Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation

Abstract

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 and 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. We showed that 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 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 (AsO), the number of PML-NBs is no longer 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 AsO-induced PML-NB 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 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 Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.

Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

“…Supporting this thought is the finding that over-activation of sumoylation is correlated with a poorer prognosis in multiple myeloma patients 70 . In addition, while SUMO levels were not examined, the increased sumoylation of select target proteins, such as promyelocytic leukemia nuclear bodies, Fas-associated protein with death domain (FADD), and β-catenin 70–72 , has been reported to be critical to the transformation of B and T lymphocytes. Therefore, we propose that all lymphomas will exhibit moderately induced SUMO levels, however, transformation by EBV, and the expression of LMP1, will result in even higher levels of SUMO expression than LMP1-negative lymphomas.…”

Intracellular hyaluronic acid-binding protein 4 (HABP4): a candidate tumor suppressor in colorectal cancer

“…However, several molecular features suggested that it might be a regulatory protein with some association with cancer [7,8]. As many other oncoproteins HABP4 showed a shuttling protein localization between the cytoplasm and the nucleus, a localization to protein bodies in the cytoplasm, especially after cellular stress, co-localization with nuclear splicing speckles and other nuclear sub-structures [13], extensive posttranslational modifications, including serine/threonine phosphorylation by PKC [20], arginine methylation [18] and SUMOylations [32]. Furthermore, the amino acid sequence of HABP4 is rich in charged amino acids and the protein seems to be part of the family of so called IUPs (intrinsically unstructured proteins) [27], proteins which are known to have many interactors, frequently with regulatory functions, that tend to occupy hub positions in complex protein networks and are frequently described for their involvement in cancer related cellular processes [33].…”

Complex interactomes and post-translational modifications of the regulatory proteins HABP4 and SERBP1 suggest pleiotropic cellular functions

“…The PTMs of HABP4 and SERBP1 have been discovered over the years mainly due to the identification of their interaction with modifying proteins. Until now, HABP4 and SERBP1 were predicted to have phosphorylation, methylation and SUMOylation sites, as shown in Figure 2[20-22]. These PTMs and their impact on the functions of HABP4 and SERBP1 are described below.…”

“…Likewise, SERBP1 exhibited fifteen potential SUMOylation sites, of which six had a higher probability of being conjugated with SUMO (Figure 2B). In vitro assays showed that HABP4 is indeed SUMOylated by SUMO-2/3 at the three main targets[22]. In vivo experiments revealed that wild-type HABP4, but not the SUMOylation-defective mutant HABP4 K213R/K276R/K336R , co-immunoprecipitated with anti-SUMO-1 and anti-SUMO-2 antibodies.…”

“…The same approach was used for SERBP1, and mutations in the three lysine residues with the highest score (K102R/K228R/K281R), resulted in SERBP1 being unavailable for modification by SUMO conjugation that was observed in the wild-type protein[22].…”