Live cell imaging reveals continuous STAT6 nuclear trafficking

Abstract

The STAT6 transcription factor is essential for the development of protective immunity; however, the consequences of its activity can also contribute to the pathogenesis of autoimmune disease. Tyrosine phosphorylation is known to activate STAT6 in response to cytokine stimulation, but there is a gap in our understanding of the mechanisms by which it enters the nucleus. In this study, live cell imaging was used in conjunction with photobleaching techniques to demonstrate the continual nuclear import of STAT6, independent of tyrosine phosphorylation. The protein domain required for nuclear entry includes the coiled coil region of STAT6 and functions similarly before or after cytokine stimulation. The dynamic nuclear shuttling of STAT6 seems to be mediated by the classical importin-alpha-importin-beta1 system. Although STAT6 is imported to the nucleus continually, it accumulates in the nucleus following tyrosine phosphorylation as a result of its ability to bind DNA. These findings will impact diagnostic approaches and strategies to block the deleterious effects of STAT6 in autoimmunity.

Figure 1. STAT6 nuclear import is independent of tyrosine phosphorylation

(A) HeLa cells were transfected with STAT6-GFP or STAT6(RY)-GFP, serum starved, and left untreated (−) or treated (+) with IL-4 for 30 minutes. Cellular localization of STAT6 was visualized by fluorescence microscopy. (B) EMSAs were performed with the radiolabeled IL-4 receptor target oligonucleotide and protein lysates from cells as treated in (A). Effects of additions of antibodies (Ab) to the reaction to GFP (G), STAT6 (S) or control (c), or 100-fold excess unlabeled oligonucleotide (DNA) are shown. (C) Western blots of protein lysates were performed with anti-phosphotyrosine 641 STAT6 (anti-pSTAT6), anti-STAT6, or anti-GFP antibodies.

Figure 2. Live cell imaging of STAT6 constitutive nuclear import

Nuclear FRAP assays were performed with cells expressing STAT6-GFP without IL-4 stimulation (upper panel) or with IL-4 stimulation (middle panel), or with cells expressing STAT6(RY)-GFP with IL-4 (lower panel). The nucleus (N) was subjected to high intensity laser to bleach the fluorescent STAT6. Subsequent fluorescence recovery with time in the nucleus was monitored and quantified relative to a site in the cytoplasm (C). The quantitative data of relative fluorescent intensity (Fl) with time are shown in the right panel. Experiments are representative of more than three independent studies.

Figure 3. Live cell imaging demonstrates decreased STAT6 nuclear export following tyrosine phosphorylation

Cytoplasmic FLIP assays were performed with cells expressing STAT6-GFP untreated (upper panel) or treated with IL-4 (lower panel). A small region in the cytoplasm (C) was subjected to continuous high intensity laser. Fluorescence loss was monitored with time in the cytoplasm and compared to the fluorescence loss in a region of the nucleus (N). The quantitative data of relative fluorescent intensity (Fl) with time is shown in the right panel. Experiments are representative of more than three independent studies.

Figure 4. DNA binding promotes nuclear accumulation of tyrosine phosphorylated STAT6

(A) The DNA binding mutant STAT6(KR)-GFP was expressed in cells and examined by fluorescence microscopy before (−) or after treatment with IL-4 (+). Western blot in the lower panel was performed with anti-phosphotyrosine 641 STAT6 (anti-pSTAT6) or anti-GFP antibodies. EMSA in the right panel was performed with the IL-4 receptor target oligonucleotide and lysates from cells expressing STAT6-GFP (wt) or STAT6(KR)-GFP (KR) without (−) or with (+) IL-4 treatment. Antibodies to STAT6 (S), GFP (G), or MOPC (c) were added to the binding reactions. (B) Live cell imaging was used with nuclear FLIP to evaluate STAT6-GFP and STAT6(KR)-GFP mobility within the nucleus. A small region in the nucleus (region 1) was subjected to continuous high intensity laser. Fluorescence loss was monitored with time in this region and a distinct region in the nucleus (region 2). Quantitative data of relative fluorescent intensity (Fl) with time is shown in the right panel. Experiments are representative of more than three independent studies.

Figure 5. Identification of sequences required for STAT6 nuclear import

(Top) Linear diagram of STAT6 functional domains and corresponding deletion mutations. Numbers indicate amino acids. (Left) Fluorescent images of STAT6-GFP deletion mutations expressed in cells serum starved (−IL-4) or stimulated with IL-4 (+IL-4). (Right) Western blot of protein lysates from cells performed with anti-phosphotyrosine 641 STAT6 (anti-pSTAT6), anti-STAT6, or anti-GFP antibodies.

Figure 6. Amino acids 136–140 are required for STAT6 nuclear import

(A) STAT6-GFP with an internal deletion of amino acids 135–140 (dl) or STAT6-GFP with a substitution of six alanine residues for amino acids 135–140 (sub6A) were expressed in cells serum starved (−) or stimulated with IL-4 (+). STAT6 localization was visualized microscopically. Bottom panel displays Western blot of protein lysates with anti-phosphotyrosine 641 STAT6 (anti-pSTAT6) or anti-GFP antibodies. (B) HeLa cells were transfected with IL-4R site-TKLuc reporter construct, pRL-null vector and different STAT6-GFP constructs. Cells were untreated or treated with hIL-4 for 8 hours before the dual-luciferase reporter assay. Luciferase result was normalized to Renilla luciferase value.

Figure 7. Evidence STAT6 import is mediated by importin-α/β system

(A) Immunoprecipitated (IP) STAT6-V5 expressed in COS1 cells was collected on protein G beads and incubated in vitro with bacterially expressed GST-importins. Western blots (WB) identified bound importins (anti-GST), and STAT6-V5 (anti-V5). 10% of importin input is shown in bottom panel (anti-GST). (B) Bacterial expressed MBP-STAT6(1–267) and MBP-STAT6(1–267dl136-140) were immobilized on amylase resin and incubated with purified GST-importin-α1 or GST-importin-α3. The binding was evaluated by Western blot with anti-GST antibody. Ponceau S (PS) staining showed the same amount of STAT6 proteins was used. (C) Fluorescent images of cells transfected with vimentin siRNA (control) or importin-β1 siRNA (impβ1) for 24 hours followed by transfection with STAT6-GFP. Cytoplasmic localization was seen in approximately 10% of cells. Lower panel displays effect of control vimentin siRNA or importin-β1 siRNA on endogenous importin-β1 mRNA or GAPDH mRNA evaluated by RT-PCR.