The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen.
We have previously demonstrated [Rihs, H.-P. and Peters, R. (1989) EMBO J., 8, 1479-1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T-antigen are fused to the amino terminus of Escherichia coli beta-galactosidase is dependent on both the nuclear localization sequence (NLS, T-antigen residues 126-132) and a phosphorylation-site-containing sequence (T-antigen residues 111-125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation-site-containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK-II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK-II sites at a distance of approximately 10-30 amino acid residues from the NLS. CK-II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK-II may exert this role by controlling the rate of nuclear protein transport.
Rihs
H P
HP
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Jans
D A
DA
Fan
H
H
Peters
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D047388:Casein Kinases; D002467:Cell Nucleus / Q000378:metabolism*; D002472:Cell Transformation, Viral; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D014709:Vero Cells; D001616:beta-Galactosidase / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D001616:beta-Galactosidase