Inducible Degradation of Ikappa Balpha by the Proteasome Requires Interaction with the F-box Protein h-beta TrCP*

Mathias KrollDagger §, Florence Margottin, Alain Kohlparallel , Patricia RenardDagger , Hervé Durand, Jean-Paul Concordet**, Françoise BachelerieDagger , Fernando Arenzana-SeisdedosDagger , and Richard BenarousDagger Dagger

From the Dagger  Unité d'Immunologie Virale and parallel  Groupe des Bunyaviridés, Unité des arbovirus et virus des fièvres hémorragiques, Institut Pasteur, 25 et 28, rue du Dr. Roux, 75724 Paris Cedex 15, France and the  Institut Cochin de Génétique Moléculaire (ICGM) INSERM CJF9703 and ** ICGM U129 INSERM, Université Paris V, 75014 Paris, France

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Activation of NF-kappa B transcription factors requires phosphorylation and ubiquitin-proteasome-dependent degradation of Ikappa B proteins. We provide evidence that a human F-box protein, h-beta TrCP, a component of Skp1-Cullin-F-box protein (SCF) complexes, a new class of E3 ubiquitin ligases, is essential for inducible degradation of Ikappa Balpha . beta TrCP associates with Ser32-Ser36 phosphorylated, but not with unmodified Ikappa Balpha or Ser32-Ser36 phosphorylation-deficient mutants. Expression of a F-box-deleted beta TrCP inhibits Ikappa Balpha degradation, promotes accumulation of phosphorylated Ser32-Ser36 Ikappa Balpha , and prevents NF-kappa B-dependent transcription. Our findings indicate that beta TrCP is the adaptor protein required for Ikappa Balpha recognition by the SCFbeta TrCP E3 complex that ubiquitinates Ikappa Balpha and makes it a substrate for the proteasome.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES

NF-kappa B transcription factor is regulated by Ikappa B proteins of which Ikappa Balpha is the main and best characterized member (1-3). Proteasome-mediated degradation of Ikappa Balpha releases NF-kappa B and allows its localization in the nucleus (4-9). Phosphorylation of Ser32-Ser36 residues and subsequent ubiquitination of Ikappa Balpha are prerequisites to make the protein susceptible to proteasome attack (10-13). While the kinase complex accounting for Ikappa Balpha phosphorylation has been recently characterized (14-20), factors necessary for ubiquitination and targeting of Ikappa Balpha to the proteasome remain unknown. Covalent attachment of polyubiquitin to substrate proteins involved a cascade of ubiquitin transfer reactions with E1,1 a ubiquitin-activating enzyme, and E2 a ubiquitin-conjugating enzyme that operates in conjunction with a specificity factor E3 (21-26). It has been suggested that E3 functions in substrate recognition and E2 positioning. Although E2 enzymes belonging to the Ubc4/Ubc5 family can ubiquitinate Ikappa Balpha in vitro, the E3 responsible for the signal-induced ubiquitination of Ikappa Balpha remains to be identified (10, 27). A novel class of E3 is represented by the Skp1-Cullin-F-box protein complexes (SCFs) (28-33). The core component of these newly identified E3s is Skp1, which assembles with different F-box proteins and has been shown in human cells to interact selectively with CUL-1, but not with other Cullin proteins belonging to the Cdc53 family (34-36). The role of the F-box proteins in these SCF complexes is to recruit phosphorylated substrate proteins to trigger their ubiquitination (28-33). Like the other members of the F-box protein family, human beta TrCP, which we recently identified (37), has a modular organization with an F-box motif involved in proteasome targeting through interaction with Skp1, and a seven WD repeats binding domain for interaction with substrate proteins (see Fig. 1A). We hypothesized that beta TrCP could be the F-box adaptor protein allowing recruitment of Ikappa Balpha by a SCF E3 ubiquitin-protein ligase complex that ubiquitinates Ikappa Balpha and makes it a substrate for degradation by the proteasome.

    EXPERIMENTAL PROCEDURES

Cell Lines, Transfections, and Infections-- Subconfluent cells of the 293 human embryo kidney cell line were transfected by LipofectAMINETM Plus (Life Technologies, Inc.) with the indicated reporter plasmids and pcDNA3 vectors expressing beta TrCP proteins or with no beta TrCP insert. Fluorigenic substrate luciferin served to quantify luciferase reporter gene expression in cytoplasmic extracts obtained by lysis in phosphate buffer containing 1% Nonidet P-40. The pcDNA3-beta TrCP or -beta TrCPDelta F constructs are described in Ref. 37. beta TrCP and beta TrCPDelta F coding sequences were amplified by polymerase chain reaction and inserted in fusion with the Myc/His double tag in the pcDNA3.1 Myc/HisA vector (Invitrogen). SV5-tagged wild type or SV5-tagged S32A/S36A phosphorylation-deficient mutant Ikappa Balpha are described in Ref. 11, 3Enh-kappa B-ConA and ConA luciferase reporter plasmids are described in Ref. 38. RSV luciferase reporter plasmid was purchased from Invitrogen. beta TrCP or Ikappa Balpha were inserted in fusion with the LexA DNA binding domain and the Gal4 activation domain, respectively, as described in Ref. 37. Construction and use of recombinant SFV was carried out as described before (39-42). Briefly, 293 cells were infected at a multiplicity of 5 for 6 h in serum-free medium with SFV particles carrying myc-tagged beta TrCP proteins. Expression of beta TrCP proteins and viral nucleocapsid protein was verified by Western blot analysis, and 100% infection efficiency was confirmed by immunofluorescence (data not shown). The same results as those shown in Fig. 2B were obtained in HeLa cells infected with SFV.

Antibodies and Reagents-- The 10B monoclonal antibody directed against the amino terminus of Ikappa Balpha was described in Ref. 43; the polyclonal antibody specifically recognizing Ikappa Balpha phosphorylated at serine residue 32 is from New England Biolabs (9241S), and the monoclonal anti-myc antibody was from Santa Cruz Biotechnology (hybridoma 9E10, SC-40). For co-immunoprecipitation experiments (Fig. 3, A and B), 200 µg of cytoplasmic extract were incubated with anti-myc-agarose conjugates (SC-40 AC from Santa Cruz) for 60 min at 4 °C. Precipitated beads were washed 10 times in phosphate-buffered saline containing 1% Nonidet P-40 and both protease and phosphatase inhibitors. Antibody-antigen complexes were disrupted by boiling in gel loading buffer (Pierce). Precipitated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes.

Electrophoretic Mobility Shift Assay-- The electrophoretic mobility shift assay was performed with 4 µg of nuclear extract incubated for 15 min at room temperature with a [gamma -32P]ATP-labeled, double-stranded oligonucleotide containing the HIV-1 long terminal repeat binding site for NF-kappa B (5'-ACAAGGGACTTTCCGCTGGGACTTTCCAGGGA-3'). Samples were analyzed in nondenaturing 6% polyacrylamide gels. Competition experiments were performed by adding a 40-fold molar excess of homologous, unlabeled oligonucleotide to each sample prior to addition of the radiolabeled probe.

    RESULTS AND DISCUSSION

To investigate the putative role of beta TrCP in the regulation of NF-kappa B activation, we first assessed the effect of wild type (beta TrCP) and a F-box deleted beta TrCP (beta TrCPDelta F) (Fig. 1A) on the transcriptional activity of a NF-kappa B-dependent (3Enh-kappa B-ConA) promoter driving a luciferase reporter gene (10, 11). Expression of beta TrCP resulted in a 2- to 3-fold increase in the activity of the 3Enh-kappa B-ConA promoter in cells of the 293 human embryo kidney cell line stimulated by either tumor necrosis factor (TNF) or okadaic acid (OKA), two well characterized inducers of NF-kappa B (Fig. 1B, left panel). Similarly, the low level of constitutive activation of NF-kappa B found in unstimulated cells (compare 3Enh-kappa B-ConA to ConA) is also enhanced by expression of beta TrCP (Fig. 1B, left panel). In sharp contrast to beta TrCP, expression of the beta TrCPDelta F mutant massively and consistently inhibited NF-kappa B-dependent transcription by more than 90% of the levels induced in TNF- or OKA-stimulated cells transfected with an insertless, control plasmid (pcDNA3) (Fig. 1B, left panel). Importantly, expression of the transdominant negative mutant beta TrCPDelta F fully prevented localization of NF-kappa B to the nucleus upon cell activation. This finding is in keeping with an increased stability of inhibitor Ikappa B proteins that anchor NF-kappa B in the cytoplasm in an inactive form. The failure of beta TrCPDelta F to modify the activity of a RSV promoter indicates that beta TrCPDelta F does not affect NF-kappa B independent mechanisms of transcription (Fig. 1B, left panel).


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Fig. 1.   beta TrCP is required for cell activation-induced NF-kappa B transcription. A, physical map of beta TrCP showing the F-box responsible for proteasome targeting through Skp1 binding and the seven WD40 repeats. The F-box deletion in the mutant beta TrCPDelta F is indicated. B, regulation of NF-kappa B-dependent transcription by beta TrCP or beta TrCPDelta F. Human 293 embryo kidney cells (2 × 106 cells) were transiently co-transfected with NF-kappa B-dependent (3Enh-kappa B-ConA luc) or -independent (ConA luc, RSV luc) DNA luciferase expression vectors (1 µg) and CMV-IE-promoter-driven (pcDNA3) vectors (3 µg) expressing either wild type (beta TrCP) or mutant (beta TrCPDelta F) forms of beta TrCP (10, 11). Equal amounts of the pcDNA3 plasmid without beta TrCP cDNA insert were transfected as a control. The amount of wild type and mutant beta TrCP proteins was assessed by Western blot analysis of cytoplasmic extracts to ensure that wild type and mutant beta TrCP proteins were expressed at comparable levels (not shown). After transfection, cells were left untreated (NS) or were stimulated with either 5 ng/ml TNF or 75 ng/ml OKA for 6 h. Experiments were repeated three times in 293 cells and confirmed in HeLa cells. Results of a representative experiment are shown. Luciferase activity is expressed as relative luciferase units (RLU) per µg of protein and results from subtracting the background signal from the values obtained for each sample. Comparative electrophoretic mobility shift assay of nuclear extracts from HeLa cells expressing either beta TrCP or the beta TrCPDelta F from SFV. A radiolabeled oligonucleotide encoding the NF-kappa B consensus was used as a probe to bind transcription factors. A SFV replicon without insert was used as a control. The asterisk indicates where competitor cold oligonucleotide was added to demonstrate the specificity of NF-kappa B/DNA interaction.

Thus, on the one hand, the capacity of beta TrCP to enhance both basal or signal-induced activation of NF-kappa B and, on the other hand, the specific inhibition of NF-kappa B dependent transcription promoted by beta TrCPDelta F, strongly suggest that beta TrCP is an essential component of the NF-kappa B activation pathway. In support of this assumption, we found that, concomitant with the enhancement of NF-kappa B-dependent transcription, overexpression of beta TrCP in 293 cells stimulated by TNF induced an accelerated degradation of Ikappa Balpha (Fig. 2A, middle panel). In contrast, expression of the beta TrCPDelta F mutant either from a eukaryotic vector (Fig. 2A, right panel) or a SFV replicon (Fig. 2B, right panel), stabilized Ikappa Balpha and delayed the kinetics of Ikappa Balpha degradation. Moreover, the presence of beta TrCPDelta F promoted the accumulation of slow migrating forms of Ikappa Balpha characteristic of the phosphorylation of residues Ser32 and Ser36 required for subsequent ubiquitination and degradation of Ikappa Balpha by the proteasome (Fig. 2, A and B, right panel). Thus, these findings exclude an inhibitory effect of beta TrCPDelta F in the transduction pathway leading to phosphorylation of Ikappa Balpha and suggest that stabilization of Ikappa Balpha is due to the blockade of a post-phosphorylation event in the metabolism of the inhibitor.


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Fig. 2.   Expression of the beta TrCPDelta F mutant inhibits degradation and promotes accumulation of Ser32-Ser36 phosphorylated Ikappa Balpha . A, stability of Ikappa Balpha analyzed in 293 cells exposed to TNF for different lengths of time in the presence of beta TrCP proteins. 293 cells were transiently transfected with beta TrCP, beta TrCPDelta F, or control pcDNA vectors. After 36 h, cells were stimulated with TNF in the presence of 100 µg/ml cycloheximide, the protein synthesis inhibitor. Cytoplasmic proteins were separated in SDS-denaturing polyacrylamide gels, transferred onto nitrocellulose membrane, and probed with the 10B monoclonal antibody directed against the amino terminus of Ikappa Balpha (top panel), a polyclonal antibody specifically recognizing Ikappa Balpha phosphorylated at serine residue 32 (alpha -Ikappa Balpha -S32-P antibody, middle panel), or a monoclonal anti-myc antibody (bottom panel) directed against the carboxyl-terminal myc tag of heterologous beta TrCP proteins (see Ref. 13 for antibodies). Immunodetection was performed using an ECL chemiluminescence kit (Amersham Pharmacia Biotech). B, stability of Ikappa Balpha in TNF-treated HeLa cells infected with Semliki forest virus replicons (SFV) (12) expressing the beta TrCPDelta F mutant (beta TrCPDelta F) or not (SFV control). After 6 h of infection, cells were treated with cycloheximide and stimulated with TNF for the indicated times. Cytoplasmic extracts were processed as described above and probed with the 10B antibody. C, specific recognition of Ser32-Ser36 phosphorylated Ikappa Balpha by the alpha -Ikappa Balpha -S32-P antibody (13). Western blot analysis of recombinant (rIkappa Balpha , 10 ng) and cytoplasmic Ikappa Balpha obtained from HeLa untreated (-) or treated with TNF for 15 min. Left, detection of Ikappa Balpha proteins with 10B antibody. Right, detection with the alpha -Ikappa Balpha -S32-P antibody.

