Phospho-Regulated SUMO Interaction Modules Connect the SUMO System to CK2 Signaling  Per Stehmeier, Stefan Muller  Molecular Cell  Volume 33, Issue 3, Pages 400-409 (February 2009) DOI: 10.1016/j.molcel.2009.01.013 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 PIAS1 Is Phosphorylated by CK2 within the SIM Module (A) Alignment of the hydrophobic-acidic SIM region of human members of the PIAS family. (B) Hydrophobic residues in PIAS1 and PIAS2 are essential for the noncovalent binding to SUMO paralogs in a directed yeast two-hybrid assay. Conjugatable (SUMOGG) or nonconjugatable (SUMOGA) versions of SUMO1 or SUMO2 were tested for interaction with wild-type PIAS1 or PIAS2 and the respective mutants. Growth of yeast was followed on control plates lacking leucine and tryptophan (−LW) and plates additionally lacking histidine (−LWH). Growth on −LWH is indicative for interaction. (C) PIAS1 is phosphorylated by CK2 at serine residues 466, 467, and 468. Recombinant proteins were purified in E. coli and incubated with recombinant CK2 in the presence of γ-32P-ATP. (D) Flag-PIAS1 and the indicated mutants were expressed in HEK293T cells, captured on anti Flag-beads, and immunoblotted by anti-Flag antibody or an antibody, which specifically recognizes phosphorylated serine residues in the context of a CK2 consensus site (mAb 9F4, Calbiochem). (E) Wild-type Flag-PIAS1 was expressed in HEK293T cells; cells were either mock treated or treated for 4 hr with the CK2 inhibitor TBB (50 μM). Phosphorylation of PIAS was monitored as in (D). Molecular Cell 2009 33, 400-409DOI: (10.1016/j.molcel.2009.01.013) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Phosphorylaton of PIAS1 Is Specific for CK2 and Induced by Cellular Stress (A) Recombinant wild-type GST-PIAS1 or GST-PIAS1S466-468A was incubated in the presence of γ-32P-ATP with a cell extract from mock-transfected cells or cells that had been transfected with the CK2α/β subunits. CK2-expressing cells were either mock treated or treated with TBB. Proteins were separated by SDS-PAGE and detected by staining either with Coomassie blue or autoradiography. (B) Experiment as in (A), but cells were either transfected with wild-type CK2α/β subunits or the wild-type β subunit together with the TBB-insensitive CK2αV66A,I174A mutant (asterisk). (C) Recombinant wild-type GST-PIAS1 or GST-PIAS1D/E469-474A was incubated with recombinant CK2 in the presence of γ-32P-ATP and processed as in (A). (D) Experiment as in (A), but GST-PIAS1 or GST-PIAS1S466-468A was incubated in the presence of γ-32P-ATP with a cell extract from mock-treated cells or cells that had been treated with 400 mM sorbitol for 1 hr or sorbitol together with TBB. Molecular Cell 2009 33, 400-409DOI: (10.1016/j.molcel.2009.01.013) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Phosphorylation Is Required for Noncovalent Interaction of PIAS1 with SUMO Paralogs (A) Wild-type PIAS1 and the indicated mutants were tested for interaction with SUMO1 or SUMO2 in the yeast two-hybrid system. To visualize the strength of interaction, serial dilutions (1:5) were spotted. (B and C) Nonconjugatable Flag-SUMO1 or Flag-SUMO2 was coexpressed with HA-tagged versions of either wild-type PIAS1 or the indicated mutants, immunoprecipitated with anti-Flag beads, and detected by either anti-HA or anti-Flag antibodies. (D) As in (B), but cells were either mock treated or treated with the CK2 inhibitor TBB. (E) GST or the respective GST-phosphoSIM versions were immobilized on glutathione Sepharose beads and incubated with 35S-labeled in vitro-transcribed/-translated SUMO1. After intensive washing, bound proteins were resolved by SDS-PAGE and visualized by autoradiography or staining with Coomassie blue. (F) Flag-PIAS1 and the indicated