Kyunglim Lee

Identification of the calcium binding sites in translationally controlled tumor protein

Translationally controlled tumor protein (TCTP), also known as lgE-dependent histamine-releasing factor, is a growth-related tumor protein. Although the primary sequence of rat TCTP does not reveal any recognizable Ca2+-binding motif, previous studies have demonstrated that rat TCTP consisting of 172 amino acids is a Ca2+-binding protein. However, the region of TCTP required for Ca2+ interaction has not been mapped to the molecule. Here, we reported that the Ca2+ binding region of TCTP, which was mapped by using a combination of deletion constructs of rat TCTP and45Ca2+-overlay assay, was confined to amino acid residues 81–112. This binding domain did not show any peculiar loop of calcium-binding motif such as CaLB domain and EF hand motif and it seems to be constituted of random coil regions neighboring the a helix. Thus, our data confirm that TCTP is a novel family of Ca2+-binding protein.

Translationally Controlled Tumor Protein Interacts with the Third Cytoplasmic Domain of Na,K-ATPase α Subunit and Inhibits the Pump Activity in HeLa Cells

Translationally controlled tumor protein (TCTP) is a growth-related protein under transcriptional as well as translational control. We screened a rat skeletal muscle cDNA library using yeast two-hybrid system and found that TCTP interacts with the third large cytoplasmic domain of α1 as well as α2 isoforms of Na,K-ATPase, believed involved in the regulation of Na,K-ATPase activity. Interaction between TCTP and Na,K-ATPase was confirmed by coimmunoprecipitation in yeast and mammalian cells. We also showed, using 86Rb+ uptake assay, that overexpression of TCTP inhibited Na,K-ATPase activity in HeLa cells. Northern and Western blotting studies of HeLa cells transiently transfected with GFP-tagged TCTP showed that overexpression of TCTP did not change mRNA and protein levels of Na,K-ATPase. Recombinant TCTP protein purified from an Escherichia coli expression system inhibited purified HeLa cell plasma membrane Na,K-ATPase in a dose-dependent manner. Using deletion analysis, we also found that the C-terminal 102–172-amino-acid region of rat TCTP that contains the TCTP homology region 2 is essential for its association with, and inhibition of, Na,K-ATPase.

Expression of translationally controlled tumor protein mRNA in human colon cancer

The translationally controlled tumor protein (TCTP) was first identified as a tumor-associated protein. Here we described the expression profile of TCTP mRNA using a panel of normal human tissues. We also investigated the cell-cycle-dependent expression of the TCTP mRNA using synchronized cultures of Saccahromyces cerevisiae. The TCTP gene was constantly expressed through the yeast cell cycle. The expression of TCTP mRNA from three human colon carcinoma cell lines and their tumors xenografted in a nude mouse was studied. Our data imply that the TCTP is a housekeeping gene and that its deregulation may be involved in the tumor development of colon carcinoma.

Interaction of the alpha subunit of Na,K-ATPase with cofilin.

The α1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,KATPase and potentially affect the function and}or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the α subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed

Dimerization of Translationally Controlled Tumor Protein Is Essential For Its Cytokine-Like Activity

Background Translationally Controlled Tumor Protein (TCTP) found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF). Human recombinant HRF (HrHRF) has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn). Methods and Findings We found that only NH2-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH2-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP) was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP). Allergenic activity of Del-N11TCTP prompted us to see whether the NH2-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice. Conclusions Dimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development.

Interaction of Cofilin with Triose-phosphate Isomerase Contributes Glycolytic Fuel for Na,K-ATPase via Rho-mediated Signaling Pathway

