Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics.
Lamina-associated polypeptide (LAP) 2 of the inner nuclear membrane (now LAP2beta) and LAP2alpha are related proteins produced by alternative splicing, and contain a common 187 amino acid N-terminal domain. We show here that, unlike LAP2beta, LAP2alpha behaved like a nuclear non-membrane protein in subcellular fractionation studies and was localized throughout the nuclear interior in interphase cells. It co-fractionated with LAP2beta in nuclear lamina/matrix-enriched fractions upon extraction of nuclei with detergent, salt and nucleases. During metaphase LAP2alpha dissociated from chromosomes and became concentrated around the spindle poles. Furthermore, LAP2alpha was mitotically phosphorylated, and phosphorylation correlated with increased LAP2alpha solubility upon extraction of cells in physiological buffers. LAP2alpha relocated to distinct sites around chromosomes at early stages of nuclear reassembly and intermediarily co-localized with peripheral lamin B and intranuclear lamin A structures at telophase. During in vitro nuclear assembly LAP2alpha was dephosphorylated and assembled into insoluble chromatin-associated structures, and recombinant LAP2alpha was found to interact with chromosomes in vitro. Some LAP2alpha may also associate with membranes prior to chromatin attachment. Altogether the data suggest a role of LAP2alpha in post-mitotic nuclear assembly and in the dynamic structural organization of the nucleus.
nstitute of Biochemistry and Molecular Cell Biology, Biocenter and Institute of Tumor Biology-Cancer Research, University of Vienna, A-1030 Vienna, Austria.
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T
Dechat
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Gotzmann
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A
Stockinger
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Harris
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Talle
J J
JJ
Siekierka
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Foisner
D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D006224:Cricetinae; D004268:DNA-Binding Proteins; D003902:Detergents; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008565:Membrane Proteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008938:Mitosis; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D012492:Salts
D004268:DNA-Binding Proteins; D003902:Detergents; D008565:Membrane Proteins; D009687:Nuclear Proteins; D012492:Salts; C093413:lamina-associated polypeptide 2