J Biol Chem, Vol. 274, Issue 40, 28169-28174, October 1, 1999

Common Pathway for the Ubiquitination of IB, IB, and IB Mediated by the F-Box Protein FWD1^*

Michiko Shirane^§, Shigetsugu Hatakeyama^§, Kimihiko Hattori^§, Keiko Nakayama^§¶, and Kei-ichi Nakayama^§¶

From the  Department of Molecular and Cellular Biology and ^¶ Laboratory of Embryonic and Genetic Engineering, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, and ^§ CREST, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

FWD1 (the mouse homolog of Drosophila Slimb and Xenopus TrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) was recently shown to interact with IB and thereby to promote its ubiquitination and degradation. This protein has now been shown also to bind to IB and IB as well as to induce their ubiquitination and proteolysis. FWD1 was shown to recognize the conserved DSGXS motif (where  represents the hydrophobic residue) present in the NH₂-terminal regions of these three IB proteins only when the component serine residues are phosphorylated. However, in contrast to IB and IB, the recognition site in IB for FWD1 is not restricted to the DSGXS motif; FWD1 also interacts with other sites in the NH₂-terminal region of IB. Substitution of the critical serine residues in the NH₂-terminal regions of IB, IB, and IB with alanines also markedly reduced the extent of FWD1-mediated ubiquitination of these proteins and increased their stability. These data indicate that the three IB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCF^FWD1 complex. FWD1 may thus play an important role in NF-B signal transduction through regulation of the stability of multiple IB proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The transcription factor nuclear factor-B (NF-B)¹ plays a central role in the activation of many genes that are important in inflammatory and immune responses as well as in the response to cellular stress (1-4). NF-B consists of homo- and heterodimeric complexes of members of the Rel family of proteins. To date, five Rel proteins have been identified in mammals as follows: p50, p52, p65 (RelA), c-Rel, and Rel-B, the first two of which are synthesized as p105 and p100 precursors, respectively (4-6). These proteins bind specifically to B motifs located in the promoters and enhancers of target genes, resulting in transcriptional activation. Furthermore, they all share an ~300-amino acid region of homology, known as the Rel homology domain, that is responsible for DNA binding, dimerization, and interaction with inhibitory proteins of the IB family.

The IB family of inhibitory proteins includes IB, IB, IB, IB, and Bcl-3 in higher vertebrates (Fig. 1A), all of which contain multiple regions of homology known as ankyrin-repeat motifs (4). Such repeats mediate protein-protein interactions, and the specific interaction between ankyrin repeats of IB proteins and the Rel homology domain of Rel proteins appears to be an important and evolutionarily conserved feature of the regulation of NF-B (7). The number of ankyrin repeats varies among the different IB proteins and appears to influence the specificity with which IB pairs with a Rel dimer. The Rel precursor proteins p100 and p105 also contain ankyrin repeats and are sometimes included in the IB family.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Structural organization of members of the IB family of proteins. A, schematic representation of the structure of IB proteins. All members of the IB family contain ankyrin repeats, the number varies from five to seven among the different proteins. IB, IB, and Bcl-3 each contain a PEST sequence in the region COOH-terminal to the ankyrin repeats, whereas IB contains a PEST-like sequence in the NH2-terminal region. Serine residues present within SXXXS and DSGXS motifs are indicated, as are the total number of amino acids in each protein. B, alignment of amino acid sequences required for association with FWD1 in IB, IB, IB, -catenin, and Vpu. Arrows indicate serine residues subjected to phosphorylation by IKK for IB proteins, by glycogen synthase kinase-3 for -catenin, and by casein kinase II for Vpu. Residue numbers are indicated. C, alignment of amino acid sequences of the SXXXS motifs in IB; the first and second sequences overlap.

