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. 2016 Dec;68(6):2659-2666.
doi: 10.1007/s10616-016-9990-1. Epub 2016 Aug 10.

Interleukin-1β effect on the endogenous ADP-ribosylation and phosphorylation of eukaryotic elongation factor 2

Ebru Hacıosmanoğlu  1   2 , Başak Varol  3 , Bilge Özerman Edis  1 , Muhammet Bektaş  1
Affiliations

Interleukin-1β effect on the endogenous ADP-ribosylation and phosphorylation of eukaryotic elongation factor 2

Ebru Hacıosmanoğlu et al. Cytotechnology. 2016 Dec.

Abstract

Eukaryotic elongation factor 2 (eEF2) plays an important role in eukaryotic polypeptide chain elongation. Adenosine diphosphate (ADP)-ribosylation is a post-translational modification reaction that catalyzes the transfer of ADP-ribose group to eEF2 and this causes the inhibition of protein synthesis. Indeed, in the absence of diptheria toxin, endogenous ADP-ribosylation can occur. eEF2 is phosphorylated by eEF2 kinase which prevents binding to ribosomes thus inhibiting its activity. Increase in endogenous ADP-ribosylation level approximately 70-75 % was observed in IL-1β treated HUVECs. Moreover, a 70 % rise of phosphorylation of eEF2 was measured. Alteration of endogenous ADP-ribosylation of eEF2 activity was related with cellular mono-ADP-ribosyltransferases (ADPrT). Increment of endogenous ADP-ribosylation on eEF2 did not seem to occur as a direct effect of IL-1β; it arises from the activation of ADPrT. This 2.5 fold increase was abolished by ADPrT inhibitors. Due to these post-translational modifications, global protein synthesis is inhibited. After dephosphorylation of phospho-eEF2, around 20 % increase in protein synthesis was observed. In conclusion, systemic IL-1β has an important role in the regulation of global protein synthesis.

Keywords: Diphtheria toxin; Endogenous ADP-ribosylation; Eukaryotic elongation factor 2; Interleukin-1β; Phosphorylation; Protein synthesis.

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Figures

Fig. 1
Fig. 1
Effect of IL-1β on ADP-ribosylation of eEF2. Cell lysates (S100) were prepared with IL-1β treatment (12 h incubation). 1 mg S100 protein was ADP-ribosylated in the presence of 5 µM [14C]NAD and 100 µg/ml DTx. a ADP-ribosylation in the presence of IL-1β after scintillation. Control is IL-1β free. b Endogenous ADP-ribosylation of eEF2 in cell lysate (1 mg S100 proteins). (Square) control (IL-1β free), (diamond) 0.1 U/ml, (triangle) 1 U/ml, (open circle) 10 U/ml, (filled circle) 100 U/ml IL-1β. Percentages are compared with ADP-ribosylation of eEF2 with DTx in 1 mg S100 proteins. c Western blot image of total eEF2 (2 mg S100 proteins). d Autoradiogram of ADP-ribosylation in the presence of IL-1β (2 mg ADP-ribosylated S100 protein). Graphs on the right side show the analysis of western blot and of the autoradiogram, respectively, using ImageJ software. The resulting values are a measure of the relative density of each sample, compared to the IL-1β free (0 U/ml)
Fig. 2
Fig. 2
Direct effect of IL-1β on ADP-ribosylation of eEF2. Autoradiograph of ADP-ribosylation in the presence of 5 µM [14C]NAD, 100 µg/ml DTx and 20 pmol (2 µg) eEF2 isolated from rat liver incubated with IL-1β (12 h). The graph on the right side shows the analysis of western blot using ImageJ software. The resulting values are a measure of the relative density of each sample, compared to the IL-1β free (0 U/ml)
Fig. 3
Fig. 3
Effect of IL-1β on phosphorylation of eEF2. a Western blot analysis of cell lysates (10 mg S100 proteins) in the presence of increasing concentrations of IL-1β (12 h incubation) using anti-phospho T56 eEF2 antibody. The graph on the right side shows the analysis of western blot using ImageJ software. The resulting values are a measure of the relative density of each sample, compared to the 100 U/ml IL-1β. b Phosphorylated-eEF2 (10 mg S100 proteins) in the presence of 100 (U/ml) IL-1β with using PP2A (5 mg/ml) for dephosphorylation. The graph on the right side shows the analysis of western blot using ImageJ software. The resulting values are a measure of the relative density of each sample, compared to the 100 U/ml IL-1β (Control)
Fig. 4
Fig. 4
Effect of IL-1β on in vitro protein synthesis. PolyPhe synthesis was performed in the presence of IL-1β (0.1–100 U/ml) for 12 hours and S100 protein (2 mg) as source for eEF2 (black bars). Control is IL-1β free. Light bars show polyPhe synthesis in presence of dephosphorylated eEF2. All data are representative of three independent experiments and statistical significance was measured using Student t test. aStatistically different from 0 U/ml IL-1β treatment group (p < 0.05). bStatistically different from 0 U/ml IL-1β and 5 µg/ml PP2A treatment group (p < 0.05). cStatistically different from 0.1 U/ml IL-1β treatment group (p < 0.05). dStatistically different from 1 U/ml IL-1β treatment group (p < 0.05). eStatistically different from 10 U/ml IL-1β treatment group (p < 0.05)
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