Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T‐antigen NLS by importin: effect of conformation

FEBS Letters

Jans, Patricia¹  Jans, David A.¹  Xiao, Chong-Yun¹ 

[1] Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia

关键词: Nuclear import kinetics;    Confocal laser scanning microscopy;    Microinjection;    Phosphorylation;    SV40 large tumor-antigen;   

DOI  :  10.1016/S0014-5793(98)01478-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown