SV40 Large Tumor Antigen Nuclear Import Is Regulated by the Double-stranded DNA-dependent Protein Kinase Site (Serine 120) Flanking the Nuclear Localization Sequence*

Nuclear localization sequence (NLS)-dependent nuclear import of SV40 large tumor antigen (T-Ag) fusion proteins is regulated by phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase Cdc2 amino-terminal to the NLS (amino acids 126–132). Between the T-Ag CKII and Cdc2 sites is a site (Ser120) for the double-stranded DNA-dependent protein kinase (dsDNA-PK), which we show here for the first time to play a role in regulating T-Ag nuclear import. We replaced Ser120 by aspartic acid or alanine using site-directed mutagenesis and assessed the effects on nuclear transport kinetics both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in HTC rat hepatoma cells. Maximal nuclear accumulation of the Asp120 and Ala120 protein derivatives was approximately 40% and 70% reduced in vivo, respectively, compared with that of the wild type protein, and similarly reducedin vitro, although to a lesser extent. This implies that the dsDNA-PK site regulates the maximal level of nuclear accumulation, normally functioning to enhance T-Ag nuclear transport; the higher accumulation of the Asp120 protein compared with the Ala120 protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import. The Asp120 protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser120 may also regulate the nuclear import rate. CKII phosphorylation of the Asp120 protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser120 and Ala120 proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser120 by approximately 80% compared with wild type, implying physical and functional interactions between the two phosphorylation sites. Quantitation of NLS recognition by the importin 58/97 subunits using an enzyme-linked immunosorbent assay indicated that while the Ala120 protein derivative had a binding affinity very similar to that of wild type, the Asp120derivative showed 40% higher affinity. In vitro CKII phosphorylation increased importin binding by about 30% in all cases. These results imply that negative charge at the dsDNA-PK site may enhance nuclear import through increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.

increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.
Nucleocytoplasmic protein transport is central to eukaryotic signal transduction and gene regulation and thereby to processes such as cell proliferation, differentiation, and transformation. Nuclear protein import is an active process (1, 2) dependent on specific targeting signals (nuclear localization sequences; NLSs) 1 (3,4), and comprises at least two steps. In the first, a heterodimeric complex, consisting of the importin 58 or ␣ (NLS-binding protein or receptor) (5,6) and 97 or ␤ (nuclear pore complex-docking protein) (7,8) subunits, targets the NLS-containing protein to the nuclear pore complex. In this process, importin 58 specifically binds NLSs while importin 97 recognizes the importin 58 subunit, as well as having affinity for particular nuclear pore complex components (9 -11). Subsequent translocation into the nucleus is mediated by the GTPbinding protein Ran/TC4 (12) and interacting factor p10/NTF2 (13)(14)(15). The final step of nuclear import may involve some sort of feedback loop, which regulates the maximal level of nuclear accumulation (see Ref. 16).
Nuclear protein import is known to be precisely regulated, with phosphorylation as one of the major mechanisms by which this is brought about (17,18). While enormous progress has been made in the last 2-3 years in terms of characterizing both the steps and factors involved in nuclear import (see Refs. 17 and 18), little is known of the molecular mechanisms by which phosphorylation regulates nuclear protein import. We have shown previously that nuclear import of simian virus SV40 large tumor antigen (T-Ag) ␤-galactosidase fusion proteins is regulated by the CcN motif (19), which comprises phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase (cdk) Cdc2 together with the NLS. While the NLS is absolutely necessary for nuclear import (20,21), the CKII site accelerates the rate of nuclear protein import about 50-fold (19,21), and phosphorylation at the Cdc2 site reduces the maximal nuclear accumulation about 70% (19). The mechanistic basis of the CKII-site enhancement of T-Ag fusion protein import appears to be through phosphorylation increasing the affinity of interaction of the T-Ag NLS with importin 58/97 (see Ref. 22), while indirect evidence suggests that Cdc2 site phosphorylation may increase T-Ag affinity for a cytoplasmic anchor or inhibitor protein (see Ref. 19).
