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. Copyright (C) 1998 Federation of European Biochemical Societies.