title: The Retinoic acid receptor alpha (RARalpha) chimeric proteins PML-, PLZF-, NPM-, and NuMA-RARalpha have distinct intracellular localization patterns.
abstract: Retinoic acid receptor alpha (RARalpha) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acute promyelocytic leukemia (APL). Disruption of RARalpha function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RARalpha expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RARalpha chimeric proteins, we engineered epitope-tagged versions of PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, and NuMA-RARalpha (X-RARalphaV5) and generated a panel of stable COS cell lines expressing X-RARalphaV5. Protein fractionation and Western analysis of these COS lines reveal that X-RARalpha proteins localize to both the cytoplasm and nucleus. NPM-RARalpha is predominantly nuclear whereas NuMA-RARalpha is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PML-RARalpha and PLZF-RARalpha share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RARalpha is also diffuse in the nucleus but, in contrast to PML-RARalpha and PLZF-RARalpha, is strongly associated with the nucleolus. Strikingly, NuMA-RARalpha predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RARalpha and NuMA-RARalpha, respectively. The distinct intracellular localization patterns and the shared ability of X-RARalpha to interact with their respective RARalpha partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis.
authors.affiliation: The Institute of Medical Sciences, University of Toronto, Ontario, M5S 1A8, Canada.
authors.forename: Jeff L
authors.initials: JL
authors.lastname: Hummel
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.forename: Richard A
authors.initials: RA
authors.lastname: Wells
authors.forename: Suzanne
authors.initials: S
authors.lastname: Kamel-Reid
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D000939:Epitopes; D005982:Glutathione Transferase / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000378:metabolism*; D011233:Precipitin Tests; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D000939:Epitopes; C120276:NPM-RARalpha protein, human; D009363:Neoplasm Proteins; C412764:NuMa-RARalpha protein, human; D015514:Oncogene Proteins, Fusion; C118318:PLZF-RARalpha fusion protein, human; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D000072482:Retinoic Acid Receptor alpha; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein; D005982:Glutathione Transferase