mutants were expressed in HEK293T cells, captured on anti Flag-beads, and immunoblotted by anti-E1B-55K antibody. Molecular Cell 2009 33, 400-409DOI: (10.1016/j.molcel.2009.01.013) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Distinct Lysine Residues on SUMO Contact the PhosphoSIM Module (A) Wild-type PIAS1 was tested for interaction with wild-type SUMO1 or SUMO2 and the indicated mutants in the yeast two-hybrid system. To visualize the strength of interaction, serial dilutions (1:5) were spotted. (B) The phospho-mimicking version PIAS1S466-468D was tested for interaction with wild-type SUMO1 or SUMO2 and the indicated mutants as in (A). (C) PIAS1S466-468K or PIAS1S466-468R was tested for interaction with wild-type SUMO1 or SUMO2 and the indicated mutants in the yeast two-hybrid system. (D) Equal expression of different SUMO forms was verified by western blotting with an antibody directed against the GAL4-DBD. Molecular Cell 2009 33, 400-409DOI: (10.1016/j.molcel.2009.01.013) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 The Integrity of the PhosphoSIM Module Is Important for the Transcriptional Coregulator Function of PIAS Family Members (A) HA-tagged wild-type PIAS1 or the indicated mutants were expressed in HeLa cells together with p53 and either empty vector or His-tagged SUMO1. His-SUMO1 conjugates were recovered on Ni-NTA beads and subjected to western blotting using anti-p53 antibodies. Expression of the respective proteins was monitored by western blotting of cell lysates. (B) C33a cells were transiently transfected with the Gal4-TK-luciferase reporter plasmid together with plasmids encoding Gal4-PIAS1 fusion proteins as indicated. Values represent the average of three independent experiments performed in triplicate, with standard deviation after normalization for the internal control renilla luciferase acitvity. The basal reporter activity was set as 1. (C) As in (B), except that the activity of the Gal4-PIAS2β was monitored in HeLa cells on a Gal4-SV40-luciferase reporter. (D) HeLa cells were transiently transfected with the pRGC luciferase reporter plasmid that harbors a p53 DNA-binding site in its promoter together with the plasmids as indicated. Values represent the average of three independent experiments (mean ± SD) after normalization for the internal control renilla luciferase activity. Molecular Cell 2009 33, 400-409DOI: (10.1016/j.molcel.2009.01.013) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 PML and PMSCL1 Possess Functional PhosphoSIM Modules (A) Alignment of the phosphoSIM module of PIAS1, PML, and PMSCL1. (B) PML is phosphorylated by CK2 at serine residues 512, 513, 514, and 517. Recombinant proteins were purified in E. coli and incubated with recombinant CK2 in the presence of γ-32P-ATP. (C) Wild-type PML and the indicated mutants were tested for interaction with SUMO1 in the yeast two-hybrid system. (D) PMSCL1 is phosphorylated by CK2 at six major residues including serines 409 and 411. PMSCL14xTS-AA has serines 305 and 353 and threonines 176 and 306 exchanged to alanine. Recombinant proteins were purified in E. coli and incubated with recombinant CK2 in the presence of γ-32P-ATP. (E) Recombinant PMSCL1 was incubated with a cell extract from mock-transfected cells or cells that had been transfected with the CK2α/β subunits. CK2-expressing cells were either mock treated or treated with TBB. Proteins were separated by SDS-PAGE and detected by staining either with Coomassie blue or autoradiography. (F) The SIM-containing region of PMSCL1WT (amino acids 342–444) and the indicated mutants were tested for interaction with SUMO1 in the yeast two-hybrid system. Molecular Cell 2009 33, 400-409DOI: (10.1016/j.molcel.2009.01.013) Copyright © 2009 Elsevier Inc. Terms and Conditions