1. SUMO-1 Protease-1 Regulates Gene Transcription through PML
Jennifer L. Best, Soula Ganiatsas, Sadhana Agarwal, Austin Changou, Paolo Salomoni, Orian Shirihai, Pamela B. Meluh, Pier Paolo Pandolfi, Leonard I. Zon 
Molecular Cell 
Volume 10, Issue 4, Pages (October 2002)
DOI: /S (02)

2. Figure 1 SuPr-1 Identification and Transcriptional Activity
(A) Amino acid sequence alignment of murine SuPr-1, SuPr-2, and SuPr-3, and human SENP1 and SENP2. Identical residues to SuPr-1 are shaded black, and similar residues are shaded gray. Asterisks mark the core catalytic residues conserved in cysteine proteases (His-396, Asp-413, and Cys-466).
(B) Nucleotide sequence comparison of the N-terminal cDNA sequences of SuPr-1, SMT3IP2, and SENP2. Putative ATG initiation codons are boxed.
(C) Northern analysis of SuPr-1 on a murine tissue blot (Clontech).
(D) Activation of Gal4-c-Jun by murine SuPr-1, SuPr-2, and SuPr T cells were transfected with SuPr-1, 2, or 3 plasmids (1 ug), Gal4-luciferase reporter plasmid (1 ug), and either Gal4-dbd alone or Gal4-c-Jun (50 ng) plasmids. Data is presented as fold activation over Gal4-c-Jun plus Gal4-Luciferase.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2 SuPr-1 Does Not Require c-Jun Phosphorylation
(A) Western blot analysis of c-Jun phosphorylation. Lysates from cells transfected with the indicated plasmids were analyzed with anti-Jun phospho-Ser-73 antisera.
(B) SuPr-1 but not MEKK1 activates Gal4-c-Jun Ala 63/73. Increasing doses of SuPr-1 or MEKK1 plasmids were cotransfected with a reporter and either Gal4-c-Jun or Gal4-c-Jun Ala 63/73 plasmids.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3 SuPr-1 Is a SUMO-1 Protease
(A) SuPr-1 hydrolyzes SUMO-1 from PML and RanGAP1 in vitro. GST-PML and GST-RanGAP1 conjugated to HA-SUMO were precipitated and incubated with purified SuPr-1 or SuPr-1 C466S mutant for the indicated time. Western blot analysis was performed with anti-GST mAb (upper panel) followed by stripping and reprobing the blot with anti-HA mAb (lower panel). Unmodified GST-PML and GST-RanGAP1 (dashes) and SUMO-1-modified forms of GST-PML (bracket) and GST-RanGAP1 (arrowhead) are indicated.
(B) SuPr-1 hydrolyzes SUMO-1-modified PML in vivo. Whole-cell lysates from 293T cells transfected with the indicated expression plasmids were analyzed.
(C) SuPr-1 does not hydrolyze SUMO-1-modified RanGAP1 (asterisk) in vivo. Note that endogenous SUMO-1 also modifies GST-PML and GST-RanGAP1 in vivo ([C], lane 5; [D], lanes 1 and 2).
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4 Binding to SUMO-1 Is Required for Activation of Transcription
(A) SuPr-1 interacts directly with PML. HA-PML plasmid was cotransfected into 293T cells with the indicated GST fusion plasmids for SuPr-1 wild-type (WT), C466S, F359A, F359A/C466S, W375A, and W375A/C466S. Cell lysates were precipitated with glutathione sepharose and analyzed by Western blotting. The blot was probed with anti-HA mAb (upper), then stripped and reprobed with anti-GST mAb (middle). A blot of the whole-cell lysates for this experiment was probed with anti-HA mAb (lower).
(B) Activation of Gal4-c-Jun by SuPr-1 mutants. Gal4-luciferase reporter plasmid (1 ug) and Gal4-c-Jun (50 ng) plasmids were cotransfected into 293T cells with GST-SuPr-1 plasmids from (A).
