INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The NF-B transcription factor family plays an evolutionarily conserved role in innate and adaptive immune responses, and its members are essential regulators of proinflammatory processes (12, 18, 33). NF-B is also an important regulator in cell fate decisions, such as programmed cell death and proliferation control, and is critical in tumorigenesis (32, 42). The vertebrate NF-B/Rel factors p50, p52, p65 (RelA), c-Rel, and RelB form various dimers and are under the control of a coevolved family of cytoplasmic IB molecules, IB, -, and -, and the nuclear IB homologue Bcl-3 (1). p50 and p52 are formed by processing from the precursor molecules p105 and p100, respectively. Prototypic heterodimeric NF-B p50-p65 is rapidly released from cytoplasmic complexes with IBs upon cellular stimulation with diverse agents, including tumor necrosis factor alpha (TNF-), interleukin-1, bacterial lipopolysaccharides (LPS), and phorbol myristate acetate, following viral infection or exposure to -irradiation (31). All these agents induce an IB kinase (IKK) complex to phosphorylate IB, -, and -. The IKK complex contains the kinases IKK and IKK and the noncatalytic IKK subunit, which is essential for signal responsiveness of the complex (see reference 17 for a review). Once phosphorylated by the IKK complex, the IBs are ubiquitinated and degraded by the 26S proteasome, resulting in nuclear translocation of NF-B. The ubiquitin ligase complex specific for IBs, SCF (Skp1, Cullin, F-box protein), has recently been identified and contains as substrate recognition molecules the F-box and WD40 domain protein TrCP1 (Slimb) (46, 53, 59) or its homologue TrCP2 (HOS) (10). Further components in this complex are Skp1, Cdc53/Cul1, and ROC1 (49). The TrCP component accounts for recognition of an IKK-phosphorylated DS(P)GLDS(P) motif containing serines 32 and 36 in IB and serines 19 and 23 in IB (45, 54). TrCP1 and TrCP2 bind as homodimers to IB (48). A similar motif in the -catenin (Armadillo) proteins of the Wnt/Wg pathways and in the human immunodeficiency virus type 1 (HIV-1)-encoded Vpu protein is phosphorylated by glycogen synthase kinase 3 (GSK3) and casein kinase II (CKII), respectively. It then confers TrCP1 binding and degradation of -catenin and the Vpu-interacting HIV receptor CD4 by the ubiquitin-proteasome system (20, 30, 53).

The NF-B precursor protein p105 (NFKB1) and its paralogue p100 (NFKB2) have unique roles in the control of NF-B activity, since both act as IB molecules when unprocessed and provide the subunits p50 and p52, respectively, upon processing. The expression of both genes is strongly autoregulated by NF-B, providing a means to replace the proteolysed molecules, similar to the situation with IB (see reference 55 for a review). Different levels of proteolytic maturation of p105 by the 26S proteasome have been described. Posttranslational p50 production from p105 was shown in mammalian and yeast cells (8, 41) and requires ubiquitination (5, 40). A glycine-rich region at the end of the amino-terminal half of p105 is required for p50 generation, perhaps by limiting the processive action of the proteasome (27). Several groups have reported that processing of p105 may be enhanced by signal-induced phosphorylation caused by agents like phorbol ester, okadaic acid, TNF-, double-stranded RNA, and hydrogen peroxide (7, 29, 34, 35, 37). In line with this, in cells stimulated with NF-B-activating agents, including TNF-, phorbol ester, and hydrogen peroxide, p105 and p100 were phosphorylated with the same kinetics as IB (37). However, the fold increase in p50 relative to p105 induced by the various conditions in these studies was modest compared to the clear decline in p105 levels.

The concept of posttranslational processing was challenged by a report that p105 processing occurs mainly as a constitutive process at the cotranslational level, leading to the production of p105-p50 heterodimers (25). This process involves a cotranslational dimerization of the Rel homology domain of the nascent polypeptides (26). Similarly, p52 production from p100 was shown to occur by a cotranslational process (15). The conditions which determine cotranslational versus posttranslational p50 formation remain to be determined. Recently, we and others demonstrated that p105 is subject to signal-induced complete degradation rather than processing (2, 13). TNF--induced p105 degradation rapidly releases p105-bound p50, which is detected in the nucleus as dimers complexed with Bcl-3 (13). Transfected IKK and IKK cause efficient phosphorylation of p105 at a carboxy-terminal region of 18 amino acids, resulting in degradation of p105 by the proteasome. The phosphorylation sites were essential for TNF--induced p105 degradation, as shown by combined mutation of all potential phosphoacceptor sites (13). Furthermore, in cells transfected with Tpl-2 (Cot), this kinase interacts with a carboxy-terminal region of p105 and causes p105 phosphorylation and degradation, but it does not phosphorylate p105 directly (2). Since Cot acts upstream of the IKK complex (28), induction of IKK may be the mechanism by which Cot triggers p105 degradation. In contrast to these studies and after completion of this work, it was recently proposed that IKK phosphorylation of p105 primarily induces enhanced processing. It was reported that IKK-induced processing is promoted by sequestration of TrCP and subsequent polyubiquitination of p105 (38).

