INTRODUCTION

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NF-B comprises a family of proteins including p50, p52, p65 or RelA, p100, p105, and c-Rel which regulate the expression of a variety of cellular and viral genes (reviewed in references 7, 75, and 79). Each of these proteins contains a region known as the Rel homology domain which is critical for the DNA binding and dimerization properties of these proteins. One of the major regulatory mechanisms which control NF-B activity is the unique cellular localization of different members of this family. In unstimulated cells, p65 or RelA is nearly exclusively localized in the cytoplasm (4-6, 13, 34), but it translocates to the nucleus upon treatment of the cells with a variety of inducers such as phorbol esters, interleukin 1, and tumor necrosis factor alpha (TNF-) (43, 73). RelA dimerizes with other NF-B family members (7, 75, 79) and activates gene expression via its potent transactivation domain (8, 67, 70). Thus, cellular proteins which regulate the nuclear translocation of NF-B are critical for the control of NF-B activation of viral and cellular genes.

The IB proteins constitute a group of cytoplasmic proteins that bind to NF-B and sequester these proteins in the cytoplasm by preventing their nuclear localization. A number of different IB proteins have been identified including IB, IB, IB (reviewed in reference 79), and IB (80). IB (41) and IB (76) are the best studied of these regulatory proteins. Treatment of cells with a variety of agents such as phorbol esters, TNF-, and UV irradiation results in the degradation of IB and IB and the nuclear translocation of NF-B (12, 17, 43, 73). IB present in the nucleus terminates the induction process in response to TNF- and other activators (2, 3, 60).

IB and IB have distinct functional domains. For example, the N terminus and the ankyrin repeats of IB are required for the cytoplasmic regulation of NF-B while C-terminal sequences are required to regulate NF-B function in the nucleus (60). The activity of IB is regulated by its phosphorylation state. The C termini of the IB and IB proteins contain PEST domains with serine and threonine residues that are phosphorylated by cellular kinases which regulate the intrinsic stability of these proteins (10, 11, 25, 57, 61, 66, 81). In addition, the amino termini of these proteins each contain two closely spaced serine residues that are also capable of being phosphorylated by cellular kinases (16, 17, 28, 32, 77). Serine residues at positions 32 and 36 of IB (16, 17, 28, 32, 77) and 19 and 23 of IB (62) are phosphorylated when cells are treated with various agents such as TNF- and phorbol esters. Phosphorylation of these residues leads to their ubiquitination and proteasome-mediated degradation (1, 23, 24, 28, 32, 58, 69, 77). Mutations of these amino-terminal serine residues in IB and IB prevent the degradation of these proteins upon treatment of cells with TNF- or phorbol esters and inhibit the nuclear translocation of NF-B (16, 28, 62, 77).

Biochemical fractionation has been performed to identify cellular kinases that are capable of phosphorylating IB. A protein complex migrating at approximately 700 kDa is capable of phosphorylating IB on serine residues 32 and 36, resulting in IB degradation by the proteasome (24, 51). Two related kinases isolated from a similar-size complex, IKK and IKK, phosphorylate serine residues 32 and 36 in IB (27, 63, 65, 83, 85). Another kinase, RSK1, also phosphorylates the amino terminus of IB (71). In contrast to IKK and IKK, RSK1 phosphorylates IB exclusively on serine residue 32. Cellular kinases are also capable of phosphorylating the carboxy terminus of IB. For example, casein kinase II phosphorylates serine and threonine residues in the carboxy terminus of IB (10, 20, 58, 59, 82). Mutation of these carboxy-terminal serine and threonine residues increases the steady-state levels of IB (57, 58). These results suggest that multiple cellular kinases are capable of phosphorylating IB and regulating different aspects of its function.

To further characterize cellular kinases that phosphorylate IB, we utilized biochemical fractionation and peptide sequence analysis by ion trap mass spectrometry (MS). This analysis demonstrated that the catalytic subunit of DNA-dependent protein kinase, DNA-PKcs, phosphorylated specific residues in the amino and carboxy termini of IB and IB. We also investigated the regulation of IB and NF-B in equine and murine severe combined immunodeficiency (SCID) cell lines deficient in DNA-PKcs. SCID cells, which lack the DNA-PKcs gene, exhibited constitutive nuclear NF-B DNA binding activity, in contrast to control cell lines. DNA-PK phosphorylation of IB increased its ability to inhibit NF-B DNA binding properties. These studies suggest that IB is a target for DNA-PK phosphorylation and that this phosphorylation regulates the DNA binding properties of NF-B in the nucleus.

MATERIALS AND METHODS

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Plasmid construction. Full-length wild-type IB cDNA and IB cDNA with mutations at the codons for serine residues 32 and 36 in a pCMV4 vector (gift from Dean Ballard) (16) were digested with BamHI and HindIII and cloned into the pGEX-KG vector at BamHI-HindIII sites. A deletion of the carboxy-terminal 74 amino acids of IB, including the PEST domain, was accomplished by digesting the IB cDNA with BamHI and HincII and cloning the resulting fragment into pGEX-KG cut with BamHI and SmaI. Another deletion of the C terminus of wild-type IB was accomplished by digesting the IB cDNA with BamHI and SacI and cloning the fragment into pGEX-KG at the BamHI-SacI site to produce an IB cDNA encoding the amino-terminal 138 amino acids of IB. These truncated IB cDNAs were then utilized to make single-stranded M13 DNA templates for mutating either serine residue 36, serine residue 32, or both serine residues to alanine or tyrosine residue 42 to phenylalanine. The oligonucleotides used for the site-directed mutagenesis mutations were as follows: for changing serine residue 32 to alanine, 5'-GAC CGC CAC GAC GCC GGC GGC CTG GAC TCC-3'; for changing serine residue 36 to alanine, 5'GCG GCC TGG ACG CCA TGA AAGA-3'; and for changing tyrosine residue 42 to phenylalanine, 5'-CGA GGA GTT CGA GCA GAT GG-3'. Finally, fragments extending from amino acids 1 to 53 for both wild-type IB and IB with mutations at serine residues 32 and 36 were generated by BamHI and XhoI digestion of these cDNAs and cloning of the resulting cDNA fragments into pGEX-KG.

Cell culture and protein extraction. Wild-type equine fibroblasts (CRL6288) and murine NIH 3T3 cells were purchased from the American Type Culture Collection ATCC. Equine SCID fibroblasts cell line 1863 (82) and murine SCID fibroblast cell line SF19 (39) were described previously. The parental (CB-17), SCID (SCID/St), and SCID-complemented (100E and 50D) cell lines have been previously described (49). HeLa spinner cells were maintained in supplemented Eagle minimal essential media with 5% newborn calf serum at a density of 4 × 10⁵ cells/ml. Tetradecanoyl phorbol acetate (TPA) (Sigma; 50 ng/ml) and/or ionomycin (Calbiochem; 2 µM) was added to either HeLa, 3T3, or SF19 cells as outlined below.

Preparation of recombinant adenoviruses. The production of recombinant adenovirus utilized previously described methodology (21, 36). The pAC/CMVplpa plasmids encoding either wild-type IB or IB with mutations at serine residues 32 and/or 36 were cotransfected with the pJM17 adenovirus plasmid into 293 cells (adenovirus E1a-transformed human embryonic kidney cells). Viruses from 293 cell supernatants of cultures showing a complete cytopathic effect were purified by cesium chloride banding. Virus titers were determined by plaque assay using serial dilution (38). The pAC/CMVplpa, pJM17, and control recombinant replication-defective adenoviruses containing -galactosidase were obtained from P. Nissen. Adenovirus infection of murine cell lines was performed by using a multiplicity of infection of 10 for 24 h.

Gel electrophoresis mobility shift assay. Nuclei from NIH 3T3, SF19 (murine SCID fibroblasts), CRL6288 (normal equine fibroblast), and 1863 (equine SCID fibroblasts) cells and the murine cell lines CB-17, SCID/St, 100E, and 50D were resuspended in buffer containing 20 mM HEPES (pH 7.5), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mg of PMSF per ml, 10% glycerol, 10 µg of leupeptin, 10 µg of aprotinin per ml and extracted at 4°C on a rocker for 30 min, followed by Eppendorf centrifugation at 14,000 rpm for 5 min. The supernatants were collected, and 3 µg of each nuclear extract was incubated with 0.1 pmol of ³²P-labeled double-stranded B binding site oligonucleotide (5'-GCTGGGGACTTTC-3') or SP1 binding site oligonucleotide (5'-ATTCGATCGGGGCGGGGCGAGC-3') in buffer containing 1 µg of poly (dI-dC), 1 µg of bovine serum albumin, 10 mM HEPES (pH 7.9), 0.5 mM DTT, 0.1 mM EDTA, 60 mM KCl, 0.2 mM PMSF, 5 mM MgCl₂, and 12% glycerol at room temperature for 15 min. Samples were analyzed by 5% native polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.

For IB inhibition experiments, bacterially produced IB protein was cleaved from the GST moiety with thrombin, followed by the addition of PMSF. This IB protein was then phosphorylated by purified DNA-PK by using cold ATP. The nonphosphorylated and phosphorylated IB proteins were added to the gel retardation reaction mixture 30 min prior to the addition of the ³²P-labeled probe. The recombinant p50 and p65 NF-B proteins were produced in baculovirus expression vectors and purified by nickel agarose chromatography.

Immunoprecipitation of DNA-PKcs. Monoclonal antibodies 42-26, 25-4, and 18-2 directed against DNA-PKcs (19, 49) or flag monoclonal antibody (Kodak) was added to column fractions containing DNA-PKcs and incubated at 4°C for 1 h. Protein G-Sepharose was then added for 1 h, and the beads were washed three times with buffer D (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10% glycerol) and one time with buffer containing 12 mM HEPES (pH 7.9), 0.6 mM EDTA, 0.6 mM DTT, 6% glycerol, 60 mM KCl, and 7.5 mM MgCl₂. Kinase reactions were then performed.

In vitro kinase assay. Kinases which bound to GST-IB fusion proteins and which were able to phosphorylate IB were assayed by a modification of the previously described methods (44). Bacterially expressed GST-IB proteins were bound to glutathione agarose matrix (Sigma). HeLa S100 cells or column fractions were incubated with GST-IB-bound matrix in buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF at 4°C for 1 h. The matrix was washed twice with buffer containing 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 5 mM DTT, and 1 mM PMSF and once with the kinase reaction buffer (50 mM Tris [pH 7.4], 5 mM MnCl₂, 5 mM DTT). The kinase assay was then performed in the kinase reaction buffer with the addition of 4 µM ATP, 10 µCi of [-³²P]ATP, 5 mM NaF, 1 mM sodium orthovanadate, 40 µM MgCl₂, a protease inhibitor cocktail (Boehringer Mannheim), and 1 mM PMSF at 30°C for 15 min. Similar results were seen when 2.5 µM okadaic acid was added to the kinase assay mixtures. The matrix was pelleted by centrifugation, 20 µl of 2× sodium dodecyl sulfate-PAGE (SDS-PAGE) sample buffer was added, and the samples were heated at 95°C for 5 min. The samples were resolved with SDS-12% polyacrylamide gels followed by autoradiography.

Purification of IB kinases. All the purification steps were conducted at 4°C. HeLa S100 extract (3 g) was prepared from 150 liters of cells (10¹¹ cells) by the method of Dignam (29) and dialyzed against 0.1 M KCl buffer D (20 mM HEPES [pH 7.9], 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT). The extract was then fractionated on a phosphocellulose column which was washed with 150 ml of 0.1 M KCl buffer D, and the kinase activity was eluted with 100 ml of buffer D containing 0.3 M or 1.0 M KCl. The IB kinase activity was assayed with GST-IB fusion proteins, which extended from amino acid 1 to 138 and contained either wild-type sequences or sequences with mutations of serine residues 32 and 36, that were bound to glutathione agarose beads. The 0.3 M KCl fraction which contained the majority of the kinase activity was dialyzed against 0.05 M KCl buffer D and fractionated on a Q-Sepharose column (1 by 2 cm) which was eluted with 1.0 M KCl in buffer D. The active kinase fractions were pooled, loaded on a Superdex 200 column (26/60; Pharmacia), and eluted with 0.05 M KCl in buffer D. The active fractions were then pooled and fractionated with a heparin agarose column (1 by 3 cm). After the column was washed with 0.05 M KCl in buffer D, it was eluted with a 20-ml linear gradient of 0.05 to 0.8 M KCl in buffer D. The fractions containing the peak kinase activity were pooled and dialyzed against 0.05 M KCl in buffer D and were fractionated on a Mono Q fast protein liquid chromatography (FPLC) column (Pharmacia) with a linear gradient of 0.05 to 1.0 M KCl in buffer D. The fractions active for kinase activity were then pooled and dialyzed against 0.05 M KCl in buffer D and fractionated by using a Mono S FPLC column (Pharmacia) with a linear gradient of 0.05 to 0.6 M KCl. The fractions containing the peak of kinase activity were dialyzed against 0.1 M KCl in buffer D, aliquoted, frozen in liquid nitrogen, and stored at 80°C.

Peptide sequencing by ion trap MS of components of the IB kinase. Purified fractions containing the IB kinase activity were pooled, loaded onto an SDS-6% polyacrylamide gel, and subjected to electrophoresis in Tris-glycine buffer. Coomassie brilliant blue-stained gel slices containing a 400-kDa protein species that correlated with the peak of kinase activity for IB were excised and subjected to in gel reduction, S-carboxyamidomethylation, and tryptic digestion (Promega). Sequence information on the digestion products was determined by capillary (Monitor C₁₈ column [0.5 by 150 mm]; Michrom BioResources) reverse-phase chromatography, coupled to the electrospray ionization source of a quadruple ion trap mass spectrometer (Finnigan LCQ, San Jose, Calif.). The instrument was programmed to acquire successive sets of three-scan modes consisting of full-scan MS over the m/z range 395 to 1,118 amu, followed by two data-dependent scans on the most abundant ion in that full scan. These data-dependent scans allowed the automatic acquisition of high-resolution (zoom scan) spectra to determine charge state and exact mass and MS/MS spectra for peptide sequence information. Interpretation of the resulting MS/MS spectra was facilitated by searching the NCBI nr and dbest databases with the algorithm Sequest (31) and was then confirmed manually. The peptide sequences TVGALQVLGTEAQSSLK, LLLQGEADQSLLTFIDK, and SLGPPQGEEDSVPR had sequence identity with the DNA-dependent protein kinase catalytic subunit (40).

RESULTS

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Identification of cellular kinases that phosphorylate IB. To characterize cellular kinases that phosphorylate IB, we prepared S100 extract from both untreated and TPA-treated HeLa cells (29). The amino-terminal 138 amino acids of IB were fused to GST so that kinases that phosphorylated the amino terminus of IB could be detected. A GST-IB fusion protein containing mutations of serine residues 32 and 36 was also assayed in an attempt to differentiate kinases with specificity for these residues (16, 17, 28, 32, 77). Following binding of cellular proteins to GST-IB coupled to glutathione agarose beads, the beads were extensively washed and kinase reactions were performed, followed by SDS-PAGE and autoradiography.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Constitutive and inducible cellular kinases that phosphorylate the amino terminus of IB. S100 extract was prepared from HeLa cells that were either untreated (A) or treated with 50 ng of phorbol ester (TPA) per ml (B) for 1 h. The extract was fractionated on a phosphocellulose column, washed with 0.1 M KCl, and eluted with 0.3 M KCl. Each of these extracts was concentrated on a Q-Sepharose column and then fractionated on a Superdex 200 column. The column fractions from the Superdex 200 column were then assayed for their ability to bind and phosphorylate a GST-IB fusion protein truncated at amino acid 138. The positions of the molecular weight markers in the Superdex 200 column fractions are indicated. (C) Fraction 19 from untreated HeLa S100 extract that eluted from the mono Q column was assayed for its ability to phosphorylate 0.5 µg of either GST (lane 1), a GST-IB fusion protein truncated at amino acid 138 that contained wild-type or mutant sequences at serine residues 32 and 36 (lanes 2 and 3, respectively), and a GST-IB fusion protein truncated at amino acid 53 containing wild-type or mutant sequences at serine residues 32 and 36 (lanes 4 and 5, respectively). Similar results were obtained with TPA-treated extract.

Purification of a cellular kinase that phosphorylates the amino terminus of IB. To purify kinases present in the 700-kDa fraction, S100 extract was prepared from 150 liters of untreated HeLa cells (Fig. 2). The purification scheme included elution of active kinase fractions from a phosphocellulose column with 0.3 M KCl, concentration of the sample on a Q-Sepharose column, and fractionation on a Superdex 200 column. The kinase activity that eluted in the 600- to 700-kDa Superdex 200 FPLC fraction was then further purified by heparin agarose, mono Q FPLC, and mono S FPLC chromatography. The fractions from the final mono S column were subjected to SDS-PAGE and Coomassie staining and were assayed for their ability to phosphorylate IB.

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Fractionation of IB kinase activity. A schematic of the purification scheme used to purify cellular kinases that phosphorylate the amino terminus of IB is shown. S100 extract was obtained from untreated HeLa cells and fractionated on a phosphocellulose column washed with buffer containing 0.1 M KCl and eluted with 0.3 M KCl. This fraction was applied to a Q-Sepharose (Q-seph) column and eluted with 1.0 M KCl, followed by fractionation on a Superdex 200 column. Proteins with kinase activity for the 138 amino-terminal residues of IB were then fractionated on heparin agarose, mono Q FPLC, and mono S FPLC and eluted with KCl gradients as indicated. FT, column flowthrough.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Purified DNA-dependent protein kinase phosphorylates the amino terminus of IB. (A) Fractions (17 to 25) from the final mono S FPLC column were subjected to SDS-PAGE and Coomassie staining. (B) Kinase assays were also performed with these column fractions by using a GST-IB fusion protein extending from amino acids 1 to 138. (C) These same column fractions were also analyzed by Western blotting using polyclonal goat antibody directed against the DNA-dependent protein kinase catalytic subunit.

DNA-PK phosphorylation of IB. DNA-PK is a serine/threonine kinase which is a member of a family of protein kinases known as phosphatidylinositol 3 (PI-3) kinases that contain a conserved kinase domain and include ATM, FRAP, and FRP1 (26, 40, 68). DNA-PK can be detected in cellular extracts prepared from both the nucleus and the cytoplasm, although the majority of this activity is found in the nucleus (19, 54). This kinase comprises two subunits, which include a regulatory subunit that is the DNA end-binding heterodimer Ku and catalytic subunit DNA-PKcs. The kinase activity of DNA-PK is stimulated by the addition of double-stranded DNA and inhibited by the addition of wortmannin (40). DNA-PK phosphorylates a number of substrates including Jun (9), DNA replication factor A (18), p53 (33, 54, 56), the RNA polymerase II C-terminal domain (CTD) (30, 64), simian virus 40 T-antigen (22), hsp90 (19, 54), SP1 (46), Oct1 (35), and the serum response factor (59).

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Antibody directed against DNA-PK immunoprecipitates IB kinase activity. (A) Full-length GST-IB (lane 1), GST-IB (lane 2), and -casein (lane 3) were used in in vitro kinase assays with mono S column fractions immunoprecipitated with either the specific monoclonal antibody 42-26 directed against DNA-PKcs (lanes 1 to 3) or the unrelated flag monoclonal antibody (lanes 4 to 6), followed by SDS-PAGE and autoradiography. (B) In vitro kinase assays were performed with GST-IB truncated at amino acid 138 and the mono S column fractions containing DNA-PK (lane 1) in the presence of 50 or 250 nM wortmannin (Wort) (lanes 2 and 3, respectively) or 50 or 250 nM rapamycin (Rap) (lanes 4 and 5, respectively). (C) Immunoprecipitation of DNA-PK activity was performed with monoclonal antibodies (42-26 and 25-4) directed against DNA-PKcs. Cytoplasmic extracts prepared by either the Dignam method (lanes 1 to 3) or the rapid-lysis method (lanes 4 to 6) were immunoprecipitated with these antibodies and analyzed in in vitro kinase assays without substrate (lanes 1 and 4), with GST (lanes 2 and 5), or with GST-IB (lanes 3 and 6).

DNA-PK phosphorylates both the amino and carboxy termini of IB. We next characterized the ability of a highly purified DNA-PK prepared by a previously described purification scheme (19) to phosphorylate GST fusion proteins containing wild-type and mutant IB (Fig. 5A) and IB (Fig. 5B) proteins. Purified DNA-PK and our mono S column fractions containing DNA-PK gave equivalent specificities for the IB and IB substrates (19). Numerous studies have demonstrated that serine and threonine phosphorylation by DNA-PK is highly dependent on the presence of free DNA ends (19, 54). Purified DNA-PK phosphorylated -casein, and the degree of this phosphorylation was greatly enhanced by the addition of sonicated double-stranded salmon sperm DNA (Fig. 6A, lanes 1 and 2). There was no phosphorylation of GST by DNA-PK (Fig. 6A, lanes 3 and 4). The GST fusion protein containing the full-length IB protein was phosphorylated by DNA-PK, and this phosphorylation was stimulated by the addition of double-stranded DNA (Fig. 6A, lanes 5 and 6). Mutations of serine residues 32 and 36 in IB only slightly decreased IB phosphorylation (Fig. 6A, lanes 7 and 8). An IB protein with its 53 N-terminal amino acids deleted was strongly phosphorylated by DNA-PK (Fig. 6A, lanes 9 and 10), indicating significant C-terminal phosphorylation of IB by DNA-PK. A deletion of the carboxy-terminal 74 amino acids of IB markedly decreased its phosphorylation (Fig. 6A, lanes 11 and 12). These results were consistent with predominant DNA-PK phosphorylation of the carboxy terminus of IB.

View larger version (29K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   FIG. 5.   Schematic structure of IB and IB fusion constructs. (A) GST fusion proteins were constructed with wild-type IB (lane 1), IB with mutations of serine residues 32 and 36 to alanine (lane 2), IB with mutations of serine residue 283 and threonine residues 291 and 299 to alanine (lane 3), IB with mutations of threonine residues 273, 291, and 299 and serine residue 283 to alanine (lane 4), IB with residues 244 to 317 deleted (lane 5), IB with residues 275 to 317 deleted (lane 6), IB with its amino-terminal 53 amino acids deleted (lane 7), IB truncated at amino acid 138 (lane 8), this construct with mutations of serine residues 32 and 36 to alanine (lane 9), serine residue 32 to alanine (lane 10), serine residue 36 to alanine (lane 11), tyrosine residue 42 to phenylalanine (lane 12), or serine residues 32 and 36 to alanine and tyrosine residue 42 to phenylalanine (lane 13). (B) GST fusion proteins were constructed with the full-length IB (lane 1), this same construct having mutations of serine residues 19 and 23 to alanine (lane 2), IB truncated at amino acid 305 (lane 3), and this protein with serine residues 19 and 23 changed to alanine (lane 4). The positions of the ankyrin repeats, the PEST domain, and serine and/or tyrosine residues that were mutated are shown.

View larger version (33K): [in this window] [in a new window]   View larger version (48K): [in this window] [in a new window]   FIG. 6.   Specificity of DNA-PK phosphorylation of IB and IB. (A) Kinase assays were performed with purified DNA-PK and substrates including -casein (lanes 1 and 2), GST (lanes 3 and 4), or GST fusion proteins containing wild-type IB (lanes 5 and 6), this construct with a mutation of serine residues 32 and 36 to alanine (lanes 7 and 8), IB with amino acids 1 to 53 deleted (lanes 9 and 10), or IB with amino acids 139 to 317 deleted (lanes 11 and 12). The kinase assays were performed in the absence () or presence (+) of sheared salmon sperm DNA (0.5 µg), as indicated. (B) GST fusion proteins containing either wild-type IB (lanes 1 and 2), IB (amino acids [aa] 1 to 275) with the majority of the PEST domain deleted (lanes 3 and 4), full-length IB with a mutation of threonine residue 291 to alanine (lanes 5 and 6), IB with mutations of serine residue 283 and threonine residues 291 and 299 to alanine (lanes 7 and 8), or IB with threonine residues 273, 291, and 299 and serine residue 283 changed to alanine (lanes 9 and 10) were analyzed in kinase assays in either the absence () or presence (+) of double-stranded DNA and purified DNA-PK. (C) GST fusion proteins containing either wild-type IB (lane 1), full-length IB with serine residues 32 and 36 changed to alanine (lane 2), IB (aa 1 to 138) with a deletion of aa 139 to 317 (lane 3), this same construct with serine residues 32 and 36 mutated to alanine (lane 4), IB (aa 1 to 138) with serine residue 32 changed to alanine (lane 5), IB (aa 1 to 138) with serine residue 36 changed to alanine (lane 6), IB (aa 1 to 138) with tyrosine residue 42 changed to phenylalanine (lane 7), and IB (aa 1 to 138) with mutations of serine residues 32 and 36 to alanine and tyrosine 42 to phenylalanine (lane 8) were analyzed in kinase assays in the presence of double-stranded DNA and purified DNA-PK. (D) GST fusion proteins containing wild-type IB (lane 1), this same construct with serine residues 19 and 23 mutated to alanine (lane 2), a truncated IB construct (aa 1 to 305) (lane 3), or a truncated IB construct with serine residues 19 and 23 changed to alanine (lane 4) were also analyzed in kinase assays with double-stranded DNA and purified DNA-PK.

Interactions between DNA-PK and IB. Since our purification scheme was based on the ability of kinases to bind to IB, we next characterized the domains of IB that were involved in interactions between these proteins. S100 extract was prepared from HeLa cells, and the presence of DNA-PK in this extract was determined by Western blot analysis (Fig. 7A, lane 1). Thus, we could determine whether DNA-PK present in the S100 extracts was able to directly interact with IB. The S100 extract was incubated with either GST alone (Fig. 7A, lane 2) or a variety of GST fusion proteins containing different portions of IB. These included GST fusion proteins with wild-type IB (Fig. 7A, lane 3), an IB protein having mutations of serine residues 32 and 36 (Fig. 7A, lane 4), an IB protein with its amino-terminal 53 amino acids deleted (Fig. 7A, lane 5), and a GST-IB protein containing either its amino-terminal 138 (Fig. 7A, lane 6) or 53 (Fig. 7A, lane 7) amino acids.

View larger version (31K): [in this window] [in a new window]   FIG. 7.   DNA-PK associates with IB. (A) HeLa S100 extract was incubated with a variety of GST-IB fusion proteins bound to glutathione agarose beads, followed by Western blot analysis with DNA-PKcs antibody. HeLa S100 extract with 50% of the input shown (lane 1) was incubated with either GST alone (lane 2), GST-wild-type IB (lane 3), GST-IB with mutations at serine residues 32 and 36 (lane 4), GST-IB with its amino terminal 53 amino acids deleted (lane 5), GST-IB containing the amino-terminal 138 amino acids of IB (lane 6), or GST-IB containing the amino terminal 53 amino acids of IB (lane 7). Western blot analysis was performed with a goat polyclonal antibody directed against DNA-PKcs. (B) The GST-IB fusion proteins used in panel A were subjected to SDS-PAGE and Western blot analysis with antibody directed against GST.

SCID cells exhibit constitutive nuclear levels of NF-B. DNA-PK is critical in the process of DNA double-strand break repair. Cells deficient in DNA-PK are hypersensitive to ionizing radiation and to other agents which induce double-strand DNA breaks (14, 55). DNA-PK is also required in resolving double-strand breaks generated in lymphocyte precursors during V(D)J rearrangement (15, 49, 72). Germ line mutations in either chain of the Ku heterodimer or in DNA-PKcs result in the SCID phenotype (15, 49, 72). Recent data suggest that DNA-PK may play a role in traversing checkpoints after cell cycle arrest (52).

View larger version (56K): [in this window] [in a new window]   FIG. 8.   IB and NF-B regulation in SCID cells. Cytoplasmic extracts were prepared from either the SCID cell line SF19 (lanes 1 to 5) or NIH 3T3 cells (lanes 6 to 10) that were untreated (lanes 1 and 6) or treated with TPA and ionomycin