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© The Rockefeller University Press, 0021-9525/1997//1621 $5.00
The Journal of Cell Biology, Volume 139, Number 7, , 1997 1621-1634

Article

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



Thomas Sternsdorf, Kirsten Jensen, and Hans Will

Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, D-20251 Hamburg, Federal Republic of Germany

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.

Abbreviations used in this paper: Ab, antibody; APL, acute promyelocytic leukemia; IFN, interferon; ND(s), nuclear dot(s) (nuclear domain(s) containing PML and Sp100 proteins); PBC, primary biliary cirrhosis; PIC1, PML interacting clone 1; PV, pellet volumes; RAR, retinoic acid receptor {alpha}; snRNP, small nuclear ribonucleoprotein; SUMO-1, small ubiquitin-related modifier.

Address all correspondence to Hans Will, Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistraße 52, D-20251 Hamburg, FRG. Tel. and Fax: 0049 40 48051221. E-mail: will{at}hpi.uni-hamburg.de


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