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. 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. Na,K-ATPase, a multimembrane-spanning enzyme, is essential for maintaining transmembrane gradients of Na+ and K+ ions and thus for cell homeostasis (1Therien A.G. Blostein R. Am. J. Physiol. 2000; 279: C541-C566Crossref PubMed Google Scholar). These ionic gradients serve to control essential cellular processes such as cell volume, membrane potential, and nutrient transport (2Ullrich K.J. Annu. Rev. Physiol. 1979; 41: 181-195Crossref PubMed Scopus (132) Google Scholar). In addition, Na,K-ATPase is involved in cell proliferation and differentiation, heart and vascular muscle contraction, and neurotransmitter and hormone secretion (3Bertorello A.M. Katz A.I. Am. J. Physiol. 1993; 265: F743-F755PubMed Google Scholar). Thus dysfunction of this enzyme can profoundly affect cell function. Na,K-ATPase is composed of a catalytic 110-kDa α subunit and a glycosylated 40–60-kDa β subunit. The α subunit contains binding sites for cations, ATP, and cardiac glycosides. It has been suggested that there might exist a diffusible cytoplasmic regulator of Na,K-ATPase activity, possibly modulated by protein kinases and hormones (4Jorgensen P.L. Andersen J.P. J. Membr. Biol. 1988; 103: 95-120Crossref PubMed Scopus (273) Google Scholar, 5Forbush B. II I Kaplan J.H. Hoffman J.F. Biochemistry. 1978; 17: 3667-3676Crossref PubMed Scopus (224) Google Scholar). The third large cytoplasmic domain (CD3) 1The abbreviations used are: CD, cytoplasmic domain; GFP, green fluorescence protein; GST, glutathione S-transferase; HA, hemagglutinin; TCTP, translationally controlled tumor protein; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. of Na,K-ATPase was proposed to be one of the domains involved in the regulation of its activity by insulin, thereby playing an important role in the catalytic function and regulation of this enzyme (6McGill D.L. Guidotti G. J. Biol. Chem. 1991; 266: 15824-15831Abstract Full Text PDF PubMed Google Scholar). Interactions between the N-terminal region of the Na,K-ATPase α subunit with phosphoinositide-3 kinase (7Yudowski G.A. Efendiev R. Pedemonte C.H. Katz A.I. Berggren P.O. Bertorello A.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6556-6561Crossref PubMed Scopus (164) Google Scholar), cytoplasmic domain 2 (CD2) and CD3 with ankyrin (8Devarajan P. Scaramuzzino D.A. Morrow J.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2965-2969Crossref PubMed Scopus (134) Google Scholar, 9Jordan C. Puschel B. Koob R. Drenckhahn D. J. Biol. Chem. 1995; 270: 29971-29975Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), CD3 with cofilin (10Lee K. Jung J. Kim M. Guidotti G. Biochem. J. 2001; 353: 377-385Crossref PubMed Scopus (47) Google Scholar), and purified Na,K-ATPase with actin (11Koob R. kraemer D. Trippe G. Aebi U. Drenckhahn D. J. Cell Biol. 1990; 53: 93-100Google Scholar) and adducin (12Ferrandi M. Salardi S. Tripodi G. Barassi P. Rivera R. Manunta P. Goldshleger R. Ferrari P. Bianchi G. Karlish S.J. Am. J. Physiol. 1999; 277: H1338-H1349PubMed Google Scholar) have also been demonstrated. We looked for other cytoplasmic agents that might interact with the CD3 of Na,K-ATPase α subunit and regulate its activity and found that translationally controlled tumor protein (TCTP) acts as a cytoplasmic repressor of Na,K-ATPase. TCTP is a growth-related protein, under tight transcriptional as well as translational control (13Bommer U.A. Lazaris-Karastzas A. De Benedetii A. Nurnberg P. Benndorf R. Bielka H. Sonenberg N. Cell. Mol. Biol. Res. 1994; 40: 633-641PubMed Google Scholar, 14Xu A. Bellamy R. Taylor J.A. Biochem. J. 1999; 342: 683-689Crossref PubMed Scopus (98) Google Scholar). It occurs as a 23-kDa protein in humans and has a 21-kDa homologue in mice but shows no significant homology with any other family of proteins. Based on structural studies of TCTP from Schizosaccharomyces pombe, it has been recently proposed that TCTP belongs to the MSS4/DSS4 superfamily of proteins, which bind to the GDP/GTP-free form of Rab proteins, described as guanine nucleotide-free chaperones (15Thaw P. Baxter N.J. Hounslow A.M. Price C. Waltho J.P. Craven C.J. Nat. Struct. Biol. 2001; 8: 701-704Crossref PubMed Scopus (165) Google Scholar). TCTP has no known primary function, but the high degree of homology of TCTP from various sources, including plants and humans, and its expression in many tissues, suggest that it may have a basic housekeeping function (16Sanchez J.C. Schaller D. Ravier F. Golaz O. Jaccoud S. Belet M. Wilkins M.R. James R. Deshusses J. Hochstrasser D. Electrophoresis. 