Interestingly, there is a phosphorylation site for the double-stranded DNA dependent protein kinase (dsDNA-PK) at Ser 120 (23,24) between the CcN motif CKII and Cdc2 sites. The dsDNA-PK is a mostly nuclear localized serine/threonine protein kinase (25,26), which has been shown to phosphorylate substrates such as the heat shock protein HSP90 (27), Ku autoantigen (26), and the tumor suppressor p53 (26,28,29). It is involved in the repair of DNA strand breaks (30,31), DNA replication (32,33), transcription (34 -36), and D(V)J recombination of immunoglobulin genes (37,38). To assess whether the dsDNA-PK site of T-Ag regulates its nuclear import, we set out to replace Ser 120 by either aspartic acid or alanine using sitedirected mutagenesis. Nuclear import measurements of the mutant derivatives both in vivo and in vitro indicate for the first time that Ser 120 is important for maximal nuclear accumulation, as well as affecting the nuclear import rate. The basis of this appears to be due to Ser 120 modulating T-Ag NLS recognition by the importin 58/97 dimer. Significantly, the CKII and dsDNA-PK phosphorylation sites appear to interact at the level of both phosphorylation and enhancement of T-Ag nuclear import, and we propose a model where the sites are in close proximity to one another and participate directly in binding to importin. Since many nuclear proteins contain dsDNA-PK sites in the vicinity of their NLSs, the dsDNA-PK may have a more general role in modulating nuclear protein import.
Cell Culture-Cells of the HTC rat hepatoma tissue culture cell line (a derivative of Morris hepatoma 7288C) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum as described previously (19,21).
Nuclear Import Kinetics-Analysis of nuclear import kinetics at the single cell level using either microinjected (in vivo) or mechanically perforated (in vitro) HTC cells in conjunction with confocal laser scanning microscopy (CLSM; Bio-Rad MRC-600) was as described previously (19,21,39,42). In the case of microinjection (Narshige IM-200 pneumatic microinjector and Leitz micromanipulator), HTC cells were fused with polyethylene glycol about 1 h prior to microinjection to produce polykaryons (21). Reticulocyte lysate (Promega) was used as the source of cytosol for the in vitro assay (42). Image analysis of CLSM files (using the NIH Image public domain software) and curve fitting were performed as described (39,42).
Phosphorylation in Reticulocyte Lysate or by Purified CKII or dsDNA-PK-Phosphorylation of T-Ag fusion proteins in reticulocyte lysate or HTC cytosolic and nuclear extracts (prepared as described; Refs. 40 and 42) was performed as described previously (40,42). In vitro phosphorylation using purified CKII was as described previously (40,42), while phosphorylation using purified dsDNA-PK was performed essentially as described by Lees-Miller et al. (26,28) using 0.5 unit/l dsDNA-PK for 1 g/l SV40 T-Ag ␤-Gal fusion protein and 100 ng/l salmon sperm DNA in the assay buffer: 12.5 mM Hepes-KOH (pH 7.5), 1 mM spermidine, 6.5 mM MgCl 2 , 10% glycerol, 0.05% Nonidet P-40, 25 mM KCl, 0.5 mM dithiothreitol, 0.2 mM ATP containing 0.5 M [␥-32 P]ATP at 30°C. dsDNA-PK activity in cell extracts was measured using the dsDNA-PK specific peptide substrate (see above) in the presence of 1 M cAMP-dependent protein kinase inhibitor 5-24 (42) and 100 ng/l salmon sperm DNA in the assay buffer: 50 mM Hepes (pH 7.5), 100 mM KCl, 10 mM MgCl 2 , 2 mM EGTA, and 0.1 mM EDTA for 30 min at 30°C. Analysis was performed as described previously (40,43) through spotting onto Whatman P-81 filters, washing with orthophosphoric acid, and scintillation counting. In the case of fusion proteins, the stoichiometry of phosphorylation was quantitated, subsequent to SDS-gel electrophoresis, using a Molecular Dynamics PhosphorImager, whereby exposure values were converted to absolute values through identical analysis of in vitro phosphorylated samples of predetermined stoichiometry of phosphorylation (40,43).
ELISA-based Binding Assay-An ELISA-based binding assay was used to examine the binding affinity between importin subunits (mouse importin 58/97-glutathione S-transferase (GST) fusion proteins, expressed as described; Refs. 5, 7, and 22) and unphosphorylated and CKII prephosphorylated T-Ag fusion proteins as described (22). Briefly, T-Ag fusion proteins were coated onto 96-well microtiter plates, blocked with BSA, hybridized with various dilutions of precomplexed importin 58/97 (the latter as a GST fusion protein), and then successive incubations carried out with goat anti-GST primary (Pharmacia Biotech Inc.) and alkaline phosphatase-coupled rabbit anti-goat secondary (Sigma) antibodies. Quantitation was performed using the substrate p-nitrophenyl phosphate (Sigma), and the change of absorbance at 405 nm followed using a plate reader (Molecular Devices), with values corrected by subtracting both the absorbance at 0 min, and the absorbance in wells incubated without importin 58/97⅐GST complex.
To correct for differences in coating, T-Ag fusion proteins were subjected to a parallel ␤-galactosidase ELISA assay (22) using a ␤-galactosidase specific monoclonal antibody (Promega) together with an antimouse alkaline phosphatase-conjugated secondary antibody and p-nitrophenyl phosphate. As above, values were corrected by subtracting the absorbance at 0 min. Measurements for importin binding were ultimately corrected for any differences in coating efficiencies quantified in the ␤-galactosidase-based ELISA assay, to enable a true estimate of bound importin to be made.

RESULTS
The dsDNA-PK Site at Ser 120 Is Phosphorylated in T-Ag Fusion Proteins and Regulates Their Nuclear Import-To assess the role of the dsDNA-PK phosphorylation site (Ser 120 ) between the CcN motif CKII and cdk sites in nuclear protein import, site-directed mutagenesis of existing T-Ag fusion protein expression plasmid constructs was performed to substitute Ser 120 by either alanine or aspartic acid (see "Materials and Methods" for construct details; see Fig. 1 for sequence details).
Phosphorylation of the proteins was tested using purified dsDNA-PK from HeLa nuclear extracts. In contrast to the Ala 120 and Asp 120 fusion protein derivatives, those containing an intact Ser 120 were phosphorylated to a marked extent, implying that phosphorylation by dsDNA-PK was specific to Ser 120 (Fig. 2, and data not shown). Interestingly, the DcN-␤-Gal (Ser 120 ) protein, which contains aspartic acid in place of Ser 112 and hence possesses negative charge at the CKII site (see Fig. 1), exhibited about 75% increased phosphorylation by dsDNA-PK compared with the wild type protein CcN-␤-Gal (Ser 120 ) (Fig. 2). Increased dsDNA-PK phosphorylation was also exhibited by a protein containing aspartic acid at position 111 (data not shown), supporting the idea that negative charge at the CKII site enhanced phosphorylation of Ser 120 . Like both CcN-␤-Gal (Ala 120 ) and CcN-␤-Gal (Asp 120 ), T-Ag fusion pro-tein CN-␤-Gal (Ala 120 ), which lacks Ser 120 (as well as Ser 123 -Thr 124 of the Cdc2 site; Ref. 21), was phosphorylated to a negligible extent by dsDNA-PK (data not shown).
The kinetics of nuclear import of the Asp 120 and Ala 120 T-Ag CcN-␤-Gal fusion protein derivatives were compared with those of the wild type CcN-␤-Gal (Ser 120 ) protein both in vivo and in vitro (Figs. 3 and 4; Table I). The maximal nuclear accumulation of the Asp 120 and Ala 120 proteins was about 60 and 30% that of wild type, respectively, in vivo, and was also reduced in vitro (by 30 and 40%, respectively, relative to wild type; see Fig. 4 and Table I). The transport rate of the Asp 120 protein was between 2 and 2.5 times faster than that of the wild type CcN-␤-Gal T-Ag fusion protein (Ser 120 ) (see Table I).
The dsDNA-PK site thus appeared to enhance T-Ag nuclear import, affecting both the maximal level and rate of nuclear accumulation.
The dsDNA-PK Site Influences Phosphorylation at the T-Ag CKII Site-Since the results for dsDNA-PK phosphorylation ( Fig. 2) implied that negative charge at the CKII site influenced phosphorylation at Ser 120 , we decided to test whether the effects of mutations at the dsDNA-PK site on nuclear import might be the direct or indirect result of inhibition of phosphorylation at the CKII site. In vitro phosphorylation experiments were accordingly carried out both using cytosolic extracts and purified rat liver CKII and a variety of T-Ag fusion proteins (Fig. 5). Significantly, CcN-␤-Gal (Asp 120 ) was found to be phosphorylated to a 30% higher extent in cytosol than both CcN-␤-Gal (Ser 120 ) and CcN-␤-Gal (Ala 120 ) (Fig. 5, left panel). The CKII site-mutated fusion protein cN-␤-Gal (Ser 120 ) exhibited negligible phosphorylation, implying that, as observed previously (40,42), more than 90% of the observed cytosolic phosphorylation was attributable to CKII. Results using purified CKII (Fig. 5, right panel) were essentially identical to those using cytosol, CcN-␤-Gal (Asp 120 ) exhibiting about 30% increased phosphorylation compared to wild type. The results clearly indicated that negative charge at position 120 did not inhibit phosphorylation at the CKII site, but instead increased it markedly, suggesting that dsDNA-PK phosphorylation at 120 could potentially regulate nuclear import through increasing CKII site phosphorylation. Asp 120 facilitation of CKII site phosphorylation thus paralleled Asp 111/112 enhancement of Ser 120 phosphorylation (previous section), strongly implying close proximity and functional interaction of the CKII and dsDNA-PK sites.
The dsDNA-PK Site Participates in NLS Binding by Importin 58/97-One possibility to explain the observed faster nuclear import kinetics of CcN-␤-Gal (Asp 120 ) compared to CcN-␤-Gal (Ser 120 ) is that the dsDNA-PK site is involved in binding FIG. 2. Specific phosphorylation of T-Ag fusion proteins at Ser 120 by purified dsDNA-PK from HeLa nuclear extracts. Proteins (see Fig. 1 for T-Ag sequence details) were phosphorylated for 10 (A) or 20 min as indicated at 30°C prior to analysis after gel electrophoresis and autoradiography (A), or analysis using a PhosphorImager (B), as described under "Materials and Methods." Results are from a single typical experiment from a series of similar experiments. dsDNA-PK possessed 12 Ϯ 3 pmol of P i /min of phosphorylation activity for the dsDNA-PK-specific peptide substrate Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys-Lys in the same experiment. All fusion proteins contain SV40 T-Ag sequences fused NH 2terminal to E. coli ␤-galactosidase (amino acids 9 -1023). The singleletter amino acid code is used, whereby the NLS is underlined and the phosphorylation sites for CKII, Cdc2, and the dsDNA-PK shaded. Uppercase letters indicate T-Ag sequence. The cN-, CN-, CcN-, and DcN-␤-Gal fusion proteins have been described previously (19,21,40).
to the importin 58/97 subunits. This is supported by the fact that a protein containing T-Ag amino acids 120 -135 binds importin about 5 times better than those containing only the NLS (amino acids 126 -135) (see Ref. 22). The CKII site and in particular phosphorylation at the site further increase the binding affinity (22). To test whether the dsDNA-PK site influences importin recognition, we employed our previously established ELISA-based binding assay (22) using unphosphorylated and CKII prephosphorylated proteins. The binding affinity of the CcN-␤-Gal (Asp 120 ) protein was 40% higher than that of the CcN-␤-Gal (Ser 120 ) protein (Fig. 6, Table I), indicating that negative charge at position 120 enhances NLS recognition by importin 58/97. This implies that phosphorylation at Ser 120 may enhance T-Ag nuclear import through facilitating recognition of the T-Ag NLS by importin 58/97. The CcN-␤-Gal (Ala 120 ) protein was comparable to wild type in its affinity (Table I).
CKII phosphorylation increased the affinity of binding of importin 58/97 to CcN-␤-Gal (Ser 120 ) consistent with previous results (see Ref. 22), as well as to CcN-␤-Gal (Asp 120 ) and CcN-␤-Gal (Ala 120 ) (Fig. 6, Table I). All proteins were thus similar in terms of the facilitating effect of CKII phosphorylation on importin-T-Ag NLS interaction. Results supported the idea that both the CKII and dsDNA-PK sites are directly involved in interaction with importin. DISCUSSION This study represents the first demonstration that the dsDNA-PK site within the CcN motif regulates T-Ag nuclear transport, and the first determination of the possible mechanism of action of phosphorylation at the site. That the dsDNA-PK site of T-Ag is important for nuclear import is shown by the fact that maximal nuclear accumulation is reduced in Asp 120 and Ala 120 mutants both in vivo and in vitro. Nuclear accumulation of the Asp 120 mutant is higher than that of the Ala 120 mutant, implying that negative charge at position 120 (normally supplied by phosphorylation of wild type Ser 120 ) is mechanistically important in the effect on nuclear import, thus being quite comparable to similar findings for the T-Ag CKII and cdk CcN motif sites (see Refs. 19 and 40). An intriguing result was that the Asp 120 mutant shows a faster import rate than the wild type Ser 120 protein, indicating that negative charge at position 120 also regulates the nuclear import rate. Phosphorylation at the CKII site either in reticulocyte lysate or using purified CKII was elevated in the case of the Asp 120 mutant, compared with wild type and the Ala 120 mutant, meaning that the increased import rate on the part of the Asp 120 mutant may in part be due to increased phosphorylation at the CKII site which is known to accelerate nuclear import (21, 40).

FIG. 4. Nuclear transport of T-Ag fusion proteins in microinjected (in vivo) or mechanically perforated (in vitro) HTC cells using CLSM.
Measurements, performed as described under "Materials and Methods" (19,39,40), represent the average of at least two separate experiments (see also Table I  The T-Ag ␤-galactosidase fusion protein sequences are shown in Fig. 1. b Raw data (see Fig. 4, and data not shown) were fitted for the function Fn/c (t) ϭ Fn/c max ϫ (1 Ϫ e Ϫkt ) (19,22,42), where t is time in minutes. Results shown are averaged over two separate experiments with the S.E. (derived from the curve fits) indicated.
c Binding of T-Ag fusion proteins to importin 58/97 was quantitated using an ELISA-based binding assay (Ref. 22; see "Materials and Methods"). Curves (see Fig. 6 and not shown) were fitted for the function B(x) ϭ B max (1 Ϫ e Ϫkx ), where x is the concentration of importin 58/97. Results represent the mean Ϯ S.E. for at least two separate experiments. d The stoichiometry of phosphorylation by CKII was between 0.7 and 1 mol of P i /mol of tetramer.
