An engineered site for protein kinase C flanking the SV40 large T-antigen NLS confers phorbol ester-inducible nuclear import.
Nuclear import of simian virus SV40 large tumour antigen (T-ag) is enhanced by the protein kinase CK2 (CK2) site flanking the nuclear localisation sequence (NLS). We report here that replacement of this site with a consensus site for protein kinase C (PK-C) can alter the regulation of T-ag nuclear import and render it inducible by phorbol ester. Measurement of nuclear import kinetics using fluorescently labelled proteins and confocal laser scanning microscopy show that the introduced PK-C site is functional in enhancing T-ag nuclear import compared to a protein lacking the CK2 site. Treatment with the PK-C activator phorbol 12-myristate 13-acetate (PMA) further increases the level of maximal nuclear accumulation and the initial nuclear import rate. This engineered PMA-responsive NLS may have application in targeting of molecules of interest to the nucleus in response to agents stimulating PK-C.
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Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT.
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D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D011493:Protein Kinase C; D001616:beta-Galactosidase