Differential Phosphorylation of the Signal-responsive Domain of IκBα and IκBβ by IκB Kinases

NF-κB activity is regulated by its association with the inhibitory IκB proteins, among which IκBα and IκBβ are the most abundant. IκB proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-κB proteins. The degradation of IκB proteins allows nuclear translocation of NF-κB and hence plays a critical role in NF-κB activation. Previous studies have demonstrated that, although both IκB proteins are phosphorylated by the same IκB kinase (IKK) complex, and their ubiquitination and degradation following phosphorylation are carried out by the same ubiquitination/degradation machinery, their kinetics of degradation are quite different. To better understand the underlying mechanism of the differences in degradation kinetics, we have carried out a systematic, comparative analysis of the ability of the IKK catalytic subunits to phosphorylate IκBα and IκBβ. We found that, whereas IKKα is a weak kinase for the N-terminal serines of both IκB isoforms, IKKβ is an efficient kinase for those residues in IκBα. However, IKKβ phosphorylates the N-terminal serines of IκBβ far less efficiently, thereby providing an explanation for the slower rate of degradation observed for IκBβ. Mutational analysis indicated that the regions around the two N-terminal serines collectively influence the relative phosphorylation efficiency, and no individual residue is critical. These findings provide the first systematic analysis of the ability of IκBα and IκBβ to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of IκBα and IκBβ.

NF-B activity is regulated by its association with the inhibitory IB proteins, among which IB␣ and IB␤ are the most abundant. IB proteins are widely expressed in different cells and tissues and bind to similar combinations of NF-B proteins. The degradation of IB proteins allows nuclear translocation of NF-B and hence plays a critical role in NF-B activation. Previous studies have demonstrated that, although both IB proteins are phosphorylated by the same IB kinase (IKK) complex, and their ubiquitination and degradation following phosphorylation are carried out by the same ubiquitination/ degradation machinery, their kinetics of degradation are quite different. To better understand the underlying mechanism of the differences in degradation kinetics, we have carried out a systematic, comparative analysis of the ability of the IKK catalytic subunits to phosphorylate IB␣ and IB␤. We found that, whereas IKK␣ is a weak kinase for the N-terminal serines of both IB isoforms, IKK␤ is an efficient kinase for those residues in IB␣. However, IKK␤ phosphorylates the N-terminal serines of IB␤ far less efficiently, thereby providing an explanation for the slower rate of degradation observed for IB␤. Mutational analysis indicated that the regions around the two N-terminal serines collectively influence the relative phosphorylation efficiency, and no individual residue is critical. These findings provide the first systematic analysis of the ability of IB␣ and IB␤ to serve as substrates for IKKs and help provide a possible explanation for the differential degradation kinetics of IB␣ and IB␤.
In most resting cells, the transcription factor NF-B is kept dormant in the cytoplasm through its interaction with IB proteins. There are several IB isoforms, among which IB␣ and IB␤ are the most abundant and well studied (for reviews, see Refs. 1 and 2). IB␣ and IB␤ share a common overall domain structure: an N-terminal signal-responsive domain, a central ankyrin repeat domain, and a C-terminal PEST domain. The two IB proteins also have similar three-dimensional structure as revealed in crystallographic studies on NF-B⅐IB complexes (3)(4)(5). In addition, both IB proteins bind predominantly to NF-B p65/p50 heterodimers in vivo (6,7).
However, these two IB proteins display different degradation kinetics in response to stimulation with NF-B inducers (6,8). For example, upon TNF-␣ 1 treatment, IB␣ undergoes rapid and complete degradation (within Ͻ15 min) before reappearing due to NF-B-induced resynthesis of IB␣ mRNA. In contrast, the degradation of IB␤ occurs more slowly, with complete degradation occurring 30 -60 min following stimulation (6,8). However, the kinetics of IB␤ degradation varies in different cell types and depends on the nature of the inducer. Thus, whereas the level of IB␤ protein is unaltered in 70Z/3, Jurkat, or EL-4 cells treated with TNF-␣ (6,8), IB␤ is partially degraded when NIH3T3 cells are stimulated over extended periods of time by TNF-␣ (this study). In certain cell lines such as 70Z/3, inducers such as lipopolysaccharide and interleukin-1 trigger complete IB␤ degradation, although the kinetics of degradation is still significantly slower than that of IB␣ (6,8). Although it has been suggested that the rapid activation of NF-B is due to the rapid degradation of IB␣, whereas a prolonged activation of NF-B occurs through the less efficient degradation of IB␤ (6, 9 -12), the mechanism responsible for the different rates of degradation of the two IB proteins has remained elusive.
