A 32-KDA PROTEOLYTIC FRAGMENT OF TRANSCRIPTION FACTOR STAT3 IS CAPABLE OF SPECIFIC DNA-BINDING

Fragments of characteristic size retaining the ability of sequence-spe cific DNA binding were generated by partial proteolysis of transcripti on factor Stat3 with trypsin, chymotrypsin, or Staphylococcus V8 prote inase, 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 acid s 406-514), the SH3 and SH2 domains, and the phosphorylated tyrosine r esidue Tyr-705, but not the C-terminal 20 amino acids, The N terminus of the 32-kDa tryptic fragment (ANCDASLIV) matched the sequence of ami no acids 424-432 deduced from cDNA. The fragments were observed after proteolytic treatment of preformed complexes between DNA and native fa ctors eluted from rat liver nuclei or recombinant, tyrosine-phosphoryl ated rat Stat3 from insect cells. It was possible to elute all three m inimal 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 th at they were probably bound to DNA as dimers, The DNA-binding domain o f Stat3 identified by these experiments overlapped the domain previous ly identified by genetic experiments as the domain controlling the seq uence specificity of DNA binding, The DNA-binding domain defined here by partial proteolysis probably represents an autonomously folding por tion of Stat3.