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The Journal of Immunology, 2001, 167: 4919-4925.
Copyright © 2001 by The American Association of Immunologists

Phosphorylation by the Protein Kinase CK2 Promotes Calpain-Mediated Degradation of I{kappa}B{alpha}1

Jian Shen*, Padmalatha Channavajhala{dagger}, David C. Seldin{dagger} and Gail E. Sonenshein2,{ddagger}

Departments of * Laboratory Medicine and Pathology, {dagger} Medicine, and {ddagger} Biochemistry, Boston University Medical School, Boston, MA 02118


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Rapid I{kappa}B{alpha} turnover has been implicated in the high basal NF-{kappa}B activity in WEHI 231 B immature IgM+ B cells. Here we show that treatment of WEHI 231 cells with apigenin, a selective inhibitor of the protein kinase CK2, decreased the rate of I{kappa}B{alpha} turnover and nuclear levels of NF-{kappa}B. Turnover of I{kappa}B{alpha} in these cells is mediated in part by the protease calpain. Since both CK2 and calpain target the proline-glutamic acid-serine-threonine (PEST) domain, we investigated the role of CK2 in the degradation of I{kappa}B{alpha} by calpain using an in vitro phosphorylation/degradation assay. CK2 phosphorylation enhanced µ-calpain-mediated degradation of wild-type I{kappa}B{alpha}, but not of mutant 3CI{kappa}B{alpha}, with S283A, T291A, and T299A mutations in phosphorylation sites within the PEST domain. Roles for CK2 and calpain in I{kappa}B{alpha} turnover were similarly shown in CH31 immature and CH12 mature IgM+ B cells, but not in A20 and M12 IgG+ B cells. These findings demonstrate for the first time that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes calpain-mediated degradation of I{kappa}B{alpha} and thereby increases basal NF-{kappa}B levels in IgM+ B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nuclear factor-{kappa}B is a family of dimeric transcription factors that have been found to play important roles in the development and activation of B lymphocytes (1, 2). NF-{kappa}B are constitutively expressed in the nucleus of mature and late immature B cells. In most non-B cells, NF-{kappa}B factors are normally sequestered in the cytoplasm via interaction with specific inhibitory proteins, termed I{kappa}Bs. I{kappa}Bs are a family of related proteins that include I{kappa}B{alpha}, I{kappa}B{beta}, I{kappa}B{epsilon}, I{kappa}B{gamma}/p105, and I{kappa}B{delta}/p100 (3). Activation of NF-{kappa}B is dependent on degradation of I{kappa}B proteins. Although the mechanisms controlling constitutive NF-{kappa}B expression in B cells are not clear, the rapid rate of degradation of I{kappa}B{alpha} was hypothesized to be a contributing factor in immature B lymphoma cells such as WEHI 231 (4). Interestingly, Doerre and Corley (5) showed more rapid decay of I{kappa}B{alpha} protein in IgM+ B cell lines (such as WEHI 231, CH31, and CH12 cells) vs IgG+ B cells (e.g., A20 and M12 cells), although the mechanism for these differences was not elucidated.

The proteasome pathway of I{kappa}B{alpha} proteolysis has been implicated in NF-{kappa}B activation via extracellular stimuli, such as TNF, LPS, and growth factors. Stimulation induces phosphorylation of I{kappa}B{alpha} on serines 32 and 36 by the I{kappa}B kinase complex (IKK)3 (6). This phosphorylation serves as a signal for subsequent ubiquitination, which promotes I{kappa}B{alpha} degradation by the 26S proteasome (7). This pathway is not universal, however, and proteasome-mediated degradation of I{kappa}B{alpha} has been ruled out in several recent cases (8, 9, 10). Turnover of I{kappa}B{alpha} mutated at the Ser32/36 was found to occur with the normal kinetics following induction of oxidative stress upon treatment with H2O2 (8). Furthermore, Miyamoto and co-workers (9) found that inhibitors of calpain, but not of the proteasome, ablated basal I{kappa}B{alpha} turnover in WEHI 231 cells.

Additional kinases have been implicated in the regulation of I{kappa}B{alpha} activity or stability. Protein kinase CK2 (previously known as casein kinase II) phosphorylates I{kappa}B{alpha} on serines and threonines in the proline-glutamic acid-serine-threonine (PEST) sequence domain, which affects the intrinsic stability of this inhibitory protein (11, 12, 13, 14). Phosphorylation of Ser283, Thr291, Ser293, and Thr299 has been reported, although some controversy exists as to the nature of the important sites. Interestingly, the PEST domain of I{kappa}B{alpha} was also shown to be the target for degradation by µ-calpain (15). This led us to hypothesize that CK2 may play a role in I{kappa}B{alpha} degradation through the µ-calpain proteolytic pathway. Consistent with this hypothesis, here we report that treatment of WEHI 231, CH31, and CH12 IgM+ B cells with inhibitors of either CK2 (apigenin) or the calcium-calpain proteolytic pathway decreased the rate of I{kappa}B{alpha} decay and thereby NF-{kappa}B levels. Furthermore, in vitro phosphorylation of I{kappa}B{alpha} by CK2 enhanced its rate of degradation by µ-calpain. Thus, these studies define an important new step in the regulatory pathway controlling basal expression of NF-{kappa}B in IgM+ B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatment conditions

