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. Copyright (C) 1998 Federation of European Biochemical Societies.
- Ca/phospholipid-dependent protein kinase C
- Confocal laser scanning microscopy
- Nuclear import kinetics
- Phosphorylation-regulated nuclear localization sequence