In this work we have analyzed the molecular determinants of p105 phosphorylation by IKK and identified the major substrate residues and a separate kinase-docking site, which is part of a death domain. We show that IKK phosphorylation of p105 is direct and that it creates a recognition site for TrCP proteins critically dependent on one of the determined phosphoacceptor serines. IKK-induced ubiquitination results in complete degradation but not enhanced processing of p105. This conclusion is supported by analysis of endogenous p105 in LPS-treated pre-B cells.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell culture. 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 100 U of penicillin-streptomycin per ml. 70Z/3 and 1.3E2 cells were maintained in RPMI medium supplemented with 10% fetal calf serum, 50 µM -mercaptoethanol, and 100 U of penicillin-streptomycin per ml. 1.3E2 cell lines were electroporated with hemagglutinin (HA)-tagged IKK (IKK-HA) pcDNA3 expression vector, and clones stably expressing IKK-HA were selected and grown in medium containing G418 (1 mg/ml; Gibco). For stimulation, cells were treated with cycloheximide at 50 µg/ml for 293 cells and 25 µg/ml for 70Z/3 and 1.3E2 cells. N-acetyl-Leu-Leu-norlencinal (ALLN) (Calbiochem) and bacterial LPS (Sigma) were added at 50 and 10 µg/ml, respectively, for the indicated periods of time prior to preparation of extracts.

Plasmids. Eucaryotic expression vectors for p105 (p105pcDNA3Flag, p105C1-C5pcDNA3Flag, and p105SSS-AAApcDNA3Flag) and IB (IBpcDNA3 and IBNpcDNA3) as well as prokaryotic expression vectors (p105NpRSET, p105NSSS-AAApRSET, IBpGEX6P, and IBS32/36pGEX6P) were described previously (13). p105C6 (amino acids 18 to 809) was generated by PCR, cloned via BamHI and SacI into pRSETB or, to generate p105C6+SRD, a p105 construct with internal deletion of amino acids 809 to 916, into p105pRSETB. The inserts were excised via BamHI and KpnI and religated into pcDNA3Flag. p105C7 (aa 18 to 776) was constructed via partial XbaI digestion, and p105C8 (amino acids 18 to 751) was constructed by PCR. Note that due to these cloning procedures, C6, C7, and C8 contain the artifical C-terminal additions ELEICSLVPPLEGPIL, EGPIL, and VVPLEGPIL, respectively. Mutant p105 (M1-M9) was generated by standard PCR techniques and cloned via SacI-KpnI digestion into procaryotic expression vector pRSETA or p105 (amino acids 18 to 968) pRSETB. The p105 cDNAs (18 to 968) coding for mutant proteins were religated after BamHI-KpnI digestion into pcDNA3, which was modified with an amino-terminal Flag tag inserted via HindIII and BamHI and a KpnI restriction site between XhoI and XbaI. All constructs were sequenced on a LiCor 4000L or with an ABI 377 sequencer (Invitek, Berlin, Germany). The eukaryotic p100 expression vector was constructed via BamHI-EcoRI digestion of p100pRSETC (13) and religation into pcDNA3Flag. Human p105 EST clones were from RZPD (Resource Center German Human Genome Project, accession numbers AL118961, AA258085, AA134618 and AW612589); human IB1 and IB2 cDNAs were described previously (16, 23). Human -TrCP1 was PCR amplified from the hE3RS^IB cDNA (59) and inserted via NotI and XbaI into pcDNA3.1 modified with an amino-terminal HA tag.

Protein purification and in vitro translation. p105 fragments (917 to 968) cloned into pRSETA were expressed in BL21/pLysS bacteria. Cell pellets were resuspended in 50 mM Tris (pH 7.4)-100 mM NaCl-5 mM -mercaptoethanol-0.4 mM Pefabloc and lysed by sonication. Expressed proteins in the lysates were bound to Ni⁺-agarose (Qiagen) for 3 h at 4°C, washed three times with lysis buffer, and eluted for 1 h at 4°C in 300 mM imidazol-300 mM NaCl-50 mM NaPO₄ (pH 6)-10% glycerol. Coupled in vitro transcription-translation was performed according to the manufacturer's protocol (Promega).

