title: Nucleocytoplasmic shuttling of soluble tubulin in mammalian cells.
abstract: We have investigated the subcellular distribution and dynamics of soluble tubulin in unperturbed and transfected HeLa cells. Under normal culture conditions, endogenous alpha/beta tubulin is confined to the cytoplasm. However, when the soluble pool of subunits is elevated by combined cold-nocodazole treatment and when constitutive nuclear export is inhibited by leptomycin B, tubulin accumulates in the cell nucleus. Transfection assays and FRAP experiments reveal that GFP-tagged beta-tubulin shuttles between the cytoplasm and the cell nucleus. Nuclear import seems to occur by passive diffusion, whereas exit from the nucleus appears to rely on nuclear export signals (NESs). Several such motifs can be identified by sequence criteria along the beta-tubulin molecule and mutations in one of these (NES-1) cause a significant accumulation in the nuclear compartment. Under these conditions, the cells are arrested in the G0-G1 phase and eventually die, suggesting that soluble tubulin interferes with important nuclear functions. Consistent with this interpretation, soluble tubulin exhibits stoichiometric binding to recombinant, normally modified and hyper-phosphorylated/acetylated histone H3. Tubulin-bound H3 no longer interacts with heterochromatin protein 1 and lamin B receptor, which are known to form a ternary complex under in vitro conditions. Based on these observations, we suggest that nuclear accumulation of soluble tubulin is part of an intrinsic defense mechanism, which tends to limit cell proliferation under pathological conditions. This readily explains why nuclear tubulin has been detected so far only in cancer or in transformed cells, and why accumulation of this protein in the nucleus increases after treatment with chemotherapeutic agents.
authors.affiliation: Department of Biochemistry, University of Crete, School of Medicine, 71 003 Heraklion, Greece.
authors.forename: Tonia
authors.initials: T
authors.lastname: Akoumianaki
authors.forename: Dimitris
authors.initials: D
authors.lastname: Kardassis
authors.forename: Hara
authors.initials: H
authors.lastname: Polioudaki
authors.forename: Spyros D
authors.initials: SD
authors.lastname: Georgatos
authors.forename: Panayiotis A
authors.initials: PA
authors.lastname: Theodoropoulos
mesh_terms: D000107:Acetylation; D021581:Active Transport, Cell Nucleus; D002453:Cell Cycle; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D049109:Cell Proliferation; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D003080:Cold Temperature; D003593:Cytoplasm / Q000378:metabolism*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015739:Nocodazole / Q000494:pharmacology; D049790:Nuclear Export Signals; D010766:Phosphorylation; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology; D000097682:Lamin B Receptor
chemical_list: D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D015739:Nocodazole; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D050257:Tubulin Modulators; D000097682:Lamin B Receptor