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.Nuclear protein transport is dependent on specific targeting signals called nuclear localization sequences (NLSs), 1 defined as the sequences sufficient and necessary for nuclear targeting (1, 2). They are typically short sequences of a single series of basic residues (resembling the NLS of the SV40 large tumor antigen (T-ag)) or of two clusters of basic residues interrupted by a 10 -12-amino acid spacer (bipartite NLSs), and they appear to be functional in various protein contexts, suggesting that NLS function is largely independent of secondary/tertiary structure (see .NLS-dependent nuclear protein import can be divided into two steps, the first of which is energy-independent and involves recognition and targeting of the NLS-bearing protein to the nuclear pore complex (NPC) by a heterodimeric protein complex. The NLS is specifically recognized by the smaller subunit of the complex or NLS receptor (6), known variously as importin 58 (7), importin ␣ (8), hSRP1/NP-1 (9), or karyopherin ␣ (10). The larger protein subunit, importin 97 (11), importin ␤ (12), karyopherin ␤ (13), p97 (14), or Kap95p (15), binds importin 58 specifically but cannot bind the NLS. Its role is to target the importin-NLS carrying protein complex to the NPC through its affinity for NPC components such as nucleoporins (13, 16 -18). The second, energy-dependent step of translocation of th...

p53 regulation by post-translational modification and nuclear retention in response to diverse stresses

“…In addition, S376, S378, and S392 are phosphorylation sites for PKC, PKA, and CK2 respectively. Each of these kinases has been implicated in regulating nuclear-cytoplasmic shuttling of other proteins, such as diacylglycerol kinase (Topham et al, 1998), dorsal (Briggs et al, 1998;Drier et al, 1999), and SV40 T-antigen (Hubner et al, 1997). In fact, PKC inhibitors induce both p53 DNA binding and nuclear accumulation (Chernov et al, 1998), both of which correlate with tetramer formation.…”

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery

“…Accordingly, before assessing HisGAL4(1-147)-NLS accessibility in complexes with DNA, we measured the affinity of recognition of the GAL4-NLS by the NLS-binding importin and karyopherin subunits using an established and specific ELISAbased binding assay. 33,34,38 Experiments were performed with all combinations of importin and karyopherin subunits as GST-fusion proteins -ie ␣-and ␤-subunits alone, or in combination (Figure 3). Surprisingly, we found that the ␣-subunit in the case of both the mouse and yeast NLS-binding subunits recognized HisGAL4(1-147) quite poorly, in contrast to the ␤-subunit, where maximal binding by the ␣-subunit was less than 15% that of the ␤-subunit in both cases ( Figure 3; see Table 1).…”

“…38 GST-free Kap60 and m58 was prepared by thrombin cleavage as described. 33,34 Electrophoretic mobility shift assays 17m was prepared by annealing (see above) and then purified by preparative polyacrylamide gel electrophoresis, before radiolabelling with ␥-32 P ATP (Bresatec, Thebarton, SA, Australia) using T4 polynucleotide kinase (Biolabs, Beverley, MA, USA). Unincorporated radionucleotide was removed using a sepharose Nick spin column (AMRAD Pharmacia Biotech, Boronia, Victoria, Australia), and incorporated radioactivity quantified by scintillation counting (Packard 1900CA-Packard, Downers Grove, IL, USA).…”

“…The dried gel was either exposed to radiographic Biomax film (Eastman Kodak, Rochester, NY, USA), or a phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA) was used to quantify the levels of bound and free radiolabelled oligomer. 33,40 For competition experiments, labelled 17m was incubated with HisGAL4(1-147) in the presence of increasing amounts of unlabelled 17m or of a sequence-unrelated oligomer (22mer: 5Ј-GATCTGATTGGGTTTCTCCCAG-TAGCGCTTGATTGGGTTTCTCCCAGT-3Ј), or with plasmids with (pSV2neo-17m and pCH110-17m) or without the 17m sequence. The latter included plasmids containing the 46-mer SOS (5Ј-GATCTGCTGTATATATA TACAGCGCTACTGTATATACACCCAGGGC-3Ј which contains specific binding sites in tandem for the DNAbinding protein lexA) inserted into the BamHI site of plasmids pSV2neo or pCH110 as for the 17m and complimentary oligomers above.…”

“…Interestingly, recognition of the GAL4 NLS was observed only by the ␤-subunits of the respective yeast and importin NLS-receptor subunits (Kap95 and importin 97, respectively), and not by the ␣-subunits (Kap60 and importin 58) which recognise conventional NLSs such as those of SV40 large tumor antigen (T-ag) and the bipartite NLS of the tumor suppressor Rb. 33,34 When included in transfection experiments, Kap95 was found to reduce GAL4 enhancement of transfection by 80%. The use of GAL4 in gene transfer conjugates should be reconsidered in the light of our findings, the observed facilitation of DNA transfection presumably not the result of enhanced nuclear targeting.…”