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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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Schematic representation of the USDDS. The SUMOylation substrate of choice is fused to one of the heterodimerization domains (FRB) and Ubc9 to the other (FKBP). When the fusion proteins are coexpressed in HEK293 cells, incubation with the membrane permeable compound AP21967 induces heterodimerization of the two fusion proteins. As a result, the SUMO-loaded conjugating enzyme Ubc9 is brought in close proximity to the substrate of interest and effective SUMO conjugation of the substrate occurs.
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Figure 1: Schematic representation of the USDDS. The SUMOylation substrate of choice is fused to one of the heterodimerization domains (FRB) and Ubc9 to the other (FKBP). When the fusion proteins are coexpressed in HEK293 cells, incubation with the membrane permeable compound AP21967 induces heterodimerization of the two fusion proteins. As a result, the SUMO-loaded conjugating enzyme Ubc9 is brought in close proximity to the substrate of interest and effective SUMO conjugation of the substrate occurs.

Mentions: Post-translational protein modifications can act separately, together or even counteract each other to integrate extracellular signals and to ensure a specific function of a protein (1). To characterize the interplay of SUMOylation with other covalent modifications in a direct and controlled way, we generated an inducible USDDS system (Figure 1). USDDS replaces the static protein fusion-immanent properties of UFDS (11) with the ‘protein matchmaker’ approach utilizing rapamycin-induced heterodimer formation between the 12 kDa-FK506-binding protein (FKBP12) and the 12 kDa-FKBP12-rapamycin-associated protein (FRB) which together form a relatively stable ternary complex (14). Accordingly, we fused the appropriate protein domains of FKBP and FRB, which can be heterodimerized by the synthetic rapamycin derivative AP21967 (15), to Ubc9 and the SUMOylation substrate of interest, respectively.Figure 1.


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

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

Schematic representation of the USDDS. The SUMOylation substrate of choice is fused to one of the heterodimerization domains (FRB) and Ubc9 to the other (FKBP). When the fusion proteins are coexpressed in HEK293 cells, incubation with the membrane permeable compound AP21967 induces heterodimerization of the two fusion proteins. As a result, the SUMO-loaded conjugating enzyme Ubc9 is brought in close proximity to the substrate of interest and effective SUMO conjugation of the substrate occurs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651805&req=5

Figure 1: Schematic representation of the USDDS. The SUMOylation substrate of choice is fused to one of the heterodimerization domains (FRB) and Ubc9 to the other (FKBP). When the fusion proteins are coexpressed in HEK293 cells, incubation with the membrane permeable compound AP21967 induces heterodimerization of the two fusion proteins. As a result, the SUMO-loaded conjugating enzyme Ubc9 is brought in close proximity to the substrate of interest and effective SUMO conjugation of the substrate occurs.
Mentions: Post-translational protein modifications can act separately, together or even counteract each other to integrate extracellular signals and to ensure a specific function of a protein (1). To characterize the interplay of SUMOylation with other covalent modifications in a direct and controlled way, we generated an inducible USDDS system (Figure 1). USDDS replaces the static protein fusion-immanent properties of UFDS (11) with the ‘protein matchmaker’ approach utilizing rapamycin-induced heterodimer formation between the 12 kDa-FK506-binding protein (FKBP12) and the 12 kDa-FKBP12-rapamycin-associated protein (FRB) which together form a relatively stable ternary complex (14). Accordingly, we fused the appropriate protein domains of FKBP and FRB, which can be heterodimerized by the synthetic rapamycin derivative AP21967 (15), to Ubc9 and the SUMOylation substrate of interest, respectively.Figure 1.

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.

Show MeSH