Nucleolar protein B23 stimulates nuclear import of the HIV-1 Rev protein and NLS-conjugated albumin.
Nucleolar phosphoprotein B23 is a putative ribosome assembly factor with a relatively high affinity for peptides containing sequences of nuclear localization signals (NLSs) of the SV40 T-antigen type [Szebeni, A., Herrera, J. E., & Olson, O. J. (1995) Biochemistry 34, 8037-8042]. The effects of protein B23 on nuclear import were determined by an in vitro assay [Dean, D. A., & Kasamatsu, H. (1994) J. Biol. Chem. 269, 4910-4916] using NLS peptide-conjugated bovine serum albumin (NLS-BSA) or the HIV-1 Rev protein as substrates for import into isolated rat liver nuclei. The import was ATP-dependent and inhibited by wheat germ agglutinin or by an antibody against p97, a component of the nuclear import system. The rate of import of either substrate was increased if protein B23 was added to the incubation medium. Similar enhancements of import were seen with both isoforms (B23.1 and B23.2). The stimulatory effect on Rev protein import was saturable with maximum stimulation (2-3-fold) at a molar ratio of protein B23:Rev of approximately 1:1. Phosphorylation of protein B23.1 by casein kinase II produced an additional doubling of the import rate. This effect was not seen if protein B23.1 was phosphorylated with a cdc2 type protein kinase. Mutant forms of protein B23.1 in which the nuclear localization signal was either deleted or altered did not stimulate import of the substrates. These results suggest that protein B23 plays a role as an accessory factor in the nuclear import of the NLS-containing proteins and that phosphorylation at sites in the highly acidic segments of the protein enhances the stimulatory effect.
Szebeni
A
A
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216, USA.
Mehrotra
B
B
Baumann
A
A
Adam
S A
SA
Wingfield
P T
PT
Olson
M O
MO
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D047390:Casein Kinase II; D002467:Cell Nucleus / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism*; D015497:HIV-1; D008099:Liver / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D000090243:Nucleophosmin; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012709:Serum Albumin / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000494:pharmacology; D054321:rev Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D000952:Antigens, Polyomavirus Transforming; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015694:Gene Products, rev; C000716967:NPM1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D014909:Wheat Germ Agglutinins; C094680:nuclear protein import factor p97; D054321:rev Gene Products, Human Immunodeficiency Virus; D000090243:Nucleophosmin; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases