The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin.

Abstract

The mechanism by which phosphorylation regulates nuclear localization sequence (NLS)-dependent nuclear protein import is largely unclear. Whereas nuclear accumulation of SV40 large tumor antigen (T-ag) fusion proteins is completely dependent on the T-ag NLS (amino acids 126-132), the rate of nuclear import is increased 50-fold by amino acid residues 111-125 and in particular a site for the protein kinase CK2 (CK2) at serine 111/112. Because the first step of nuclear protein import involves the binding of the NLS by an NLS-receptor complex such as the importin 58/97 heterodimer, we established a novel enzyme-linked immunosorbent assay to test whether NLS recognition is influenced by amino acids amino-terminal to the NLS and the CK2 site. We found that recognition of the T-ag NLS by importin 58/97 was enhanced 10-fold in the presence of amino acid residues 111-125 and strongly dependent on importin 97. A T-ag fusion protein in which the spacer between the CK2 site and the NLS was decreased showed 30% reduced binding by importin 58/97. Maximal nuclear accumulation of this protein was reduced by more than 50%, indicating the physiological importance of the correctly positioned CK2 site. Phosphorylation by CK2 increased the T-ag NLS binding affinity for importin 58/97 by a further 40%. We conclude that flanking sequences and in particular phosphorylation at the CK2 site are mechanistically important in NLS recognition and represent the basis of their enhancement of T-ag nuclear import. This study thus represents the first elucidation of the mechanistic basis of the regulation of nuclear protein import through phosphorylation within a phosphorylation-regulated NLS.