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Modification of Daxx by small ubiquitin-related modifier-1
Moon-Sun Jang1, Seung-Wook Ryu, Eunhee Kim
1Research Center for Biomedicinal Resources and Division of Life Science, PaiChai University, 439-6 Doma-2-dong, Seo-gu, Daejon 302-735, Republic of Korea.
Abstract:
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-1 conjugating enzyme, as well as with SUMO-1. In BOSC23 cells expressing FLAG-Daxx together with HA-SUMO-1, 110 and 130kDa 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-1. Substitution of K630 and K631 abrogated the modification of Daxx by SUMO-1, 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.
Insights
Small ubiquitin-related modifier-1 (SUMO-1) covalently modifies Daxx, a protein crucial for cell survival. SUMO-1 modification of Daxx is essential for its function but does not impact its localization within PML oncogenic domains.
Area of Science:
- Molecular Biology
- Cell Biology
- Protein Biochemistry
Background:
- Small ubiquitin-related modifier-1 (SUMO-1) is a post-translational modification involved in protein regulation and cellular protection.
- Daxx is a protein known to interact with SUMO-1 conjugating enzymes and SUMO-1 itself.
- Previous studies suggested a role for Daxx in cellular defense mechanisms.
Purpose of the Study:
- To investigate the covalent modification of Daxx by SUMO-1.
- To identify the specific sites on Daxx responsible for SUMO-1 conjugation.
- To determine the effect of SUMO-1 modification on Daxx localization and interaction with PML.
Main Methods:
- Co-immunoprecipitation assays using FLAG-tagged Daxx and HA-tagged SUMO-1 in BOSC23 cells.
- Site-directed mutagenesis to substitute lysine residues (K630, K631, K634, K636, K637) in Daxx.
- Immunofluorescence microscopy to assess Daxx and PML co-localization in PML oncogenic domains (PODs).
Main Results:
- Daxx undergoes covalent modification by SUMO-1, resulting in 110 and 130 kDa bands.
- Lysine residues K630 and K631 were identified as essential for Daxx sumoylation.
- Mutations disrupting putative C-terminal nuclear localization signals did not prevent nuclear entry.
- The sumoylation-defective Daxx mutant (K630, 631A) successfully interacted with and co-localized with PML in PODs.
Conclusions:
- Daxx is sumoylated at lysine residues K630 and K631.
- The sumoylation status of Daxx does not influence its presence or interaction within PML oncogenic domains.
- These findings elucidate the role of SUMO-1 modification in Daxx function and localization.