Photocontrol of STAT6 dimerization and translocation.
Phosphorylation of signal transducers and activators of transcription (STAT) proteins is a key step in activation of the JAK-STAT signal transduction cascade. Here we describe an approach to selectively analyze the impact of STAT6 phosphorylation on STAT dimerization, DNA binding and nuclear import by accessing six types of semisynthetic STAT6 proteins via expressed protein ligation (EPL). All STAT6 analogs carry a modified C-terminus, which comprises the key phosphorylation site (Tyr641) responsible for homodimerization via reciprocal SH2 domain binding, a fluorescence label and a hexahistidine tag. Protecting the phosphate moiety of tyrosine residue 641 by a photolabile protection (caged) group in two of the STAT6 variants allowed control over STAT6 dimerization. Using electrophoretic mobility shift assay (EMSA) techniques, comparative studies with STAT6 analogues proved the need for dimerization for the successful binding to oligonucleotides carrying the gamma-activated site (GAS) sequence. Furthermore, we demonstrated that light-induced decaging fully restored dimerization and DNA binding of STAT6. In addition to binding experiments, we focused on the influence of caged Tyr641 on nuclear import by microinjection of STAT6 into the cytoplasm of living cells. Decaging of monomers resulted in a rapid increase of the fluorescence signal inside the nucleus and a simultaneous decrease in the cytoplasm. We have established a powerful light-controllable system by EPL for analyzing STAT6 in vitro and in live cells that can be used for studying phosphorylation-dependent signal transduction as well as for setting up screening systems for inhibitors of STAT6 dimerization and translocation.
Lahiri
Sunanda
S
Max-Planck-Institute of Molecular Physiology, Dept. of Physical Biochemistry, Otto-Hahn-Str. 11, 44227 Dortmund, Germany.
Seidel
Ralf
R
Engelhard
Martin
M
Becker
Christian F W
CF
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D053612:Janus Kinases / Q000378:metabolism; D008027:Light; D050505:Mutant Proteins / Q000378:metabolism; D055503:Protein Multimerization / Q000528:radiation effects*; D021381:Protein Transport / Q000528:radiation effects; D050800:STAT6 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000528:radiation effects
D050505:Mutant Proteins; D050800:STAT6 Transcription Factor; D053612:Janus Kinases