Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.
Post-translational modifications control the physiological activity of the signal transducer and activator of transcription STAT1. While phosphorylation at tyrosine Y701 is a prerequisite for STAT1 dimerization, its SUMOylation represses the transcriptional activity. Recently, we have demonstrated that SUMOylation at lysine K703 inhibits the phosphorylation of nearby localized Y701 of STAT1. Here, we analysed the influence of phosphorylation of Y701 on SUMOylation of K703 in vivo. For that reason, an Ubc9/substrate dimerization-dependent SUMOylation (USDDS) system was developed, which consists of fusions of the SUMOylation substrate and of the SUMO-conjugating enzyme Ubc9 to the chemically activatable heterodimerization domains FKBP and FRB, respectively. When FKBP fusion proteins of STAT1, p53, CRSP9, FOS, CSNK2B, HES1, TCF21 and MYF6 are coexpressed with Ubc9-FRB, treatment of HEK293 cells with the rapamycin-related dimerizer compound AP21967 induces SUMOylation of these proteins in vivo. For STAT1-FKBP and p53-FKBP we show that this SUMOylation takes place at their specific SUMOylation sites in vivo. Using USDDS, we then demonstrate that STAT1 phosphorylation at Y701 induced by interferon-beta treatment inhibits SUMOylation of K703 in vivo. Thus, pY701 and SUMO-K703 of STAT1 represent mutually exclusive modifications, which prevent signal integration at this molecule and probably ensure the existence of differentially modified subpopulations of STAT1 necessary for its regulated nuclear cytoplasmic activation/inactivation cycle.
Zimnik
Susan
S
Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.
Gaestel
Matthias
M
Niedenthal
Rainer
R
D002460:Cell Line; D019281:Dimerization; D006801:Humans; D011499:Protein Processing, Post-Translational; D050794:STAT1 Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
C539629:AP 21967; D050794:STAT1 Transcription Factor; D025841:Small Ubiquitin-Related Modifier Proteins; D014443:Tyrosine; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D020123:Sirolimus