A critical step in the activation of NF-B is the phosphorylation of IB proteins by the IB kinase (IKK) complex. Characterization of the 700 -900-kDa IKK complex has led to the identification of two catalytic subunits, IKK␣ and IKK␤, and a regulatory subunit, IKK␥/NEMO (13)(14)(15)(16)(17)(18)(19). IKK␣ and IKK␤ dimerize via their leucine zipper motifs while binding to IKK␥/ NEMO with the NEMO-binding domain to form the core IKK complex (17,20,21). The IKK complex may also include other components such as a kinase-specific chaperone (Cdc37/ HSP90) that appears to play a role in shuttling the complex from the cytoplasm to the membrane during TNF-␣-induced NF-B activation (22).
Both in vitro and in vivo studies have proven that IKK␣ and IKK␤ can phosphorylate IB proteins at specific serine residues, including Ser 32 and Ser 36 of IB␣ and Ser 19 and Ser 23 of IB␤ (13,14,16). Phosphorylation at these conserved serine residues leads to ubiquitination and degradation of the IB proteins. Although it is generally accepted that IKK phosphorylates both IB␣ and IB␤, the efficiency of the two IB isoforms as substrates has never been carefully examined. Because IKK activity is likely rate-limiting for IB degradation and NF-B activation, since a 2-fold reduction in IKK␤ causes nearly 80% decrease in NF-B activation (23), we compared the phosphorylation of IB␣ and IB␤ by IKK␣ and IKK␤ in an attempt to better understand the mechanism of differential regulation of IB degradation. * This work was supported by Grant R37-AI33443 from National Institutes of Health and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Site-directed Mutagenesis-The pGEX-IB␣N deletion mutant was generated by PCR using specific primers for the 5Ј-and 3Ј-terminal sequences. pGEX-IB␤N point mutations were generated by PCR according to point mutagenesis protocol from Clontech. Escherichia coli DH5␣ bacterial cells were used for cloning, and the mutants were verified by sequencing.
Preparation of GST-tagged Proteins-Plasmids were transformed into BL21 bacterial cells and grown in ampicillin-containing LB medium to A ϭ 0.6. Isopropyl-␤-D-thiogalactopyranoside was added to a final concentration of 0.1 mM to induce production of GST-tagged proteins. The cultures were grown overnight at room temperature. Cells were collected and resuspended in phosphate-buffered saline plus protease inhibitors and then sonicated using a Virsonic sonicator (Virtis). The supernatant was clarified by centrifugation in a microcentrifuge at 14,000 rpm for 30 min and incubated with glutathione-conjugated agarose beads for 30 min. GST-tagged proteins were eluted by 10 mM glutathione in 50 mM Tris buffer (pH 8.0) and dialyzed in 50 mM Tris buffer (pH 8.0).
Transfection-Cells were grown in 10-cm plates to 40% confluence and transfected with the indicated DNAs using FuGENE TM 6 (Roche Applied Science). For transient transfections, cells were harvested after 36 -48 h. For stable transfections, puromycin (1 g/ml final concentration) was added to the medium. Resistant colonies formed after ϳ1 week and were moved to 24-well plates to expand to single clones. Positive stable transfectants were verified for expression of the cloned proteins using specific antibodies.