IB, the best characterized member of the IB family, is a 37-kDa protein with a tripartite structural organization that is also apparent in IB and which consists of (i) an NH₂-terminal domain that is phosphorylated in response to extracellular signals, (ii) a central ankyrin-repeat domain, and (iii) a COOH-terminal PEST domain that is important in the basal turnover of the protein (6, 8, 9). The defining functional characteristic of IB is its ability to confer rapid but transient induction of NF-B activity as a result of its participation in an autoregulatory feedback loop. Activation of NF-B leads to up-regulation of transcription of the IB gene, and the consequent increase in the amount of IB serves to shut off the activation signal (10-15).

IB is a 45-kDa protein and, like IB, binds p50-p65 and p50-c-Rel complexes (16, 17). Functionally, IB and IB differ with regard both to the nature of the incoming signal as well as to the timing of the onset of and the duration of the response (18). Both IB and IB are rapidly degraded after exposure of cells to an appropriate stimulus; however, whereas transcription of the IB gene is induced as a result of NF-B activation that of the IB gene is not (16). Thus, the abundance of IB remains low until the NF-B-activating signal is attenuated.

IB is degraded with slower kinetics than are IB and IB (17, 19). Unlike IB and IB, IB contains a cluster of serine residues at the NH₂ terminus and lacks a PEST domain at the COOH terminus (Fig. 1A), suggesting that these differences in structure may underlie the differences in function. The NH₂-terminal region of IB is rich in Pro, Glu, Asp, Ser, and Thr residues (61 out of 118 residues; 51.7%) and resembles a PEST sequence (17). IB does not interact with either p50 or p52; instead, it associates exclusively with p65-p65 and p65-c-Rel complexes and therefore likely regulates the transcription of genes, such as that for interleukin-8, whose promoters bind preferentially to p65 and c-Rel complexes (19, 20).

The dimeric NF-B complex is normally sequestered in an inactive form in the cytoplasm through interaction with IB. In response to signals such as tumor necrosis factor, interleukin-1, lipopolysaccharide, ultraviolet irradiation, and viral infection, IB proteins are rapidly phosphorylated by a protein kinase complex known as IKK (IB kinase) (21-29). The IKK complex contains at least four kinases (IKK, IKK, IKK (or NEMO), and NIK) and a scaffold protein (IKAP) (30). IKK phosphorylates the 2 serine residues of the DSGXS motif (where  represents the hydrophobic residue) in the NH₂-terminal regions of IB (Ser-32 and Ser-36), IB (Ser-19 and Ser-23), and IB (Ser-18 and Ser-22) (Fig. 1B). Such phosphorylation of IB triggers its rapid degradation by ubiquitin-mediated proteolysis, thereby allowing translocation of NF-B to the nucleus and activation of target genes (31-33). However, it has remained unknown whether other members of the IB family are also ubiquitinated in response to cell stimulation and, if so, whether the phosphorylation of the DSGXS motifs of IB and IB is also required for their ubiquitin-dependent degradation.

The ubiquitin-proteasome pathway of protein degradation is an important mechanism by which the abundance of specific cellular proteins is regulated (34-36). The formation of ubiquitin-protein conjugates is mediated by three enzymes that participate in a series of ubiquitin transfer reactions: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). The specificity of protein ubiquitination often derives from the E3 ubiquitin ligases. Proteins that are polyubiquitinated by these enzymes are subjected to degradation by the 26 S proteasome (36). Recent genetic and biochemical studies in yeast have identified a class of E3 ligases, termed SCF complexes, that are required for degradation of cyclins and their inhibitors as well as for that of an increasing number of other proteins (37-39). SCF complexes consist of the invariable components Skp1 and Cdc53 (Cullin or Cul1) as well as a variable component known as an F-box protein, which binds to Skp1 through the F-box motif. F-box proteins serve as receptors for the target protein, which is usually phosphorylated.

