Role of SUMO in RNF4-mediated promyelocytic leukemia protein (PML) degradation: sumoylation of PML and phospho-switch control of its SUMO binding domain dissected in living cells.
Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and ... maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.
Mesh Terms:
Antineoplastic Agents, Arsenicals, Bacterial Proteins, Cell Line, Fluorescence Resonance Energy Transfer, Humans, Kidney, Leukemia, Promyelocytic, Acute, Luciferases, Renilla, Luminescent Measurements, Luminescent Proteins, Mutagenesis, Site-Directed, Nuclear Proteins, Oxides, Protein Binding, Protein Structure, Tertiary, SUMO-1 Protein, Transcription Factors, Transfection, Tumor Suppressor Proteins
Antineoplastic Agents, Arsenicals, Bacterial Proteins, Cell Line, Fluorescence Resonance Energy Transfer, Humans, Kidney, Leukemia, Promyelocytic, Acute, Luciferases, Renilla, Luminescent Measurements, Luminescent Proteins, Mutagenesis, Site-Directed, Nuclear Proteins, Oxides, Protein Binding, Protein Structure, Tertiary, SUMO-1 Protein, Transcription Factors, Transfection, Tumor Suppressor Proteins
J. Biol. Chem.
Date: Jun. 12, 2009
PubMed ID: 19380586
View in: Pubmed Google Scholar
Download Curated Data For This Publication
140560
Switch View:
- Interactions 2
- PTM Genes 1