We reported previously that cofilin, an actin-binding protein, interacts with Na,K-ATPase and enhances its activity (Lee, K., Jung, J., Kim, M., and Guidotti, G. (2001)Biochem. J. 353, 377–385). To understand the nature of this interaction and the role of cofilin in the regulation of Na,K-ATPase activity, we searched for cofilin-binding proteins in the rat skeletal muscle cDNA library using the yeast two-hybrid system. Several cDNA clones were isolated, some of which coded for triose-phosphate isomerase, a glycolytic enzyme. The interaction of cofilin with triose-phosphate isomerase as well as Na,K-ATPase was confirmed by immunoprecipitation and confocal microscopy in HeLa cells. Cofilin was translocated to the plasma membrane along with triose-phosphate isomerase by the Rho activator lysophosphatidic acid but not by the p160 Rho-associated kinase inhibitor Y-27632, suggesting that the phosphorylated form of cofilin bound to TPI interacts with Na,K-ATPase. Ouabain-sensitive 86Rb+ uptake showed that Na,K-ATPase activity was increased by the overexpression of cofilin and lysophosphatidic acid treatment, but not by the overexpression of mutant cofilin S3A and Y-27632 treatment. Pretreatment with the glycolytic inhibitor iodoacetic acid caused a remarkable reduction of Na,K-ATPase activity, whereas pretreatment with the oxidative inhibitor carbonyl cyanidem-chlorophenylhydrazone caused no detectable changes, suggesting that the phosphorylated cofilin is involved in feeding glycolytic fuel for Na,K-ATPase activity. These findings provide a novel molecular mechanism for the regulation of Na,K-ATPase activity and for the nature of the functional coupling of cellular energy transduction.

A protein transduction domain located at the NH2-terminus of human translationally controlled tumor protein for delivery of active molecules to cells.

Protein transduction domains (PTDs) are small peptides, able to penetrate biological membranes and deliver various types of cargo both in vitro and in vivo. Because use of PTDs originating from viral origins resulted in undesired effects, PTDs originating from non-viral origins are needed. Here, we report that a 10-amino acid peptide (MIIYRDLISH) derived from the NH(2)-terminus of human translationally controlled tumor protein (TCTP) functions as a PTD. This peptide was internalized through lipid raft-dependent endocytosis and partial macropinocytosis, and did not enter lysosome and nucleus. Beta-galactosidase fused to TCTP-PTD, when injected into mice, was efficiently delivered to liver, kidney, spleen, heart, and lungs of the animals. Preincubation of TCTP-PTD with adenovirus increased adenoviral mediated-gene expression in cells and also improved immune response to intranasally administered adenovirus expressing the triple repeat of G glycoprotein of respiratory syncytial virus (RSV), rAd/3×G. These findings suggest that TCTP-PTD might overcome the limitations of polycation-mediated transduction and serve as an efficient vehicle for drug delivery.

Effects of mutation at a conserved N‐glycosylation site in the bovine retinal cyclic nucleotide‐gated ion channel

Bovine retinal cyclic nucleotide‐gated (CNG) ion channel contains an evolutionary conserved N‐glycosylation site in the external loop between the fifth transmembrane segment and the pore‐forming region. The effect of tunicamycin treatment and the site‐specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N‐glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N‐glycosylation site did not affect expression and functionality of the CNG channel in oocytes.

Translationally Controlled Tumor Protein induces epithelial to mesenchymal transition and promotes cell migration, invasion and metastasis

Translationally controlled tumor protein (TCTP), is a highly conserved protein involved in fundamental processes, such as cell proliferation and growth, tumorigenesis, apoptosis, pluripotency, and cell cycle regulation. TCTP also inhibits Na,K-ATPase whose subunits have been suggested as a marker of epithelial-to-mesenchymal transition (EMT), a crucial step during tumor invasiveness, metastasis and fibrosis. We hypothesized that, TCTP might also serve as an EMT inducer. This study attempts to verify this hypothesis. We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers. Conversely, depletion of TCTP reversed the induction of these EMT phenotypes. TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9. Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes. Overall, these findings support our hypothesis that TCTP is a positive regulator of EMT and suggest that modulation of TCTP expression is a potential approach to inhibit the invasiveness and migration of cancer cells and the attendant pathologic processes including metastasis.

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra‐ and intracellular functions. Our group recently reported that TCTP interacts with Na,K‐ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K‐ATPase. However, when overexpressed, it restores the Na,K‐ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K‐ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K‐ATPase activity, but activates Na,K‐ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K‐ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K‐ATPase activity.