We and others (40-48) recently showed that FWD1, the mouse homolog of Drosophila Slimb and Xenopus TrCP and a member of the F-box and WD40 repeat family of proteins, is specifically associated with IB and -catenin only when the latter is phosphorylated at the serine residues in the DSGXS motif. FWD1 also interacts with the Skp1-Cul1 complex through its F-box domain, thereby forming an SCF complex, SCF^FWD1. Thus, the phosphorylation of IB results in the recruitment of FWD1, which links IB to the ubiquitination machinery. SCF^FWD1 therefore plays an important role in transcriptional regulation by NF-B as a result of its control of IB protein stability.

We have now investigated whether FWD1 contributes to ubiquitin-mediated proteolysis of IB family members other than IB. Our data show that IB and IB are also ubiquitinated and that FWD1 mediates the ubiquitination of these proteins in response to their phosphorylation by the IKK complex, thereby triggering their degradation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- 293T cells were grown at 37 °C and under an atmosphere of 5% CO₂ in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.).

Construction of Expression Plasmids and Mutagenesis-- Molecular cloning of the FWD1 and FWD2 cDNAs was described previously (40, 41). Complementary DNAs encoding FWD1, FWD2, and FWD1(F), each protein tagged at the NH₂ terminus with the FLAG or Myc epitope, were generated with the use of the polymerase chain reaction, as performed with the high fidelity thermostable DNA polymerase KOD (Toyobo, Tokyo, Japan); they were then sequenced and subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). FWD1(F) contains residues 1-140 of FWD1 fused to residues 194-569. IB, IB, IB, and IKK cDNAs were kindly provided by H. Nakano (27). The IB cDNAs were subcloned into pcDNA3 with the FLAG or Myc epitope tag, and the cDNA encoding FLAG- or Myc-tagged IKK was cloned with the TA Cloning System (Invitrogen).

Transfection, Immunoprecipitation, and Immunoblot Analysis-- 293T cells were transfected by the calcium phosphate method or with the use of the LipofectAMINE reagent (Life Technologies, Inc.). After 48 h, the cells were lysed with a solution containing 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 0.5% Triton X-100 (v/v), aprotinin (10 µg/ml), leupeptin (10 µg/ml), 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 0.4 mM Na₃VO₄, 0.4 mM EDTA, 10 mM NaF, and 10 mM sodium pyrophosphate. The cell lysates were pretreated with 50 µl of protein G-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C and were then incubated with 5 µg of the appropriate antibodies and protein G-Sepharose beads for 4 h at 4 °C. The resulting immunoprecipitates were then washed thoroughly four times with ice-cold lysis buffer, fractionated by SDS-polyacrylamide gel electrophoresis (PAGE), and subjected to immunoblot analysis with antibodies (1 µg/ml) to Myc (9E10; Roche Diagnostics, Tokyo, Japan), to the FLAG epitope (M5; Sigma), or to ubiquitin (1B3; MBL, Nagoya, Japan).

Pulse-Chase Experiments-- Transfected 293T cells were metabolically labeled with Tran³⁵S-label (ICN, Costa Mesa, CA) at a concentration of 100 µCi/ml for 1 h. After incubation for various times in the absence of isotope, the cells were lysed and subjected to immunoprecipitation with antibodies to Myc (9E10) and protein G-Sepharose. The immunoprecipitates were fractionated by SDS-PAGE and subjected to quantitative analysis with a BAS-2000 image analyzer (Fuji Film, Kanagawa, Japan).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

