FBXO25-associated nuclear domains: a novel subnuclear structure.
Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.
Manfiolli
Adriana O
AO
Departments of Biochemistry and Immunology and Cellular and Molecular Biology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, São Paulo 14049-900, Brazil.
Maragno
Ana Leticia G C
AL
Baqui
Munira M A
MM
Yokoo
Sami
S
Teixeira
Felipe R
FR
Oliveira
Eduardo B
EB
Gomes
Marcelo D
MD
D000682:Amyloid / Q000378:metabolism; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D022003:Cell Nucleus Structures / Q000378:metabolism*; D002478:Cells, Cultured; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D003609:Dactinomycin / Q000494:pharmacology; D044783:F-Box Proteins / Q000378:metabolism*; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012313:RNA / Q000235:genetics; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D054875:Ubiquitination / Q000187:drug effects
D000682:Amyloid; D044783:F-Box Proteins; C510759:FBXO25 protein, human; C512115:FBXO25 protein, mouse; D009419:Nerve Tissue Proteins; D010455:Peptides; D003609:Dactinomycin; C097188:polyglutamine; D012313:RNA; D012321:DNA-Directed RNA Polymerases