Kinase Assay-293 cells were treated with TNF-␣ for 10 min or transfected with the indicated IKK constructs for 48 h before harvesting. Cells were lysed in buffer containing 0.1% Triton X-100, a mixture of protease inhibitors (Roche Applied Science), and phosphatase inhibitors. Supernatants were collected and incubated with anti-IKK␥ polyclonal antibody with protein G-Sepharose beads or anti-FLAG antibody M2-agarose beads for 3 h. The beads were washed with TNT buffer (20 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 1% Triton X-100) and phosphate-buffered saline, before incubation with kinase reaction buffer containing [␥-32 P]ATP and the indicated GST-substrate IB proteins for the indicated times. The reaction was stopped by dilution in TNT buffer, followed by pull-down of the GST-tagged proteins using glutathioneconjugated agarose beads. The pelleted beads were washed three times with TNT buffer, and the samples were analyzed by SDS-PAGE. The gels were fixed and vacuum-dried before exposure to BioMax films (Eastman Kodak Co.). After exposure, the gels were rehydrated and stained with Coomassie Blue.
Immunoprecipitation and Immunoblotting-Cells were lysed in TNT buffer supplemented with protease inhibitors. In immunoprecipitation experiments, cell lysates were incubated with 30 l of anti-FLAG antibody M2 affinity gel for 3 h at 4°C. Immobilized immunocomplexes were washed with TNT buffer three times, boiled in SDS loading buffer, and resolved by 10% SDS-PAGE. Proteins were transferred to Immobilon transfer membrane (Millipore Corp.) and blotted with the indicated primary antibodies for 3 h at room temperature, followed by the appropriate secondary antibody for 1 h. Immunoreactive bands were visualized by ECL.

Differential Degradation Kinetics of IB␣ and IB␤-When
cells such as the mouse pre-B cell line 70Z/3 are treated with TNF-␣, the cytosolic protein levels of IB␣ and IB␤ change with distinct patterns (6,8,9). Whereas IB␣ disappears and reappears over a period of 3 h, IB␤ levels remain unaltered. Activated NFB induces the resynthesis of IB␣ via the three B sites in the IB␣ promoter (24 -28). Although the IB␤ promoter region harbors a single B site, it is minimally responsive to NF-B binding (29). In the presence of cycloheximide, IB␣ did not reappear after 30 min, whereas IB␤ levels were unaffected (Fig. 1A, left panel), thus excluding the possibility that IB␤ protein levels were maintained through a balance of degradation/turnover and resynthesis. The non-responsiveness of IB␤ to TNF-␣ treatment was, however, cell FIG. 1. IB degradation kinetics in response to TNF/lipopolysaccharide treatment. A and B, left panels, 70Z/3 and NIH3T3 cells, respectively, were treated with TNF-␣ (10 ng/ml) for the indicated times. Cellular IB␣ and IB␤ protein levels were examined by Western blotting. As indicated, cells were treated with cycloheximide (ϩCHX; 10 g/ml) for 1 h before TNF-␣ was added. Right panels, 70Z/3 and NIH3T3 cells, respectively, were treated with lipopolysaccharide (LPS; 10 g/ml) for the indicated times. Cellular IB␣ and IB␤ protein levels were examined by Western blotting. type specific. In NIH3T3 cells, IB␤ did respond to TNF-␣, although the rate and extent of degradation were noticeably less than those of IB␣ (Fig. 1B, left panel). When both cell types were treated with lipopolysaccharide, the level of IB␤ degradation was greater, but the overall pattern remained the same (Fig. 1, A and B, right panels).
