Real time analysis of STAT3 nucleocytoplasmic shuttling.
The transcription factor STAT3 is most important for the signal transduction of interleukin-6 and related cytokines. Upon stimulation cytoplasmic STAT3 is phosphorylated at tyrosine 705, translocates into the nucleus, and induces target genes. Notably, STAT proteins are also detectable in the nuclei of unstimulated cells. In this report we introduce a new method for the real time analysis of STAT3 nucleocytoplasmic shuttling in living cells which is based on the recently established fluorescence localization after photobleaching (FLAP) approach. STAT3 was C-terminally fused with the cyan (CFP) and yellow (YFP) variants of the green fluorescent protein. In the resulting STAT3-CFP-YFP (STAT3-CY) fusion protein the YFP can be selectively bleached using the 514-nm laser of a confocal microscope. This setting allows studies on the dynamics of STAT3 nucleocytoplasmic transport by monitoring the subcellular distribution of fluorescently labeled and selectively bleached STAT3-CY. By this means we demonstrate that STAT3-CY shuttles continuously between the cytosol and the nucleus in unstimulated cells. This constitutive shuttling does not depend on the phosphorylation of tyrosine 705 because a STAT3(Y705F)-CY mutant shuttles to the same extent as STAT3-CY. Experiments with deletion mutants reveal that the N-terminal moiety of STAT3 is essential for shuttling. Further studies suggest that a decrease in STAT3 nuclear export contributes to the nuclear accumulation of STAT3 in response to cytokine stimulation. The new approach presented in this study is generally applicable to any protein of interest for analyzing nucleocytoplasmic transport mechanisms in real time.
Pranada
Albert L
AL
Institut für Biochemie, Universitätsklinikum Rheinisch-Westfälische Technische Hochschule Aachen, Pauwelsstrasse 30, Aachen 52057, Germany.
Metz
Silke
S
Herrmann
Andreas
A
Heinrich
Peter C
PC
Müller-Newen
Gerhard
G
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D007834:Lasers; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D016207:Cytokines; D004268:DNA-Binding Proteins; D015850:Interleukin-6; D008164:Luminescent Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D014443:Tyrosine