1997; 18: 150-155Crossref PubMed Scopus (146) Google Scholar, 17Chitpatima S.T. Makrides S. Bandyopadhyay R. Brawerman G. Nucleic Acids Res. 1988; 16: 2350Crossref PubMed Scopus (130) Google Scholar). Yeast Two-hybrid Screen Assay—The cDNA library was constructed as described previously (10Lee K. Jung J. Kim M. Guidotti G. Biochem. J. 2001; 353: 377-385Crossref PubMed Scopus (47) Google Scholar). cDNAs of rat Na,K-ATPase α1 and α2 subunits were obtained from Dr. Jerry Lingrel (University of Cincinnati College of Medicine). The CD3 region (Lys-352–Val-l756) of the α2 subunit of Na,K-ATPase was fused into the LexA DNA-binding domain and used as a bait to screen the cDNA library from rat skeletal muscle. Potential positive clones activating the reporter genes were analyzed by restriction mapping and sequencing. One of these clones had DNA sequences that correspond to the gene for rat TCTP, based on a nucleotide BLAST search in NCBI homepage. The sequences coding for the CD3 region (Lys-354–Val-l759) of the α1 subunit were cloned into the pEG202 vector of the LexA fusion plasmid. The PCR fragments of the following were amplified and cloned into the pEG202 vector: the N-terminal portions of α1 (Met-1–Cys-93) and α2 (Met-1–Cys-91); α1CD2 (Glu-151–Ile-292) and α2CD2 (Glu-149–Ile-290) encoding the H2–H3 loop; α1CD4 (Glu-825–Arg-848) and α2CD4 (Glu-822–Arg-845) encoding the H6–H7 loop; the C-terminal portions of α1 (Ile-935–Tyr–1023) and α2 (Ile-932–Tyr-1020); and the N-terminal portion of β subunit (Ala-1–Lys-33). Yeast cells, EGY48/pSH18–34, containing LexAop-LEU2 and Lex-Aop-LacZ reporters were transformed simultaneously with both one of the resulting LexA fusions and a rat TCTP clone using the high efficiency transformation method. Protein-protein interaction results in trans-activation of the LexAop-LEU2 and LexAop-LacZ reporter genes (10Lee K. Jung J. Kim M. Guidotti G. Biochem. J. 2001; 353: 377-385Crossref PubMed Scopus (47) Google Scholar). Therefore, individual transformants of the yeast cells were tested on glucose Ura–His–Trp–Leu–, galactose Ura–His–Trp–Leu–, glucose Ura–His–Trp– X-Gal and galactose Ura–His–Trp– X-Gal plates. β-galactosidase activity was determined as reported previously (18Himmelfarb H.J. Pearlberg J. Last D.H. Ptashne M. Cell. 1990; 63: 1299-1309Abstract Full Text PDF PubMed Scopus (112) Google Scholar) using the formula: β-galactosidase unit = A420 × 1000/t (min) × volume of extract (ml) × protein (μg/ml). Co-immunoprecipitations—Co-immunoprecipitations were performed as described previously (10Lee K. Jung J. Kim M. Guidotti G. Biochem. J. 2001; 353: 377-385Crossref PubMed Scopus (47) Google Scholar). Briefly, yeast cells cultured in glucose Ura–His–Trp– and galactose Ura–His–Trp– media were collected at 3,000 × g for 5 min and resuspended by vortexing with yeast lysis buffer (YLB) (50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 150 mm NaCl, 50 mm NaF, 2 mm ZnCl2, and protease inhibitor mixture) and glass beads. After adding radioimmune precipitation buffer (10 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS), the mixtures were centrifuged at 10,000 × g for 30 min. Affinity-purified anti-hemagglutinin (HA) 12CA5 antibody was added to the supernatants, and the mixtures were incubated for 3 h at 4 °C. Fifty percent protein A-agarose solution (Roche Applied Science) was then added, and incubation continued overnight at 4 °C. After a 5-s centrifugation at 12,000 × g, the pellets were washed twice with the radioimmune precipitation buffer and then with a wash buffer (1 m NaCl, 10 mm Tris-HCl, pH 8.0, and 0.1% Nonidet P-40). The pellets were then resuspended in 2× SDS sample buffer and subjected to SDS-PAGE. The blots obtained were incubated with the anti-LexA antibody. Immunoprecipitations from extracts of HeLa cells were performed according to Jung et al. (19Jung J. Yoon T. Choi E.C. Lee