WEHI 231, CH31, and CH12 cells were maintained in DMEM as described previously for WEHI 231 (16). A20 and M12 cells were maintained in RPMI with 10% FBS, 50 µM {beta}-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Where indicated, apigenin (Sigma, St. Louis, MO), 1,2-BAPTA-AM (Novabiochem, La Jolla, CA), calpeptin (Calbiochem, La Jolla, CA), E-64d (Peptide International, Louisville, KY) dissolved in DMSO, or a similar dilution of DMSO as a control was added. For half-life studies, 15 µg/ml cycloheximide was added, and whole cell extracts (WCEs) were prepared in lysis buffer (20 mM Tris (pH 8.0), 500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM {beta}-glycerophosphate, 10 mM NaF, 10 mM p-nitrophenyl phosphate, 300 µM Na3VO4, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM DTT, and 0.25% Nonidet-P40) (17).

Immunoblot analysis of I{kappa}B proteins

Samples of extracts (30 µg) were subjected to immunoblot analysis as previously described (18). The Abs used were I{kappa}B{alpha} (C-21), I{kappa}B{beta} (C-20), and p27 (C-19; Santa Cruz Biotechnology, Santa Cruz, CA). An mAb specific for the {beta}-actin (AC-15) was purchased from Sigma. Resulting autoradiograms of decay were quantified by densitometry, and linear regression analysis was used to calculate the t1/2. Shown are representative data from a minimum of two experiments.

EMSA

Nuclear extracts were prepared and subjected to gel electrophoresis as previously described (19). The sequence of the NF-{kappa}B-containing oligonucleotide from the c-myc gene is 5'- GATCCAAGTCCGGGTTTTCCCCAACC-3', where the underlined region indicates the core binding element (20). The octomer-1 (Oct-1) oligonucleotide has the sequence 5'- TGTCGAATGCAAATCACTAGAA-3'.

Transient transfection analysis

Exponentially growing WEHI 231 cells were electroporated with 30 µg of an NF-{kappa}B element-driven luciferase construct (21), provided by Georges Rawadi (Hoescht-Marion-Roussel, Romainville, France) and 5 µg SV40 {beta}-galactosidase ({beta}-gal) reporter construct. After electroporation, cells were incubated at 37°C for 8 h in the absence or the presence of 20 or 40 µM apigenin or 50 µg/ml of the calpain inhibitor E-64d. Cells were harvested, and luciferase activity was measured and normalized with {beta}-gal activity.

CK2 kinase assay

For evaluation of I{kappa}B{alpha} phosphorylation directed by CK2, WCEs were prepared, and samples (10 µg) were adjusted to a 5-µl final volume with the CK2 kinase buffer (100 mM Tris (pH 8.0), 100 mM NaCl, 50 mM KCl 20 mM MgCl2, and 100 µM Na3VO4). Following addition of 15 µl incubation buffer, including CK2 kinase buffer, 50 µCi [{gamma}-32P]GTP, and 200 ng wild-type (wt) I{kappa}B{alpha}-GST or 3CI{kappa}B{alpha}-GST, with three point mutations at S283A, T291A, and T299A (20), provided by J. Hiscott (McGill University, Institut Lady Davis de Recherches Medicales, Montréal, Canada) protein, reactions were incubated at 30°C for 30 min. These fusion proteins were prepared as we have previously described (22). Where indicated, the CK2-specific peptide substrate RRREEETEEE or apigenin was added to the kinase reaction. Samples were subjected to gel electrophoresis and autoradiography. To assess equal loading of protein extracts or GST fusion proteins, the gel was rehydrated and stained with Coomassie blue. Alternatively, 1 mM of the CK2-specific peptide RRREEETEEE (Genosys Biotechnologies, The Woodlands, TX) was used as substrate, as described above, except that 5 µg WCEs were used, and the reactions were stopped by adding 25 µl 100 mM ATP in 4 N HCl. Samples were spotted onto a P81 Whatman filter (Clifton, NJ) and washed four times in 150 mM H3PO4, and incorporated radioactivity was measured by scintillation counting.

