Together with recent data from other laboratories, they extend the view that Siz/PIAS proteins can function as SUMO ligases in yeast and mammals (7C9, 25). on glutathione-Sepharose 4B beads (Amersham Pharmacia) according to the manufacturer’s protocol. MBP-Ubc9 was purified on an amylose resin (New England Biolabs) according to the manufacturer’s protocol. The heterodimeric E1 was purified from bacteria expressing His-tagged Uba2 together with GST-tagged Aos1 as explained (20). For sumoylation, the substrates were translated by using the TNT quick-coupled reticulocyte lysate system (Promega); 1.5 l of translation product was incubated for 2 h at 30C in a 20-l reaction (50 mM Tris, pH 7.5/5 mM MgCl2/2 mM ATP) containing 100 ng E1 (Aos1/Uba2), 50 ng of Ubc9, and 2 g of SUMO-GG. GST-PIAS proteins were added in a concentration range from 10 to 100 ng. GST-Pulldown Assays. [35S]Methionine-labeled translated proteins were incubated with the relevant GST-fusion proteins loaded on glutathione-Sepharose 4b beads for 2 h at 4C in GST binding buffer (120 mM NaCl/50 mM Tris, pH 8/0.25% Nonidet P-40/1 mM PMSF/1 mM DTT). Beads were washed three times with binding buffer, and bound proteins were Gemilukast eluted with SDS sample buffer and analyzed by gel electrophoresis, followed Gemilukast by autoradiography. Reporter Gene Assays. Cells were transfected in six-well dishes as explained above by using 200 ng of a firefly luciferase reporter gene plasmid (pRGC, provided by M. Oren) that harbors a p53 DNA binding site in its promoter. To normalize for transfection efficiency, 50 ng of a pRL-SV40 renilla luciferase control vector (Promega) were cotransfected. Twenty-four hours after transfection, cells were lysed in 500 l of passive lysis buffer, and 20 l of cell extract were analyzed with the Dual Luciferase Reporter Assay (Promega) by using a Berthold Lumat LB9507 apparatus. Results PIAS1 and PIASx Stimulate Sumoylation of p53 and c-Jun sumoylation assay, where a 35S-labeled substrate, generated by translation, serves as a target for SUMO modification in the presence of recombinant E1 (the Aos1/Uba2 heterodimer), recombinant Ubc9, SUMO-1, and ATP. In this system, quite a strong sumoylation of p53 had been observed previously without any additional components (4C6, 21). However, these experiments were performed in the presence of relatively high amounts of Ubc9. When reducing the amount of Ubc9, only minimal sumoylation of p53 was observed (Fig. ?(Fig.11 and translated and incubated either in the absence (?) or presence (+) of the assay mix made up of recombinant E1 (Aos1/Uba2), Rabbit Polyclonal to RPS6KC1 Ubc9, and SUMO-1. Where indicated, GST-PIAS1 (and sumoylation experiment. As shown in Fig. ?Fig.11was utilized for sumoylation as explained in Fig. ?Fig.1.1. GST-p53 was used at a concentration of 50 ng and GST-PIASx at 250 ng. p53 was detected by immunoblotting by using an anti-p53 antibody. To further examine whether the Gemilukast SUMO ligase activity of PIAS proteins is restricted to p53, we tested various other SUMO targets in this system. PIAS did not exert any effects on sumoylation of the IB protein (Fig. ?(Fig.3A3translated and incubated either in the absence (?) or presence (+) of the assay mix as explained in Fig. ?Fig.1.1. In GST-pulldown assays. Consistent with the p53CPIAS1 interactions found in yeast-two-hybrid conversation assays, translated 35S-labeled p53 bound GST-PIAS1. In addition, we observed binding to GST-PIASx but not the GST-control beads or beads loaded with a fragment of PIASx made up of only the zinc-finger domain name (amino acids 336C397) (Fig. ?(Fig.44translated p53 (translated 35S-labeled PIASx with GST-Ubc9, GST-p53 as a positive control, and GST as a negative control. As shown in Fig. ?Fig.55translated PIASx (translated 35S-labeled PIASx was used in the sumoylation assay without additional substrate proteins. As shown in Fig. ?Fig.6,6, PIASx is strongly sumoylated when incubated with E1, Ubc9, and SUMO-1 indicated by the appearance of at least three higher molecular excess weight SUMO conjugates. The identity of these bands was verified by immunoblotting with an anti-SUMO antibody (not shown). Sumoylation of PIASxC362S was severely compromised, as only minimal conjugation occurs. Intriguingly, addition of recombinant wild-type PIASx protein could at least partially restore the modification of PIASxC362S. This indicates that PIAS proteins not only mediate the transfer of SUMO to heterologous substrates but also to themselves. Consistent with the data from these experiments, we.