The initial step of nuclear import involves binding by the NLS-binding importin heterodimer (5)(6)(7)(8)(9)(10)(11). Results from our ELISA-based binding assay, which enables direct measurement of the affinity of NLS recognition by importin (see also Ref. 22), indicate that the Asp 120 mutant has a higher affinity for importin 58/97. This implies that Ser 120 may be directly involved in contacting importin 58 in conjunction with the NLS and that negative charge at the site increases the binding affinity. Importin binding can be further increased by CKII phosphorylation, consistent with our previous observations (see Ref. 22). The clear implication is that the amino acids flanking the T-Ag NLS, including the dsDNA-PK and CKII sites, determine the affinity of NLS binding by importin 58/97. Negative charge at position 120 is thus able to enhance the nuclear protein import rate through directly increasing the affinity of binding of the NLS by importin 58/97, as well as through facilitating phosphorylation at the CKII site and thereby further increasing the affinity of binding between the NLS and importin 58/97. This suggests that phosphorylation at Ser 120 could also regulate T-Ag nuclear import through modulation of NLS recognition. Although the dsDNA-PK is known to be largely nuclear (see also Ref. 44) and phosphorylation at Ser 120 may therefore mostly take place in the nucleus, we observed significant phosphorylation of a specific dsDNA-PK peptide substrate in cytosol (see "Materials and Methods"), implying that Ser 120 phosphorylation prior to nuclear entry, thereby enhancing CKII site phosphorylation and interaction with importin 58/97, may occur under physiological conditions. Consistent with this, T-Ag has been shown to be phosphorylated at Ser 120 to some extent in the cytosol in SV40 virus infected cells (see Refs. 45 and 46 with respect to data for phosphopeptides 7 and 11). Although we are formally unable at this stage to assert that the cytosolic phosphorylation activity is due to dsDNA-PK rather than to another kinase of similar properties and specificity (e.g. a dsDNA-PK of a different subunit make-up; see Ref. 44), what is clear from this study is that the dsDNA-PK site positively regulates both T-Ag NLS recognition by importin and T-Ag nuclear import. Nuclear Fig. 1 for T-Ag sequences) were incubated for 4 h at 30°C, after which the stoichiometry of phosphorylation was determined as described under "Materials and Methods." Results are from a single experiment representative of two separate experiments, where the S.E. for the raw data for this experiment was not greater than 8.7 and 4.5% of the value of the mean for cytosol and purified CKII, respectively. Numbers on top of the columns represent the results as a percentage relative to that for CcN-␤-Gal (Ser 120 ).  Table I for pooled data). The stoichiometry of phosphorylation by CKII for CcN-␤-Gal (Ser 120 ) and CcN-␤-Gal (Asp 120 ) was around 1 mol of P i /mol of tetramer. phosphorylation of Ser 120 by dsDNA-PK may also play a role in nuclear import in the light of the fact that, based on our results here, CKII site phosphorylation in the cytoplasm would make Ser 120 a better phosphorylation site for dsDNA-PK in the nucleus. This might constitute the feedback communication between nucleus and cytoplasm indicated by our previous study (16). The state of dsDNA-PK site phosphorylation may conceivably be important for the import step of translocation into the nucleus, during which the NLS-containing protein enters the nucleus complexed to the importin 58 subunit alone (10,47) rather than to the higher affinity importin 58/97 dimer; in this case, higher affinity binding to importin 58 through negative charge at Ser 120 may be critical. Dephosphorylation at the dsDNA-PK site seems unlikely to be mechanistically important in regulating T-Ag nuclear import (see also Refs. 19 and 40), since the nuclear import properties of the Asp 120 fusion protein, containing a non-dephosphorylatable residue at position 120, resemble those of the wild type Ser 120 -containing protein reasonably well. The fact that the wild type protein is accumulated more efficiently than the Asp 120 protein is attributable to the fact that aspartic acid is not functionally equivalent to phosphoserine, consistent with similar results for the CKII and Cdc2 sites (19,40).

FIG. 5. In vitro phosphorylation of T-Ag fusion proteins in cytosol and by rat liver CKII. Fusion proteins (see
dsDNA-PK is implicated in regulating gene expression and growth (26,44), as well as a number of other cellular processes (see Introduction). Nuclear transcription factors phosphorylated by dsDNA-PK include Sp1, c-Myc, Oct-1, serum response factor, c-Fos, c-Jun, and estrogen and progesterone receptors, as well as high mobility group proteins 1 and 2 (see Ref. 44). Our findings here indicate that the dsDNA-PK may have a role in regulating T-Ag nuclear import, and it is perhaps not insignificant that a number of other NLS-containing nuclear proteins, some of which are listed in Table II, contain either putative or confirmed sites for dsDNA-PK (see also Ref. 44). In the case of most of these, with the exceptions of c-Jun and VirE2, the dsDNA-PK sites are quite large distances from the NLSs of the respective proteins, implying that it is unlikely that, if they do influence nuclear import, they do so by directly modulating NLS recognition. Of significance, however, in view of the results here may be the fact that several of the dsDNA-PK sites listed in Table II, intriguingly including those of c-Jun and VirE2, are in close proximity to consensus CKII sites (see legend to Table II). Whether these sites indeed regulate nuclear import of their respective proteins will require direct experimentation.
