Regulation of oncogenic transcription factor hTAF(II)68-TEC activity by human glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Tumour-specific chromosomal rearrangements are known to create chimaeric products with the ability to generate many human cancers. hTAF(II)68-TEC (where hTAF(II)68 is human TATA-binding protein-associated factor II 68 and TEC is translocated in extraskeletal chondrosarcoma) is such a fusion product, resulting from a t(9;17) chromosomal translocation found in extraskeletal myxoid chondrosarcomas, where the hTAF(II)68 NTD (N-terminal domain) is fused to TEC protein. To identify proteins that control hTAF(II)68-TEC function, we used affinity chromatography on immobilized hTAF(II)68 (NTD) and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and isolated a novel hTAF(II)68-TEC-interacting protein, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). GAPDH is a glycolytic enzyme that is also involved in the early steps of apoptosis, nuclear tRNA export, DNA replication, DNA repair and transcription. hTAF(II)68-TEC and GAPDH were co-immunoprecipitated from cell extracts, and glutathione S-transferase pull-down assays revealed that the C-terminus of hTAF(II)68 (NTD) was required for interaction with GAPDH. In addition, three independent regions of GAPDH (amino acids 1-66, 67-160 and 160-248) were involved in binding to hTAF(II)68 (NTD). hTAF(II)68-TEC-dependent transcription was enhanced by GAPDH, but not by a GAPDH mutant defective in hTAF(II)68-TEC binding. Moreover, a fusion of GAPDH with the GAL4 DNA-binding domain increased the promoter activity of a reporter containing GAL4 DNA-binding sites, demonstrating the presence of a transactivation domain(s) in GAPDH. The results of the present study suggest that the transactivation potential of the hTAF(II)68-TEC oncogene product is positively modulated by GAPDH.
Kim
Sol
S
Department of Life Science, Sogang University, Seoul 121-742, Korea.
Lee
Jungwoon
J
Kim
Jungho
J
D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000201:enzymology; D035381:TATA-Binding Protein Associated Factors / Q000502:physiology*; D015534:Trans-Activators / Q000502:physiology
D017931:DNA Primers; C102041:TAF15 protein, human; D035381:TATA-Binding Protein Associated Factors; D015534:Trans-Activators; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases