©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Critical Role for Lysines 21 and 22 in Signal-induced, Ubiquitin-mediated Proteolysis of IB- (*)

(Received for publication, October 13, 1995)

Lucia Baldi Keith Brown Guido Franzoso Ulrich Siebenlist (§)

From the Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1876

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The NF-kappaB transcription factor induces rapid transcription of many genes in response to a variety of extracellular signals. NF-kappaB is readily activated from normally inhibited cytoplasmic stores by induced proteolytic degradation of IkappaB-alpha, a principal inhibitor of this transcription factor. Following the inhibitor's degradation, NF-kappaB is free to translocate to the nucleus and induce gene transcription. The IkappaB-alpha inhibitor is targeted for degradation by signal-induced phosphorylation of two closely spaced serines in its NH(2) terminus (Ser and Ser). Proteolytic degradation appears to be carried out by proteasomes which can recognize ubiquitinated intermediates of the IkappaB-alpha inhibitor. We provide evidence which supports a ubiquitin-mediated mechanism. Amino acid substitutions of two adjacent potential ubiquitination sites in the NH(2) terminus of IkappaB-alpha (Lys and Lys) almost completely block the rapid, signal-induced degradation of the mutant protein, while they do not interfere with induced phosphorylation. The mutant IkappaB-alpha also does not permit signal-induced activation of NF-kappaB bound to it. The data suggest that ubiquitination at either of the two adjacent lysines (21 and 22) is required for degradation following induced phosphorylation at nearby serines 32 and 36. Such dependence on ubiquitination of specific sites for protein degradation is unusual. This mechanism of degradation may also apply to IkappaB-beta, an inhibitor related to and functionally overlapping with IkappaB-alpha, as well as to cactus, an IkappaB homolog of Drosophila.


INTRODUCTION

The transcription factor complexes known collectively as NF-kappaB function primarily as mediators of inducible transcription in response to a variety of environmental signals (for recent reviews, see (1, 2, 3, 4, 5) ). Stress- and pathogen-related signals in particular are known to activate NF-kappaB, leading to induced expression of a large number of genes, including many genes which encode functions relevant to immune responses. In most cell types, p50-p65 heterodimers represent the vast majority of the rapidly inducible NF-kappaB complexes, although several other NF-kappaB dimers may coexist and may become activated also. All NF-kappaB dimers are composed of members of the Rel/NF-kappaB family of polypeptides, and in vertebrates this family is comprised of p50 (NF-kappaB1), p65 (RelA), c-Rel, p52 (NF-kappaB2), and RelB. The various NF-kappaB dimers usually lie dormant in the cytoplasm of cells, kept there by inhibitory ankyrin-containing members of the IkappaB family of proteins, in particular IkappaB-alpha. IkappaB-alpha strongly associates with p50/p65 heterodimers and appears to shield the nuclear localization sequences contained in both subunits; this is presumed to be the mechanism by which this protein retains the heterodimers in the cytoplasm(6, 7, 8, 9) . Activation of NF-kappaB proceeds via rapid, signal-induced proteolytic degradation of the inhibitor, liberating the transcription factor which is now free to translocate to the nucleus (10, 11, 12, 13, 14, 15, 16, 17) . Degradation is carried out by proteasomes and is preceded by signal-induced phosphorylation of IkappaB-alpha itself(18, 19, 20, 21, 22, 23, 24) . Induced phosphorylation occurs on two closely spaced serines in the NH(2) terminus of the protein (amino acids 32 and 36), mediated by an as yet unknown kinase(s)(25, 26, 27, 28) . It has recently been shown that signal-induced phosphorylation can lead to ubiquitination of IkappaB-alpha(29) . Since ubiquitin-tagged proteins are generally subject to proteasome-mediated proteolysis(30) , these observations suggest that degradation of IkappaB-alpha is triggered by ubiquitination. However, a ubiquitin-independent mechanism of degradation is not necessarily excluded, since only a fraction of the total pool of IkappaB-alpha could be shown to be ubiquitinated (under conditions of proteolysis inhibition). Furthermore, precedents exist for a ubiquitin-independent, but proteasome-dependent degradation mechanism(31) . Therefore, we sought to demonstrate ubiquitin-dependence by investigating the requirement for lysines in IkappaB-alpha degradation, because lysines are the sites at which ubiquitin is ligated(30) . Here we provide evidence which strongly suggests that rapid, signal-regulated degradation of IkappaB-alpha proceeds primarily via a ubiquitin-dependent mechanism. An IkappaB-alpha mutant bearing conservative substitutions at two potential ubiquitination sites is remarkably resistant to signal-regulated degradation. The results imply that two adjacent NH(2)-terminal lysines (Lys and Lys) are the primary targets of signal-induced ubiquitination. That specific lysines play such an important role in ubiquitin-mediated protein degradation is uncommon(30) .


MATERIALS AND METHODS

Site-directed Mutagenesis

Mutations in full-length, human IkappaB-alpha cDNA (32) were generated essentially as described previously(25, 33) . In each case, lysine codons were substituted with arginine codons (for positions 21, 22, 47 and 67: AAG was changed to AGG; for 38: AAA was changed to CGA; for 87: AAG was changed to CGG). The mutated IkappaB-alpha cDNAs were excised from a Bluescript vector (Stratagene, La Jolla, CA) with EcoRI (1550 base pairs) and subcloned into the PMT2T mammalian expression vector(34) . Mutations were confirmed by subsequent DNA sequence analysis.

Transient Transfections

Ntera-2, human embryonal carcinoma cells were transfected via calcium phosphate-mediated transfer as described elsewhere (35) with the following vectors: a CAT (^1)reporter plasmid containing the tandemly repeated kappaB sites of human immunodeficiency virus (5 µg)(35) ; the p65-PMT2T expression vector (0.2 µg)(35) ; PMT2T vectors carrying the wild-type or mutant IkappaB-alphas (see above) (depending on the experiment, between 0.15 and 0.6 µg were found to be required for near-maximal inhibition, which was the point used in the experiments). Cells were stimulated with PMA (10 ng/ml) for 6 h prior to harvesting, stimulation starting at about 36 h post-transfection, and CAT activity was measured as previously described(25, 35, 36, 37, 38) .

Permanent Transfections

EL-4 murine T lymphoma cells were maintained in RPMI medium in the presence of 10% FCS (Life Technologies, Inc.) and were stably transfected with the various PMT2T-IkappaB-alpha vectors, together with a plasmid conferring neomycin resistance, as described elsewhere(25) . Briefly, 20 µg of PMT2T-IkappaB-alpha DNA and 2 µg of neomycin-resistant plasmid DNA were electroporated into 10^7 EL-4 cells using the Bio-Rad Gene Pulser, set at 250 V, 960 microfarads. Stable neomycin-resistant transfectants were selected after 3-4 weeks of incubation with geneticin (G418, Life Technologies, Inc.), used at 400 µg/ml during the first week and at 200 µg/ml thereafter. The resulting cell lines were screened for expression of human IkappaB-alpha using a rabbit polyclonal antibody directed against full-length IkappaB-alpha (10, 25) and ECL technology (Amersham Corp.). Cells were stimulated with PMA (40 ng/ml) and ionomycin (2 µM), and calpain inhibitor I was used at 100 µM, starting 30 min prior to stimulation.


RESULTS

Since ubiquitin is ligated to proteins through lysine residues, we substituted the lysines in IkappaB-alpha by site-directed mutagenesis and tested the resulting mutant proteins for defects in signal-dependent degradation. Human IkappaB-alpha (Mad-3) contains lysine at positions 21, 22, 38, 47, 67, 87, 98, 177, and 238 (32) . Lysines 22, 38, 87, and 238 are perfectly conserved in pig, rat, and chicken (pp40) IkappaB-alpha; lysines 21, 47, 67, and 98 are absent in chicken; and lysine 177 is not conserved at all(39) . We substituted each of the NH(2)-terminal lysines (21, 22, 38, 47, 67, and 87) and the pair, 21 + 22, with arginine residues to block ubiquitination of these sites which all lie near the inducibly phosphorylated serines 32 and 36. Arginine was chosen so as not to change the charge of the protein. As an initial test of the mutant proteins, we transiently transfected expression constructs for the various IkappaB-alpha mutants into Ntera-2 embryonal carcinoma cells, together with an expression construct for NF-kappaB/p65. Undifferentiated Ntera-2 cells do not express significant levels of endogenous NF-kappaB or IkappaB proteins and are thus ideal for evaluating the activities of the transfected proteins without interference by endogenous counterparts(33, 35, 36, 37, 38, 40) . Transfected p65 potently transactivated a cotransfected kappaB-dependent CAT reporter, while coexpression of wild-type IkappaB-alpha or any of the lysine-substituted mutants severely inhibited p65-mediated transactivation (25) (data not shown). Therefore, these mutations did not interfere with the inhibitory activity of the protein, which was expected, since inhibition does not require the NH(2)-terminal domain of IkappaB-alpha(25, 41, 42, 43) . We then tested whether the lysine mutations interfered with the signal-induced degradation of the proteins bearing them, as measured by the ability of a PMA stimulus to relieve inhibition and thus allow p65-mediated transactivation of the CAT reporter (Fig. 1; inducibility of mutants is shown as percent of inducibility of wild-type IkappaB-alpha; inducibility is measured as the ratio of CAT activity of PMA-stimulated cells/unstimulated cells). None of the individual lysine mutations significantly interfered with signal-induced transactivation, suggesting that no single lysine is critical for the signal-induced degradation of IkappaB-alpha (Fig. 1; column 1, wild-type (wt); columns 2, 3, 5-8, mutants K21R, K22R, K38R, K47R, K67R, and K87R (R, R, R, R, R, and R, respectively). However, an IkappaB-alpha mutant bearing substitutions at both lysines 21 and 22 (K21R/K22R (RR mutant) had a dramatic effect; this mutant did not allow significant activation of NF-kappaB in PMA-stimulated, transfected Ntera-2 cells (Fig. 1, column 4). The data suggest that at least one of the two lysines at positions 21 and 22 has to be present for rapid signal-induced degradation of IkappaB-alpha to occur, since elimination of both lysines effectively blocked NF-kappaB activation, while substitution of either lysine alone was of little consequence.


Figure 1: Inducibility of a kappaB-dependent CAT reporter in the presence of wild-type (wt) or mutant IkappaB-alphas in transiently transfected NTera-2 cells. NTera-2 cells were transfected with PMT2T-NF-kappaB/p65, wild-type or mutant PMT2T-IkappaB-alpha expression vectors and the kappaB-dependent CAT reporter plasmid (see ``Materials and Methods''). (p65, transfected alone, potently stimulated CAT activity, and cotransfection of the IkappaB-alpha vectors inhibited this transactivation to near-background levels(25) .). The p65/IkappaB-alpha cotransfected cells were stimulated with PMA, and inducibility was calculated as the ratio of PMA-stimulated CAT activity to unstimulated activity. The inducibilities are shown as a percent of that seen with matched, wild-type IkappaB-alpha, which represents an at least 10-fold stimulation. In several independent experiments, only the K21R/K22R (RR) mutant blocked PMA induction of CAT activity.



To confirm these interpretations and to rule out a potential defect in phosphorylation of the K21R/K22R mutant, we directly evaluated mutants for phosphorylation and degradation. Murine EL-4 T cells were permanently transfected with the various IkappaB-alpha mutants, and then the cells were stimulated with PMA and ionomycin. We showed previously that exogenously derived human IkappaB-alpha is subject to the same signal-induced phosphorylation and degradation as the endogenous murine IkappaB-alpha(25) . Endogenous murine IkappaB-alpha serves as an internal positive control in these experiments. Since it migrates slightly faster than the transfected human protein, both proteins could be simultaneously visualized(25) . Among the mutants tested, only the K21R/K22R mutant was resistant to signal-induced degradation in the EL-4 cells (Fig. 2), while all other transfected human (h) mutant proteins bearing individual lysine substitutions appeared to be degraded as efficiently as the endogenous murine (m) IkappaB-alpha (Fig. 2, K21R, K22R, K38R, K47R, K67R, and K87R (R, R, R, R, R, and R). All mutant proteins, including the double mutant K21R/K22R, were rapidly phosphorylated in response to signals, as indicated by the shift in mobility of IkappaB-alpha in the presence of calpain inhibitor I, which inhibits proteasomes (20, 21, 22, 23, 24) (Fig. 2, data not shown for K38R, K47R, K67R, and K87R). This is as expected for the rapidly degraded IkappaB-alpha proteins. In the case of the double mutant (K21R/K22R), the proteasome inhibitors were not needed to see the phosphorylation, since this mutant was not efficiently degraded. The presence of the proteasome inhibitor did, however, increase the amount of the K21R/K22R IkappaB-alpha mutant observed, suggesting that these mutations may not completely block induced degradation. Nonetheless, the K21R/K22R mutation afforded this IkappaB-alpha significant protection from degradation, and the results are consistent with the Ntera-2 experiments shown in Fig. 1.


Figure 2: Signal-induced degradation and phosphorylation of wild-type and mutant IkappaB-alpha expressed in stably transfected EL-4 cells. EL-4 murine T cells were permanently transfected with wild-type (wt) or mutated (mt), human (h) IkappaB-alpha expression vectors (see ``Materials and Methods''), as indicated. Cells were stimulated with PMA and ionomycin (Iono) for 15 min in the presence (+) or absence of calpain inhibitor I (Calp. Inhib.), which inhibits proteasome activity. Both transfected human and endogenous murine (m) IkappaB-alpha, as well as the inducibly phosphorylated form of human IkappaB-alpha (IkappaB-alpha^P) were visualized by Western analysis (see ``Materials and Methods''). (The inducibly phosphorylated mutated IkappaB-alpha migrates to almost the same position as the uninduced form and is not readily distinguished here(25) .) Only the K21R/K22R (RR) mutation in human IkappaB-alpha blocked rapid degradation (while the murine wild-type protein was degraded in the same cells), which resulted in an accumulation of the inducibly phosphorylated form, even in the absence of proteasome inhibitors.




DISCUSSION

We have demonstrated a critical requirement for the presence of either of two lysines at positions 21 and 22 in signal-induced degradation of IkappaB-alpha and, as a consequence, in signal-induced transactivation by NF-kappaB/p65. The substitution of both lysines 21 and 22 with arginines in IkappaB-alpha (K21R/K22R) caused a severe block to rapid signal-induced degradation of that IkappaB-alpha in stably transfected EL-4 cells. The mutant protein was, however, still phosphorylated at nearby serine sites (S and S), indicating that the defect lies downstream of the phosphorylation step; it also suggests that the protein was not grossly altered by these conservative substitutions, since the kinase(s) acitivity on this substrate appears unaffected. The K21R/K22R mutant prevented the signal-induced, p65-mediated transactivation of a kappaB-dependent reporter in transient transfection experiments using Ntera-2 cells. In contrast to the K21R/K22R mutant, mutants bearing substitutions of individual lysines, including those at residue 21 or 22, had no measurable effect and behaved like wild type. Taken together these data demonstrate that either of the two lysines at positions 21 and 22 is necessary for rapid degradation (but not for phosphorylation) and they provide a compelling argument for obligatory ubiquitination prior to degradation of IkappaB-alpha: at least one of the two potential ubiquitination sites must be present for rapid signal-induced degradation to proceed. Although degradation is dramatically inhibited, it appears not to be absolutely blocked (see Fig. 2); this may indicate that ubiquitination can also occur at other sites, albeit less efficiently, or that another mechanism allows for a slower degradation. Finally, the data do not tell us if Lys or Lys are sufficient for ubiquitin-mediated degradation. It is possible, for example, that some other, not necessarily specific, lysine is necessary also. To formally test this less likely possibility would require a mutant IkappaB-alpha bearing substitutions of all lysines other than 21 and 22.

Individual ubiquitination sites do not usually play a dominant role in protein degradation, where often multiple functional ubiquitination sites exist and targeted mutations have little effect(30) , although the degradation of Mos may be another exception to this rule(44) . It is possible that ubiquitination of IkappaB-alpha may be specifically directed to lysines 21 and 22, or, alternatively, that these lysines are the only ones accessible for ligation (no other lysines exist NH(2)-terminal to the phosphorylation sites). This latter possibility may be supported by the observation that ubiquitination occurs with IkappaB-alpha still bound to NF-kappaB(29) , which should partially shield the inhibitor. The central part of IkappaB-alpha consists of 6 ankyrin repeats whose primary function is to interact with NF-kappaB; this part may be largely buried in the cleft between the two NF-kappaB subunits, as suggested by x-ray crystallographic data of p50 homodimers(45, 46) . The fairly short COOH-terminal region of IkappaB-alpha is required for inhibition of DNA binding by NF-kappaB, implying that it too may interact with NF-kappaB proteins(25, 43) . By contrast, the NH(2)-terminal part of the protein is not required for these functions, rather, it must be accessible to a kinase(s) to allow inducible phosphorylation. An as yet undetermined protein may then recognize the phosphorylated protein, presumably by recognizing the phosphorylated serines or local changes induced in the protein as a consequence of phosphorylation (no major conformational changes are expected, since the phosphorylated species remains tightly bound to NF-kappaB and continues to inhibit)(18, 19, 20, 21, 22, 23, 24) . Thus, the two lysines NH(2)-terminal to the two serines may present the only obvious targets. It remains to be shown whether highly ubiquitinated IkappaB-alpha is removed from the complex just prior to degradation, or if degradation is initiated while ubiquitinated IkappaB-alpha is still in the complex. In contrast to bound IkappaB-alpha, the free unbound form may present additional sites for ubiquitination.

Chicken IkappaB-alpha (pp40) contains only one of the two lysine residues important for degradation (the Lys equivalent to that at position 22 in the human protein), suggesting that a single substitution of that lysine may be sufficient to block rapidly inducible degradation of pp40. Recently IkappaB-beta was cloned and shown to be inducibly degraded in response to certain signals, such as interleukin-1 and lipopolysaccharide(47) . While IkappaB-alpha and IkappaB-beta share high overall similarity, their NH(2)-terminal regions are surprisingly different, save for a few conserved amino acids; however, these few amino acids appear to be highly significant in that they suggest shared regulatory features of these proteins (Fig. 3). Both inducibly phosphorylated serines and a few surrounding residues are conserved as is the lysine equivalent to that at position 22 in IkappaB-alpha. (This is the only lysine in the entire NH(2)-terminal part of the IkappaB-beta protein, which may suggest that it is absolutely required for signaling in that protein). This limited but significant conservation of functional sites can also be found in cactus, the Drosophila homolog of IkappaB proteins (48, 49) (see Fig. 3), suggesting that all three proteins may be regulated in a similar fashion. Cactus, which contains a much larger NH(2)-terminal domain than either IkappaB-alpha or IkappaB-beta, may offer additional sites for regulation.


Figure 3: Sequence comparisons of IkappaB-alpha, IkappaB-beta, and cactus. Functionally important residues of IkappaB-alpha are conserved in IkappaB-beta and cactus. This includes both inducibly phosphorylated serines, 32 and 36, a few surrounding residues, and at least one of the two NH(2)-terminal lysines important for degradation.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dr. Ulrich Siebenlist, NIH, Bldg. 10, Rm. 11B16, Bethesda, MD 20892-1876. Tel.: 301-496-7662; Fax: 301-402-0070; us3n@nih.gov.

(^1)
The abbreviations used are: CAT, chloramphenicol acetyltransferase; PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We are grateful to A. S. Fauci for support and review of the manuscript.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.