The human regulatory protein Ki-1/57 is a target of SUMOylation and affects PML nuclear bodies formation

• Published in: Journal of proteome research 2017-07, Vol.16 (9), p.3147-3157
• Main Authors: Saito, Ângela, Souza, Edmarcia E., Costa, Fernanda C., Meirelles, Gabriela V., Gonçalves, Kaliandra A., Santos, Marcos T., Bressan, Gustavo C., McComb, Mark E., Costello, Catherine E., Whelan, Stephen A., Kobarg, Jörg
• Format: Article
• Language: English
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.7b00001

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin’s lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins, undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery: the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo . FLAG-Ki-1/57 wild-type and mutant Lys to Arg proteins were overexpressed in HEK293T cells, immunoprecipitated with the antibody anti-FLAG and probed with anti-SUMO-1 or anti-SUMO-2/3. SUMOylation of Ki-1/57 occurred on lysines 213, 276 and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type or their non-SUMOylated triple-mutants, the average number of PML-nuclear bodies (PML-NBs) is reduced compared to the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As 2 O 3 ) the mean number of PML-NBs is no more reduced when the non-SUMOylated triple-mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As 2 O 3 -induced PML-NBs formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated to cellular metabolism maintenance and cell cycle.