Calpain treatment

For CK2 phosphorylation, wtI{kappa}B{alpha}-GST or 3CI{kappa}B{alpha}-GST protein (50–100 ng) was adjusted to either 50 µCi [{gamma}-32P]GTP or 1 mM ATP in 10 µl CK2 kinase buffer and treated with either 10 or 20 U recombinant CK2 (New England Biolabs, Beverly, MA), respectively, at 30°C for 15–30 min. After the kinase incubation, 10 µl of 2x calpain incubation buffer containing 1.5 mM DTT, 1.5 mM CaCl2, and the indicated concentration of calpain I from human erythrocytes (Calbiochem) was added, and the mixture was incubated at 30°C for 15 min as described previously (15). The reaction was terminated by addition of an equal volume of 2x loading buffer and boiling for 5 min, and the proteins were subjected to autoradiography or immunoblot analysis, as appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Apigenin inhibits CK2 activity in WEHI 231 cells

CK2 phosphorylates I{kappa}B{alpha} in the PEST domain, and mutation of S283A, T291A, and T299A was previously shown to reduce phosphoryl group transfer by CK2 in vitro (20). Thus, a CK2 kinase assay was developed using WEHI 231 WCEs and either wtI{kappa}B{alpha}-GST or mutant 3CI{kappa}B{alpha}-GST, with point mutations in critical serine and threonine residues, as substrate. Since either ATP or GTP can be used as a phosphate donor by CK2, while the IKKs can only use ATP, assays were performed in the presence of [{gamma}-32P]GTP. Phosphorylation of the wt was substantially higher than that of the mutant I{kappa}B{alpha} fusion protein, consistent with CK2 kinase activity (Fig. 1GoA). The 3CI{kappa}B{alpha}-GST was phosphorylated in an in vitro IKK kinase assay to the same extent as wtI{kappa}B{alpha}-GST (data not shown). Addition of the CK2-specific peptide substrate RRREEETEEE or the selective inhibitor apigenin (23, 24) reduced this activity in a dose-dependent fashion (Fig. 1GoA). Equal I{kappa}B{alpha} and protein loading was confirmed by Coomassie staining. These findings verify that this assay monitors CK2 activity using I{kappa}B{alpha} as substrate.



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  		FIGURE 1. Apigenin inhibits CK2 activity in WEHI 231 cells. A, Kinase assay. WEHI 231 WCEs (10 µg) were tested in a CK2 kinase assay using either 200 ng wtI{kappa}B{alpha}-GST or 3CI{kappa}B{alpha}-GST as substrate and 50 µCi [{gamma}-32P]GTP as phosphate donor (upper panel). Where indicated, 0.18, 0.36, or 0.72 M CK2-specific peptide substrate RRREEETEEE or 20 or 40 µM apigenin was added to the kinase reaction. Coomassie staining verified equal loading of I{kappa}B{alpha} (middle panel) and proteins (lower panel, illustrating the region of 46 kDa, and data not shown). B, Time course of apigenin inhibition. WEHI 231 cells were incubated with 20 µM apigenin or carrier DMSO alone for 3 or 8 h. WCEs were prepared and subjected to a CK2 kinase assay using wtI{kappa}B{alpha}-GST as described above. Coomassie staining verified equal loading of I{kappa}B{alpha} (middle panel) and proteins (lower panel, illustrating region of 93 kDa, and data not shown). C, Concentration curve of apigenin inhibition using I{kappa}B{alpha} as substrate. WEHI 231 cells were incubated with 0 (carrier DMSO), 10, 20, 30, or 40 µM apigenin for 3 h. WCEs were prepared and subjected to a CK2 kinase assay using wtI{kappa}B{alpha}-GST as described above. Coomassie staining verified equal loading of I{kappa}B{alpha} (middle panel) and proteins (lower panel, illustrating region of 49–61 kDa, and data not shown). D, Concentration curve of apigenin inhibition using CK2-specific peptide as substrate. The WCEs prepared in C were subjected to a CK2 kinase assay using the CK2-specific peptide substrate RRREEETEEE as substrate.

 
To measure the effects of CK2 activity on NF-{kappa}B levels in WEHI 231 cells, we used the CK2 inhibitor apigenin. We first confirmed the effects of apigenin on CK2 activity in WEHI 231 cells. At a low dose of 20 µM, apigenin has displayed selective inhibition of CK2 in different cell lines (24), so this concentration was chosen initially. WEHI 231 cells were grown in the presence of 20 µM apigenin or an equivalent volume of carrier DMSO as a control. After either 3 or 8 h WCEs were isolated and subjected to an in vitro CK2 kinase assay using wtI{kappa}B{alpha}-GST protein as the substrate (Fig. 1GoB). Compared with DMSO-treated controls, a significant down-regulation of CK2 activity was measured by 3 h, and no additional decrease in activity was noted at the 8 h point. Thus, apigenin can effectively inhibit CK2 activity in WEHI 231 cells by 3 h of treatment.

