The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin
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1Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, ACT 2601, Australia.
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.
Insights
Phosphorylation of protein kinase CK2 (CK2) sites near nuclear localization sequences (NLS) enhances protein import into the nucleus. This study reveals how flanking sequences and CK2 phosphorylation mechanistically regulate NLS recognition by importins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear protein import is crucial for cellular function, regulated by nuclear localization sequences (NLS).
- The precise mechanisms by which phosphorylation influences NLS-dependent nuclear import remain largely undefined.
- SV40 large tumor antigen (T-ag) nuclear import is NLS-dependent but modulated by flanking residues and protein kinase CK2 (CK2) phosphorylation sites.
Purpose of the Study:
- To investigate how amino acids N-terminal to the NLS and CK2 phosphorylation sites affect NLS recognition by importin receptors.
- To elucidate the mechanistic basis of phosphorylation-mediated regulation of nuclear protein import.
Main Methods:
- Development of a novel enzyme-linked immunosorbent assay (ELISA) to quantify NLS-importin binding.
- Utilized SV40 T-ag fusion proteins with modifications to flanking sequences and CK2 phosphorylation sites.
- Assessed the impact of CK2 phosphorylation on NLS binding affinity and nuclear accumulation.
Main Results:
- Amino acid residues 111-125, including the CK2 site, enhanced T-ag NLS recognition by importin 58/97 tenfold.
- Altering the spacer between the CK2 site and NLS reduced importin binding by 30% and nuclear accumulation by over 50%.
- CK2 phosphorylation increased T-ag NLS binding affinity for importin 58/97 by 40%.
Conclusions:
- Flanking sequences and CK2 phosphorylation at specific sites are mechanistically critical for NLS recognition.
- These modifications enhance T-ag nuclear import by improving NLS binding to importin receptors.
- This study provides the first mechanistic explanation for phosphorylation-regulated nuclear protein import via a phosphorylation-regulated NLS.