Abstract
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.
Highlights
Stat3 is a transcription factor mediating the effects of a variety of cytokines including interleukin 6 (IL6)1 and IL6related cytokines on the transcriptional induction of their target genes [1,2,3,4,5,6]
Nuclear protein extracts from rat livers excised 6 h after induction of an experimental acute phase response were used as the initial source of activated Stat3
Earlier studies had demonstrated that Stat3 was the most abundant activated Stat factor in liver nuclei at this stage of the acute phase response
Summary
Gel Mobility Shift Experiments—Nuclear extracts from rat livers were prepared and electrophoretic mobility shift assays (EMSA) were performed as described previously (17, 24 –26). Subsequent digestion of DNA-bound Stat was performed for 10 min at 25 °C with trypsin or chymotrypsin and at 37 °C with V8 proteinase in 500 l of CP buffer supplemented with 100 mM KCl. Trypsin (14 units), chymotrypsin (8 units), and V8 proteinase (375 units) were used to treat 250 g of crude insect cell extract containing recombinant rat Stat. N-terminal Amino Acid Sequence Analysis—Ten mg of crude extract from Sf21 insect cells coinfected with baculoviral expression constructs for Stat and JAK2 and harvested 50 h after infection were incubated with the double-stranded biotinylated JR2 oligonucleotide bound to streptavidin/magnetic porous glass beads. Tryptic digestion of the DNA-bound Stat was performed as described above, and the eluted minimal fragment was electrophoretically separated in an SDS-polyacrylamide gel. N-terminal amino acid sequencing was performed by automated Edman degradation using an Applied Biosystems 492A sequencer
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