The protein inhibitor of activated STAT1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. Here we used quantitative SUMO proteomics to identify potential substrates of PIAS1 in a system-wide manner. We identified 983 SUMO sites on 544 proteins, of which 62 proteins were assigned as putative PIAS1 substrates. In particular, vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, was SUMOylated by PIAS1 at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly and cells expressing a non-SUMOylatable VIM mutant showed a reduced level of migration. Our approach not only enables the identification of E3 SUMO ligase substrates but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.

Protein inhibitor of activated STAT (PIAS) proteins are E3 SUMO ligases playing important roles in protein stability and signaling transduction pathways. PIAS proteins are overexpressed in the triple-negative breast cancer cell line MDA-MB-231, and PIAS knockout (KO) results in a reduction in cell proliferation and cell arrest in the S phase. However, the molecular mechanisms underlying PIAS functions in cell proliferation and cell cycle remain largely unknown. Here, we used quantitative SUMO proteomics to explore the regulatory role of PIAS SUMO E3 ligases upon CRISPR/Cas9 KO of individual PIAS. A total of 1422 sites were identified, and around 10% of SUMO sites were regulated following KO of one or more PIAS genes. We identified protein substrates that were either specific to individual PIAS ligase or regulated by several PIAS ligases. Ki-67 and TOP2A, which are involved in cell proliferation and epithelial-to-mesenchymal transition, are SUMOylated at several lysine residues by all PIAS ligases, suggesting a level of redundancy between these proteins. Confocal microscopy and biochemical experiments revealed that SUMOylation regulated TOP2A protein stability, while this modification is involved in the recruitment of Ki-67 nucleolar proteins containing the SUMO interacting motif. These results provide novel insights into both the redundant and specific regulatory mechanisms of cell proliferation and cell cycle mediated by PIAS SUMO E3 ligases.

29The Protein Inhibitor of Activated STAT 1 (PIAS1) is an E3 SUMO ligase that plays important roles 30 in various cellular pathways, including STAT signaling, p53 pathway, and the steroid hormone 31 signaling pathway. PIAS1 can SUMOylate PML (at Lys-65 and Lys-160) and PML-RARα promoting 32 their ubiquitin-mediated degradation. Increasing evidence shows that PIAS1 is overexpressed in 33 various human malignancies, including prostate and lung cancers. To understand the mechanism 34 of action of PIAS1, we developed a quantitative SUMO proteomic approach to identify potential 35 substrates of PIAS1 in a system-wide manner. Our analyses enabled the profiling of 983 SUMO 36 sites on 544 proteins, of which 204 SUMO sites on 123 proteins were identified as putative PIAS1 37 substrates. These substrates are involved in different cellular processes, such as transcriptional 38 regulation, DNA binding and cytoskeleton dynamics. Further functional studies on Vimentin 39 (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell 40 motility, revealed that PIAS1 exerts its effects on cell migration and cell invasion through the 41 SUMOylation of VIM at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its 42 dynamic disassembly, and cells expressing a non-SUMOylatable VIM mutant showed reduced 43 levels of proliferation and migration. Our approach not only provides a novel strategy for the 44 identification of E3 SUMO ligase substrates, but also yields valuable biological insights into the 45 unsuspected role of PIAS1 and VIM SUMOylation on cell motility. 46 47 48The small ubiquitin-like modifier (SUMO) protein is an ubiquitin-like (UBL) protein that is 49 highly dynamic and can reversibly target lysine residues on a wide range of proteins involved in 50 several essential cellular events, including protein translocation and degradation, mitotic 51 chromosome segregation, DNA damage response, cell cycle progression, cell differentiation and 52 apoptosis 1 . SUMO proteins are highly conserved through evolution, and the human genome 53 encodes 4 SUMO genes, of which 3 genes (SUMO-1, SUMO-2 and SUMO-3) are ubiquitously 54 expressed in all cells 1,2 . Prior to conjugation, the immature SUMO proteins are C-terminally 55 processed by sentrin-specific proteases (SENPs) 3 . These proteases also cleave the isopeptide 56 bond formed between the -amino group of the acceptor lysine residues and the C-terminus 57 residue of the conjugated SUMO proteins. The conjugation of SUMO to target proteins requires 58 an E1 activating enzyme (SAE1/2), an E2 conjugating enzyme (UBC9) and one of several E3 SUMO 59 ligases 4 . Unlike ubiquitination, in vitro SUMOylation can occur without E3 SUMO ligases, although 60 enhanced substrate specificity is conferred by E3 SUMO ligases 5 . It is believed that SUMOylation 61 events occurring without the aid of E3 SUMO ligases arise primarily on the consensus motif 62 composed of ψKxE, where ψ represents a large hydrophobic residue and x, any amino acid 6 . To 63 ...