We then performed a dose-response curve of the effects of apigenin on CK2 activity using either wtI{kappa}B-{alpha}-GST or the consensus CK2 peptide as substrate. WEHI 231 cells were treated with 0, 10, 20, 30, or 40 µ M apigenin for 3 h, and then WCEs were prepared. Using these extracts in kinase assays, a dose-dependent decrease in activity was seen using I{kappa}B{alpha}-GST as a substrate (Fig. 1GoC). Equal I{kappa}B{alpha} and protein loading was confirmed by Coomassie staining. Similarly, apigenin was found to cause a dose-dependent decrease in CK2 activity when the CK2-specific peptide substrate RRREEETEEE was used (Fig. 1GoD). Thus, apigenin inhibits CK2 activity in WEHI 231 cells in a dose-dependent fashion.

Apigenin selectively decreases I{kappa}B{alpha} stability and basal levels and activity of NF-{kappa}B

To assess the effects of the inhibition of CK2 on the rate of decay of I{kappa}B{alpha} and I{kappa}B{beta}, WEHI 231 cells were treated with 20 µM apigenin or carrier DMSO for 4 h before addition of the protein synthesis inhibitor cycloheximide. Cytoplasmic proteins were isolated after 0, 30, 60, 120, and 180 min and subjected to immunoblot analysis (Fig. 2GoA), and the results of this and a duplicate experiment were quantified by densitometry (Fig. 2GoB). A slower rate of decay of I{kappa}B{alpha} protein was noted in the apigenin-treated cells (t1/2 = 101.4 ± 12 min) compared with DMSO-treated control (t1/2 = 50.4 ± 4.8 min) cells. In contrast, the stability of I{kappa}B{beta} was not altered in the same samples (t1/2 = 108.4 vs 100.7 min in control vs apigenin-treated cells, respectively; Fig. 2Go, A and B). To determine whether the increase in half-life of I{kappa}B{alpha} indeed correlated with a drop in NF-{kappa}B binding, WEHI 231 cells were treated with 20 µM apigenin or carrier DMSO for 8 h, and nuclear extracts were subjected to EMSA (Fig. 2GoC). NF-{kappa}B, but not control Oct-1, binding activity was down-regulated in nuclear extracts from cells treated with apigenin compared with control DMSO-treated cells. Previous Ab supershift analysis indicated that band 1 contains p50 homodimers, while band 2 consists predominantly of p50/c-Rel with lesser amounts of p50/RelA complexes (16). The down-regulation of NF-{kappa}B activity was also confirmed by lower luciferase activity in an NF-{kappa}B reporter assay in 8-h apigenin-treated WEHI 231 cells compared with untreated control cells (Fig. 2GoD). Taken together, the above observations suggest that apigenin can inhibit CK2 phosphorylation of I{kappa}B{alpha}, prolong I{kappa}B{alpha} half-life, and selectively down-regulate NF-{kappa}B levels and activity in WEHI 231 cells.



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  		FIGURE 2. Inhibition of CK2 by apigenin decreases the rate of decay of I{kappa}B{alpha} and levels of NF-{kappa}B binding and activity in WEHI 231 cells. A, Immunoblot analysis. WEHI 231 cells were incubated with 20 µM apigenin or carrier DMSO alone for 4 h. Then 15 µg/ml cycloheximide (CHX) was added, and WCEs were prepared at the indicated times and subjected to immunoblot analysis for I{kappa}B{alpha} and I{kappa}B{beta}. The t1/2 values obtained in B for I{kappa}B{alpha} are given below. B, Half-life of decay. The immunoblots in A were subjected to densitometry, and the results are presented. C, EMSA. nuclear extracts were prepared from WEHI 231 cells incubated with 20 µM apigenin or carrier DMSO alone for 8 h, and samples (3 µg) were used in EMSA with NF-{kappa}B or Oct-1 probes. D, Activity. Exponentially growing WEHI 231 cell were electroporated in duplicate with 30 µg NF-{kappa}B element luciferase reporter DNA and 5 µg SV40 {beta}-gal DNA to normalize for transfection efficiency. After electroporation, either 20 or 40 µM apigenin or the equivalent volume of DMSO was added, and the cultures were incubated for 8 h. Cells were harvested, and extracts were subjected to luciferase and {beta}-gal reporter activity assays. Luciferase activity was normalized to {beta}-gal, and results are presented as the average ± SD.

