysine and arginine (Lys4, Arg6), and heavy-labeled lysine and arginine (Lys8, Arg10) were subjected different DON treatments in two sets of experiments. Nuclear and cytoplasmic proteins were extracted, mixed equally among different groups based on protein amount and trypsin digested. Phosphopeptides were enriched by titanium dioxide chromatography and analyzed by high-resolution mass spectrometry. Each set was repeated in three independent experiments (n=3).

Fig. 3

DON-induced RSR involves extensive protein phosphorylation changes. The numbers of significantly regulated (grey) phosphorylated proteins (A) and peptides (B) as determined by Significance B corrected for FDR≤0.05 among all unique proteins and peptides identified (grey + black).

Fig. 4

Confirmation of phosphoproteome quantification. Phosphorylation levels were measured using Western blot by normalizing the signaling intensity of the bands from phosphosite-specific antibody with those from total antibody. Normalized intensities (blue, shown as mean± SEM) were then correlated with the SILAC ratios (red, shown as median).

Fig. 5

Gene Ontology associated with significantly regulated phosphoproteins as determined by DAVID Biological Process terms (DAVID EASE score < 0.05). Data are presented as a histogram of the relevant biological processes identified and shown as a percentage of the total identified proteins that fall within each category.

Fig. 6

Phosphorylation changes of transcription factors (A), cofactors (B), and epigenetic regulators (C) during DON-induced RSR. The heat map depicts the log 2 transformed relative abundance (red to green) of the protein phosphorylation.

Fig. 7

Phosphorylation changes of proteins involved in the MAPK (A), NFκB (B), AKT and AMPK pathways (C) during DON-induced RSR. The heat map depicts the log 2 transformed relative abundance (red to green) of the protein phosphorylation.

Fig. 8

Signaling pathways mediating DON-induced RSR, including the MAPK (A, blue), NFκB (B, red), AKT and AMPK pathways (C, green). Dark-filled boxes indicate novel mediators based on this study, light-filled boxes indicate known mediators identified here, open boxes with solid outline indicate known mediators not identified here, and open boxes with dashed outline indicate known mediators in canonical pathway and not identified here.

Fig. 9

DON-regulated phosphosites can be categorized into several temporal profiles. Based on the phosphorylation dynamics, Fuzzy c-means clustering analysis generated clusters representing early, intermediate and late responders with phosphorylation levels up- or down-regulated were selected from 40 stable clusters based on the average probability (A–F). Each trace depicts the natural log value of relative phosphorylation level as a function of time, and is color coded according to its membership value (i.e. the probabilities that a profile belongs to different clusters) for the respective cluster. Each cluster is designated by the function of prominent members. Examples of such members are given for each cluster.

Fig. 10

Summary of pathways and biological processes involved in DON-induced RSR. In response the ribosome damage caused by ribotoxic stressors, signaling pathways including MAPK, NFκB, AKT and AMPK pathways are activated which mediate key biological processes in the cytoplasm and the nucleus to adapt and respond to RSR. Proteins in bold indicate novel mediators identified in the present study, proteins in bold and italics indicate previously known mediators confirmed here, and proteins in italics indicate previously known mediators but not identified here.