The IKK Complex Differentially Phosphorylates IB␣ and IB␤-Because phosphorylation is the first step that marks IB proteins for degradation, we examined whether IB␣ and IB␤ can be phosphorylated equally well by the IKK complex. We therefore performed in vitro kinase assays using recombinant GST-IB␣ and GST-IB␤ proteins as substrates and immunoprecipitated the IKK complex from TNF-␣-stimulated HEK293 cells as the kinase. It has been previously shown that, upon stimulation by TNF-␣, IKK activity peaks within 5-15 min before declining to ϳ25% of its peak value by 30 min (15, 16, 30) (data not shown). We therefore used 293 cells stimulated with TNF-␣ for 10 min as a source of active IKK in our assays. Since IKK␥/NEMO exists as a complex with both IKK␣ and IKK␤ in vivo (17), we immunoprecipitated the IKK complex using anti-NEMO polyclonal antibody. Surprisingly, we observed a significant difference in the level of phosphorylation of IB␣ and IB␤ ( Fig. 2A, upper panel), even though the amount of GST-IB proteins was similar (lower panel). The IKK complex immunoprecipitated from unstimulated cells also phosphorylated the IB proteins weakly ( Fig. 2A, lanes 1 and  3), indicating that some constitutive activation of IKK occurred in these cells.
Although both IKK␣ and IKK␤ can phosphorylate the Nterminal serine residues in IB proteins, we wanted to test whether they exhibit any preference for IB␣ or IB␤. Both IKK␣ and IKK␤ contain an activation loop, and phosphorylation of specific serine residues in the activation loop leads to activation of the kinases (14,31,32). It has been shown that substitution of the serine residues (Ser 176 and Ser 180 in IKK␣ and Ser 177 and Ser 181 in IKK␤) with glutamic acid (which mimic phosphoserine) results in constitutively active IKK␣ and IKK␤ (14). We therefore transfected constitutively active FLAG-tagged IKK␣ and IKK␤ into 293 cells and immunoprecipitated them using anti-FLAG antibody-conjugated agarose beads. The cloning vector was used as a negative control (labeled as Ϫ in the figures). The majority of the overexpressed IKK␣ or IKK␤ formed homodimers, with very small amounts of heterodimers being formed between transfected and endogenous IKK subunits (data not shown). As shown in Fig. 2 (B and  C), both constitutively active IKK␣ and IKK␤ homodimers phosphorylated IB␣ more efficiently than IB␤, as seen previously for the endogenous TNF-␣-stimulated IKK complex ( Fig. 2A).
IKK␤ Differentially Phosphorylates IB␣ and IB␤ N Termini-Some prior reports have indicated that, besides Ser 32 and Ser 36 in IB␣ and Ser 19 and Ser 23 in IB␤, the IKK complex can also phosphorylate the C terminus of IB␣ (16,33). To check whether the differential phosphorylation between full-length IB␣ and IB␤ is due to additional C-terminal sites of phosphorylation in IB␣, we compared the efficiency of phosphorylation of full-length IB␣ and IB␤ versus truncated IB proteins containing only the N-terminal serine residues (IB␣N containing the N-terminal 54 residues and IB␤N containing the N-terminal 44 residues). Using TNF-␣-activated IKK immunoprecipitated with anti-NEMO antibody, we found no significant difference in the efficiency of phosphorylation between the full-length and N-terminal IB␣ and IB␤ substrates (Fig. 3A).
We then tested whether there is any difference in the ability of IKK␣ and IKK␤ to phosphorylate full-length IB proteins

FIG. 2. The IKK complex differentially phosphorylates IB␣ and IB␤.