Antibodies. The IKK (FL-419), IKK (H-744), IB (C-21), HA (Y-11), p50 (D-17), and Cot (M-20) antibodies were obtained from Santa Cruz. Monoclonal antiubiquitin antibody (1B3; MoBiTec) and IKK antibody 10AG2 (Biosource) were used for Western blotting. Anti-Flag tag antibody M2 or M5 (Sigma) and anti-T7 tag antibody (Novagen) were used for immunoprecipitation or immunoblotting.

Extracts, EMSA, and Western blotting. Whole-cell extracts were analyzed by electrophoretic mobility shift assay (EMSA) and Western blotting essentially as described previously (21).

293 cell transfection and preparation of extracts. Plates (10 cm) of 293 cells were transiently transfected using the calcium phosphate precipitation method with 10 µg of total DNA. Cells were lysed 24 to 36 h after transfection. For detection of ubiquitinated proteins, one plate was lysed in 1 ml of lysis buffer containing 50 mM Tris (pH 7.6)-300 mM NaCl-0.5% Triton X-100-10 mM iodocetamide-0.4 mM NaVO₄-0.4 mM EDTA-10 mM NaF-10 mM sodium pyrophosphate-1 mM dithiothreitol (DTT)-8 mM -glycerophosphate and the complete protease inhibitor cocktail (Boehringer). Equal volumes of lysates (20 µl) were used for detection in Western blots and (1 ml) immunoprecipitation.

Immunoprecipitation. Immunoprecipitation with cell extract equivalents of 10-cm plates were performed with anti-Flag antibody (M2; Sigma) or anti-gene 10 antibody (Novagen) in 1 ml of the indicated lysis buffers. The cell extracts were cleared for 1 h at 4°C with 20 µl of protein A-Sepharose (Pharmacia) and precipitated with antibodies and protein A-Sepharose for 3 h at 4°C. The precipitates were washed three times with 1 ml of the individual buffer used for immunoprecipitation, boiled in sodium dodecyl sulfate (SDS) loading buffer, and separated on 8 to 10% SDS-polyacrylamide gel electrophoresis (PAGE).

IKK assay. Kinase assays were performed as described previously (13) using 1 µg of recombinant substrate protein and 25 ng of purified IKK.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

IKK phosphorylates p105 directly at residues located within a DSXXDS motif. We previously identified IKK phosphoacceptor sites between amino acids 920 and 936 of the p105 precursor molecule. Less efficient IKK phosphorylation was also observed in a region between amino acids 850 and 891, but deletion of this site had no effect on p105 stability (13). Phosphorylation was analyzed using transfected IKKs after immunoprecipitation in an in vitro kinase reaction. To formally rule out the possibility that p105 is not a direct target of IKKs, we now used recombinant IKK purified from baculovirus-infected insect cells in an in vitro kinase reaction (Fig. 1A). Recombinant IKK (Fig. 1B) phosphorylated p105 and IB with similar efficiency, while phosphorylation of IBS32,36A and p105NAAA was abolished and strongly reduced, respectively. Similar results were obtained for recombinant IKK (data not shown). Residual phosphorylation of the p105 mutant is likely due to the cryptic phosphorylation site between amino acids 850 and 891 (13). Thus, phosphorylation of p105 by IKKs is direct and does not need further kinases acting downstream of IKK. Next, we wanted to examine the phosphoacceptor sites in p105. Intriguingly, the published human p105 sequences contain either a threonine or a serine at position 927 (3, 14, 19, 36), but serine in rat and mouse p105 and either serine or threonine in chicken p105 (various cDNA and expressed sequence tag [EST] sequences in the database [not shown]). Similarly, human IB was reported to contain a threonine at position 19, while in murine IB the first potential acceptor position was determined to be a serine (23, 50). Importantly, it was shown that a threonine instead of a serine is not tolerated as a phosphoacceptor site in IB (6, 24). We resequenced several human p105 EST and cDNA clones and IB1 and IB2 cDNA clones in this region to determine whether the obvious differences in the published sequences are due to polymorphisms or arise from sequencing problems in this region, which indeed revealed compression effects upon sequencing. We found that both human p105 and human IB in fact contain a serine instead of a threonine at positions 927 and 19, respectively (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Purified recombinant IKK phosphorylates the signal response domain of p105 directly. (A) Coomassie brilliant blue-stained SDS-PAGE showing the purification of IKK. His-tagged IKK was expressed in SF9 cells with the baculovirus system. IKK protein from the lysates of infected cells (lane 1) was bound to Ni+ -agarose and cleaved off by thrombin digestion (lane 2). The supernatant was bound in 50 mM Tris (pH 7)-1 mM DTT-1 mM EDTA to a MonoQ column and eluted with a 10-ml gradient of 0 to 1 M NaCl (lanes 4 to 9). The protein eluted at about 500 mM NaCl. Sizes are shown in kilodaltons. (B) The purified protein (fraction 9) (lane 5, lower panel) was used in an in vitro kinase assay with IB S32/36A, wild-type IB, p105N, and p105NAAA, as indicated. p105NAAA contains serine-to-alanine mutations at amino acids 921, 923, and 932 and a serine-to-threonine mutation at residue 927. Top, kinase assay (KA); bottom, Coomassie brilliant blue-stained (CS) SDS-PAGE of IB, p105, and IKK proteins.

