Transfer-RNA-mediated enhancement of ribosomal proteins S6 kinases signaling for cell proliferation

Abstract

While transfer-RNAs (tRNAs) are known to transport amino acids to ribosome, new functions are being unveiled from tRNAs and their fragments beyond protein synthesis. Here we show that phosphorylation of 90-kDa RPS6K (ribosomal proteins S6 kinase) was enhanced by tRNA^Leu overexpression under amino acids starvation condition. The phosphorylation of 90-kDa RPS6K was decreased by siRNA specific to tRNA^Leu and was independent to mTOR (mammalian target of rapamycin) signaling. Among the 90-kDa RPS6K family, RSK1 (ribosomal S6 kinase 1) and MSK2 (mitogen-and stress-activated protein kinase 2) were the major kinases phosphorylated by tRNA^Leu overexpression. Through SILAC (stable isotope labeling by/with amino acids in cell culture) and combined mass spectrometry analysis, we identified EBP1 (ErbB3-binding protein 1) as the tRNA^Leu-binding protein. We suspected that the overexpression of free tRNA^Leu would reinforce ErbB2/ErbB3 signaling pathway by disturbing the interaction between ErbB3 and EBP1, resulting in RSK1/MSK2 phosphorylation, improving cell proliferation and resistance to death. Analysis of samples from patients with breast cancer also indicated an association between tRNA^Leu overexpression and the ErbB2-positive population. Our results suggested a possible link between tRNA^Leu overexpression and RSK1/MSK2 activation and ErbB2/ErbB3 signaling.

Keywords: EBP1; MSK; RSK; cell proliferation; tRNA.

Figure 1.

Overexpression of tRNAs enhanced the cell proliferation. (A) Relative levels (percentage) of cell number, thymidine incorporation, and methionine incorporation after overexpression of each tRNA were compared with those in EV (empty vector)-transfected control. Values are presented as means ± standard deviations (n = 3). *, P < 0.05; ***, P < 0.001. (B) Change in cell cycle after tRNA transfection was compared with that in control. (C) Real-time proliferation of HEK 293T cells transfected with different tRNAs was monitored under different culture conditions. Values are presented as means ± standard errors (n = 9). Values for tRNA^Leu _CAG and negative control (NC) are repeated in all the graphs for the comparison. Cell viability was calculated as the relative percentage of confluency. CM, complete media; SF, serum free; AA, amino acids.

Figure 2.

Overexpression of tRNA^Leu induced the phosphorylation of 90-kDa RPS6K under amino acid starvation. (A and B) Western blot analysis of HEK 293T cells transfected with different tRNAs. Phosphorylation of S6K and 4E-BP, P-S6K and P-4E-BP, respectively, were analyzed without media change (A) and after incubation under amino acid-deprivation for 3 h as well as after addback of amino acids and further incubation for 1.5 h (B). Phosphorylation signals in the 90-kDa (p90) and 70-kDa (p70) sizes detected by p70 S6K specific antibody (P-S6K) are shown. tRNA-like pseudo RNA was also included. −AA, amino acid starvation; +AA, amino acid supplementation. Tubulin, total S6K, or total 4E-BP were used as controls. (C) P-S6K was analyzed via western blot with HEK 293T cell lysates transfected with tRNA for 24 h, and then incubated with leucine-free media for 3 h. (D) Western blot and RT-PCR analyses for the cell lysates transfected with tRNA^Leu isotypes. Two tRNA^Leu _AAG vectors with different flanking sequences, but the same tRNA gene, were included. P-S6K and ERK phosphorylation (P-ERK) were detected after overexpression of each tRNA. Tubulin and GAPDH were used as controls. (E) Western blot analysis for the tRNA^Leu _CAG-overexpressing cell lysates transfected with tRNA^Leu _CAG-specific siRNA (si-Leu). Two different si-Leu sequences were used here. −AA, amino acid starvation; +AA, amino acid supplementation. (F) Gel shift assay to monitor the formation of the complex between tRNA^Leu _CAG and si-Leu. si-Cont, si-Control.

Figure 3.

Phosphorylation of 90-kDa RPS6K was mediated by mature tRNA^Leu, and it was independent of mTOR activation. (A) Northern blot analysis to detect both mature tRNA^Leu and its tRFs with 5′ and 3′ radioactive short probes. Expression levels of tRNAs in HEK 293T cells after tRNA^Leu transfection under different culture conditions were compared with that in the controls. Arrows show the mature tRNA. Upper, short exposure; middle, long exposure; and bottom, EtBr staining of the gel to visualize the loading amounts of total RNA. HeLa cells were used as controls for tRFs. (B and C) Western blot analysis to investigate the effect of LRS knockdown (B) or rapamycin treatment (C) on the tRNA-mediated p90 RPS6K phosphorylation.

Figure 4.

RSK1 and MSK2 were the 90-kDa RPS6K phosphorylated by tRNA^Leu _CAG overexpression. (A) Sequence alignment of 8 different RPS6 family kinases. The hydrophobic motif and representative phosphorylation sites are shown in gray and indicated by arrows, respectively. The 502 aa and 525 aa sizes in S6K1 indicate p70 S6K and p85 S6K, respectively. (B and C) Effect of tRNA overexpression on the ERK phosphorylation (B) and qRT-PCR analysis to detect tRNA overexpression (C). Values are presented as means ± standard deviations (n = 3). (D) The effect of ERK (U0126), p38 MAPK (SB203580), and JNK (SP600125) inhibitors on the tRNA^Leu _CAG-mediated p90 RPS6K phosphorylation. (E-G) Western blot analysis for the phosphorylation of RPS6K after overexpression of tRNA^Leu and the different RPS6 family kinases (E), after siRNA transfection specific to p85 S6K and RSK1 (F) as well as RSK2 and MSK2 (G).

Figure 5.

Interaction between EBP1 (ErbB3-binding protein 1) and tRNA^Leu. (A) Schematic diagram for the process of SILAC and LC-MS/MS analysis. Synthesized tRNA^Leu _CAG was biotinylated by incubation with biotin-Phe-DME and flexizyme. After isolating the tRNA^Leu _CAG-associated protein complexes via biotin-streptavidin interaction, the proteins were analyzed by LC-MS/MS. F, Phenylalanine. (B) qRT-PCR analysis to detect the tRNAs associated with immunoprecipitated EBP1 from the HEK 293T cells incubated in amino acid-free media. Values are presented as means ± standard deviations (n = 3). (C) Gel shift assay to detect the formation of the complex between EBP1 and radioactively synthesized tRNA^Leu _CAG. (D) Western blot analysis for the ErbB3 signal pathway of MCF10A cell lines transfected with tRNA^Leu. −AA, amino acid starvation; +AA, amino acid supplementation.

Figure 6.

Characteristics of stable tRNA^Leu _CAG-overexpressing cells. (A) Real-time monitoring of the proliferation of stable NIH3T3 MEF cells expressing tRNA^Leu _CAG. Values are presented as means ± standard deviations (n = 9). (B and C) Western blot analysis of the stable NIH3T3 MEF cells under different culture conditions. Activation of RSK and MSK signaling was compared under SF (serum free) and −AA (amino acid starvation) (B) or under CM (complete media) and −AA (amino acid starvation) conditions (C). (D) Levels of the different tRNA^Leu isotypes in several cancer cell lines were analyzed using qRT-PCR and then compared with those in normal cell lines (CCD18C0, NHA, M13SV1, and WI-26). Values are presented as means ± standard deviations (n = 3). (E) Comparison of RSK and MSK signaling between MCF7 and BT20 cell lines under amino acid-deprived conditions (−AA).

Figure 7.

Schematic representation for the role of free tRNA^Leu. Under amino acid starvation condition, free tRNA^Leu interacts with EBP1 and enhances the activation of ErbB2/ErbB2 pathway. Finally, the downstream signaling mediated by RSK1/MSK2 facilitates cancer cell proliferation.