A, 293 cells were treated with TNF-␣ (10 ng/ml) for 10 min (ϩ) or left untreated (Ϫ) before harvesting. Endogenous IKK complex was purified using anti-NEMO polyclonal antibody and then incubated with GST-tagged full-length IB␣ and IB␤ in kinase reaction buffer. GST-IB proteins were then pulled down with glutathioneconjugated agarose, resolved by SDS-PAGE, fixed, and exposed to film (kinase activity (KA)). Equal amounts of GST-IB proteins were used in the experiment, as shown by Coomassie Blue (CB) staining of the gel. The lower band in all lanes of the Coomassie Blue-stained panel marked with an asterisk represents the GST fragment alone (ϳ26 kDa). B and C, 293 cells were transfected with constitutively active FLAG (F)-IKK␣(CA) or FLAG-IKK␤-(CA), respectively, or empty vector (Ϫ) and incubated for 36 h before harvesting. The IKK complex was purified using anti-FLAG monoclonal antibody M2 and then incubated with GST-tagged full-length IB␣ and IB␤ in the kinase assay. The band at ϳ97 kDa represents autophosphorylated FLAG-tagged IKK␣(CA) or IKK␤(CA). The phosphorylation of the IB␣ substrate seen in lane 1 in B is probably due to immunoprecipitation of some endogenous IKK from the untransfected cells by the FLAG-conjugated beads. and their N-terminal portions. As shown in Fig. 3B, whereas IKK␤(constitutively active) homodimers strongly phosphorylated both full-length IB␣ and IB␣N, the equivalent IB␤ constructs were phosphorylated poorly. In contrast, whereas IKK␣(constitutively active) homodimers were able to efficiently phosphorylate both full-length IB constructs, it was unable to phosphorylate IB␣N and IB␤N. To determine whether the inability of IKK␣ to phosphorylate the IB N terminus might be influenced by IKK␥/NEMO, the regulatory subunit of the IKK complex, we transfected wild-type IKK␣ with or without cotransfection of NEMO to generate the kinase used to phosphorylate IB␣ and IB␤ N termini. However, in both cases, overexpressed wild-type IKK␣ efficiently phosphorylated full-length IB␣ and, to a lesser extent, full-length IB␤, but was unable to phosphorylate the N-terminal IB substrates (Fig. 3C).
Comparison of the Phosphorylation and Degradation Kinetics of IB Proteins-To explore the possibility that inefficient IB␤ phosphorylation by IKK␤ may be responsible for the slower and incomplete degradation of IB␤, we compared the kinetics of IB␣N and IB␤N phosphorylation by IKK␤. As mentioned above, the activity of IKK␤ in most cell lines treated with TNF-␣ peaks between 5 and 15 min and then decreases significantly, most likely due to extensive autophosphorylation of its C-terminal region (30). As described above, we used immunoprecipitated IKK␤(CA) and incubated it with IB␣ or IB␤ N termini for different time points. Interestingly, the autophosphorylation of IKK␤(CA) increased in a linear manner over time (Fig. 4A). Whereas the phosphorylation of both IB␣N and IB␤N also increased over time, the dramatic difference between IB␣ and IB␤ remained even after 1 h of incubation with IKK␤ (Fig. 4A). Quantitation of the degree of phosphorylation by a PhosphorImager indicated a roughly linear correlation between time and degree of phosphorylation for both IB␣N and IB␤N. We also noticed that the phosphorylation of IB␤ at 1 h was less than that of IB␣ at 5 min. Interestingly, the level of IB␤ phosphorylation increased only slightly even after 3 h of incubation (data not shown). These results suggest that the inefficiency of IB␤ phosphorylation by IKK␤ cannot be explained as a simple kinetic difference and probably reflects a more fundamental difference in the ability of the two proteins to serve as substrates for IKK␤. Differential Phosphorylation of IB N Termini Is Linked to Protein Stability of IB Proteins-To further establish the link between phosphorylation efficiency of IB proteins and their degradation pattern, we transiently transfected HeLa cells with IB␣ and IKK␤. Cotransfection of IKK␤(CA) dramatically decreased the protein level of wild-type IB␣, but did not affect the level of the non-phosphorylatable IB␣AA mutant, in which the two conserved serine residues at positions 32 and 36 had been mutated to alanines. Therefore, this experiment establishes that the instability of IB␣ protein caused by IKK␤ cotransfection was dependent on phosphorylation of Ser 32 and Ser 36 . However, cotransfection of IKK␤ did not affect the protein stability of IB␤, which likely correlates with the inability of IKK␤ to efficiently phosphorylate IB␤ (Fig. 5B, lower panel,  third and fourth lanes). To further establish the connection between phosphorylation efficiency and stability of transfected IB proteins, we generated two chimeric proteins, IB␣␤ and IB␤␣, by swapping the N-terminal signal peptide region between IB␣ and IB␤ (Fig. 5B, upper panel). Because the N termini of IB proteins determine the efficiency of phosphorylation by IKK␤, GST-IB␣␤ was efficiently phosphorylated in in vitro kinase assays, whereas GST-IB␤␣ was not (data not shown). Consistent with this difference in phosphorylation efficiency, the level of transfected IB␣␤ protein was significantly reduced by IKK␤ cotransfection, whereas the level of IB␤␣ was affected only marginally. Thus, the N termini of IB proteins determine their ability to be phosphorylated by IKK␤, which in turn is reflected in their rate of degradation.
