Immunoaffinity purification and characterization of CACGTG sequence-binding proteins from cultured mammalian cells using an anti c-Myc monoclonal antibody recognizing the DNA-binding domain.
c-Myc protein contains a basic helix-loop-helix and leucine zipper (bHLHLZ) structure in its carboxyl terminal region, forms heterodimers with Max, and binds to the CACGTG sequence in DNA. A number of bHLHLZ proteins are present in cells, and some of them bind to the same DNA sequence. Using an anti c-Myc monoclonal antibody, MYC5, raised against a bacterially synthesized c-Myc protein, we have carried out immunoaffinity purification of c-Myc proteins from cultured mammalian cells. The immunoaffinity-purified fraction was found to contain not only c-Myc but also other CACGTG sequence-binding proteins, such as Max, Mad, and USF, indicating a wide cross-reaction to CACGTG sequence-binding proteins. The MYC5 antibody recognized the common structural motif in their basic region required for sequence-specific DNA binding and was shown to inhibit their DNA-binding activities. The immunoaffinity-purified N-Myc, Max, Mad, and, presumably, c-Myc were highly phosphorylated, and phosphatase treatment increased the DNA-binding activity of Myc, suggesting that the DNA-binding activity of c-Myc was regulated by phosphorylation in vivo. From these results, we can conclude that the MYC5 antibody constitutes a powerful tool for the purification and characterization of c-Myc and other CACGTG sequence-binding proteins.
Nozaki
N
N
Department of Molecular Biology, School of Science, Nagoya University, Chikusa-ku. nnozaki@st.rim.or.jp
Naoe
T
T
Okazaki
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D010744:Phosphoric Monoester Hydrolases