FWD1-induced Ubiquitination of IB, IB, and IB-- We and others (40, 42-44, 46, 47) have recently shown that the FWD1 component of the ubiquitin ligase complex SCF^FWD1 serves as an intracellular receptor for IB that has been phosphorylated at the DSGXS motif by IKK. We therefore investigated whether FWD1 also associates with IB and IB through their phosphorylated DSGXS motifs. Expression plasmids encoding FLAG-tagged IB, IB, IB, or their SA mutants, in which serine was replaced with alanine at positions 32 and 36 (S32A/S36A), 19 and 23 (S19A/S23A), or 18 and 22 (S18A/S22A), respectively, were introduced into 293T cells with or without plasmids encoding Myc-tagged IKK, AU1-tagged MEKK1, and Myc-tagged FWD1. Immunoprecipitation assays revealed that Myc-tagged FWD1 was present in the IB, IB, and IB immunoprecipitates prepared with antibodies to FLAG (Fig. 2). The SA mutants of IB and IB did not interact with FWD1, indicating that FWD1 recognizes the serine-phosphorylated DSGXS motif. In contrast, association of the SA mutant of IB with FWD1 was apparent. The observed pattern of ubiquitination of the IB proteins was consistent with the pattern of FWD1 binding. FWD1 markedly increased the ubiquitination of wild-type IB and IB but not that of the corresponding SA mutants. In contrast, both wild-type IB and its SA mutant were ubiquitinated in the presence of FWD1, although the extent of ubiquitination of the SA mutant was less than that apparent with the wild-type protein. These results indicate that FWD1 interacts with all three IB proteins and thereby promotes their ubiquitination, although the mode of binding appears to differ between IB and IB on the one hand and IB on the other, probably as a result of the corresponding structural differences between these proteins: IB contains additional serine residues present within SXXXS motifs in the NH₂-terminal region and lacks a COOH-terminal PEST domain (Fig. 1).

View larger version (53K): [in this window] [in a new window]   Fig. 2.   FWD1-induced ubiquitination of phosphorylated IB, IB, and IB. 293T cells were transfected with expression plasmids encoding FLAG-tagged wild-type (wt) IB proteins or the corresponding SA mutants either alone or together with plasmids encoding Myc-IKK, AU1-MEKK1, and Myc-FWD1 as indicated. Cell lysates were prepared and subjected to immunoprecipitation (IP) with antibodies to the FLAG epitope, and the resulting immunoprecipitates were subjected to immunoblot (IB) analysis with antibodies to FLAG, to Myc, or to ubiquitin (Ub), as indicated. Polyubiquitinated IB proteins are indicated by IB-Ubn. A portion of the cell lysates corresponding to 10% of the input for immunoprecipitation was also subjected to immunoblot analysis with antibodies to Myc in order to indicate the levels of expression of IKK and FWD1.

Phosphorylation-induced Ubiquitination of IB Mediated by FWD1-- To investigate further the role of FWD1 in IB ubiquitination, we examined whether IKK activity is required for this reaction. Expression of IKK alone with IB in 293T cells resulted in a small increase in the extent of IB ubiquitination (Fig. 3), which was likely mediated by endogenous FWD1. However, expression of both IKK and FWD1 together with IB induced a marked increase in IB ubiquitination. The interaction of IB with FWD1 was observed only in the presence of exogenous IKK. Another mammalian F-box and WD40 repeat protein, FWD2 (or MD6), neither interacted with IB, even in the presence of IKK, nor increased the extent of its ubiquitination, confirming that the interaction between FWD1 and IB is specific. Furthermore, FWD1 did not bind to the S19A/S23A mutant of IB in the presence of IKK. These results suggest that FWD1-induced ubiquitination requires prior phosphorylation of IB on Ser-19 and Ser-23 by the IKK complex.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Phosphorylation-induced ubiquitination of IB mediated by FWD1. 293T cells were transfected with expression plasmids encoding Myc-tagged wild-type IB or its S19A/S23A mutant either alone or together with plasmids encoding FLAG-FWD1, FLAG-FWD2, or FLAG-IKK, as indicated. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to Myc, and the resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to Myc, to FLAG, or to ubiquitin as indicated. A portion of the cell lysates corresponding to 10% of the input for immunoprecipitation was also subjected to immunoblot analysis with antibodies to FLAG in order to indicate the levels of expression of IKK, FWD1, and FWD2.