View larger version (67K): [in this window] [in a new window]   FIG. 2.   Mutational analysis of IKK phosphorylation sites in p105. p105 cDNA fragments encoding amino acids 917 to 968 of p105 were cloned into pRSETA. Purified proteins (see Coomassie-stained SDS gel [CS], left panel) were used in an in vitro kinase assay with purified IKK (KA, right panel). Mutated amino acids in the p105 destruction box are shown in bold letters.

IKK phosphorylation triggers interaction of p105 with the F-box proteins TrCP1 and HOS/TrCP2. The IKK phosphorylated destruction box of IB is recognized by the F-box proteins TrCP1 and TrCP2 to trigger degradation (10, 45, 47, 53, 54, 59). Since IKK phosphorylation of p105 coincides with proteasome-dependent p105 proteolysis, a likely possibility is that phosphorylated p105 could associate with TrCPs, as suggested earlier (13). As expected, in vitro-translated TrCP1 could be coimmunoprecipitated with IB from extracts of cells expressing full-length IB and IKK (Fig. 3A, lane 3), but not when the destruction box was deleted (in IBN, lane 6) or the kinase was inactive (in IKKK/A, lane 2). In a virtually identical manner, TrCP1 could be precipitated with p105 (lane 9) strictly dependent on both the C-terminal destruction box (missing in p105C5, lane 12) and active IKK (lane 8 versus 9). Likewise, TrCP2 bound to p105 phosphorylated by IKK (Fig. 3B, lane 3), but did not bind to p105 mutants with deletion or point mutations of all serines in the destruction box (lanes 6 and 9). Thus, the similarity between the recognition sites in IB and p105 suggests that both proteins utilize the same TrCP1- or -2-containing SCF complexes for signal-dependent ubiquitination.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   SCF ligase receptor subunits TrCP1 and TrCP2 recognize IKK-phosphorylated p105. (A) Plates of 293 cells were transfected with 1 µg of IB (lanes 1 to 3), 1 µg of IBN (lanes 4 to 6), 2 µg of p105 (lanes 7 to 9), or 2 µg of p105C5 (lanes 10 to 12) together with kinase inactive IKK (lanes 2, 5, 8, and 11) or wild-type IKK (lanes 3, 6, 9, and 12). Cells were extracted with 200 mM NaCl-50 mM HEPES (pH 7.5)-0.5% NP-40-1 mM EDTA-10% glycerol. The extracts were diluted to 100 mM NaCl-50 mM HEPES (pH 7.5)-0.5% NP-40-1 mM EDTA- 5% glycerol and incubated for preclearance and immunoprecipitation with 4.5 µl of in vitro-translated, 35S-labeled TrCP1-HA. Immunoprecipitations were performed with anti-gene 10 (IB) or anti-Flag (p105) antibodies. Coprecipitated TrCP1 (top panels), expression (Western blots) of IB constructs and endogenous IB (anti-IB antibody; middle panel, left), of p105 constructs (anti-Flag antibody, right panel) as well as of expressed wild-type and mutant IKK are shown as indicated. (B) A similar experiment was performed using p105 (lane 1 to 3) p105C5 (lanes 4 to 6) and p105AAA (lanes 7 to 9) expression constructs cotransfected with IKK K/A (lanes 2, 5, and 8) or IKK (lanes 3, 6, and 9). Cell lysis and immunoprecipitation were performed as described above, using anti-Flag antibodies and in vitro translated TrCP2-HA/Hos protein.

IKK and TrCP induce ubiquitination and subsequent proteasomal degradation of p105. The effect on ubiquitination and proteolysis of p105 in response to IKK phosphorylation and TrCP recognition was analyzed in intact cells. When p105 was cotransfected along with IKK and TrCP1, polyubiquitination was observed (Fig. 4, lane 3, top panel). Ubiquitination was enhanced when degradation was blocked with a proteasome inhibitor (lane 4). Mutation of residues critical for p105 phosphorylation by IKKs greatly reduced ubiquitin conjugation (lanes 7 and 8). IKK and TrCP1-mediated ubiquitination was paralleled by a loss of p105 (compare lane 3 with lanes 1 and 2, second panel), which was abolished by proteasome inhibition (lane 4) or by mutation of IKK phosphorylation sites in p105 (lane 7). Thus, in an IKK-dependent fashion, p105 is targeted by TrCP/SCF, polyubiquitinated, and degraded by the proteasome.

View larger version (59K): [in this window] [in a new window]   FIG. 4.   Specific phosphorylation of p105 triggers TrCP1-dependent ubiquitination and proteasomal degradation. Plates of 293 cells were transfected with 3 µg of p105 (lanes 1 to 4) or p105AAA (lanes 5 to 8) together with 2 µg of IKK (lanes 2, 3, 4, 6, 7, and 8) and 1 µg of -TrCP1-HA (lanes 3, 4, 7, and 8) or with TrCP1-HA alone (lane 9). Cells were treated for 2 h with ALLN as indicated, extracts were prepared and immunoprecipitation was performed with anti-Flag antibodies. Top panel, antiubiquitin Western blot of precipitated proteins. Second panel, anti-Flag Western blot of cell extracts; third panel, anti-Flag Western blot of precipitates, lower panel, Western blot detection of IKK in extracts.

IKK and TrCP trigger posttranslational complete degradation but not processing of p105; critical role of serine 927. A matter of debate is the relation of p105 processing versus complete p105 degradation in response to IKK signaling (13, 38). In order to uncouple de novo synthesis and cotranslational processes from posttranslational events, the effect of IKK and TrCP on p105 processing and degradation was analyzed in the presence of cycloheximide (Fig. 5A). When p105 was expressed alone, the total amounts of p105 and p50 as well as the precursor-product ratio did not change over time following translation inhibition (lanes 1 to 4). Coexpression of IKK resulted in increased production of both p105 and p50, perhaps by enhancing transcription of the p105 expression vector, but did not change the precursor-product ratio (lanes 5 to 8). However, when TrCP1 was coexpressed with p105 and IKK, p105 amounts were gradually diminished after cycloheximide addition and nearly disappeared after 2 h (lanes 9 to 12). In stark contrast, the amounts of p50 were unaffected and did not increase, as expected if any posttranslational processing were regulated by a concerted action of IKK and TrCP. Taken together, these results strongly suggest that complete degradation but not posttranslational processing of p105 is regulated by IB kinases and TrCP/SCF complexes.

View larger version (61K): [in this window] [in a new window]   FIG. 5.   IKK and TrCP1 trigger serine 927-dependent degradation of p105. (A) Transfections were performed in quadruplicate. One fourth of the CaPO4 DNA precipitate was used to transfect a 6-cm plate of 293 cells. Each transfection contained a total of 20 µg of DNA with 2 µg of p105 expression vector (lanes 1 to 12) or 2 µg of p105AAA (lanes 13 to 24). For cotransfection, 4 µg of IKK (lanes 5 to 12 and 17 to 24) and 2 µg of TrCP1-Flag (lanes 9 to 12 and 21 to 24) were used. Cells were treated with cycloheximide (CHX) for the indicated times, and extracts were prepared in RIPA buffer containing the complete protease inhibitor cocktail (Boehringer). (B) 293 cells were cotransfected with IKK, TrCP1, and wild-type p105 or various p105 proteins (residues 18 to 968) carrying point mutations as shown in Fig. 2 or with an expression vector encoding p100. Transfection, stimulation, and lysis of cells were performed as described for panel A.

