Abstract
Thioredoxin-binding protein-2 (TBP-2)/vitamin D(3) up-regulated protein 1 is an endogenous molecule interacting with thioredoxin (TRX), negatively regulating TRX function, and being implicated in the suppression of tumor development and metastasis. We found that TBP-2 ectopically expressed in the breast cancer cell line MCF-7 was localized predominantly in the nucleus exhibiting growth suppressive activity. The nuclear accumulation of endogenous TBP-2 protein was also demonstrated when the cells were treated with an anti-cancer drug, suberoylanilide hydroxamic acid. To investigate the mechanism underlying the nuclear localization, we performed a yeast two-hybrid screening and identified importin alpha(1) (Rch1) as a protein interacting with TBP-2. The physical interaction between TBP-2 and Rch1 was confirmed with a glutathione S-transferase pull-down assay. The interaction of TBP-2 was specific to Rch1 among other importin alpha subfamilies (Qip1 and NPI-1), and amino acids 1-227 of TBP-2 were sufficient for both the interaction with Rch1 and the nuclear localization, although there is no typical nuclear localization signal in this sequence. The expression of short interfering RNA of Rch1 suppressed suberoylanilide hydroxamic acid-induced nuclear accumulation of TBP-2. Collectively, our results strongly suggest that an interaction with importin system is required for TBP-2 nuclear translocation and growth control tightly associated with TRX-dependent redox regulation of transcription factors.
Highlights
We recently identified thioredoxin binding protein-2 (TBP2),1 identical to vitamin D3 up-regulated protein 1 [1], as a regulatory molecule interacting with thioredoxin (TRX) [2]
The Thioredoxin-binding protein-2 (TBP-2)-transfected clones, A1 and D3, showed marked growth inhibition in culture, whereas a control clone, EG1, expressing EGFP alone showed no significant difference in its cell growth rate (Fig. 1B)
We demonstrated that when TBP-2 was overexpressed, cell growth was markedly suppressed in a breast cancer cell line, MCF-7 (Fig. 1B)
Summary
We recently identified thioredoxin binding protein-2 (TBP2),1 identical to vitamin D3 up-regulated protein 1 [1], as a regulatory molecule interacting with thioredoxin (TRX) [2]. We found that TBP-2 ectopically expressed in the breast cancer cell line MCF-7 was localized predominantly in the nucleus exhibiting growth suppressive activity. To investigate the mechanism underlying the nuclear localization, we performed a yeast two-hybrid screening and identified importin ␣1 (Rch1) as a protein interacting with TBP-2.
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