Requirement of the F-Box Domain in FWD1 for IB Ubiquitination-- We previously showed that FWD1 links IB to the Skp1-Cul1 complex as a result of the interaction of Skp1 with the F-box motif of FWD1. We therefore examined whether the F-box domain of FWD1 is also essential for the ubiquitination of IB. The FWD1(F) mutant, which lacks the F-box domain (amino acids 148 to 192), did not induce ubiquitination of IB in the presence of IKK, although FWD1(F) did interact with IB (Fig. 4A). A pulse-chase experiment indicated that expression of FWD1 increased the rate of degradation of IB relative to that apparent in cells expressing FWD2 (Fig. 4B). The FWD1(F) mutant had no such effect, consistent with its inability to induce ubiquitination of IB. Together, these observations suggest that FWD1, acting as a component of the SCF^FWD1 complex, functions as an intracellular receptor for IB and thereby promotes the ubiquitination and degradation of this protein.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Requirement of the F box motif of FWD1 for ubiquitination and degradation of IB. A, 293T cells were transfected with expression plasmids encoding Myc-IB and FLAG-IKK alone or together with plasmids encoding FLAG-tagged wild-type (wt) FWD1 or FWD1(F), as indicated. Cell lysates were subjected to immunoprecipitation (IP) with antibodies to Myc, and the resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to Myc, to FLAG, or to ubiquitin. A portion of the cell lysates corresponding to 10% of the input for immunoprecipitation was also subjected to immunoblot analysis with antibodies to FLAG in order to indicate the levels of expression of IKK, FWD1, and FWD1(F). B, 293T cells were transfected with expression plasmids encoding Myc-tagged IB, FLAG-tagged IKK, and either FLAG-tagged FWD2 (top), FWD1 (middle), or FWD1(F) (bottom). After pulse labeling with [35S]methionine and [35S]cysteine, the cells were incubated for the indicated chase periods in the absence of isotope and then lysed. Cell lysates were subjected to immunoprecipitation with antibodies to Myc, and the resulting IB precipitates were subjected to SDS-PAGE and autoradiography.

FWD1-induced Ubiquitination of IB-- We have shown that FWD1 promotes the ubiquitination of IB (Fig. 2). However, unlike IB and IB, the recognition motif for FWD1 in IB was not restricted to the phosphorylated DSGXS sequence because the S18A/S22A mutant of IB was also ubiquitinated, although to a reduced extent compared with that apparent with the wild-type protein. We therefore investigated whether phosphoserine residues located within other SXXXS motifs in the NH₂-terminal region of IB contribute to FWD1-mediated ubiquitination of this protein. FWD1 bound to IB and elicited marked ubiquitination in the absence of exogenous IKK (Fig. 5A), whereas the ubiquitination of IB and IB requires exogenous IKK activity. Furthermore, an IB mutant (SA-all) in which all nine serine residues within SXXXS motifs were replaced with alanines also associated with FWD1 and was ubiquitinated, although to a reduced extent relative to that observed with the wild-type protein (Fig. 5A). An IB mutant (N) that lacked the entire NH₂-terminal domain (residues 1-118) neither bound to FWD1 nor was ubiquitinated. Thus, the susceptibility to ubiquitination of the various IB proteins examined decreases in the rank order wild-type > S18A/S22A > SA-all > N. These results suggest that FWD1 interacts predominantly with Ser-18 and Ser-22 within the DSGXS motif of IB but is also able to bind to other serine residues in the NH₂-terminal region of the protein.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   FWD1-induced ubiquitination of IB. A, requirement of the NH2-terminal region of IB for both the binding of IB to FWD1 and its ubiquitination. 293T cells were transfected with expression plasmids encoding FLAG-p27 (negative control) or FLAG-FWD1, in combination with vectors encoding Myc-tagged wild-type (wt) IB, IB(SA-all), or IB(N). Cell lysates were subjected to immunoprecipitation (IP) with antibodies to Myc, and the resulting precipitates were subjected to immunoblot (IB) analysis with antibodies to Myc, to FLAG, or to ubiquitin as indicated. A portion of the cell lysates corresponding to 10% of the input for immunoprecipitation was also subjected to immunoblot analysis with antibodies to FLAG in order to indicate the levels of expression of p27 and FWD1. B, effect of a kinase-negative IKK mutant (K44M) on the binding of IB to FWD1 and its ubiquitination. 293T cells were transfected with expression plasmids encoding Myc-IB and FLAG-FWD1, in the absence or presence of a vector encoding IKK(K44M) at increasing concentrations, as indicated. Cell lysates were subjected to immunoprecipitation with antibodies to Myc, and the resulting precipitates were subjected to immunoblot analysis with antibodies to Myc, to FLAG, or to ubiquitin. A portion of the cell lysates corresponding to 10% of the input for immunoprecipitation was subjected to immunoblot analysis with antibodies to FLAG to indicate the levels of expression of IKK(K44M) and FWD1.