 
Calpain-mediated degradation of I{kappa}B{alpha}

Recently, basal degradation of I{kappa}B{alpha} protein in immature B cells has been reported to be mediated via calpain (9, 10). To confirm this observation WEHI 231 cells were incubated in the presence of either 40 µg/ml calpeptin or 50 µg/ml E-64d, two specific inhibitors of calpain, or carrier DMSO as a control. After 30 min, cycloheximide was added, and cytoplasmic extracts were isolated after 2.5 h. Addition of either calpain inhibitor significantly decreased the extent of I{kappa}B{alpha} decay (Fig. 3GoA). Densitometry of this and a duplicate experiment indicated that the normal decay, which leaves 36.0 ± 5.7% of the I{kappa}B{alpha} remaining after 2.5 h, is ablated upon treatment with either calpeptin or E64d (88.9 ± 5.1 and 74.5 ± 10.7%, respectively). Equal loading was confirmed by analysis of {beta}-actin levels on the blot. Furthermore, addition of intracellular and extracellular calcium chelators (30 µM BAPTA-AM plus 5 mM EGTA (B/E)), which inhibits calpain activity (10), completely blocked I{kappa}B{alpha} degradation (106.1 ± 8.5%; Fig. 3GoA). To verify that these inhibitors were not working through the proteasome pathway, we examined the decay of the p27Kip1 protein, which is mediated via the proteasome (25). Normal degradation of this protein was seen in the presence of the calpeptin, E-64d, or calcium chelators. To assess NF-{kappa}B levels, WEHI 231 cells were incubated in the absence or the presence of E-64d (Fig. 3GoB). The level of NF-{kappa}B/Rel binding was reduced in a manner consistent with the increased stability of the I{kappa}B{alpha} protein. For the upper, transcriptionally active NF-{kappa}B complexes, approximately 60% remained following E64d treatment compared with the DMSO control. As expected, addition of the calcium chelators had a more substantial effect on NF-{kappa}B binding, consistent with their effects on I{kappa}B{alpha} half-life. Similarly, NF-{kappa}B reporter activity was also decreased in cells treated with 50 µg/ml E-64d for 8 h after transfection (Fig. 3GoC). Taken together, these results indicate calpain plays an important role in basal degradation of I{kappa}B{alpha} and thereby NF-{kappa}B levels in WEHI 231 cells.



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  		FIGURE 3. Inhibition of calpain decreases the rate of I{kappa}B{alpha} decay and levels of NF-{kappa}B and activity in WEHI 231 cells. A, Decay of I{kappa}B{alpha}. WEHI 231 cells were incubated for 30 min with DMSO alone, B/E, 40 µg/ml calpeptin, or 50 µg/ml E-64d. Where indicated, 15 µg/ml cycloheximide (CHX) was added, and cultures were incubated for an additional 2.5 h. WCEs were prepared, and samples (30 µg) were subjected to immunoblot analysis for I{kappa}B{beta}, I{kappa}B{alpha}, p27Kip1, and {beta}-actin. B, EMSA. WEHI 231 cells were incubated for 6 h with DMSO alone, B/E, or 50 µg/ml E-64d, and nuclear extracts were subjected to EMSA for NF-{kappa}B. C, Activity. Exponentially growing WEHI 231 cell were electroporated in duplicate with 30 µg NF-{kappa}B element luciferase reporter and 5 µg SV40 {beta}-gal DNA. After electroporation, 50 µg/ml E-64d or the equivalent volume of DMSO was added. After 8 h, cells were harvested and subjected to assays for luciferase and {beta}-gal activities. Luciferase activity was normalized to {beta}-gal, and results are presented as the average ± SD.

 
CK2 promotes degradation of I{kappa}B{alpha} by calpain

Since CK2 phosphorylates I{kappa}B{alpha} in the C-terminal PEST domain, which is the target of calpain protease, we asked whether phosphorylation of I{kappa}B{alpha} by CK2 affects its rate of degradation by calpain. We first sought to determine the appropriate dose ofµ-calpain needed. The wtI{kappa}B{alpha}-GST was phosphorylated using [{gamma}-32P]GTP and recombinant CK2, and then the mixture was incubated for 15 min with 30, 90, or 180 nM µ-calpain in the presence or the absence of 750 µM calcium. Degradation of the radiolabeled protein was assessed by gel electrophoresis and autoradiography (Fig. 4GoA). In the presence of calcium, treatment with 30 nM µ-calpain resulted in a substantial decrease in radiolabeled wtI{kappa}B{alpha}-GST, and with 90 nM µ-calpain little remaining I{kappa}B{alpha} was detected. As expected, degradation by µ-calpain required calcium, and, hence, in the absence of this divalent cation, no degradation was detected even with the higher dose of 180 nM µ-calpain. Thus, phosphorylated I{kappa}B{alpha}-GST protein is efficiently degraded by calpain in vitro. Doses of 60 and 90 nM calpain were selected for the analysis.



