title: Nuclear γ-tubulin associates with nucleoli and interacts with tumor suppressor protein C53.
abstract: γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Czech Republic.
authors.forename: Barbora
authors.initials: B
authors.lastname: Hořejší
authors.forename: Stanislav
authors.initials: S
authors.lastname: Vinopal
authors.forename: Vladimíra
authors.initials: V
authors.lastname: Sládková
authors.forename: Eduarda
authors.initials: E
authors.lastname: Dráberová
authors.forename: Vadym
authors.initials: V
authors.lastname: Sulimenko
authors.forename: Tetyana
authors.initials: T
authors.lastname: Sulimenko
authors.forename: Věra
authors.initials: V
authors.lastname: Vosecká
authors.forename: Anatoly
authors.initials: A
authors.lastname: Philimonenko
authors.forename: Pavel
authors.initials: P
authors.lastname: Hozák
authors.forename: Christos D
authors.initials: CD
authors.lastname: Katsetos
authors.forename: Pavel
authors.initials: P
authors.lastname: Dráber
mesh_terms: D001253:Astrocytes / Q000378:metabolism; D001932:Brain Neoplasms / Q000378:metabolism; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D016147:Genes, Tumor Suppressor; D005909:Glioblastoma / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D013058:Mass Spectrometry; D016253:Microscopy, Immunoelectron; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction; D059008:Time-Lapse Imaging; D014404:Tubulin / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
chemical_list: C405928:CDK5RAP3 protein, human; D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D014404:Tubulin; D025521:Tumor Suppressor Proteins