Direct interaction of CREB protein with 21 bp Tax-response elements of HTLV-ILTR.
The three 21-bp repeats (Tax-responsive elements) of the long terminal repeat (LTR) of the human T-cell leukemia virus (HTLV-I) mediates the response of the Tax protein. All three Tax-responsive elements (TREs) contain a TGACG motif, reminiscent of the CREB/ATF-binding site TGACGTCA. DNA-affinity chromatography with the 5'-TRE resulted in a previous study in proteins of about 32, 36 to 42, 50 and 110 kDa. Here we demonstrate that the 42 kDa protein is the cAMP-response element-binding (CREB) protein. This is shown by phosphorylation of the proteins eluted from the DNA-affinity column with protein kinase A (PKA) in vitro and subsequent indirect immunoprecipitation with a CREB-specific antiserum raised against an internal CREB-specific peptide. This method allows detection of phosphorylated proteins by autoradiography with high sensitivity and is superior to metabolic labeling. One of the phosphorylated proteins co-migrates with immuno-affinity-purified CREB protein--also phosphorylated in vitro--and competes with the peptide antigen, which proves the specificity of the reaction. The purified CREB protein leads to specific DNA-protein complexes in DNA mobility-shift analyses with all three TREs. Comparison of these TRE-CREB complexes with those formed by nuclear extracts from the HTLV-I-transformed T-cell line C81-66-45 indicates that additional cellular factors contribute to the complexes, especially to the middle TRE. This is also shown by using CREB-depleted instead of complete nuclear extracts for DNA mobility-shift assays. Antibodies against CREB but not Tax affect the mobility of the DNA-protein complex.
Beimling
P
P
Max-Planck-Institut für Molekulare Genetik, Abt. Schuster, Berlin, Germany.
Moelling
K
K
D001483:Base Sequence; D002846:Chromatography, Affinity; D017362:Cyclic AMP Response Element-Binding Protein; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
D017362:Cyclic AMP Response Element-Binding Protein; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides