Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1

Abstract

PML and Sp100 proteins are associated with nuclear domains, known as nuclear dots (NDs). They were discovered in the context of leukemic transformation and as an autoantigen in primary biliary cirrhosis, respectively. Both proteins are expressed in the form of many COOH-terminally spliced variants, and their expression is enhanced by interferons (IFN). The recent finding that PIC1/SUMO-1, a small ubiquitin-like protein, is covalently linked to the RanGAP1 protein of the nuclear pore complex and also binds PML in yeast cells led us to determine whether PML is covalently modified by PIC1/SUMO-1 and whether the same is true for Sp100. We found an immune reaction of PML and Sp100 proteins with a PIC1/SUMO-1–specific monoclonal antibody by immunoblotting when using cell extracts prepared from stably transfected cells inducibly expressing one isoform of each protein as well as from nontransfected cells. In contrast, both proteins did not react when synthesized in vitro. Immunofluorescence staining showed that PIC1/SUMO-1 colocalized with Sp100 and PML in NDs except in mitotic cells, in which PML and Sp100 are dissociated. Cell fractionation and immunoblotting demonstrated that PIC1/SUMO-1 immunoreactive Sp100 in IFN-treated and untreated cells was exclusively nuclear, whereas nonmodified Sp100 was also found in the cytoplasm. Taken together, these data strongly suggest covalent modification of specific nuclear isoforms of Sp100 and PML by PIC1/SUMO-1. This modification may play a regulatory role in ND structure, composition, and function.

Cross Talk between PML and p53 during Poliovirus Infection: Implications for Antiviral Defense

“…After infection, Sp100 was further recruited and found to be colocalizing with PML in the large NBs. As previously reported (41), in noninfected cells, two Sp100 forms were detected by Western blotting (Fig. 3B).…”

Body language: the function of PML nuclear bodies in apoptosis regulation

“…37,38 SUMO-1 (also termed sentrin, GMP1, SMT3C or ULP1) is a small polypeptide that is covalently attached to PML and several other PML-NB components, including Sp100, 40 Daxx, 41 p53 42,43 and CBP, 44 through an enzymatic machinery similar to that of ubiquitin modification. 45 SUMOlation requires an activating E1 SUMO-1 activating enzyme (SAE)1/SAE2 heterodimer, 46 the conjugating E2 enzyme Ubc9 47,48 and the recently identified E3 SUMO ligases.…”

A role for PML and the nuclear body in genomic stability

“…PML is covalently conjugated to small ubiquitin-like modi®er 1 (SUMO-1, also known as PIC1) (Sternsdorf et al, 1997;MuÈ ller et al, 1998). SUMO-1 is conjugated to various proteins that localize to di erent cellular compartments.…”

“…SUMO-1 is conjugated to various proteins that localize to di erent cellular compartments. In the nucleus, it concentrates in the NB where, besides PML, it covalently modi®es other NB proteins such as Sp100 (Sternsdorf et al, 1997).…”