IKK physically interacts with a docking site on p105 which is part of a death domain and separate from the substrate site. We previously showed that both IKK and IKK can physically associate with p105 (13). To test the requirement of the phosphorylation sites for binding of IKK and to delineate the interacting region, p105 constructs with progressively deleted C-terminal sequences were generated (Fig. 6A, left panels). All deletion mutants underwent processing (Fig. 6A) and interacted with p65 (not shown). p105 wild-type and mutant proteins were coexpressed with IKK and subjected to coimmunoprecipitation (Fig. 6A, right panel). Intriguingly, point mutation of the IKK substrate serine residues in p105AAA did not affect the robust physical interaction of IKK (lanes 1 and 2). The p105 deletion mutants C1 to C5, either still containing all phosphorylation sites (C1), lacking the major IKK phosphorylation sites (C2), or devoid of all IKK sites, including cryptic sites (C5), all interacted with IKK with the same efficiency as the wild-type protein (lanes 1 compared to 2 to 6). However, further deletion of sequences N-terminal to amino acid 850 (C6, C7, and C8) resulted in an almost complete loss of interaction (lanes 1 to 6 compared to 8 to 10). The signal response domain, containing the IKK phosphorylation sites, could not restore IKK interaction when fused to C6 (lane 7 versus 8). These results reveal that IKK binds to sequences in p105 located more than 70 amino acids amino terminal to the major phosphorylation sites and that the substrate serines do not contribute to efficient physical interaction. Similar findings were obtained for IKK (data not shown). Thus, substrate recognition of p105, and probably of other substrates, by IKKs is not determined solely by specificity-determining residues in close proximity to the substrate serines, but also by the quality of a separate docking site on the substrate.

View larger version (53K): [in this window] [in a new window]   FIG. 6.   (A) Physical interaction of IKK with p105 is conferred by the N-terminal half of a death domain. (Left panel) p105 and C-terminal deletion constructs were expressed in 293 cells and analyzed in a Western blot for expression and basal processing levels. Bottom, schematic summary of deletion constructs. RHD, Rel homology domain; ARD, ankyrin repeat domain; DD, death domain (residues 805 to 892); SRD, signal response domain. Amino acids encoded by the deletion mutants are indicated. Right panel, 293 cells were transfected with 4 µg of p105, p105AAA, p105C1-p105C8, or p105C6+SRD, as indicated, along with 3 µg of IKK expression vector (lanes 1 to 10). IKK, coprecipitated with Flag-tagged p105 proteins, is shown in an anti-IKK Western blot (top panel), expression of p105 proteins and IKK is shown by Western blotting of precipitated proteins or extracts with the respective antibodies, as indicated (lower panels). Cell lysis and immunoprecipitation was performed with RIPA buffer. Extracts or precipitated proteins were resolved by SDS-PAGE, probed with anti-IKK antibody, stripped, and reprobed with anti-Flag antibody. (B) Left panel, effect of kinases on processing efficiencies of p105, p105 mutants, and p100. In a total of 10 µg of DNA, Flag-tagged p105, p105AAA, and p100 (2 µg of each) were transfected alone (lanes 1, 6, and 11) or along with 4 µg of the different kinase expression vectors for IKK (lanes 2, 7, and 12), IKKK/A (lanes 3, 8, and 13), Cot (lanes 4, 9, and 14), or CotK/R (lanes 5, 10, and 15), as indicated. Top panels, precursor and processing products in Western blots revealed by anti-Flag antibody. Middle and bottom panels, Western blots of extracts with anti-IKK and anti-Cot antibodies. Right panel, Flag-p105C5 and -p105C6 were transfected either alone (lanes 1 and 4) or together with IKK (lanes 2 and 5) or IKKK/A (lanes 3 and 6) and analyzed as described above.

LPS induces degradation but not processing of cellular p105; requirement for a functional IKK complex containing IKK. The data obtained from in vitro experiments and from transfected cells strongly suggest a model for activation of cytoplasmic p105 complexes to release sequestered NF-B subunits, including the p105 processing product p50. IKKs phosphorylate a destruction box equivalent to that of the small IBs and generate a recognition site for TrCP-containing SCF ubiquitin ligases. Subsequent polyubiquitination of p105 leads to complete degradation.

