Abstract
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 alpha1 as well as alpha2 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 (86)Rb(+) 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.
Highlights
Na,K-ATPase, a multimembrane-spanning enzyme, is essential for maintaining transmembrane gradients of Naϩ and Kϩ ions and for cell homeostasis [1]
We looked for other cytoplasmic agents that might interact with the CD3 of Na,K-ATPase ␣ subunit and regulate its activity and found that translationally controlled tumor protein (TCTP) acts as a cytoplasmic repressor of Na,K-ATPase
Translationally controlled tumor protein (TCTP) Interacts with the Third Cytoplasmic Domain of Na,K-ATPase ␣ Subunit—Using the LexA DNA-binding domain CD3 of the Na,K-ATPase ␣2 subunit fusion in a yeast two-hybrid screening, several cDNA clones were isolated from the rat skeletal muscle library
Summary
Yeast Two-hybrid Screen Assay—The cDNA library was constructed as described previously [10]. cDNAs of rat Na,K-ATPase ␣1 and ␣2 subunits were obtained from Dr Jerry Lingrel (University of Cincinnati College of Medicine). After transient transfections with HA-tagged TCTP, GFP, GFPTCTP, and GFP-TCTP-(1–101) and GFP-TCTP-(102–172) constructs in the LipofectAMINE PLUSTM 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 CompleteTM protease inhibitor mixture tablets (Roche Applied Science) for 30 min on ice and homogenized with a Pyrex glass homogenizer. 0.2 mg/ml of plasma membrane fraction at 30% sucrose was incubated with various concentrations of ouabain (10Ϫ9–10Ϫ3 M), recombinant TCTP (0.1–100 g/ml), or recombinant TCTP alone in assay buffer containing 18 mM histidine, 18 mM imidazole, 80 mM NaCl, 15 mM KCl, 3 mM MgCI2, and 0.1 mM EGTA, pH 7.4, for 20 min at 37 °C. The binding activity of the deletion mutants was determined by -galactosidase assay
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
