Human Regulatory Protein <i>K</i>i-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation

Abstract

K i-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. K i-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that K i-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that K i-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on K i-1/57 sequence and observed that K i-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of K i-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG- K i-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As 2 O 3 ), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant K i-1/57 is expressed, suggesting that the SUMOylation of K i-1/57 has a role in the control of As 2 O 3 -induced PML-NB formation. A proteome-wide analysis of K i-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of K i-1/57 with the 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 K i-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.