View larger version (47K): [in this window] [in a new window]   FIG. 7.   Requirement of a functional cellular IKK complex for rapid LPS-induced proteolysis of p105. 70Z/3 cells (lanes 1 to 13), 1.3E2 cells (lanes 14 to 25), or 1.3E2 cells stably expressing HA-tagged IKK (lanes 26 to 29) were LPS stimulated for the indicated times in either the presence or absence of cycloheximide (CHX) or CHX and ALLN (30 min of incubation), as indicated. As a control, cells were treated with CHX or CHX and ALLN alone for 150 min (lanes 11, 12, 24, and 25). An EMSA with NF-B DNA-binding activities is shown in the top panel. Migration of p50-p65 as well as of p50-p50 complexes is indicated. Detection of p105, p50, IB, IKK, and IKK by Western blotting is shown below, and the migration of the specific bands is indicated. Please note that a nonspecific band migrates slightly slower than p105, as determined by peptide competition (lane 13).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The NF-B protein p105 acts in a dual fashion as a cytoplasmic IB molecule, able to associate with other NF-B subunits, and as the precursor for the p50 subunit. We have shown previously that p105 is subject to signal-dependent proteolysis, which gives rise to induced release of p50 homodimers and is mediated by the IKK complex (13). In this work we have investigated specificity-determining residues for the action of IKK and provide evidence for IKK-dependent ubiquitination and complete degradation of p105. By using purified recombinant IKK, we have shown first that IKK directly phosphorylates p105. This experiment is important, since it excludes the formal possibility that unknown kinases downstream of IKK could account for the observed p105 phosphorylation. Using this purified kinase, we have determined that serines 923 and 927 are the major substrate sites. Serines 923 and 927 are in the same spacing (SXXXS) as the phosphorylation sites in IB, -, and - and are both preceded by acidic amino acids (Fig. 8). The p105 sequence in fact bears extended similarity with the sequences surrounding IKK sites in the small IBs. Serine 927 is part of a conserved DSG (where  is a hydrophobic residue) motif. All small IBs and p105 share at the 5 position of this serine an acidic amino acid and at 4 an acidic residue or serine. The 2 and +3 positions bear further IB-specific preferences, cysteine and acidic residues, respectively. However, compared to the small IBs, the phosphorylated residues in p105 (Fig. 8, underlined) are in nonequivalent positions, 4 and +1 versus +1 and +5 relative to the generally conserved central DSG motif (Fig. 8). The sequence conservation suggests that IKK may utilize solely serines in the +4 spacing, preferentially when both are preceded by acidic residues. This, however, is not sufficient to create a bona fide IKK substrate site, since Vpu is not phosphorylated by IKK (D. Krappmann, not shown). Perhaps the 4 and 5 positions of the IBs are specificity-determining residues.

View larger version (64K): [in this window] [in a new window]   FIG. 8.   Phylogenetic conservation (h, human; m, murine; r, rat; ch, chicken; p, porcine) of destruction boxes in p105, in IB,-, and -, in -catenin, and in HIV-1 Vpu. Position numbers of the critical serine residues in the respective full-length proteins are indicated. Serines where stimulated phosphorylation has been shown experimentally are underlined.

Functionally, the conserved serines differ in the various IBs. Unlike the situation in IB, where single mutation of either serine 32 or 36 did not fully abrogate inducible phosphorylation (6, 51), mutation of S927 almost completely abrogated phosphorylation of p105. In IB, any mutations of serines in the conserved motif had little or no effect on phosphorylation (45), although degradation was abolished by mutation of serines 18 and 22 to alanine (52). Perhaps the relatively high number of serines close to the conserved substrate site in IB provide alternative phosphorylation sites for the IKK complex in the mutant proteins.

Our analysis of physical interaction of IKK with p105 revealed that the kinase binds to a region that is nonoverlapping with the destruction box and that mutation or deletion of the substrate serines does not affect interaction strength. The docking site was delineated to the N-terminal half of a death domain and is separated by 70 amino acids from the destruction box (Fig. 6A). The interaction of IKK with a docking site may contribute to substrate recognition in addition to specificity-determining residues flanking the substrate serines.

The death domain confers homo- and heterotypic protein interactions and is mostly found in receptors or adaptors that signal cell death. It is also conserved in signaling molecules which are not associated with apoptotic pathways but regulate NF-B activation, such as Drosophila Pelle and Tube, which are part of the Toll-Dorsal pathway (9, 56). The fact that the death domain is phylogenetically conserved in p105 and in p100 (reference 44 and data not shown) indicates an essential function for these molecules. The physical IKK-death domain interaction may indicate that IKKs also interact with death domains of other molecules and that this interaction could be relevant for recruitment of IKKs to activated receptor-adapter complexes. Similarly, the death domain could engage the precursors into heterotypic complexes with other signaling molecules.

We have also analyzed the physical interaction of IKKs with IB and IB, which do not contain a death domain. Compared to the robust IKK-p105 interaction, IB was only weakly bound by IKK (V. Heissmeyer, unpublished data). However, human IB revealed a stronger interaction with IKK, which was conferred by a C-terminal PEST sequence shared by the IB1 and IB2 splicing isoforms (V. Heissmeyer, unpublished data).

