Fig. 6: Mitotic proteins specifically SUMOylated by PIAS2b include proteasomal and spindle proteins.

a SUMO-capture strategy to find PIAS2b SUMOylation targets at mitosis (DT/R-6 h) in ns- or PIAS2b-dsRNAi treated 8305C cells. b PIAS2b-dsRNAi has a greater anti-cancer effect in mitotic synchronized than in asynchronous anaplastic cells. c, d Time-course of mitotic proteins reveal key alterations induced by PIAS2b-dsRNAi: fully phosphorylated AURKA (c); tubulin gamma (c) and PLK1 (c) markedly decrease; fully active CDK1, negative for inhibitory pTyr15-CDK1 while a single band in whole protein detection (d); d cyclin B and pSer10-HH3 reduction at mitosis; and PPP2CA shifts to 130 kDa when detected with F-8 antibody, respect to normal protein (detected with 0.T.118). e Proteins identified by LC-MS/MS and quantified by Spectrum count: from ns-dsRNAi cells, retained SUMOylated (pink), and flow-through not SUMOylated (blue); from PIAS2b-dsRNAi cells, retained SUMOylated (darker pink), and flow-through not SUMOylated (yellow). White circles: proteins unaltered in both treatments. f 132 proteins (pink bar) identified in both treatments, but with significant SUMO-retention in ns-dsRNAi compared to PIAS2b-dsRNAi; 52 proteins (yellow bar) presented less SUMO-retention in ns-dsRNAi. In proteins that lost SUMOylation in absence of PIAS2b, GO pathways highlights G2/M transition, mitosis and proteasome (red). g Venn diagram cross-analysis of Proteomic Assays 1 + 2 + 3. Shown the 20 proteins that are significantly bound to PIAS2b (Proteomic Assays 1 + 2) and loss SUMOylation in the absence of PIAS2b; 30 other PIAS2b-bound proteins losing SUMOylation are also remarkable although were not detected in the less sensitive Proteomic Assay 2. Highlighted in red, proteasome and green, spindle proteins. h Validation of PSMC5 as a PIAS2b-dependent SUMOylated protein in the SUMO-affinity column urea extracts as compared to flow-through. Direct association of FLAG-hPIAS2b to PSMC5 and TUBB3 (i), and of FLAG-PSMC5 to PIAS2b (j). k Specific association of PIAS2b to PSMC5 at mitosis (DT/R-6 h), compared to asynchronous (Asyn) or DT/R-0 h. A TUBB3–PIAS2b association increased at mitosis. l In PIAS2b-dsRNAi, TUBB3 and PSMC5 accumulated at mitosis, especially post-translationally modified (PTM)-PSMC5 (p70-p95). b n = 3 independent experiments, two-sided two-way ANOVA with repeated measures. Bar indicate means ± SEM; exact p value is indicated in the Fig. c, d n = 3 independent experiments. For e and h Proteomic Assay 3, n = 3 independent experiments, also extracts used in h; quantifications in Supplementary Data 4. i–l n = 3 independent experiments. Source Data provided.