(C) Increasing amounts of SUMO-1 expression plasmid were cotransfected with Gal4-luciferase reporter plasmid (1 ug), Gal4-c-Jun (50 ng) plasmid, and the indicated HA-SuPr-1 or HA-SuPr-1 C466S plasmids.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5 SuPr-1 Is Localized to Nuclear PODs and Disperses SUMO-1
(A) HeLa cells were transfected with GFP-tagged SuPr-1 or SuPr-1 C466S plasmids, and expression was detected by direct fluorescence (green). Cell nuclei were visualized by DAPI staining (blue). Localization of SuPr-1 to the nucleus is shown (merge).
(B) SuPr-1 affects endogenous SUMO-1 expression. Endogenous SUMO-1 for the same field of cells was detected using anti-SUMO-1 mAb (anti-GMP1) and anti-mouse IgG, rhodomine-conjugated secondary antibodies (red). Colocalization of endogenous SUMO-1 with GFP-tagged SuPr-1 C466S mutant is indicated in yellow (bottom merge). Scale represents 10 μm.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
SuPr-1 Expression Alters the Distribution of PML and Nuclear POD-Associated Proteins, CBP and Daxx
(A) HeLa cells were transfected with the indicated HA-tagged SuPr-1 or SuPr-1 mutant plasmids, C466S, F359A, F359A/C466S, W375A, and W375A/C466S. Endogenous PML (red) and HA (green) for the same fields of cells were visualized by immunofluorescence using polyclonal rabbit anti-PML Ab and anti-HA mAb with rhodamine- and fluorescein-conjugated secondary antibodies, respectively.
(B and C) HeLa cells were transfected with HA-tagged SuPr-1 or SuPr-1 C466S plasmids. Endogenous CBP (B) and endogenous Daxx (C) were detected using rabbit polyclonal antisera and rhodamine-conjugated secondary antibodies (red).
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7 PML and CBP Contribute to SuPr-1 Activation of Transcription
(A) PML activates Gal4-c-Jun. Gal4-Luc reporter plasmid was transfected into 293T cells with Gal4-c-Jun plasmid and increasing doses of PML plasmid.
(B) PML enhances SuPr-1 activation of Gal4-c-Jun. Doses of PML plasmid were cotransfected with empty vector or SuPr-1 plasmid. In order to demonstrate the enhancement of SuPr-1 activity by PML, each dose of PML alone (without SuPr-1) was set at 1, and data are expressed as fold activation for each dose. The data are representative of three independent experiments.
(C) Activation of Gal4-c-Jun by SuPr-1 is inhibited in PML−/− cells. Primary murine embryonic fibroblasts (MEF) were transfected with the Gal4-Luciferase reporter construct and indicated combinations of Gal4-c-Jun, SuPr-1, and PML plasmids. Luciferase activity is normalized to an internal Renilla luciferase control.
(D) PMLΔSUMO mutant does not activate Gal4-c-Jun. Increasing amounts of wild-type PML or PML K65/160/490R plasmids were transfected with Gal4-c-Jun plasmid and the Gal4-Luc reporter plasmid.
(E) PML enhances hydrolysis of SUMO-1-modified proteins. 293T cells were transfected with the indicated plasmids in addition to SUMO-1 plasmid. Cells were lysed in RIPA buffer at 24 hr posttransfection, precipitated with glutathione sepharose beads, and analyzed by Western blotting with anti-SUMO-1 mAbs. The blots for the precipitated proteins (upper panel) and the whole lysate (lower panel) are shown.
(F) CBP activates Gal4-c-Jun. 293T cells were transfected with Gal4-c-Jun, Gal4-Luc reporter, and increasing amounts of CBP plasmid.
(G) CBP synergizes with SuPr-1 to activate Gal4-c-Jun. Doses of CBP plasmid were cotransfected with empty vector or SuPr-1 as in (B) and graphed as fold activation over CBP alone for each dose. Data is presented as fold activation over control with reporter plasmid.
(H) 293T cells were transfected with either pSRE-Luc or C-TRE-Luc reporter plasmids, and either SuPr-1 or control plasmids.
(I) RA-dependent induction of RARB2 promoter is enhanced by SuPr-1. Cos-1 cells were transfected with pRARB2-luc reporter plasmid and increasing does of SuPr-1. Cells were treated with or without RA (1 μm) for 48 hr. Data is presented in light units.
Molecular Cell  , DOI: ( /S (02) )