K. J. Biol. Chem. 2002; 277: 48931-48937Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The deletion mutants, TCTP-(1–101) and TCTP-(102–172), were constructed with Exsite PCR-based site-directed mutagenesis kit (Stratagene). The deletion cDNA constructs were PCR-amplified from GFP-TCTP using the following 5′-phosphorylated primers: for TCTP-(1–101), 5′-GCCCTTGAGTGATTTCATGTAGTCTTT-3′ (antisense) and phospho-5′-CAGTCGACGGTACCGCGGGCCCGGGAT-3′ (sense), and for TCTP-(102–171), phospho-5′-CATGGTGACGGAATTCGAAGCTTGAG-3′ (antisense) and 5′-AAACTTGAAGAACAGAAACCAGAAAGG-3′ (sense). After transient transfections with HA-tagged TCTP, GFP, GFPTCTP, and GFP-TCTP-(1–101) and GFP-TCTP-(102–172) constructs in the LipofectAMINE PLUS™ reagent (Invitrogen), HeLa cells were incubated with ice-cold lysis buffer containing 20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 2 mm EGTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm NaF, 2 mm Na3VO4, and Complete™ protease inhibitor mixture tablets (Roche Applied Science) for 30 min on ice and homogenized with a Pyrex glass homogenizer. The cell lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatants (1 mg/ml) were preabsorbed with protein G-agarose or protein A-agarose (Roche Applied Science) for 3 h at 4 °C. Anti-HA antibody or anti-GFP antibody (Santa Cruz Biotechnology) was added to the reaction mixture and incubated at 4 °C for 3 h and incubated with protein G-agarose or protein A-agarose overnight on a rocking platform at 4 °C. The bound immune complexes so obtained were pelleted and washed three times with ice-cold phosphate-buffered saline, eluted with 2× SDS sample buffer, and separated on SDS-PAGE. The blots obtained were incubated with anti-Na,K-ATPase α1 monoclonal antibody (Upstate Biotechnology) and anti-GFP antibody. of cells were transiently transfected with GFP, and GFP-TCTP-(1–101) and GFP-TCTP-(102–172) constructs and in at 4 × After the cells were washed with and incubated for 2 h at The cells were washed three times with buffer mm NaCl, 5 mm 10 mm 1 mm mm and mm pH and incubated with or 1 mm mm in the buffer for min at °C. 86Rb+ was added to a of 2 and the reaction was to for min at °C. 86Rb+ uptake was by with The cells were then with lysis buffer (50 mm 1% and incubated for 5 min at The of the cell lysates was determined adding mixture well was determined according to of the M. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and cells in three were the the of Northern was from HeLa cells using reagent according to the The was separated on a containing membrane and was performed using solution Na,K-ATPase α1 a to of Na,K-ATPase α1 cDNA was cloned using the obtained as as and with After and the were analyzed by system and with of Na,K-ATPase in plasma membrane was obtained as described in a Guidotti G. Biochem. 1997; PubMed Scopus Google Scholar). of plasma membrane at was incubated with various of TCTP or TCTP in buffer containing mm mm mm NaCl, mm 3 mm and mm EGTA, pH 7.4, for 20 min at °C. The was then with the of to a of 5 mm in 100 of a incubation for min at °C, the of from was using the of and C.H. J. Biol. Chem. Full Text PDF Google Scholar). The were on the at The obtained were to activity by using a of sodium that was in the Na,K-ATPase activity was determined by the of resulting from incubation with TCTP from that with Na,K-ATPase activity of Na,K-ATPase was of of of deletion constructs of TCTP, and were by the sequences from the of rat TCTP and to clones and described previously T. Jung J. Kim M. Lee Choi E.C. Lee K. Biochem. 2000; PubMed Scopus (44) Google Scholar). was clone by yeast two-hybrid in the and were constructed by PCR and into the library vector to the deletion constructs of The N-terminal of constructs were confirmed by DNA and the expression of fusion was confirmed by with the 12CA5 antibody the The binding activity of the deletion was determined by β-galactosidase TCTP with the of Na,K-ATPase α the LexA DNA-binding domain CD3 of the Na,K-ATPase α2 subunit fusion in a yeast two-hybrid cDNA clones were from the rat skeletal muscle One of these clones a of with a of as The cytoplasmic domains of Na,K-ATPase were amplified by PCR and cloned into the LexA fusion the cytoplasmic of Na,K-ATPase that interact