Results: 4

1.
Figure 1.

Figure 1. From: Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

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.

Susan Zimnik, et al. Nucleic Acids Res. 2009 March;37(4):e30-e30.
2.
Figure 3.

Figure 3. From: Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

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.

Susan Zimnik, et al. Nucleic Acids Res. 2009 March;37(4):e30-e30.
3.
Figure 2.

Figure 2. From: Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

AP21967-induced in vivo SUMOylation of STAT1 and p53. (A) STAT1-FRB and EGFP-SUMO1 or (B) p53-FRB and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (−) or together with Ubc9-FKBP (+). (C) STAT1-FKBP and EGFP-SUMO1 or (D) p53-FKBP and EGFP-SUMO1 or (E) CRSP9-FKBP and EGFP-SUMO1 or (F) TCF21-FKBP and EGFP-SUMO1 or (G) FOS-FKBP and EGFP-SUMO1 or (H) CSNK2B-FKBP and EGFP-SUMO1 or (I) MYF6-FKBP and EGFP-SUMO1 or (J) HES1-FKBP and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (−) or together with Ubc9-FRB (+). After 24 h, the cells were stimulated with the AP21967 (1 µM) for the indicated times. Fusion proteins in the extracts of the transfectants were detected by western blot using (A and C) a STAT1 antibody (α-STAT1), (B and D) a p53 antibody (α-p53) or (E–J) a FKBP antibody (α-FKBP). E-S1-STAT1-FRB(FKBP) = STAT1-FRB(FKBP) fusion protein conjugated with coexpressed EGFP-SUMO1; E-S1-p53-FRB(FKBP) = p53-FRB(FKBP) conjugated with coexpressed EGFP-SUMO1; E-S1-CRSP9-FKBP = CRSP9-FKBP conjugated with coexpressed EGFP-SUMO1; E-S1-TCF21-FKBP = TCF21-FKBP conjugated with coexpressed EGFP-SUMO1; E-S1-FOS-FKBP = FOS-FKBP conjugated with coexpressed EGFP-SUMO1; E-S1-CSNK2B-FKBP = CSNK2B-FKBP conjugated with coexpressed EGFP-SUMO1; E-S1-MYF6-FKBP = MYF6-FKBP conjugated with coexpressed EGFP-SUMO1; E-S1-HES1-FKBP = HES1-FKBP conjugated with coexpressed EGFP-SUMO1; and E = EGFP-Tag.

Susan Zimnik, et al. Nucleic Acids Res. 2009 March;37(4):e30-e30.
4.
Figure 4.

Figure 4. From: Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation.

Mutually exclusive in vivo phosphorylation of Y701 and SUMOylation of K703 in STAT1. (A–C) For USDDS, STAT1-FRB or STAT1-FKBP was coexpressed with Ubc9-FKBP or Ubc9-FRB and EGFP-SUMO1 in HEK293 cells. After 24 h, the transfectants were stimulated with interferon-β (1/2 h or 1 h) or left unstimulated (–) and were subsequently treated with AP21967 (1 µM). (B and C) Where indicated transfectants were treated (2 h) with AP21967 (1 µM) first and subsequently stimulated with interferon-β (1 h). The proteins of the transfectants were immunoblotted with a phospho (p)Y701 STAT1 antibody (α-pY701 STAT1), stripped and re-probed with a STAT1 antibody (α-STAT1) to detect also non-phosphorylated and SUMOylated STAT1. (D) Schematic representation of the role of mutually exclusive STAT1 modifications. The phosphorylation site Y701 of STAT1 and the SUMOylation site K703 are in close proximity. Receptor activation, e.g. by interferon-β induces phosphorylation at Y701. This is a prerequisite for the STAT1 dimerization, nuclear import and transcriptional activation and inhibits SUMOylation at K703. STAT1 is inactivated by a nuclear phosphatase (PPase). Dephosphorylated STAT1 is then a potential substrate for SUMOylation that inhibits nuclear re-phosphorylation of Y701 of STAT1 and is possibly involved in transcriptional reprogramming, nuclear export or regulation of further STAT1 modifications such as acetylation. SUMOylation of cytoplasmic STAT1 inhibits Y701 phosphorylation and could be involved in nuclear import and regulation of preceding STAT1 modifications like acetylation. E-S1-STAT1-FRB or -FKBP = STAT1-FRB or -FKBP fusion protein conjugated with coexpressed EGFP-SUMO1, P-STAT1-FRB or -FKBP = STAT1-FRB or -FKBP phosphorylated at Y701, E = EGFP-Tag. E-S1-STAT1-FRB or -FKBP, P-STAT1-FRB or -FKBP, endogenous P-STAT1, STAT1-FRB or -FKBP and endogenous STAT1 are indicated by black arrow heads. In the upper blot, the open arrow head indicates the positions of the E-S1-STAT1-FRB or -FKBP that are not decorated by the pY701-STAT1 antibody (α-pY701-STAT1).

Susan Zimnik, et al. Nucleic Acids Res. 2009 March;37(4):e30-e30.

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