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Volumn 279, Issue 5353, 1998, Pages 1048-1052

Structure of the amino-terminal protein interaction domain of STAT-4

Author keywords

[No Author keywords available]

Indexed keywords

CYTOKINE; STAT4 PROTEIN; TRANSCRIPTION FACTOR;

EID: 0032512661     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5353.1048     Document Type: Article
Times cited : (222)

References (42)
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    • 2, 50 mM KCI, and 5 mM DTT. In vitro phosphorylation was done as described (9).
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    • Gel-shift experiments and determination of tetramer stability were done as described (9) with an oligonucleotide containing two copies of the STAT recognition element from the c-fos gene (26) spaced by 10 base pairs (5′-GCCAGTCAGTTCCCGTCAATGCATCAGGTTCCCGTCAATGCAT-3′, binding sites underlined). Both protein preparations (Tyr-phosphorylated wild-type STAT-1α and W37A mutant) were titrated in gel-shift experiments with an oligonucleotide containing a single M67 (26) site (5′-GCCGATTTCCCGTAAATCAT-3′) to assure similar loading of active protein.
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    • Transient transfections were done on six-well plates with 50% confluent U3A cells using the calcium phosphate method as instructed by the manufacturer (Stratagene) with the following modifications. Transfection reactions contained 4.5 μg per well of either wild-type STAT-1α or the W37A mutant in plasmid pcDNA3 (Invitrogen), 4 μg of luciferase reporter plasmid pLuc (C. M. Horvath), and 0.4 μg of β-galactosidase reporter plasmid (Stratagene). The luciferase reporter contained in its Bam HI site as an enhancer element two tandemly arranged weak STAT-1 binding sites (5′-GATCAGTTCCCGTCAATCATGATCCAGTTCCCGTCAATGATCCCCGGGATC-3′) from the human c-fos promoter. Thirty-six hours after transfection, cells were treated with interferon-γ (5 ng/ml, Amgen) for 10 hours or left untreated. Luciferase assays (Promega) and β-galactosidase assays (Stratagene) were done according to the manufacturer's protocol. Protein expression and Tyr-phosphorylation were checked in gel-shift experiments with whole-cell extracts for both wild-type and mutant protein and were comparable. All results shown are luciferase activities normalized to β-galactosictase activity.
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    • Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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    • We thank H. Viguet and M. M. Allen for expert technical support, X. Zhu and C. M. Horvath for reagents, and the staff at BNL, especially C. Ogata, for advice in collecting MAD data. U.V. expresses special thanks to C. Harrison and F. Sicheri for valuable and generous help with DENZO and X-PLOR, and to D. E. Drake for assistance with figures. Supported by NIH grants AI34420 and AI32489 to J.E.D.


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