Involvement of PIAS1 in the Sumoylation of Tumor Suppressor p53 Tomoaki Kahyo, Tamotsu Nishida, Hideyo Yasuda  Molecular Cell  Volume 8, Issue 3, Pages 713-718 (September 2001) DOI: 10.1016/S1097-2765(01)00349-5

Figure 1 Interaction of Wild-Type PIAS1 and PIAS1 Mutants with SUMO-1 and Ubc9 Wild-type PIAS1 and its mutants (C350A, C350S, and 1-318) were cloned into pGAD424 as GAL4 activation domain fusion constructs. Two-hybrid assay was performed with Y190 cells transformed with a pGAD424-PIAS1 (wild-type or mutant) and a bait plasmid expressing GAL4 DNA binding domain fused with the indicated protein. All the transformants were plated on selective medium lacking tryptophan, leucine, and histidine but containing 25 mM 3-amino-1, 2, 4-triazole to test cell growth. (A) Wild-type. (B) Mutants (C350A, C350S, and 1-318) Molecular Cell 2001 8, 713-718DOI: (10.1016/S1097-2765(01)00349-5)

Figure 2 Interaction of PIAS1 with p53 In Vitro Sf-9 cell extract expressing His-PIAS1 or GST-p53 was mixed together with 10 mM Tris-HCl (pH 7.4) containing 3 mM MgCl2, 70 mM NaCl, and 1 mM PMSF, and incubated at 25°C for 10 min. GST-p53 was trapped by Glutathione Sepharose 4B beads (Pharmacia-Amersham). After the beads had been washed thoroughly, the proteins bound to them were subjected to SDS-PAGE and Western blotted with anti-His antibody. In lanes 4, 6, and 8, Sf-9 cells transfected with empty vectors were used for the binding assay Molecular Cell 2001 8, 713-718DOI: (10.1016/S1097-2765(01)00349-5)

Figure 3 Effect of PIAS1 on the Modification of p53 by SUMO-1 in Intact Cells and In Vitro (A and B) U2OS cells were transfected with the indicated plasmids. After a 24 hr incubation, the cells were lysed with SDS-PAGE sample buffer followed by boiling at 100°C for 90 s. The samples were subjected to SDS-PAGE and Western blotted with anti-p53 (DO-1) or anti-GFP antibody. (A) Effect of wild-type PIAS1 on the sumoylation of p53. In lane 5, Flag-Flag (two Flags)-tagged SUMO-1 was used for transfection. (B) Effect of PIAS1 mutants on the sumoylation of p53. In lane 1, empty vector was used for transfection. (C and D) Enhancement of the sumoylation of p53 by the addition of PIAS1 in vitro. GST-p53 purified by Glutahione Sepharose 4B resin was subjected to the in vitro sumoylation assay described in the Experimental Procedures. The results of Western blotting using anti-SUMO-1 are shown in the upper panels, and the membranes stained with Coomasie brilliant blue R-250 (CBB) are shown in the bottom panels. (C) Sumoylation of p53 in the presence (lanes 1, 2, 4, 5, and 6) and absence (lane 3) of wild-type PIAS1. Lanes 1, 2, and 6 contained 24 ng GST-PIAS1. Lanes 4 and 5 contained 6 and 12 ng, respectively. Lane 7 contained 24 ng of C350S mutant. Lane 1 did not contain E1 and lane 2 did not contain Ubc9. (D) Sumoylation of p53 in the presence of wild-type PIAS1 (expressed in sf-9 cells, lanes 2 and 3, and expressed in E. coli, lanes 7 and 8), and 1-318 mutant (lanes 4 and 5). Lane 1 did not contain PIAS1. Lanes 2 and 4 contained 6 ng, and lanes 3 and 5 contained 24 ng. Only in the presence of wild-type PIAS1 was p53 modified by SUMO-1 Molecular Cell 2001 8, 713-718DOI: (10.1016/S1097-2765(01)00349-5)

Figure 4 Sequence Comparison of RING Finger-Like Domains of the PIAS Family All are human proteins except for the putative yeast homolog, YDR409W (accession number NC_001136). The PIAS family consists of PIAS1, PIAS3, PIASxα, PIASxβ, and PIASy (accession numbers AAC36702, NP_006090, AAC36704, XP_008706, and AAC36703, respectively). One more protein (accession number CAB66507) has a similar sequence in the RING finger domain. The conserved residues are shaded, and identical sequences are shown by black boxes. The asterisks indicate cysteine and histidine in the consensus sequence of the RING finger domain. Dotted amino acids correspond to the cysteine residues in the consensus sequence. The bars above residues show the positions of eight residues forming the RING finger domain of c-Cbl and MDM2 (accession number BAA86298 and XP_012197, respectively), which are well-known ubiquitin ligases Molecular Cell 2001 8, 713-718DOI: (10.1016/S1097-2765(01)00349-5)