With regard to the observation that FWD1-mediated ubiquitination of IB did not require exogenous IKK, it was possible that FWD1 interacted with IB in an IKK-independent manner. Alternatively, the small amount of endogenous IKK might have been sufficient to promote the FWD1-IB interaction. Immunoblot analysis indeed revealed the presence of endogenous IKK in 293T cells (data not shown). We investigated these two possibilities by introducing a kinase-negative mutant of IKK (K44M) into 293T cells to inhibit the endogenous IKK activity. This mutant markedly inhibited the interaction between FWD1 and IB as well as FWD1-induced ubiquitination of IB in a dose-dependent manner (Fig. 5B), suggesting that endogenous IKK is required for both these events. Thus, whereas signal-induced activation of the IKK complex is required for the proteolysis of IB and IB, IB appears to be constitutively degraded as a result of the low level of endogenous IKK activity.

FWD1-induced Degradation of IB, IB, and IB-- To investigate further whether the phosphorylation-induced interaction with FWD1 promotes the degradation of IB, IB, and IB, we performed pulse-chase experiments (Fig. 6). In the presence of FWD1 and IKK, both IB and IB were rapidly degraded, although the kinetics of IB degradation were slightly slower that those of IB degradation. The rates of degradation of the IB(S32A/S36A) and IB(S19A/S23A) mutants were markedly reduced relative to those of the corresponding wild-type proteins, indicating that FWD1 promotes degradation of both IB and IB after specific phosphorylation of the DSGXS motif. The half-life of IB was longer than those of IB and IB, as previously demonstrated (17). The IB(SA-all) mutant was more stable than was wild-type IB, consistent with our observation that the extent of ubiquitination of the mutant was markedly reduced compared with that of the wild-type protein (Fig. 5A). Thus, phosphorylation of serine residues present within SXXXS motifs in its NH₂-terminal region appears to influence the stability of IB.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   FWD1-induced degradation of phosphorylated IB, IB, and IB. 293T cells transfected with vectors encoding the indicated Myc-tagged IB proteins, FLAG-IKK, and FLAG-FWD1 were pulse-labeled with [35S]methionine and [35S]cysteine, and then incubated in the absence of isotope for the indicated chase periods. Cell lysates were then subjected to immunoprecipitation with antibodies to Myc, and the resulting precipitates were subjected to SDS-PAGE, autoradiography, and scanning densitometry. The amount of each IB protein is expressed as a percentage of that present at the beginning of the chase period. , IB; , IB(S32A/S36A); , IB; , IB(S19A/ S23A); , IB; , IB(SA-all).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We and others (40-48) previously showed that FWD1 mediates the ubiquitination of IB and -catenin by functioning as an intracellular receptor that links these substrates to the core complex of the SCF E3 ubiquitin ligase. Human FWD1 was also shown to interact with the Vpu protein of human immunodeficiency virus-type 1 (49). IB, IB, IB, -catenin, and Vpu all share a DSGXS motif (Fig. 1B), the two serines in which undergo signal-induced phosphorylation. This shared property suggested that FWD1 might recognize the phosphorylated DSGXS motif in each of these proteins, as we showed was the case for IB. The phosphorylated DSGXS motif thus might constitute a signal for FWD1-mediated ubiquitination. In the present study, we therefore investigated whether FWD1 also mediates the ubiquitination of IB and IB. Indeed, our data show that FWD1 also serves to link these two proteins to the core complex of the SCF ubiquitin ligase in a phosphorylation-dependent manner.

