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Figure 1 | BMC Cell Biology

Figure 1

From: Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA

Figure 1

Substitution of two residues in the DNA-binding domain of STAT1 transcription factor leads to increased tyrosine phosphorylation and prolonged GAS binding. (A) Localization of critical glutamyl residues 411 and 421 in the DNA-binding domain adjacent to the DNA molecule. Depicted is a part of the crystal structure of truncated STAT1 bound to DNA[3] with the side chains of E411 and E421 pointing towards the backbone of DNA (on the left; molecular surface, on the right; detail from ribbon diagram with glutamyl residues marked in yellow). (B) Hyperphosphorylation of STAT1-E411A upon stimulation of cells with increasing concentrations of IFNγ. Equal cell numbers of HeLa cells expressing either wild-type or mutant STAT1, both tagged with green fluorescent protein (GFP), were exposed to increasing concentrations of IFNγ for 45 min before tyrosine phosphorylation was tested for in cell lysates. A representative result from a Western blot experiment with a STAT1-specific phospho-tyrosine antibody (top panel) and the corresponding re-blot after the stripping off of bound immunoreactivity and re-incubation with pan-STAT1 antibody C-24 (bottom panel) is shown. The upper band on each blot marks recombinant GFP-tagged STAT1, whereas the lower band corresponds to native STAT1. (C) Decreased tyrosine dephosphorylation of STAT1-E411A upon inhibition of kinase activity. Equal numbers of U3A cells expressing wild-type or mutant STAT1 were prestimulated for 45 min with 5 ng/ml IFNγ and subsequently exposed to 500 nM of the kinase blocker staurosporine for the durations indicated. Note that staurosporine treatment resulted in the rapid loss of tyrosine-phosphorylated STAT1-WT, whereas the mutant was partially resistant to the inactivating staurosporine effect. (D) Prolonged GAS binding of STAT1-E411A following treatment of IFNγ−prestimulated cells with staurosporine. U3A cells expressing either wild-type or mutant STAT1 were stimulated for 45 min with IFNγ and thereafter the medium was replaced and the cells treated with 500 nM staurosporine. Cell lysates were equilibrated with radioactively labeled DNA containing a high-affinity STAT binding site (GAS) before being loaded onto a gel. A representative gel-shift including two lanes with supershifts using either anti-STAT3 (αS3) or anti-STAT1 (αS1) antibody, respectively, is shown. (E) STAT1-E421K resists dephosphorylation as determined by gelshift assay using extracts from IFNγ−prestimulated U3A cells that have been exposed to staurosporine for the indicated times. (F) Tetramer stabilization of the E411A mutant. Extracts from IFNγ−stimulated U3A cells expressing either wild-type or mutant STAT1 were incubated in vitro with [32P]-labeled DNA containing a tandem GAS site. The reactions were either left unchallenged (−) or challenged for 30 min with a 750-fold molar excess of a single, unlabeled GAS site (+ competition). At the margin of the EMSA gel the positions of tetrameric (top arrow) and dimeric (bottom arrow) STAT1 are marked.

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