with the TCTP, individual transformants were glucose and galactose and for β-galactosidase activity of and with the of the reporter genes It is not the N-terminal portion of Na,K-ATPase interacts with TCTP the yeast cells transformed with or reporter genes in the of glucose or yeast cells were transformed with and there was no of the reporter gene in a on that and the C-terminal portion of the α subunit and the cytoplasmic region of the β subunit not interact with These results suggest that TCTP to the CD3 region of the α1 and α2 subunit of Na,K-ATPase, and the binding is not α of TCTP with the CD3 of Na,K-ATPase α1 and α2 subunits in yeast two-hybrid media activity β-galactosidase unit = A420 × (min) × volume of extract (ml) × protein The β-galactosidase unit = A420 × (min) × volume of extract (ml) × protein (μg/ml). in a TCTP with Na,K-ATPase from Yeast and the 12CA5 antibody TCTP, we TCTP with the α2 subunit and the region of Na,K-ATPase in shows results obtained by with anti-LexA antibody to the CD3 region of Na,K-ATPase. The 12CA5 antibody which the in media but not in The of the were to of in the yeast extracts that fusion proteins, which were by These results that TCTP with a protein, with the of TCTP and Na,K-ATPase interact in we transiently transfected HeLa cells with TCTP Western blotting using antibody confirmed that the cells TCTP not were from HeLa cell extracts with antibody and with anti-Na,K-ATPase α subunit antibody. HA-tagged TCTP the α subunit of Na,K-ATPase from cells but not from cells that the interaction between the α subunit of Na,K-ATPase and TCTP occurs in of TCTP in HeLa Na,K-ATPase the role of TCTP in the regulation of the Na,K-ATPase activity, we transiently TCTP in HeLa cells and Na,K-ATPase activity using 86Rb+ uptake of 86Rb+ uptake in HeLa cells is for by Na,K-ATPase and the system G. J. Cell. Physiol. 1990; PubMed Scopus Google Scholar), we 1 mm inhibitor of the and mm inhibitor of the can be in the activity of Na,K-ATPase of the cells was that of cells. cells or GFP-TCTP showed a and activity. These results suggest that the of TCTP in HeLa cells 86Rb+ uptake and that this inhibition is to its on the Na,K-ATPase but not on the of TCTP in HeLa Na,K-ATPase at mRNA or blotting using Na,K-ATPase that the mRNA of Na,K-ATPase α1 subunit was the in both and cells, that of TCTP in cells We also determined the of expression of Na,K-ATPase α1 subunit using monoclonal The expression of α1 subunits was the in as well as cells that an in TCTP protein not affect the expression of Na,K-ATPase α1 TCTP in in HeLa Cell in a of TCTP on Na,K-ATPase was using HeLa cell plasma membrane with Na,K-ATPase by of The purified HeLa cell plasma membrane Na,K-ATPase activity with of = × The of to the purified plasma membrane in a significant in Na,K-ATPase activity in a dose-dependent = the of and did not not It that TCTP protein HeLa cell plasma membrane Na,K-ATPase activity. C-terminal 2 of TCTP for with, and of, the of TCTP that interact with the CD3 of α1 and α2 subunits of Na,K-ATPase, we TCTP and its constructs and into yeast cells reporter genes and the of these in glucose and galactose The by these constructs were confirmed by Western blotting in yeast extracts transformed with not We β-galactosidase assay, the activity of the reporter gene in cells in or in constructs of TCTP, and showed β-galactosidase that not involved in the interaction with CD3 of Na,K-ATPase. β-galactosidase activity of the was with that of TCTP, that for the association between TCTP and the CD3 of Na,K-ATPase and for the expression of the enzyme activity. we constructed containing and determined its on β-galactosidase activity, there was an of β-galactosidase activity. It that of TCTP for the interaction between TCTP and the CD3 region of the α1 as well as the α2 subunit of Na,K-ATPase. that the C-terminal region of TCTP interacts with Na,K-ATPase in mammalian cells, we deletion that and and in HeLa cells. with anti-GFP antibody were from HeLa cell extracts the deletion and with anti-Na,K-ATPase α subunit and in TCTP and C-terminal GFP-TCTP complexes with the of the α subunit of Na,K-ATPase but not the and N-terminal These results that the C-terminal region of TCTP is essential for its interaction with α subunit