Fig 2: SUMOylation of PIAS1 promotes its PML localization.a Distribution of SUMO sites identified on PIAS1. Three SUMO sites (K137, K237, and K315) were identified in the dataset. K137 and K238 are located in the PINIT domain, whereas K315 is located in the SP-RING domain. b HEK293-SUMOm cells were co-transfected with PIAS1-GFP-WT, PIAS1-GFP-3xKR, PIAS1-GFP-K137R, PIAS1-GFP-K238R, or PIAS1-GFP-K315R and immunofluorescence was performed with anti-PML. Scale bar: 2.5 µm. c Scatter graph showing the Pearson’s correlation coefficient for the PIAS1–PML colocalization. Data represent the mean ± SD, error bars represent SD, ns nonsignificant, *p < 0.05, **p < 0.01, Student’s t-test, n = 20 biologically independent cells/condition. Source data are provided as a Source Data file.

Fig 3: SUMOylation of vimentin regulates its dynamic assembly.a HeLa cells were transfected with an empty Flag vector as a negative control, Flag-VIMwt, and Flag-VIMmt, and were separated into RIPA-soluble and -insoluble fractions. VIM protein levels were examined by western blotting. b MCF-7 cells were transfected with Emerald wild-type vimentin (VIMwt) and Emerald vimentin K439/445R, double mutant (VIMmt), and the proportion of unit-length filament (ULF), vimentin intermediate filament (VIF), and cytosolic vimentin between VIMwt and VIMmt were calculated under a microscope. Data represent the mean ± SD, error bars represent SD, ns nonsignificant, *p < 0.05, **p < 0.01, Student’s t-test, n = 4 biologically independent samples. c Fluorescence recovery after photobleaching (FRAP) assay of Emerald wild-type vimentin (VIMwt) and Emerald vimentin K439/445R, double mutant (VIMmt) in MCF-7 cells. Line plot shows average fluorescence at each time point ± SD. Differences between values for VIMwt and VIMmt were not statistically significant at all time points by Student’s t-test. n = 7 biologically independent cells. d Model of the VIM dynamic assembly and disassembly. Vimentin is maintained in equilibrium between unit-length filament (ULF) and soluble tetramers (subunit exchange step). Vimentin filaments elongate by end-to-end annealing of ULF to form mature vimentin intermediate filament (VIF; annealing step). The phosphorylation-dependent shortening of VIF (severing step) involves phosphorylation on Ser-39 and Ser-56 of vimentin by several kinases, including Akt1. The short filaments can re-anneal with another ULF to form new VIF (re-annealing step). However, the phosphorylated ULF is not amenable to the re-annealing process. These phosphorylated ULF products are subject to PIAS1-mediated SUMOylation, stimulating the dephosphorylation of the phosphorylated ULF, and subsequently re-enter to either subunit exchange process or VIF maturation process. Source data are provided as a Source Data file.

Fig 4: Protein–protein interaction network of PIAS1 substrates.STRING network of PIAS1 substrates and their interacting partners. Proteins are grouped according to their GO terms.

Fig 5: Functional effects of PIAS1 expression on HeLa cells.a HeLa cells were transfected with Myc-PIAS1 (PIAS1-OE) or Empty vector (NC-vector) for 48 h. PIAS1 overexpression efficiency was determined by western blotting. Actin was used as a loading control. b PIAS1 KO HeLa cells were generated by CRISPR/Cas9. PIAS1 KO was determined by western blotting. Actin was used as a loading control. c PIAS1 overexpression in HeLa cells significantly promotes cell growth, n = 3 biologically independent samples. d PIAS1 KO HeLa cells (PIAS1-sgRNA) showed impeded cell growth compared with the negative control cells (NC-sgRNA), n = 3 biologically independent samples. e PIAS1 overexpression increased cell migration as determined by a wound-healing assay. Scale bar: 200 µm, n = 5 biologically independent samples. f PIAS1 KO cells displayed decreased cell migration compared with the sgRNA-negative control cells, as determined by a wound-healing assay. Scale bar: 200 µm, n = 5 biologically independent samples. Data represent the mean ± SD, error bars represent SD, **p < 0.01, Student’s t-test. Source data are provided as a Source Data file.