Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T-antigen NLS by importin: effect of conformation.
SV40 large tumor-antigen (T-ag) nuclear import is enhanced by the protein kinase CK2 (CK2) site (Ser111Ser112) flanking the nuclear localization sequence (NLS). Here we use site-directed mutagenesis to examine the influence of negative charge and conformation at the site on T-ag nuclear import and recognition by the NLS-binding importin subunits. Negative charge through aspartic acid in place of Ser111 simulated CK2 phosphorylation in enhancing nuclear accumulation to levels well above those of proteins lacking a functional CK2 site. This was shown to be through enhancement of T-ag NLS recognition by importin using an ELISA-based assay. Asp112-substituted mutants containing proline at positions 109, 110 (wild-type position) or 111 were compared to assess the role of conformation at the CK2 site. Maximal nuclear import of the protein with Pro109 was lower than that of the Pro110 derivative, with the Pro111 variant even lower, these differences also being attributable to effects on importin binding. All results indicate a correlation of the initial nuclear import rate with the importin binding affinity, demonstrating that NLS recognition by importin is a key rate-determining step in nuclear import.
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Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT.
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D000818:Animals; D000957:Antigens, Viral, Tumor / Q000276:immunology / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001692:Biological Transport; D047390:Casein Kinase II; D028884:Karyopherins; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D013539:Simian virus 40 / Q000276:immunology; D014407:Tumor Cells, Cultured
D000957:Antigens, Viral, Tumor; D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D001224:Aspartic Acid; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases