Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A

Abstract

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bbeta, as TCTP-interacting partners. In vitro and in vivo studies confirmed that TCTP bound specifically eEF1Bbeta and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bbeta. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.

Fig. 1.

Association of TCTP with either eEF1Bβ or eEF1A. (A) TCTP interacts with the C terminus of eEF1Bβ and eEF1A in yeast. Diploids carrying the constructs HM12, Bcl-x, eEF1A, eEF1Bβ, or truncated form of eEF1Bβ (Bβ-Nter residues 1–152 or Bβ-Cter residues 153–281) fused to B42 domain (activation domain) and LexA-TCTP were streaked onto either glucose (Glu) or galactose (Gal) plates and assayed for growth. (B and C) In vitro interaction of TCTP with either eEF1Bβ or eEF1A. (B) The indicated GST-fusion proteins immobilized on glutathione beads were incubated with AIP1, TCTP, or eEF1Bβ IVT. Radiolabeled proteins bound to the GST proteins were visualized by autoradiography. Inputs for each experiment are indicated. The negative controls, AIP1 and NKTR, are 120- and 150-kDa proteins, respectively. (C) GST-NKTR or GST-TCTP were incubated with eEF1A purified from rabbit liver. Eluted eEF1A was detected with the anti-eEF1A antibody. Arrow, full-length eEF1A. *, a degraded product of eEF1A.

Fig. 4.

TCTP preferentially stabilizes the GDP form of eEF1A. The time course of GDP exchange was assayed at 0°C in the presence of 350 nM eEF1A-[³H]GDP without TCTP (▪) or with increasing amounts of TCTP (final concentrations of 200, 300, 500, and 750 nM, and 1, 2, and 3 μM, which are indicated by red and blue symbols in A and B, respectively). The reaction was monitored in the presence of 150 μM unlabeled GDP (A)or150 μM unlabeled GTP (B). In the absence of unlabeled nucleotide, the eEF1A–[³H]GDP complex remained stable (•). All reactions were performed in triplicate. (C) The inhibition of GDP exchange on eEF1A observed in A and B in the presence of GDP or GTP is plotted as a function of TCTP added.

Fig. 2.

Endogenous interaction of TCTP with either eEF1Bβ or eEF1A. TCTP was immunoprecipitated from either 293T or HeLa cell extracts with a rabbit anti-TCTP or an isotype-matched control antibody (IgG). Immunoprecipitates (IP) were analyzed by Western blot using anti-eEF1Bβ (A) or anti-eEF1A (C) antibodies. (B) Anti-eEF1Bβ antibodies or preimmune serum (IgG) were used to immunoprecipitate eEF1Bβ from 293T or HeLa cell extracts. Western blot analysis on the immune complexes using either anti-TCTP (B) or anti-eEF1A (D) antibodies revealed a specific association. Total cell lysates (TL) are indicated. Arrows highlight IP proteins. *, a degraded product of eEF1A; Hc and Lc, heavy and light chain of IgG, respectively.

Fig. 3.

Endogenous colocalization of TCTP with either eEF1Bβ or eEF1A in HeLa cells. (A) Immunofluorescence analysis of anti-TCTP (green) and anti-eEF1Bβ (red) staining. Merge shows a partial colocalization of TCTP and eEF1Bβ (yellow). A higher magnification of cell is shown in Lower. (B) Immunofluorescence analysis of anti-TCTP (green) and anti-eEF1A (red) staining. Merge indicates a partial colocalization of TCTP and eEF1A. A higher magnification of cell is shown in Lower. Note the enhanced colocalization of TCTP with either eEF1Bβ or eEF1A within the perinuclear region of the cell. Each confocal image represents a similar plane through the cell.

Fig. 5.

TCTP inhibits the eEF1Bβ-mediated exchange reaction. Kinetics of GDP exchange promoted by 10 nM of the eEF1Bα–eEF1Bγ complex (EF1Bαγ) (A) or by 50 nM of eEF1Bβ (EF1Bβ) (B) were conducted in the presence (+TCTP) or in the absence (–TCTP) of 2 μM TCTP. In the absence of unlabeled nucleotide, the eEF1A–[³H]GDP complex remained stable (•). The time course of GDP exchange was initiated by addition of 150 μM unlabeled GDP.