Fig. 1

Hyper-phosphorylation of the STAT1-E169A mutant. A Domain structure of the STAT1 protein including the localization of residues H158 and E169 and phosphorylation site Y701. SH2 = Src-homology 2, TAD = transactivation domain. B Crystal structure of the STAT1 tetramer, with side chains H158 and E169 in the coiled-coil domain highlighted in yellow. Protein crystal structure renderings from the Protein Data Bank file 1YVL [25] were generated using the PyMOL software (DeLano Scientific). C Close-up view of the investigated interface of the front-facing protomer. Residues H158A and E169A in the coiled-coil domain (green) are marked in yellow and the putative interaction partners in the amino-terminus (red) are shown in blue. D The interface at the back of the tetrameric structure (as shown in B) between the coiled-coil domain (cyan) and the N-terminus (dark blue). E Representative immunoblot from whole cell extracts expressing STAT1-GFP constructs. U3A cells expressing fusions of green fluorescent protein with WT or mutant STAT1 were stimulated with 50 ng/ml IFNγ for 45 min, followed by incubation with the kinase inhibitor staurosporine (1 µM) for the indicated times. Immunoblotting was performed using phospho-tyrosine-specific (α-pSTAT1) and pan-STAT1 (α-STAT1) antibodies. F Quantification of immunoblotting results from three independent transfection experiments as shown in (E). Asterisks indicate significant difference between the WT protein and the respective mutant. G, H Representative Western blot from whole cell extracts expressing the indicated untagged STAT1 proteins (G), including quantification of results thereof from three independent transfection experiments (H)