Residues 28 -42 in IB␣ and Residues 15-31 in IB␤ Influence Substrate Phosphorylation Efficiency-To determine which specific features of the IB␣ N terminus sequence make it a better substrate for IKK␤, we carried out a comprehensive mutational analysis of the N-terminal regions of IB␣ and IB␤. We initially focused on the first 12 amino acids in IB␣, which is a sequence unique to IB␣ (Fig. 6A). We generated an IB␣NϪ12 mutant by deleting these 12 amino acids and compared the phosphorylation efficiency of this mutant and the wild-type IB proteins. In vitro kinase assays revealed no significant difference between this mutant and wild-type IB␣, indicating that these 12 unique residues of IB␣ do not determine the phosphorylation efficiency of IB proteins (Fig. 6B).
We then divided the remaining homologous region in the N termini of IB proteins (residues 13-54 in IB␣ and residues 1-42 in IB␤) into three parts and swapped the different parts between IB␣ and IB␤ to produce a set of chimeric GSTtagged N-terminal IB proteins, IBN␣␤␣, IBN␤␣␤, IBN␤␣␣, IBN␤␤␣, IBN␣␤␤, and IBN␣␣␤ (Fig. 6C). Only one of the GST-IBN chimeric proteins, GST-IBN␤␣␣, was phosphorylated to the same level as GST-IB␣N in in vitro kinase assays (Fig. 6D). Because both IBN␤␣␤ and IBN␤␤␣ were poorly phosphorylated by IKK␤, we believe that sequence elements present in residues 28 -54 of IB␣ are responsible for the high phosphorylation efficiency of IB␣.
To further narrow down the sequences responsible for determining the efficiency of IB␣ as a substrate for IKK␤, we made additional chimeras by fusing different lengths of the sequence from residues 39 to 54 of IB␣ with the central core of amino acids 29 -38 from IB␣ (data not shown). Analysis of these chimeras indicated that attaching residues 29 -43 to the central core in the IBN␤␣␤Ј chimera (where residues 29 -43 are derived from IB␣, with the flanking sequences from IB␤) allowed it to be phosphorylated as efficiently as IB␣N (Fig. 6, E and F, third lane). Since residues 29 -43 of IB␣ appear to be the critical sequence responsible for efficient phosphorylation of IB␣, we generated a set of IB␤N mutants by replacing smaller clusters of residues in this region with their counter- parts in IB␣ to see if the phosphorylation efficiency of this IB␤N substrate could be enhanced (Fig. 6E) (individual point mutations were also made (data not shown)). It is important to note that aspartic acid at position 35 in IB␣ and glycine at position 22 in IB␤ are the only residues that differ between IB␣ and IB␤ within the conserved core sequence recognized by IKK, viz. DSGL(D/G)S. However, the substitution of aspartic acid for glycine in IB␤ (mutant IB␤Nd) did not significantly improve its substrate efficiency, and other mutations in this region did not individually reconstitute the high phosphorylation efficiency of IB␣N. Therefore, it is likely that the overall structure of the region from amino acids 29 to 43 in IB␣ (and the corresponding region in IB␤, amino acids 15-31) determines its efficiency as a substrate for IKK␤ phosphorylation.

