title: Green fluorescent protein-tagging reduces the nucleocytoplasmic shuttling specifically of unphosphorylated STAT1.
abstract: Fluorescence recovery after photobleaching (FRAP) and related techniques using green fluorescent protein (GFP)-tagged proteins are widely used to study the subcellular trafficking of proteins. It was concluded from these experiments that the cytokine-induced nuclear import of tyrosine-phosphorylated (activated) signal transducer and activator of transcription 1 (STAT1) was rapid, while the constitutive shuttling of unphosphorylated STAT1 was determined to be inefficient. However, unrelated experiments came to different conclusions concerning the constitutive translocation of STAT1. Because these discrepancies have not been resolved, it remained unclear whether or not unphosphorylated STAT1 is a relevant regulator of cytokine-dependent gene expression. This study was initiated to examine the influence of GFP-tagging on the nucleocytoplasmic shuttling of phosphorylated and unphosphorylated STAT1. In accordance with previous findings our results confirm the undisturbed rapid nuclear import of GFP-tagged activated STAT1. However, we reveal an inhibitory influence of GFP specifically on the constitutive nucleocytoplasmic cycling of the unphosphorylated protein. The decreased shuttling of unphosphorylated STAT1-GFP significantly reduced the activation level while nuclear accumulation was prolonged. Importantly, despite unimpaired nuclear import of activated STAT1 the transcription of a STAT1-dependent reporter gene was more than halved after GFP-tagging, which could be linked directly to reduced nucleocytoplasmic shuttling. In conclusion, it is demonstrated that GFP-based techniques considerably underestimate the actual shuttling rate of unphosphorylated native STAT1. The results confirm that the activation of STAT1 and hence its transcriptional activity is proportional to the nucleocytoplasmic shuttling rate of the unphosphorylated protein. Moreover, our data indicate that GFP-tagging may differently affect the mechanistically distinct translocation pathways of a shuttling protein.
authors.affiliation: Abteilung Psychosomatische Medizin und Psychotherapie, Philipps-Universität Marburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Meyer
authors.forename: Andreas
authors.initials: A
authors.lastname: Begitt
authors.forename: Uwe
authors.initials: U
authors.lastname: Vinkemeier
mesh_terms: D021581:Active Transport, Cell Nucleus; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; D049452:Green Fluorescent Proteins; D014443:Tyrosine