title: PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation.
abstract: Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment.
authors.affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Hands
authors.forename: Delphine
authors.initials: D
authors.lastname: Cuchet-Lourenco
authors.forename: Roger D
authors.initials: RD
authors.lastname: Everett
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Hay
mesh_terms: D001151:Arsenic / Q000494:pharmacology*; D015153:Blotting, Western; D022003:Cell Nucleus Structures / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D020033:Protein Isoforms; C114070:RNF4 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D001151:Arsenic
keywords: Arsenic; PML; RNF4; SUMO