Modification of Daxx by small ubiquitin-related modifier-1

Small ubiquitin-related modifier-1 (SUMO-1) is a protein that is covalently modified to various cellular proteins and protects cells against both anti-Fas and TNF-induced cell death. Previously, we reported that the C-terminus of Daxx interacted with Ubc9, an E2 type SUMO-I conjugating enzyme, as well as with SUMO-1. In BOSC23 cells expressing FLAG-Daxx together with HA-SUMO-1, 110 and 130 kDa Daxx appeared and the 130kDa band bound to both anti-HA and anti-FLAG antibodies. This means that Daxx can be covalently modified by SUMO-L Substitution of K630 and K631 abrogated the modification of Daxx by SUMO1, implying that K630 and K631 were essential for sumoylation. Daxx (K630, 631A) and Daxx (K634, 636, 637A) in which the putative C-terminal nuclear localization signals (NLSs) were disrupted appeared in the nucleus, suggesting that the C-terminal NLS was not functional. Daxx (K630, 631A), the sumoylation defective mutant, was able to interact with PML and co-localized with PML in the PML oncogenic domains (PODs). Thus, our data show that sumoylation status of Daxx does not affect its presence in PODs. (C) 2002 Elsevier Science (USA). All rights reserved.