In contrast to p105, p100 is not phosphorylated by IKKs (13), consistent with the fact that the carboxy-terminal amino acids of p100, downstream of the death domain, show no conservation with p105. Accordingly, p100 was not degraded upon coexpression with IKK and TrCP (Fig. 5B and 6A). Thus, p100 is the only cytoplasmic IB protein not directly phosphorylated by IKKs. However, IKK binds to p100 (V. Heissmeyer, data not shown). It is thus possible that IKK, once bound to the death domain of p100, activates a further, unknown kinase to phosphorylate p100.

The overall similarity of the sequence context of IKK phosphorylation sites in p105 and IB suggested that p105 should interact with the same type of ubiquitin ligase as IB. In fact, the interaction efficiency of TrCP1 with IB and p105 was virtually identical. Furthermore, when comparing the related F-box proteins TrCP1 and TrCP2, which bind equally well to phosphorylated IB (47), both also interact with phosphorylated p105 with comparable efficiency. The interaction with TrCP2 was lost completely when the major IKK sites were mutated (p105AAA), indicating that the phosphorylated minor sites in p105 (between residues 850 and 891) cannot attract the F-box protein. The binding of both TrCPs again underscores the similarity of the destruction boxes in p105 and the small IBs and discriminates these proteins from -catenin, which, upon GSK3 phosphorylation, can attract TrCP1 but not TrCP2 (11). Our data also reveal that the last residue in the DSGXS consensus sequence for TrCP recognition is not maintained for p105, which contains a threonine, a very poor IKK substrate. This is intriguing, since the DSGXS motif is strictly conserved in all other proven and potential TrCP substrates (Fig. 8), including armadillo and plakoglobin (not shown). The last serine in the motif is functionally important in IB, since single mutation of this residue (serine 36) completely abolishes induced degradation (4, 6). Yaron et al. (58) have shown that short IB competitor peptides with singly phosphorylated serine 36 or 32 have strongly impaired inhibitory effects on IB ubiquitination compared to their doubly phosphorylated counterparts. It is therefore possible that TrCPs recognize the phosphorylated signal sequences in IB and p105 in a slightly different manner.

We have shown that coexpression of IKK and TrCP1 triggers p105 polyubiquitination which results in complete proteasomal degradation but not in enhanced processing of p105 (Fig. 4 and 5). This result is in contrast to the conclusions drawn by Orian et al. (38), who reported that IKK predominantly enhanced processing. We demonstrated that the expression of IKK alone led to an increase in p50, but this effect is ascribed to IKK-induced expression of p105, resulting in increased amounts of p50 produced by processing and loss of p105 by simultaneous IKK-induced degradation. This conclusion is also supported by the observation that IKK, which does not phosphorylate p100, enhances p52 production along with p100 expression. Likewise, Cot, a kinase which does not phosphorylate p105, enhanced production of p50 and p52 as well as of p105 and p100, most likely by acting on the expression vector. Importantly, by the use of cycloheximide, we have shown that at the posttranslational level, and thus independent of any effects of the kinase on the expression vector, IKK (TrCP) triggered complete degradation but not processing of p105. The observed degradation was fully dependent on serine 927, in agreement with the pivotal role of this residue as an IKK phosphoacceptor site. We also showed that in mouse pre-B cells, LPS triggered degradation but not processing of endogenous p105. LPS-induced degradation but not basal processing required a functional endogenous IKK complex.

Our data fully support the notion that p105 contains a carboxy-terminal destruction box that, like the N-terminal domain in IB, upon IKK phosphorylation, is recognized by an SCF^TrCP E3 ubiquitin ligase which mediates polyubiquitination and complete degradation by the proteasome. Thus, p105 is degraded by the same mechanism as IB. It is also interesting to note that both proteins can obviously be degraded when complexed with Rel factors (p50-p65 and the processing product of p105 or other p105-associated Rel factors, respectively).

The basal processing reaction, in contrast, has been shown to require a glycine-rich region (residues 372 to 394) and an acidic domain (residues 446 to 454) (27, 39), both located at the end of the first half of the precursor. That the basal processing reaction does not require carboxy-terminal sequences containing the destruction box described here is also supported by the fact that deletion or mutation of the IKK phosphorylation sites in p105 does not affect basal processing and that processing was not reduced in cells lacking a functional IKK complex.

IKK-regulated and ubiquitin-mediated p105 degradation is an important bifurcation in NF-B signaling downstream of the IKK complex. This bifurcation provides a means to regulate p50 homodimers, which may act as inhibitors to limit transcriptional responses of p50-p65 or as activators, depending on the availability of Bcl-3. To dissect the regulation of p50 and Bcl-3 is important for understanding the function of these molecules in the immune response and in oncogenesis.