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Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase - PubMed

  • ️Sat Jan 01 2011

Methylation of eukaryotic elongation factor 2 induced by basic fibroblast growth factor via mitogen-activated protein kinase

Gyung Ah Jung et al. Exp Mol Med. 2011.

Abstract

Protein arginine methylation is important for a variety of cellular processes including transcriptional regulation, mRNA splicing, DNA repair, nuclear/cytoplasmic shuttling and various signal transduction pathways. However, the role of arginine methylation in protein biosynthesis and the extracellular signals that control arginine methylation are not fully understood. Basic fibroblast growth factor (bFGF) has been identified as a potent stimulator of myofibroblast dedifferentiation into fibroblasts. We demonstrated that symmetric arginine dimethylation of eukaryotic elongation factor 2 (eEF2) is induced by bFGF without the change in the expression level of eEF2 in mouse embryo fibroblast NIH3T3 cells. The eEF2 methylation is preceded by ras-raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK1/2)- p21Cip/WAF1 activation, and suppressed by the mitogenactivated protein kinase (MAPK) inhibitor PD98059 and p21Cip/WAF1 short interfering RNA (siRNA). We determined that protein arginine methyltransferase 7 (PRMT7) is responsible for the methylation, and that PRMT5 acts as a coordinator. Collectively, we demonstrated that eEF2, a key factor involved in protein translational elongation is symmetrically arginine-methylated in a reversible manner, being regulated by bFGF through MAPK signaling pathway.

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Figures

Figure 1
Figure 1

Symmetric dimethylation of arginine on a 95-kDa protein is induced by bFGF in NIH3T3 cells. Protein arginine methylation profiles of NIH3T3 and HCT116 cells produced at (A) 30 min, (B) 8 h, and (C) 24 h after the administration of EGF, bFGF, or HGF with concurrent changes in the levels of p-ERK and p-Akt activation. Symmetric dimethylation of arginine on a 95-kDa protein is induced by bFGF 24 h after administration into NIH3T3 cells. ERK1/2 was activated 30 min after bFGF administration, which decreased gradually thereafter, while the level of Akt activation remained unchanged during the period. Cells were cultured in the absence or presence of EGF, bFGF, or HGF, and cell lysates were prepared at 30 min, 8 h, and 24 h. Western blot analysis was performed with anti-symmetric dimethylarginine antibody SYM10 and anti-p-ERK1/2 or anti-p-Akt antibodies. GAPDH was used as a loading control. Protein bands were visualized using enhanced chemiluminescence. (D) Bar graph showing the change in levels of symmetric arginine dimethylation of a 95-kDa protein after the administration of growth factors. Mean and standard deviations were obtained from three independent experiments. Statistically significant differences as determined by the Wilcoxon test were set at *P < 0.001. (E) The change in the levels of symmetric arginine dimethylation of a 95-kDa protein and eEF2 expression in NIH3T3 cells during the three-day period after the administration of bFGF. Cell lysates were obtained at the indicated time points of 1, 2 and 3 days, and Western blots were conducted using SYM10 and anti-eEF2 antibody as in (A). Arrows indicate the symmetrically arginine-dimethylated 95-kDa protein.

Figure 2
Figure 2

eEF2 contains symmetrically dimethylated arginines induced by bFGF. (A) bFGF induced the level of symmetric arginine dimethylation but neither asymmetric arginine dimethylation nor expression of eEF2. NIH3T3 cell lysates were immunoprecipitated with anti-eEF2 antibody, and immunoprecipitation with normal goat anti-rabbit IgG was performed as a negative control. Western blotting was performed with anti-eEF2 antibody, SYM10 and ASYM24, and protein bands were visualized using enhanced chemiluminescence. GAPDH was used as a loading control. In put, whole cell lysates; IP, immunoprecipitation; WB, western blot. (B) The symmetrically dimethylated arginine-containing 95-kDa protein is eEF2. bFGF induced symmetric arginine dimethylation of eEF2 but did not affect the level of eEF2 expression. NIH3T3 cell lysates were prepared, immunoprecipitated with SYM10, and analyzed using anti-eEF2 antibody and SYM10 Western blots, respectively as in (A).

Figure 3
Figure 3

The ras-raf-MEK-ERK1/2-p21Cip/WAF1 pathway mediates the effect of bFGF on symmetric arginine dimethylation of eEF2. (A) The ras-raf-MEK-ERK1/2-p21Cip/WAF1 pathway was activated in the process of bFGF-induced symmetric dimethylation of arginine on eEF2, while the levels of p-Akt and eEF2 expression were not changed. NIH3T3 cells were treated with bFGF, and cell lysates were prepared after 24 h incubation. Western blot analysis was conducted with anti-pan-ras, p-raf, anti-MEK, anti-p-ERK1/2, anti-p21Cip/WAF1, or anti-p-Akt antibodies for signal transduction analysis; SYM10 and anti-eEF2 antibodies were used for eEF2 symmetric arginine dimethylation or expression analysis. Anti-GAPDH antibody was used as a loading control. (B) The inhibition of ERK1/2 by the MAPK inhibitor PD98059 almost completely blocked symmetric arginine dimethylation of eEF2 induced by bFGF with a concurrent decrease in the level of p21Cip/WAF1. NIH3T3 cells were treated with bFGF in the presence or absence of PD98059, and cell lysates were prepared after 24 h incubation. Western blot analysis was conducted with anti-p-ERK, anti-p21Cip/WAF1 and SYM10 antibodies as in (A). (C) The inhibition of p21Cip/WAF1 by siRNA almost completely blocked the bFGF-induced symmetric arginine dimethylation of eEF2 but did not affect bFGF-induced down-regulation of the fibroblast differentiation marker, α-SMA. NIH3T3 cells were transfected with p21Cip/WAF1 siRNA and treated with bFGF. Cell lysates were prepared after 24 h incubation, and Western blot analysis was conducted with anti-p21Cip/WAF1, SYM10 and anti-α-SMA antibodies as in (A).

Figure 4
Figure 4

PRMT7 is responsible for symmetric dimethylation of arginine on eEF2 induced by bFGF, while PRMT5 appears to play a coordinating role. (A) The inhibition of PRMT7 by siRNA resulted in the complete loss of the bFGF-induced symmetric arginine dimethylation of eEF2. The levels of symmetric dimethylation of arginine on eEF2 and α-SMA expressions were not correlated to each other. NIH3T3 cells were transfected with PRMT7 siRNA for 4 days and treated with bFGF for 24 h. Cell lysates were prepared, and the RNA level of PRMT7 was analyzed using real-time quantitative PCR. Western blot analysis was conducted with SYM10 and anti-α-SMA antibodies. Anti-α-SMA Western blot was used to determine the interrelationship between the levels of symmetric dimethylation of arginine on eEF2 and the expression of the fibroblast differentiation marker, α-SMA. Anti-GAPDH antibody was used as a loading control. (B) PRMT5 inhibition remarkably increases the amount of sDMA on eEF2 and negates the regulatory effect of bFGF on the symmetric arginine dimethylation of eEF2. NIH3T3 cells were transfected with PRMT5 siRNA for 4 days and treated with bFGF for 24 h. Cell lysates were prepared, and Western blot analysis was conducted as in (A). (C) The simultaneous inhibition of PRMT7 and PRMT5 suppressed the bFGF-induced symmetric arginine dimethylation of eEF2. NIH3T3 cells were co-transfected with PRMT7 and PRMT5 siRNAs for 4 days and treated with bFGF for 24 h. Cell lysates were prepared, and Western blot analysis was conducted as in (A).

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