Figure 1.
Phenylalanine 172 in the coiled-coil domain and threonine 385 in the DNA-binding domain (the latter colored in orange and yellow) are part of the interface surface formed by two STAT1 monomers oriented in antiparallel alignment.
(A) Crystal structure of a truncated STAT1 dimer showing the localization of F172 (in magenta) and T385 (in dark blue), including a closer view of the ribbon representation (B), demonstrates the spatial orientation of the functional groups of F172 and T385, which are required for interaction with the partner monomer in the antiparallel dimer conformation. (C) Substitution of alanine for threonine 385 resulted in prolonged and elevated tyrosine phosphorylation of mutant STAT1 in cells stimulated with interferon-γ. Equal numbers of HeLa cells expressing either wild-type (WT) or mutant STAT1, both tagged with green fluorescent protein (GFP), were stimulated with 5 ng/ml of IFNγ for 45 min, before the kinase inhibitor staurosporine (1 µM) was added for the indicated times. Whole cell extracts were assessed for the time course of tyrosine phosphorylation by means of Western blotting using a STAT1-specific phospho-tyrosine antibody followed by a secondary antibody (αp-STAT1, top panel). The same membrane was stripped off bound immunoreactivity and re-probed with the pan-STAT1 antibody C-24 (αSTAT1, bottom panel). The upper arrowhead marks GFP-tagged STAT1 and the lower one indicates endogenous STAT1. (D, E) Gelshift assays confirm the increased DNA-binding activity of the T385A mutant which resulted from enhanced tyrosine phosphorylation. STAT1-negative U3A cells reconstituted with either wild-type or mutant STAT1 were stimulated with IFNγ (5 ng/ml) and subsequently treated with staurosporine for the indicated times, before whole cell lysates were incubated with [33P]-labeled duplex oligonucleotides containing a GAS site (M67) and loaded onto a native polyacrylamide gel. The asterisk at the right-hand margin of the gel marks a non-specific band and the arrowhead corresponds to recombinant STAT1 bound to M67. (E) Time of DNA binding activity of STAT1-WT and T385A to M67 as quantified from EMSA experiments such as in (D).
Figure 2.
The T385A and F172W alterations are associated with prolonged interferon-γ-induced nuclear accumulation.
(A, B) Within one hour of addition of staurosporine to IFNγ-pretreated HeLa cells, nuclear accumulation of GFP-tagged STAT1-WT has collapsed, while nuclear retention is unaffected by staurosporine treatment in cells expressing STAT1-T385A and -F172W. (A) Microscopic images show the intracellular localization of STAT1-GFP and the corresponding Hoechst-stained nuclei. (B) The ratios of cytoplasmic-to-nuclear fluorescence intensities in differently treated cells are depicted as means and standard deviations. (C, D) Exposure of reconstituted U3A cells to staurosporine resulted in a normal loss of cytokine-induced nuclear accumulation of untagged wild-type STAT1, but significantly retarded the nuclear residence time of mutant STAT1. (C) Immunocytochemical staining using a STAT1-specific primary and Cy3-labeled secondary antibody as well as Hoechst-stained nuclei are depicted. (D) Cytosolic/nuclear fluorescence intensities were determined and presented as histograms. All experiments were performed at least three times with similar results.
Figure 3.
The STAT1 mutant T385A shows unaltered DNA-binding affinity to GAS elements.
