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Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

Zimnik S, Gaestel M, Niedenthal R - Nucleic Acids Res. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.

ABSTRACT
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.

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USDDS results in SUMOylation of STAT1 and p53 at their specific SUMOylation sites. EGFP-SUMO1 and (A) STAT1-FRB or STAT1-K703R-FRB, (B) p53-FRB or p53-K386R-FRB were coexpressed in HEK293 cells either alone (−) or together with Ubc9-FKBP (+). After 24 h, the cells were stimulated with AP21967 (1 µM) for the indicated times. Fusion proteins in the extracts of the transfectants were detected by western blot using (A) a STAT1 antibody (α-STAT1) or (B) a p53 antibody (α-p53). After stripping the Ubc9-FKBP was detected with the Ubc9 antibody (α-Ubc9) and after a second stripping EGFP-SUMO1 and SUMOylated proteins (EGFP-S1 proteins) were detected with the SUMO1 antibody (α-SUMO1). E-S1-STAT1-FRB = STAT1-FRB fusion protein conjugated with coexpressed EGFP-SUMO1; E-S1-p53-FRB = p53-FRB conjugated with coexpressed EGFP-SUMO1; and E = EGFP-Tag.
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Figure 3: USDDS results in SUMOylation of STAT1 and p53 at their specific SUMOylation sites. EGFP-SUMO1 and (A) STAT1-FRB or STAT1-K703R-FRB, (B) p53-FRB or p53-K386R-FRB were coexpressed in HEK293 cells either alone (−) or together with Ubc9-FKBP (+). After 24 h, the cells were stimulated with AP21967 (1 µM) for the indicated times. Fusion proteins in the extracts of the transfectants were detected by western blot using (A) a STAT1 antibody (α-STAT1) or (B) a p53 antibody (α-p53). After stripping the Ubc9-FKBP was detected with the Ubc9 antibody (α-Ubc9) and after a second stripping EGFP-SUMO1 and SUMOylated proteins (EGFP-S1 proteins) were detected with the SUMO1 antibody (α-SUMO1). E-S1-STAT1-FRB = STAT1-FRB fusion protein conjugated with coexpressed EGFP-SUMO1; E-S1-p53-FRB = p53-FRB conjugated with coexpressed EGFP-SUMO1; and E = EGFP-Tag.

Mentions: To prove that USDDS displays specificity for the in vivo SUMOylation sites of p53 and STAT1, we coexpressed the mutant proteins p53K386R-FRB and STAT1K703R-FRB with Ubc9-FKBP and EGFP-SUMO1. In HEK293 cells, we could not identify any SUMOylation of STAT1K703R-FRB (Figure 3A) but only weak second site SUMOylation of p53K386R-FRB (Figure 3B) which was also detected with the UFDS system (11). Obviously, the induced heterodimerization of the Ubc9 fusion protein with the substrate fusion proteins leads preferentially to a modification at their specific SUMOylation sites. This let us suppose that USDDS is a useful tool to analyse the dynamic interplay between SUMOylation and other protein modifications.Figure 3.


Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

Zimnik S, Gaestel M, Niedenthal R - Nucleic Acids Res. (2009)

USDDS results in SUMOylation of STAT1 and p53 at their specific SUMOylation sites. EGFP-SUMO1 and (A) STAT1-FRB or STAT1-K703R-FRB, (B) p53-FRB or p53-K386R-FRB were coexpressed in HEK293 cells either alone (−) or together with Ubc9-FKBP (+). After 24 h, the cells were stimulated with AP21967 (1 µM) for the indicated times. Fusion proteins in the extracts of the transfectants were detected by western blot using (A) a STAT1 antibody (α-STAT1) or (B) a p53 antibody (α-p53). After stripping the Ubc9-FKBP was detected with the Ubc9 antibody (α-Ubc9) and after a second stripping EGFP-SUMO1 and SUMOylated proteins (EGFP-S1 proteins) were detected with the SUMO1 antibody (α-SUMO1). E-S1-STAT1-FRB = STAT1-FRB fusion protein conjugated with coexpressed EGFP-SUMO1; E-S1-p53-FRB = p53-FRB conjugated with coexpressed EGFP-SUMO1; and E = EGFP-Tag.
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Figure 3: USDDS results in SUMOylation of STAT1 and p53 at their specific SUMOylation sites. EGFP-SUMO1 and (A) STAT1-FRB or STAT1-K703R-FRB, (B) p53-FRB or p53-K386R-FRB were coexpressed in HEK293 cells either alone (−) or together with Ubc9-FKBP (+). After 24 h, the cells were stimulated with AP21967 (1 µM) for the indicated times. Fusion proteins in the extracts of the transfectants were detected by western blot using (A) a STAT1 antibody (α-STAT1) or (B) a p53 antibody (α-p53). After stripping the Ubc9-FKBP was detected with the Ubc9 antibody (α-Ubc9) and after a second stripping EGFP-SUMO1 and SUMOylated proteins (EGFP-S1 proteins) were detected with the SUMO1 antibody (α-SUMO1). E-S1-STAT1-FRB = STAT1-FRB fusion protein conjugated with coexpressed EGFP-SUMO1; E-S1-p53-FRB = p53-FRB conjugated with coexpressed EGFP-SUMO1; and E = EGFP-Tag.
Mentions: To prove that USDDS displays specificity for the in vivo SUMOylation sites of p53 and STAT1, we coexpressed the mutant proteins p53K386R-FRB and STAT1K703R-FRB with Ubc9-FKBP and EGFP-SUMO1. In HEK293 cells, we could not identify any SUMOylation of STAT1K703R-FRB (Figure 3A) but only weak second site SUMOylation of p53K386R-FRB (Figure 3B) which was also detected with the UFDS system (11). Obviously, the induced heterodimerization of the Ubc9 fusion protein with the substrate fusion proteins leads preferentially to a modification at their specific SUMOylation sites. This let us suppose that USDDS is a useful tool to analyse the dynamic interplay between SUMOylation and other protein modifications.Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physiological Chemistry/Biochemistry, Medical School Hannover, Carl-Neuberg Strasse 1, 30625 Hannover, Germany.

ABSTRACT
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.

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