title: 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.
abstract: 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.
authors.affiliation: From the CNRS FRE2937, Institut André Lwoff, Villejuif 94801, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Percherancier
authors.affiliation: Département de Biochimie, Université de Montréal, Montréal H3C 3J7, Canada.
authors.forename: Delphine
authors.initials: D
authors.lastname: Germain-Desprez
authors.affiliation: From the CNRS FRE2937, Institut André Lwoff, Villejuif 94801, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Galisson
authors.affiliation: Département de Biochimie, Université de Montréal, Montréal H3C 3J7, Canada.
authors.forename: Xavier H
authors.initials: XH
authors.lastname: Mascle
authors.affiliation: From the CNRS FRE2937, Institut André Lwoff, Villejuif 94801, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Dianoux
authors.affiliation: Département de Biochimie, Université de Montréal, Montréal H3C 3J7, Canada.
authors.forename: Patricia
authors.initials: P
authors.lastname: Estephan
authors.affiliation: From the CNRS FRE2937, Institut André Lwoff, Villejuif 94801, France. Electronic address: mchelbi@vjf.cnrs.fr.
authors.forename: Mounira K
authors.initials: MK
authors.lastname: Chelbi-Alix
authors.affiliation: Département de Biochimie, Université de Montréal, Montréal H3C 3J7, Canada. Electronic address: muriel.aubry@umontreal.ca.
authors.forename: Muriel
authors.initials: M
authors.lastname: Aubry
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism* / Q000473:pathology; D049410:Luciferases, Renilla / Q000235:genetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010087:Oxides / Q000494:pharmacology; D000071156:Promyelocytic Leukemia Protein; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D001152:Arsenicals; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D010087:Oxides; D000071156:Promyelocytic Leukemia Protein; C114070:RNF4 protein, human; D025842:SUMO-1 Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C054562:yellow fluorescent protein, Bacteria; C070576:PML protein, human; D049410:Luciferases, Renilla; D000077237:Arsenic Trioxide