DISCUSSION
In a continuing effort to understand the basis for the differential kinetics of IB␣ and IB␤ degradation, we have been searching for cis-and trans-acting factors that might influence the behavior of the two IB isoforms (6,34). The findings reported in this study suggest that the amino acid sequence of the N-terminal region that includes the actual sites of phosphorylation determines the efficiency with which the two IB proteins can be phosphorylated by IKK␤. This intrinsic difference in phosphorylation efficiency would therefore likely contribute to the differential kinetics of degradation of the two isoforms observed in response to some inducers in some cells (6,8). It is important to note that this difference is unlikely to fully explain all observations regarding differential regulation of these isoforms. In particular, it has been reported that, whereas IB␤ is degraded in response to TNF signaling in some cells, it is unaffected in many other cells (6,8). Such variability in the behavior of IB␤ in different cells is difficult to explain on the basis of intrinsic differences in the ability of IKK␤ to phosphorylate IB␣ and IB␤. It is also not completely clear how the differences between IB␣ and IB␤ seen in this analysis can be reconciled with the results of IB␤ knock-in mice (35). In that study, a tagged IB␤ was knocked into the IB␣ locus, and the finding that IB␤ in these cells rescued the lethality seen in IB␣ knockout mice suggested that IB␣ and IB␤ are interchangeable and that their differential responses to NF-B inducers are not due to intrinsic differences in the proteins (35). It is likely that trans-acting factors such as B-Ras proteins also contribute to the observed differences in the degradation kinetics of the two IB isoforms since B-Ras binds preferentially to IB␤ in cells (34,36). 2 We observed a surprising and unexpected difference in the ability of IKK␣ to phosphorylate full-length IB proteins versus the N-terminal fragments of IB proteins. Whereas IKK␤ was able to phosphorylate both the full-length and truncated IB substrates equally well, IKK␣ displayed a strong preference for the full-length proteins and was almost inactive on the Nterminal fragments. The inability of IKK␣ to efficiently phosphorylate the N-terminal IB substrates also raises the possibilities that, in vivo, IKK␣ might not phosphorylate IB proteins and that the lower level of phosphorylation observed with the full-length IB proteins as substrates may be an in vitro phenomenon since it is known that many kinases lose their selectivity when tested in vitro at high concentrations. Such an explanation would also predict that IKK␤, rather than IKK␣, is responsible for phosphorylating the two conserved serines in IB proteins and transducing inflammatory signals, such as from TNF-␣. This observation corroborates previous genetic studies that have disputed the apparent commonality of the two subunits of IKK (37). IKK␣ Ϫ/Ϫ mice died perinatally due to limb and skin abnormalities; however, no impairment in 2 C. Wu and S. Ghosh, unpublished data. IB degradation in response to signaling by cytokines such as TNF-␣ and interleukin-1 was observed (38,39). More recently, further analysis of mice in which a catalytically inactive form of IKK␣ was knocked in led to the discovery that IKK␣ functions in different biological processes, including breast development, keratinocyte differentiation, and lymph node development, not all of which are NF-B-dependent (40 -42). In instances where IKK␣ has been shown to influence NF-B, the effect appears to be due to an alternate pathway that leads to the activation of p52⅐RelB complexes following the processing of the p100 subunit (41). Therefore, it is possible that the inability of IKK␣ to phosphorylate the N-terminal IB substrates hints at the pleiotropic nature of IKK␣, whose more important targets for phosphorylation might be NF-B2 p100, histone H3, and NF-B p65.
The major finding of this study is, however, the clear demonstration of the difference in the ability of the two IB isoforms to serve as phosphorylation substrates for IKK␤. Comparison of the sequence around the target serine residues in the N-terminal signal-responsive domains of IB␣ and IB␤ indicates the absence of an aspartic acid immediately upstream of the second serine residue in IB␤ (Asp 35 in IB␣ versus Gly 22 in IB␤). However, as shown in Fig. 6, changing the glycine in IB␤ to aspartic acid did not enhance the ability of IKK␤ to phosphorylate the IB␤ protein. Instead, it appears that a larger segment from amino acids 29 to 43 is responsible for allowing efficient phosphorylation of IB␣. Unfortunately, the existing crystal structures of IB proteins do not include this segment; and therefore, it is difficult to propose a model to explain the role played by this region of IB␣. Ultimately, a co-crystal of the IB N-terminal domain with IKK␤ will be necessary to understand the selectivity displayed by IKK␤ for IB␣.