The structural organization of IB differs from that of IB and IB (4). Whereas IB and IB contain a PEST domain in the COOH-terminal region, IB contains a putative PEST domain in the NH₂-terminal region, upstream of the ankyrin repeats (Fig. 1A) (17). The DSGXS motif is present in the NH₂-terminal regions of all three proteins; however, IB also contains four additional SXXXS motifs clustered in this region (Fig. 1C). Although FWD1 interacts with IB, IB, and IB, the mode of interaction with IB and IB appears to differ from that for IB. Thus, mutation of the two serines in the DSGXS motifs of IB and IB to alanines prevented both the interaction of these proteins with FWD1 and their FWD1-mediated ubiquitination as well as increased their stability. These data indicate the absolute requirement for the DSGXS motif in the interaction of IB or IB with FWD1. In contrast, mutation of the two serines in the DSGXS motif of IB did not prevent its binding to FWD1. Furthermore, the IB(SA-all) mutant, in which the serines in all five SXXXS motifs were replaced by alanines, also interacted with FWD1, although the extent of its ubiquitination was substantially reduced compared with that of the wild-type protein. Hence, FWD1 appears to recognize motifs other than SXXXS in IB. Together, our data indicate that FWD1 is important for the ubiquitination of these three IB proteins, although the manner of association appears to differ between IB and IB on the one hand and IB on the other hand.

Another difference between IB and IB versus IB is that although FWD1 binding to and efficient ubiquitination of IB and IB in 293T cells required introduction of both FWD1 and IKK, IB interacted with FWD1 and underwent ubiquitination in the presence of exogenous FWD1 alone (without introducing exogenous IKK). Our observation that a kinase-negative mutant of IKK (K44M) inhibited both the interaction of IB with FWD1 and its ubiquitination suggests that endogenous IKK activity was necessary and sufficient for these reactions to proceed. However, we also showed that IKK phosphorylated IB specifically on Ser-18 and Ser-22 in transfected 293T cells (data not shown). This result appears inconsistent with the observation that the IB(S18A/S22A) mutant retained the ability to undergo ubiquitination, but it might be explained by IKK phosphorylation of other kinases or modulators that promote the interaction of FWD1 with IB.

Although IB, IB, and IB all appear to undergo FWD1-mediated ubiquitination, and at least IB and IB seem to be phosphorylated in a similar manner, the three proteins exhibited differential stabilities. It is possible that factors other than phosphorylation of the DSGXS motif affect the interaction of FWD1 with the IB proteins. Simeonidis et al. (7) showed that the structural differences in the NH₂-terminal regions of IB, IB, and IB may contribute to the differential stabilities of the three proteins. The COOH-terminal structures, which are thought to control basal turnover, also differ among the three proteins (6, 8, 9). Thus, regulatory mechanisms other than the FWD1-dependent pathway also may contribute to control of the stability of IB proteins (50). Although the underlying mechanisms responsible for the differential down-regulation of IB proteins remain to be fully characterized, it is clear that FWD1 is an important regulator of NF-B signaling.

	ACKNOWLEDGEMENTS

We thank Drs. H. Nakano, K. Okumura, and S. Tanaka for the plasmids and cell lines used in this study; S. Matsushita, K. Shimoharada, N. Nishimura, R. Yasukochi and other laboratory members for technical assistance; and M. Kimura for secretarial assistance.

	FOOTNOTES

^* This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan. Tel.: 81-92-642-6815; Fax.: 81-92-642-6819; E-mail: nakayak1@bioreg.kyushu-u.ac.jp.

	ABBREVIATIONS

The abbreviations used are: NF-B, nuclear factor-B; IKK, IB kinase; PAGE, polyacrylamide gel electrophoresis; E3, ubiquitin ligase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES