Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1147 Is Specific for eIF4G1 Bound to eIF4E in Delayed Neuronal Death after Ischemia

Abstract

Ischemic strokes are caused by a reduction in cerebral blood flow and both the ischemic period and subsequent reperfusion induce brain injury, with different tissue damage depending on the severity of the ischemic insult, its duration, and the particular areas of the brain affected. In those areas vulnerable to cerebral ischemia, the inhibition of protein translation is an essential process of the cellular response leading to delayed neuronal death. In particular, translation initiation is rate-limiting for protein synthesis and the eukaryotic initiation factor (eIF) 4F complex is indispensable for cap-dependent protein translation. In the eIF4F complex, eIF4G is a scaffolding protein that provides docking sites for the assembly of eIF4A and eIF4E, binding to the cap structure of the mRNA and stabilizing all proteins of the complex. The eIF4F complex constituents, eIF4A, eIF4E, and eIF4G, participate in translation regulation by their phosphorylation at specific sites under cellular stress conditions, modulating the activity of the cap-binding complex and protein translation. This work investigates the phosphorylation of eIF4G1 involved in the eIF4E/eIF4G1 association complex, and their regulation in ischemia-reperfusion (IR) as a stress-inducing condition. IR was induced in an animal model of transient cerebral ischemia and the results were studied in the resistant cortical region and in the vulnerable hippocampal CA1 region. The presented data demonstrate the phosphorylation of eIF4G1 at Ser¹¹⁴⁷, Ser¹¹⁸⁵, and Ser¹²³¹ in both brain regions and in control and ischemic conditions, being the phosphorylation of eIF4G1 at Ser¹¹⁴⁷ the only one found in the eIF4E/eIF4G association complex from the cap-containing matrix (m⁷GTP-Sepharose). In addition, our work reveals the specific modulation of the phosphorylation of eIF4G1 at Ser¹¹⁴⁷ in the vulnerable region, with increased levels and colocalization with eIF4E in response to IR. These findings contribute to elucidate the molecular mechanism of protein translation regulation that underlies in the balance of cell survival/death during pathophysiological stress, such as cerebral ischemia.

Keywords: cerebral ischemia; confocal microscopy; eIF4E; eIF4G1; neuronal death; protein phosphorylation; protein synthesis regulation; vulnerable regions.

Figure 1

Phosphorylation sites in eIF4G1 sequence. (A) Alignment of human and rat eIF4G1 sequences in the region of the phosphorylation sites studied in this work. Amino acid positions with homology between human and rat are shaded in grey and serine phospho-sites studied are marked in green. (B) eIF4G1 protein domains. Serine phospho-sites studied are marked in yellow in the interdomain linker (IDL) region. Labels of initiation factors (eIFs) and other proteins indicate the binding region to eIF4G1.

Figure 2

Identification of eIF4G1 phosphorylation sites induced by ischemia-reperfusion (IR) stress. (A) Samples of the cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were analyzed by Western blotting with anti-eIF4G1 N-20 (eIF4G1_(N-20)), anti-eIF4G1 H-2 (eIF4G1_(H-2)), anti-phospho-eIF4G1 Ser¹¹⁴⁷ (p-Ser1147), anti-phospho-eIF4G1 Ser¹¹⁸⁵ (p-Ser1185), anti-phospho-eIF4G1 Ser¹²³¹ (p-Ser1231), and anti-β-tubulin (β-tubulin) antibodies. The α and β forms of eIF4G1 were indicated; numbers on the right indicate the apparent molecular mass in kDa from protein markers. The figures are representative results of 4–6 independent experiments from 4–6 animals. Full original images of the Western blots are shown in the Supplementary Material (Figure S2) (B) Quantification of the eIF4G1 levels in Western blots using eIF4G1_N-20 (upper) or eIF4G1_H-2 (lower) antibodies. (C) Quantification of eIF4G1 phosphorylated (phospho-eIF4G1) at Ser¹¹⁴⁷, Ser¹¹⁸⁵ and Ser¹²³¹ residues with respect to total eIF4G1 levels (ratios) detected with anti–eIF4G1_(N-20) antibody. Bar graphs represent the mean of 4–6 independent experiments from 4–6 animals; error bars indicate SE. No statistical significance was found.

Figure 3

eIF4G1 phosphorylated at Ser¹¹⁴⁷ is bound to eIF4E. (A) Samples of cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were bound to a cap-containing matrix (m⁷GTP-Sepharose) and eIF4E and eIF4E-associated proteins were analyzed by SDS-PAGE followed by Western blotting for anti-phospho-eIF4G1 Ser¹¹⁴⁷ (p-Ser1147), anti-phospho-eIF4G1 Ser¹¹⁸⁵ (p-Ser1185), anti-phospho-eIF4G1 Ser¹²³¹ (p-Ser1231), anti-eIF4G1_(N-20) (eIF4G1), and anti-eIF4E (eIF4E) antibodies. Among phospho-eIF4G1 forms, only eIF4G1 phosphorylated at Ser¹¹⁴⁷ was detected. Molecular mass (kDa) of protein markers is stated in the right. The figures are representative results of 4–6 independent experiments from 4–6 animals. Full original images of the Western blots are shown in the Supplementary Material (Figure S3) (B) Quantification of eIF4G1 bound to eIF4E, and (C), quantification of eIF4G1 phosphorylated at Ser¹¹⁴⁷ bound to eIF4E with respect to eIF4E levels (ratios). (D) Relative levels of eIF4G1 phosphorylated at Ser¹¹⁴⁷ bound to eIF4E with respect to eIFG1 levels. In all experiments, eIF4E was detected with anti-eIF4E antibody, and no significant differences between eIF4E levels were found. Bar graphs represent the mean of 4–6 independent experiments from 4–6 animals; error bars indicate SE. Statistical significance were performed by Newman–Keuls post-test (# p < 0.05; ## p < 0.01) or by Student’s t-test (* p < 0.05), after significant ANOVA (p < 0.05), compared with their respective control, or between the cerebral cortex and CA1 samples (indicated by lines).

Figure 4

Association of eIF4E to eIF4G1 phosphorylated at Ser¹¹⁴⁷ in eIF4G1 immunoprecipitates. (A) Samples of cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were immunoprecipitated with anti–eIF4G1_(N-20) antibody and eIF4G1-associated proteins analyzed by SDS-PAGE followed by Western blotting for anti-phospho-eIF4G1 Ser¹¹⁴⁷ (p-Ser1147), anti-phospho-eIF4G1 Ser¹¹⁸⁵ (p-Ser1185), anti-phospho-eIF4G1 Ser¹²³¹ (p-Ser1231), anti-eIF4G1_(H-2) (eIF4G1) and anti-eIF4E (eIF4E) antibodies. Among eIF4G1 phospho-forms, only eIF4G1 phosphorylated at Ser¹¹⁴⁷ was detected. Numbers on the right indicate the apparent molecular mass in kDa, from protein markers. The figures are representative results of 4–6 independent experiments from 4–6 animals. Full original images of the Western blots are shown in the Supplementary Material (Figure S4). (B) Quantification of eIF4E levels, and (C) quantification of eIF4G1 phosphorylated at Ser¹¹⁴⁷, with respect to immunoprecipitated eIF4G1 levels (ratios). (D) Relative levels of eIF4G1 phosphorylated at Ser¹¹⁴⁷ with respect to eIF4E levels in eIF4G1 immunoprecipitates. Bar graphs represent the mean of 4–6 independent experiments from 4–6 animals; error bars indicate SE. Statistical significance were performed by Newman–Keuls post-test (## p < 0.01; ### p < 0.001) or by Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001), after significant ANOVA (p < 0.05), compared with their respective control, or between the cerebral cortex and CA1 samples (indicated by lines). IP, immunoprecipitated fraction.

Figure 5

Colocalization of eIF4G1 phosphorylated at Ser¹¹⁴⁷ and eIF4E in the cerebral cortex and hippocampal CA1 regions induced by ischemia-reperfusion stress. (A) Brain sections of cerebral cortex (C) or hippocampal CA1 region from control (SHC3d) and ischemic animals with reperfusion (R3d), were used for eIF4E and eIF4G1 phosphorylated at Ser¹¹⁴⁷ colocalization study by confocal fluorescence microscopy. eIF4E was visualized using Alexa Fluor 568 secondary antibody (red) while phospho-eIF4G1 at Ser¹¹⁴⁷ was visualized with Alexa Fluor 488 secondary antibody (green). Cell nuclei were stained with Hoechst 33342 dye (blue). Green and red channels were merged and colocalized components are shown in yellow (eIF4E +phospho-eIF4G1, central images). Merged images display the eIF4E (red), phospho-eIF4G1 (green) and Hoechst (blue) signal. Images are representative results from four to six different animals and are full original scanned images. Scale bar, 50 μm. (B) Quantification of eIF4G1 phosphorylated at Ser¹¹⁴⁷ and eIF4E colocalization. The degree of colocalization is expressed by the percentage of green objects colocalizing with red objects in the scanned area. Data are from four to six different animals; error bars indicate SE. ## p < 0.01 by Newman–Keuls post-test after significant ANOVA (p < 0.05), compared with their control and with the cerebral cortex.