Specific Interaction with Transcription Factor IIA and Localization of the Mammalian TATA-binding Protein-like Protein (TLP/TRF2/TLF)

• Published in: The Journal of biological chemistry 2004-02, Vol.279 (9), p.7447-7455
• Main Authors: Nakadai, Tomoyoshi, Shimada, Miho, Shima, Daisuke, Handa, Hiroshi, Tamura, Taka-aki
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Cell Nucleus - chemistry; Cross-Linking Reagents; Cytoplasm - chemistry; Dimerization; DNA - metabolism; Electrophoresis, Polyacrylamide Gel; Gene Expression; Glutathione Transferase - genetics; Humans; Kinetics; Macromolecular Substances; Mice; Mutagenesis; NIH 3T3 Cells; Recombinant Fusion Proteins; TATA Box - genetics; TATA Box Binding Protein-Like Proteins - analysis; TATA Box Binding Protein-Like Proteins - genetics; TATA Box Binding Protein-Like Proteins - metabolism; TATA-Box Binding Protein - chemistry; TATA-Box Binding Protein - metabolism; TFIIA protein; TLF protein; TLP protein; Transcription Factor TFIIA - metabolism; Transcription Factor TFIIA - pharmacology; Transfection; TRF2 protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305412200

TBP-like protein (TLP) is structurally similar to the TATA-binding protein (TBP) and is thought to have a transcriptional regulation function. Although TLP has been found to form a complex with transcription factor IIA (TFIIA), the in vivo functions of TFIIA for TLP are not clear. In this study, we analyzed the interaction between TLP and TFIIA. We determined the biophysical properties for the interaction of TLP with TFIIA. Dissociation constants of TFIIA versus TLP and TFIIA versus TBP were 1.5 and 10 nm, respectively. Moreover, the dissociation rate constant of TLP and TFIIA (1.2 × 10–4/m·s was significantly lower than that of TBP (2.1 × 10–3/m·s). These results indicate that TLP has a higher affinity to TFIIA than does TBP and that the TLP-TFIIA complex is much more stable than is the TBP-TFIIA complex. We found that TLP forms a dimer and a trimer and that these multimerizations are inhibited by TFIIA. Moreover, TLP mutimers were more stable than a TBP dimer. We determined the amounts of TLPs in the nucleus and cytoplasm of NIH3T3 cells and found that the molecular number of TLP in the nucleus was only 4% of that in the cytoplasm. Immunostaining of cells also revealed cytoplasmic localization of TLP. We established cells that stably express mutant TLP lacking TFIIA binding ability and identified the amino acids of TLP required for TFIIA binding (Ala-32, Leu-33, Asn-37, Arg-52, Lys-53, Lys-78, and Arg-86). Interestingly, the level of TFIIA binding defective mutant TLPs in the nucleus was much higher than that of the wild-type TLP and TFIIA-interactable mutant TLPs. Immunostaining analyses showed consistent results. These results suggest that the TFIIA binding ability of TLP is required for characteristic cytoplasmic localization of TLP. TFIIA may regulate the intracellular molecular state and the function of TLP through its property of binding to TLP.