A 32-kDa proteolytic fragment of transcription factor Stat3 is capable of specific DNA binding.
Fragments of characteristic size retaining the ability of sequence-specific DNA binding were generated by partial proteolysis of transcription factor Stat3 with trypsin, chymotrypsin, or Staphylococcus V8 proteinase. The molecular masses of the smallest DNA-binding fragments were 75, 48, and 32 kDa after digestion with V8 proteinase, chymotrypsin, and trypsin, respectively. The fragments contained major parts of the domain controlling the sequence specificity of DNA binding (amino acids 406-514), the SH3 and SH2 domains, and the phosphorylated tyrosine residue Tyr-705, but not the C-terminal 20 amino acids. The N terminus of the 32-kDa tryptic fragment (ANCDASLIV) matched the sequence of amino acids 424-432 deduced from cDNA. The fragments were observed after proteolytic treatment of preformed complexes between DNA and native factors eluted from rat liver nuclei or recombinant, tyrosine-phosphorylated rat Stat3 from insect cells. It was possible to elute all three minimal fragments from their complexes with DNA and to obtain specific re-binding. The minimal fragments eluted from complexes with DNA still contained the phosphorylated Tyr-705 and the SH2 domain suggesting that they were probably bound to DNA as dimers. The DNA-binding domain of Stat3 identified by these experiments overlapped the domain previously identified by genetic experiments as the domain controlling the sequence specificity of DNA binding. The DNA-binding domain defined here by partial proteolysis probably represents an autonomously folding portion of Stat3.
Dreier
B
B
Chair of Genetics, University of Erlangen-Nürnberg, D-91058 Erlangen, Federal Republic of Germany.
Fritz
S
S
Volkert
G
G
Brachvogel
B
B
Lottspeich
F
F
Fey
G H
GH
D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018604:Epitope Mapping; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D051381:Rats; D050796:STAT3 Transcription Factor; D018411:Spodoptera; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D000209:Acute-Phase Proteins; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D015534:Trans-Activators; D004247:DNA