(A, B) Gelshift experiments demonstrated that STAT1-T385A and wild-type protein exhibit a similar dissociation rate from a single STAT1-binding site (M67). Cell lysates from IFNγ-stimulated U3A cells (5 ng/ml) expressing either wild-type or mutant STAT1 were incubated with [33P]-labeled M67 for 15 min and subsequently a 750-fold molar excess of unlabeled M67 was added for the durations indicated, before the reactions were loaded onto a native polyacrylamide gel. Shown is a typical gel, with the arrowhead at its right-hand margin corresponding to dimeric STAT1, including a densitometric quantification thereof. Asterisks mark unspecific bands. (C) The T385A mutant displays cooperative DNA binding due to tetramer stabilization. Extracts from an equal number of IFNγ-stimulated U3A cells expressing either wild-type or mutant STAT1 (5 µl in each lane) were incubated in vitro with [33P]-labeled DNA containing two GAS sites separated by 10 bp (2xGAS). The reactions were either left unchallenged (−) or challenged for 30 min with a 750-fold excess of a single, unlabeled GAS site (+ Competition). Note that the tetrameric, but not the dimeric occupancy of the probe resisted competition with excess unlabeled DNA. (D) Gelshift experiments demonstrate the propensity of tetrameric STAT1-T385A to exchange dimers. Shown is a representative gel using [33P]-labeled 2xGAS and cellular extracts from IFNγ/vanadate-co-stimulated U3A cells expressing either GFP-tagged or untagged STAT1. Supershifts with either anti-STAT3 (lane 1 and 3) or anti-STAT1 (lane 2 and 4) antibodies identified bands corresponding to DNA-bound STAT1. For the identification of dimeric STAT1 complexes, a competition experiment using 750-molar excess of unlabelled GAS was included in the last lane. Similar amounts of GFP-tagged and untagged homodimers were either immediately mixed and incubated together for 45 min (lanes 6 and 8) or incubated separately for 45 min before being loaded together onto the gel (lanes 5 and 7). Note that newly formed bands corresponding to STAT-GFP/STAT1 heterotetramers appeared only in extracts that had been co-incubated. (E) The T385A mutant binds to GAS sequences as a tetramer. Whole cell extracts from reconstituted, IFNγ-prestimulated U3A cells were incubated with various [33P]-labeled DNA probes containing either two (2xGAS), one (GAS-nonGAS) or no (2xnonGAS) GAS sites, respectively. In the first lane, a non-specific anti-STAT3 antibody and in the second lane, anti-STAT1 antibody C-24 was present in the EMSA reaction used for identification of STAT1-DNA complexes (marked with arrowheads). (F) The F172W and T385A mutants are defective in tyrosine dephosphorylation as revealed by an in vitro dephosphorylation assay using purified Tc45 phosphatase. Cell extracts from reconstituted U3A cells expressing either wild-type or mutant STAT1 (10 µl in each reaction) were incubated with 2 U of the STAT1-specific Tc45 phosphatase and tyrosine dephosphorylation was monitored with time by means of Western blotting.
Figure 4.
Differential gene activation by the STAT1-F172W and -T385A mutants.
(A) Expression of the 3xLy6E reporter gene by mutant and wild-type STAT1 in unstimulated (gray columns) and IFNγ-stimulated (black columns) reconstituted U3A cells. (B) Inhibited down-regulation of 3xLy6E reporter expression following staurosporine treatment by the dimer interface mutants. U3A cells reconstituted with the indicated STAT1 variants were stimulated with 5 ng/ml IFNγ for 3 h and subsequently exposed to 1 µM staurosporine for 0, 2, and 3 h, respectively. Expression rates for each STAT1 variant were normalized to samples not treated with staurosporine. (C) Activation of five endogenous STAT1 target genes, and for control the transfected stat1 gene, in U3A cells expressing either mutant or wild-type STAT1, as determined by real-time RT-PCR. Expression levels of irf1 (C), gbp1 (D), mig1 (E), cxcl10 (F) and mcp1 (G), and for control stat1 (H), before and 6 hours after stimulation with 5 ng/ml of IFNγ are shown. Expression data, normalized to the levels of the house-keeping gene gapdh, are presented as means and standard deviations. Significant differences in the IFNγ-induced gene activation between the two STAT1 variants are marked by asterisks. All experiments were repeated at least three times with similar results.
Figure 5.
Transcriptionally active STAT1-F172W and -T385A bind to a TTC/GAA motif 10 bp upstream of a GAS site.
(A–C) STAT1-T385A and -F172W, but to a much lesser extent wild-type STAT1, bound as tetrameric complexes to a [33P]-labeled DNA fragment from the mcp1 promoter, which comprised a STAT1-binding site containing the one-and-a-half-GAS element. Significant binding of mutant STAT1 was observed by EMSA, when the GAS site was mutated, while leaving the TCC element intact (MCP1-C). Depicted are a drawing of the three MCP1 probes used in this study (A), a typical EMSA gel (B), and a quantification thereof showing densitometric signal intensities of tetramer-bound STAT1(C).