of Na,K-ATPase in We also the 86Rb+ uptake of HeLa cells transfected with TCTP with that of HeLa cells transfected with deletion The 86Rb+ uptake in cells transfected with the and C-terminal was and in cells, that the C-terminal region of TCTP is to the activity of Na,K-ATPase in HeLa cells the other the and in GFP, and GFP-TCTP-(102–172) cells were These also suggest that the C-terminal region of TCTP is essential for interaction with Na,K-ATPase α subunit and is to the Na,K-ATPase activity. TCTP has been in the of tumor B. M. H. Bielka H. Nucleic Acids Res. 17: PubMed Scopus (112) Google Scholar) and has been reported to be in cell with the of cells (16Sanchez J.C. Schaller D. Ravier F. Golaz O. Jaccoud S. Belet M. Wilkins M.R. James R. Deshusses J. Hochstrasser D. Electrophoresis. 1997; 18: 150-155Crossref PubMed Scopus (146) Google Scholar). we TCTP in cells and rat by Western M. J. J. M. and K. and it that TCTP in cell TCTP is and a high degree of homology various that this protein have essential cellular function. In the we TCTP as a protein and as a cytoplasmic repressor of Na,K-ATPase activity. One might that the basic function of TCTP may be to cell by the Na,K-ATPase activity. of by Na,K-ATPase is an important by which cell differentiation, and in has been obtained that inhibition of Na,K-ATPase a in to its inhibition of the enzyme cell T. K. R. S. K. K. A. J. PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar). TCTP has been to bind to the cell a homologue D. F. D. K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). et al. F. D. K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) showed that transient overexpression of TCTP in HeLa cells that TCTP cell its and regulation of Therefore, it that of Na,K-ATPase activity resulting from the overexpression of TCTP in HeLa cells is to cell and A. J. Biochem. 2002; PubMed Scopus Google Scholar) that Na,K-ATPase is a with but One the inhibition by the in the that a of and in cardiac It is that inhibition of Na,K-ATPase activity by TCTP an in which in the to an in HeLa cells not have we cardiac to TCTP is to both and regulation by a of Na,K-ATPase or regulation on the of the enzyme or of Na,K-ATPase to the plasma The of Na,K-ATPase activity described in the studies is to binding of Na,K-ATPase to TCTP and not to in Na,K-ATPase of the role that Na,K-ATPase in and it is important to the that the regulation of Na,K-ATPase activity by and PubMed Scopus Google Scholar) were the to suggest that protein kinase an protein to its on the Na,K-ATPase in rat It has also been reported that a protein kinase in plasma membrane Na,K-ATPase activity is not with A. N. Acad. Sci. 1997; PubMed Scopus Google Scholar). TCTP is a protein with one for protein kinase and three sites for kinase according to a In addition, it was reported that TCTP is at and by a protein kinase that function Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). Thus it is that TCTP might be by kinases to the Na,K-ATPase activity. and Guidotti (6McGill D.L. Guidotti G. J. Biol. Chem. 1991; 266: 15824-15831Abstract Full Text PDF PubMed Google Scholar) reported that both α1 and α2 isoforms of Na,K-ATPase in were by and that this was the of the the of a diffusible repressor of Na,K-ATPase, by It be of to also a role in the regulation of TCTP binding to Na,K-ATPase in It has been reported that TCTP has a region and that TCTP transiently with the cell S. Lee M. A. T. U.A. J. Cell Sci. 1999; PubMed Google Scholar). We reported previously that the C-terminal region of TCTP is involved in interaction with T. Jung J. Kim M. Lee Choi E.C. Lee K. Biochem. 2000; PubMed Scopus (44) Google Scholar). In the we found that the C-terminal region of is essential for its interaction with Na,K-ATPase α subunit and is to Na,K-ATPase activity. Thus it that TCTP by with of In this is the that TCTP is a protein as an inhibitor of Na,K-ATPase activity. TCTP acts on the cytoplasmic of the cell in to which the enzyme by binding to the of the cell of regulation of Na,K-ATPase activity by TCTP a for the regulation of the of the cell and for the of is under to the role of We for with yeast two-hybrid and deletion We also Lee of of of and for with activity

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