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  		FIGURE 4. CK2 promotes calpain-mediated degradation of I{kappa}B{alpha}. A, Radiolabeled I{kappa}B{alpha}. Recombinant CK2 (10 U) was incubated with 100 ng wtI{kappa}B{alpha}-GST as substrate and 50 µCi [{gamma}-32P]GTP as phosphate donor. After 15 min, µ-calpain was added to a final concentration of 30, 90, or 180 nM in the presence or the absence of 750 mM CaCl2, as indicated, and the reaction was incubated for an additional 15 min. Proteins were subjected to SDS-PAGE and visualized by autoradiography. B, Immunoblot analysis of I{kappa}B{alpha}. The wtI{kappa}B{alpha}-GST protein (100 ng) was adjusted to 1 mM ATP in 10 µl CK2 kinase buffer and incubated in the absence or the presence of 20 U recombinant CK2 for 30 min. I{kappa}B{alpha} protein was then digested with the indicated concentration of µ-calpain in the presence or the absence of CaCl2 for 15 min and subjected to immunoblot analysis. C, Immunoblot analysis of GST protein. GST protein (100 ng) was subjected to digestion with µ-calpain as described in B. D, In vitro CK2 kinase assay. Samples of wtI{kappa}B{alpha}-GST or 3CI{kappa}B{alpha}-GST (100 ng) were subjected to a kinase assay using 10 U recombinant CK2 with 50 µCi [{gamma}-32P]ATP as phosphate donor and analyzed as described in A. E, Immunoblot analysis of 3CI{kappa}B{alpha}-GST. Samples of either 3CI{kappa}B{alpha}-GST or wtI{kappa}B{alpha}-GST (100 ng) were subjected to CK2 kinase assay as described in B.

 
To assess the effects of phosphorylation by CK2 on calpain-mediated degradation, wtI{kappa}B{alpha}-GST was incubated in the absence or the presence of CK2 and then subjected to degradation with 60 or 90 nM µ-calpain in the presence of CaCl2 as described above (Fig. 4GoB). Alternatively, 90 nM µ-calpain was added in the absence of CaCl2. Incubation with µ-calpain effectively led to the degradation of I{kappa}B{alpha}, but only in the presence of CaCl2, as expected. At both doses of µ-calpain, the extent of degradation of the I{kappa}B{alpha}-GST protein was greater following phosphorylation by CK2. GST protein alone was resistant to treatment with 90 nM µ-calpain (Fig. 4GoC), which confirmed that calpain degradation of the fusion protein was mediated via the I{kappa}B{alpha} sequences. Lastly, the 3CI{kappa}B{alpha} mutant, which cannot be efficiently phosphorylated by CK2 in cellular extracts (Fig. 1GoA) or in vitro (Fig. 4GoD), was used to confirm the role of phosphorylation in enhancing degradation of I{kappa}B{alpha} by µ-calpain. The effect of CK2 treatment on the extents of degradation of wtI{kappa}B{alpha}-GST and 3CI{kappa}B{alpha}-GST by calpain were compared during a 15-min incubation in the presence of 750 µM calcium (Fig. 4GoE). Incubation of the 3CI{kappa}B{alpha}-GST fusion protein with CK2 had no effect on the basal extent of degradation mediated by 90 nM µ-calpain (Fig. 4GoE). In contrast, as shown above, CK2 phosphorylation of wtI{kappa}B{alpha}-GST enhanced the extent of degradation upon a similar treatment with µ-calpain. Thus, the enhanced rate of I{kappa}B{alpha} degradation by µ-calpain described above is dependent upon CK2 phosphorylation of sites S283, T291, and T299.

Inhibition of CK2 or calpain increase I{kappa}B{alpha} half-life in CH31 and CH12 IgM+, but not in A20 and M12 IgG+, B cells

As discussed above, the half-life of I{kappa}B{alpha} decay was previously shown to be much shorter in IgM+ vs IgG+ B cells (5). To determine whether our findings could be extended to other IgM+ B cell lines, the effects of inhibition of CK2 and calpain on the half-life of I{kappa}B{alpha} decay were assessed in CH31 and CH12 cells. A slower rate of decay of I{kappa}B{alpha} protein was similarly noted in apigenin-treated CH31 and CH12 cells compared with the control cells treated with carrier DMSO (Fig. 5GoA). In this and a duplicate experiment I{kappa}B{alpha} protein decayed with a t1/2 of 3.2 ± 0.95 h in the apigenin-treated vs 1.56 ± 0.1 h in the DMSO-treated control CH31 cells and a t1/2 of 5.2 ± 1.66 h in the apigenin-treated vs 2.28 ± 0.42 h in the control CH12 cells. To assess the role of calpain, cultures were next incubated in the presence of DMSO, B/E, E-64d, or calpeptin for 30 min, then cycloheximide was added, and cytoplasmic extracts were isolated at the times indicated (Fig. 5GoB). Addition of either calpain inhibitor significantly decreased the extent of I{kappa}B{alpha} decay, whereas degradation of p27 was unaffected (Fig. 5GoB). These results confirm the role of calpain in I{kappa}B{alpha} turnover in IgM+ B cells.