Based on our results here and our previous study demonstrating the role of the CKII site in NLS binding by importin (22), it is possible to speculate that the dsDNA-PK and CKII sites are both very close to one another in spatial terms and are directly involved in importin recognition, as shown schematically in Fig. 7. Close proximity of the sites as depicted in Fig. 7A explains the fact that negative charge at Ser 120 increases phosphorylation at the CKII site; the concentration of negative charge through both the dsDNA-PK site and the CKII site recognition site makes the CKII site an even higher affinity CKII site (see Ref. 51). The finding that negative charge at the CKII site increases dsDNA-PK phosphorylation of Ser 120 supports the idea that the two phosphorylation sites are in close proximity, and that negative charge at either site can influence events at the other. Recognition of the T-Ag NLS by the importin 58 homolog karyopherin ␣ 1 is known to be dependent on the acidic carboxyl terminus (residues 501-510; Ref. 52), which is homologous to amino acids 492-503 of mouse importin 58 (including Glu-Glu-Glu-Glu-Asp). Since the T-Ag NLS includes five positively charged residues, it seems reasonable to postulate that interactions between the two regions occur through the opposite charge. Analogously, the phosphorylated CKII/ dsDNA-PK sites may also interact with importin through charge interactions. Consistent with this idea, substitution of Asp-Asp-Glu (amino acids 113-115) of the CKII recognition site severely reduce importin binding (22), while CKII phosphorylation, increasing negative charge at the site, markedly increases the affinity of binding. Additionally, CKII phosphorylation increases importin binding in the case of a T-Ag fusion protein containing the complete NH 2 -terminal flanking region but retaining a mutated (non-functional) NLS (22), implying that the phosphorylated CKII site in the presence of the dsDNA-PK site, may have low but significant affinity for im- portin. As shown here, substitution of Ser 120 by aspartic acid also increases importin binding, implying that the dsDNA-PK site also participates directly in NLS binding by importin. Consistent with this, a fusion protein lacking the CKII site but retaining amino acids 120 -125 in addition to the NLS shows 5 times higher binding to importin 58/97 than a protein containing only the NLS (22). Intriguingly, the NH 2 terminus of importin 58 contains a basic region (amino acids 16 -51, including two blocks of positive charge resembling a bipartite NLS; see Ref. 52), which would be a possible candidate as a binding region for the CKII and dsDNA-PK sites (see Fig. 7B), although this region also appears to be essential for binding to an acidic region (amino acids 329 -342) of the importin 97 homolog karyopherin ␤ (53). Mutagenic analysis to determine the importin 58 residues involved in interaction with the CKII and dsDNA-PK sites is currently under way in this laboratory. An alternative hypothesis with respect to the role of the CKII and dsDNA-PK sites on importin recognition is that phosphorylation/negative charge at the sites alters T-Ag conformation (possibly through charge repulsion between the two sites), thereby enhancing the accessibility of the NLS; in the absence of phosphorylation, the NLS might be largely masked by the amino-terminal flanking region. This, however, does not seem to explain the fact that in the complete absence of the flanking regions, both NLS recognition (22) and nuclear transport (20,21) are severely impaired, even the presence of the dsDNA-PK site alone drastically increasing both markedly (22). This is also supported by measurements for importin binding to NLS-containing peptides, implying that the results for the T-Ag fusion proteins are valid and not a product of masking or other effects by ␤-galactosidase residues. Additionally, circular dichroism measurements of peptides comprising the CcN motif 2 or secondary structure predictions provide no indication as to any significant structural element, so that we presently tend to the view expressed in Fig. 7 that the simplest model to explain our results involves direct interaction of the CKII and dsDNA-PK sites with importin 58. Detailed structural analysis of importin-NLS complexes, and thereby definitive determination of the mechanistic basis and molecular details of the enhancement of NLS binding by negative charge at the CKII and dsDNA-PK sites, is the focus of future work in this laboratory.