SUMO Proteomics Analyses Identify Protein Inhibitor of Activated STAT-Mediated Regulatory Networks Involved in Cell Cycle and Cell Proliferation

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.