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  		FIGURE 5. Inhibition of CK2 or calpain decreases the rate of decay of I{kappa}B{alpha} in CH31 and CH12 IgM+ B cells. A, Apigenin treatment. CH31 and CH12 cells were incubated with 20 µM apigenin or carrier DMSO alone for 4 h. Then 15 µg/ml cycloheximide (CHX) was added, and WCEs were prepared at the indicated times and subjected to immunoblot analysis for I{kappa}B{alpha} and {beta}-actin, as a control for equal loading. The t1/2 values obtained for I{kappa}B{alpha} are given below. B, Inhibition of calpain. Cells were incubated for 30 min with DMSO alone, B/E, 50 µg/ml E-64d, or 40 µg/ml calpeptin. Where indicated, 15 µg/ml cycloheximide (CHX) was added, and cultures were incubated for an additional 2 or 3 h. WCEs were prepared, and samples (30 µg) were subjected to immunoblot analysis for I{kappa}B{beta}, I{kappa}B{alpha}, p27Kip1, and {beta}-actin.

 
We next asked whether this pathway might account in part for the differences in I{kappa}B{alpha} half-life observed previously between IgG+ vs IgM+ B cells (5). To test this possibility, two IgG+ B cell lines A20 and M12, were analyzed. In agreement with previous work A20 and M12 cells displayed a longer basal half-life of decay for I{kappa}B{alpha} than the IgM+ B cell lines (Fig. 6Go, DMSO control lanes). Treatment with either E-64d or apigenin had little effect on I{kappa}B{alpha} turnover in A20 or M12 IgG+ B cells (Fig. 6Go). In contrast, addition of the proteasome inhibitor MG-132 prevented the decay of I{kappa}B{alpha} in both A20 or M12 B cell lines (Fig. 6Go). Equal loading was confirmed by analysis of {beta}-actin levels on the blots. Thus, these findings indicate that the CK2/calpain pathway plays little role in the degradation of I{kappa}B{alpha} in either A20 or M12 IgG+ B cells.



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  		FIGURE 6. Inhibition of CK2 or calpain has little effect on the decay of I{kappa}B{alpha} in A20 and M12 IgG+ B cells. Cultures were incubated for 30 min with carrier DMSO, 20 µM MG-132 or 50 µg/ml E-64d (upper panel), or 20 µM apigenin (lower panel). Following addition of CHX (15 µg/ml), cells were harvested at the indicated times, and WCEs were analyzed by immunoblot analysis for I{kappa}B{alpha} and {beta}-actin protein levels. A, A20 cells; B, M12 cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we show for the first time that basal phosphorylation of serine/threonine residues in the PEST domain of I{kappa}B{alpha} by CK2 facilitates degradation of this inhibitory protein via a calcium- and calpain-dependent pathway in IgM+ B cells, thereby controlling NF-{kappa}B levels and activity. This pathway was active in the WEHI 231 and CH31 immature and CH12 mature IgM+ B cell lines, but not in A20 and M12 IgG+ mature B cells. This suggests that the more rapid degradation of I{kappa}B{alpha} seen in IgM+ vs IgG+ B cells previously (5) can be attributed at least in part to this calcium- and calpain-dependent pathway. Interestingly, an important relationship was reported in Drosophila between CK2 and the stability of Cactus, the selective inhibitor of the NF-{kappa}B/Rel-related Dorsal protein, which is responsible for dorsal patterning (26). In particular, serine to alanine mutations in the PEST domain of Cactus prevented CK2 phosphorylation, increasing the stability of Cactus while not affecting its ability to bind to Rel. In mammalian cells experiments have similarly shown that phosphorylation by CK2 of Ser283, Thr291, Ser293, and Thr299 in the C-terminus plays a significant role in the regulation of I{kappa}B{alpha} stability (11, 12, 13, 14, 15). Our studies provide data on the mechanism by showing that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes degradation of I{kappa}B{alpha} via calpain.

While a majority of studies indicate that I{kappa}B{alpha} degradation during signal-induced activation of NF-{kappa}B is mediated via a ubiquitin-proteasome pathway (7, 27), several lines of evidence suggest that alternative pathways operate under basal as well as inducible situations. As discussed above, a proteasome-independent proteolytic pathway was reported to mediate basal degradation of I{kappa}B{alpha} in WEHI 231 cells and other primary B cells (9, 10). Turnover of I{kappa}B{alpha} mutated in the PEST domain, but not at Ser32/36, was found to occur with the reduced kinetics following induction of oxidative stress upon treatment with H2O2 (8). Furthermore, TNF-{alpha}-inducible proteolysis of I{kappa}B{alpha} in human liver and airway epithelial cells was mediated by cytosolic m-calpain (28). Overexpression of the intracellular calpain inhibitor calpastatin was shown to block both basal and silica-induced NF-{kappa}B activation in human bronchial epithelial cell lines (29, 30). Calpain inhibitor I was also shown to inhibit NO synthesis by blocking I{kappa}B{alpha} degradation (31). Finally, mice deficient in the skeletal muscle-specific calpain 3 were found to display myonuclear apoptosis and profound perturbation of the I{kappa}B{alpha}/NF-{kappa}B pathway typical of limb-girdle muscular dystrophy type 2A (32). These findings solidify the initial in vitro observation made by Shumway et al. (15), which indicated that I{kappa}B{alpha} is a potential proteolytic target of µ-calpain, and that the PEST domain of this protein is necessary and sufficient for this process to occur. In our study we extend this observation by linking phosphorylation of I{kappa}B{alpha}-GST by recombinant CK2 with its degradation via µ-calpain. This enhanced calpain-mediated degradation following phosphorylation of CK2 in the PEST domain probably contributes to the observed intracellular role of calpain in control of the intrinsic stability of I{kappa}B{alpha}.

Elevated CK2{alpha} expression in T cells of transgenic mice led to development of T cell lymphomas (33). More recently, transgenic overexpression of CK2{alpha} in the mammary gland was found to lead to breast tumors in mice, and cells derived from these mammary tumors contained functional NF-{kappa}B activity (34). Consistent with these observations, human breast cancer cell lines and multiple primary breast cancer specimens displayed constitutive CK2 activity; inhibition of this activity in culture with either apigenin or emodin resulted in reduced NF-{kappa}B binding and activity (35). The potential role of CK2 in the regulation of aberrant NF-{kappa}B expression that typifies many tumors will require further investigation.

To date, two degradation pathways for I{kappa}B{alpha} have been identified: proteasome and calpain. Miyamoto (9) reported that the pathway in WEHI 231 cells was mediated exclusively via calcium and calpain, rather than the proteasome. Interestingly, we observed that treatment of WEHI 231 cells with MG-132, a selective proteasome inhibitor, also reduced the extent of basal turnover of I{kappa}B{alpha} (t1/2 = 114 vs 55 min in control DMSO-treated cells) and of control p27Kip1 protein as in CH31 and CH12 cells (data not shown). These results suggest that both calpain- and proteasome-mediated degradation may contribute to the rapid I{kappa}B{alpha} turnover seen in IgM+ B cells (5). The differences between the previous studies (9) and our findings with respect to WEHI 231 cells probably represent clonal variations between the lines. Thus, while our experiments make a strong case for phosphorylation by CK2 playing a role in degradation of I{kappa}B{alpha} by µ-calpain, they do not rule out the possibility that CK2 also plays a role in proteasome-mediated degradation in these cells. However, the data do indicate that CK2 phosphorylation does not play a role in proteasome-mediated degradation in IgG+ cells, since apigenin had no effect on I{kappa}B{alpha} decay in either M12 or A20 cells.

In summary, our findings begin to provide a mechanism for the previously observed role of CK2 in half-life of decay of I{kappa}B{alpha} protein and for the differences in control of NF-{kappa}B in IgM+ vs IgG+ B cells. Interestingly, CK2 was recently also implicated in signal-dependent (e.g., TNF-{alpha}) degradation of I{kappa}B{alpha} in association with NF-{kappa}B (36). The reason why cells bearing different isotypes of B cell Ag receptor use different mechanisms of I{kappa}B{alpha} turnover is not clear. It would be interesting to determine whether developmental regulation of either the level or the activity of CK2 or calpain exists in B cells. Furthermore, engagement of the surface IgM B cell Ag receptor on mature vs immature B cells leads to dramatically different effects on turnover of I{kappa}B proteins and NF-{kappa}B levels (16, 18, 37, 38). Whether the effects on I{kappa}B{alpha} turnover are mediated via CK2 or calpain is under investigation.


   Acknowledgments
 
We thank J. Hiscott for generously providing I{kappa}B{alpha} cloned DNAs; R. Corley, T. Rothstein, and G. Bishop for CH31, CH12, and A20 cells, respectively; and J. Foster for use of the densitometer. The assistance of Darin Sloneker in the preparation of this manuscript is gratefully acknowledged.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants T32HL07501 (to P.C.), RO1CA36355 (to G.E.S.), and RO1CA71796 (to D.C.S.). D.C.S. is a scholar of the Leukemia and Lymphoma Society of America. Back

2 Address correspondence and reprint requests to Dr. Gail E. Sonenshein, Department of Biochemistry, Boston University Medical School, 715 Albany Street, Boston, MA 02118. E-mail address: gsonensh{at}bu.edu Back

3 Abbreviations used in this paper: IKK, I{kappa}B kinase complex; CK2, protein kinase CK2 (formerly casein kinase II); PEST, proline-glutamic acid-serine-threonine; {beta}-gal, {beta}-galactosidase; Oct-1, octomer-1; WCE, whole cell extract; wt, wild type. Back

Received for publication March 13, 2001. Accepted for publication August 23, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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