Slow migrating forms of Ikappa Balpha predominated following induction with TNF but could even be detected in unstimulated cells (Fig. 2, A and B, right panel, time 0). This latter phenomenon likely reflects the inhibitory effect of beta TrCPDelta F on basal breakdown of Ikappa Balpha and is in keeping with the low and constitutive NF-kappa B-dependent transcription observed in 293 cells (Fig. 1B, left panel).

Characterization of the slow migrating band of Ikappa Balpha (Fig. 2A, right top panel), as a phosphorylated form of Ikappa Balpha accumulated in beta TrCPDelta F expressing cells, was accomplished (Fig. 2A, middle row of panels) using a polyclonal antibody that does not recognize the unphosphorylated form of Ikappa Balpha but specifically reacts with Ser32-Ser36-phosphorylated Ikappa Balpha which accumulates in the presence of the proteasome inhibitor Z-LLL-H following induction with TNF (Fig. 2C) (44).

To ascertain whether the regulatory effect of beta TrCP on Ikappa Balpha metabolism requires association with Ikappa Balpha , carboxyl-terminal c-myc-tagged variants of either beta TrCP or beta TrCPDelta F were expressed in HeLa cells from SFV (Fig. 3A) or in 293 cells from eukaryotic expression vectors (Fig. 3B), and their co-precipitation with Ikappa Balpha was investigated. Cytoplasmic extracts of cells treated or not with TNF and the proteasome inhibitor Z-LLL-H, were incubated with an anti-myc tag antibody bound to protein A-coated agarose beads. Proteins precipitated by the anti-myc antibody were probed with anti-Ikappa Balpha antibodies (Fig. 3A, middle and bottom panels). In cells expressing either the wild type or the mutated counterpart of beta TrCP, a single band of co-precipitated Ikappa Balpha was recognized by a monoclonal antibody that detects both native and phosphorylated forms of Ikappa Balpha (Fig. 3A, bottom panel, lanes 4-6). This band migrates with a pattern characteristic of the typical upshift induced by phosphorylation of Ikappa Balpha Ser32 and Ser36. Western blot analysis using the antibody specifically recognizing the phosphoserine Ser32-Ser36 Ikappa Balpha confirmed that the proteins detected are phosphorylated at the critical residues that permit subsequent ubiquitination of Ikappa Balpha (Fig. 3A, middle panel, lanes 4-6). No unphosphorylated form of Ikappa Balpha was detected in immunoprecipitates (Fig. 3A, lower panel). Detection of phosphorylated Ikappa Balpha did not require TNF induction when the mutant beta TrCPDelta F was expressed (Fig. 3A, middle panel, lane 5). Moreover, larger amounts of endogenous phosphorylated Ikappa Balpha co-precipitated with beta TrCPDelta F, compared with wild type beta TrCP (Fig. 3A, compare lanes 6 with lane 4), confirming that the beta TrCPDelta F mutant acts as a transdominant negative regulator of both constitutive and TNF-induced proteolysis of Ikappa Balpha .


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Fig. 3.   beta TrCP interacts with Ser32-Ser36 phosphorylated but not with unmodified or phosphorylation-deficient S32A/S36A Ikappa Balpha . A, beta TrCP selectively co-precipitates phosphorylated Ikappa Balpha proteins. HeLa cells were infected as described in Fig. 2B with SFV expressing a beta -galactosidase (beta Gal), wild type (beta TrCP), or Delta F (beta TrCPDelta F) myc-tagged proteins (11). Before harvesting, cells were exposed to TNF for 15 min in the presence of proteasome inhibitor (Z-LLL-H + TNF) or left untreated (-). Proteins from total cytoplasmic lysates were either separated in denaturing gels and probed with alpha -Ikappa Balpha -S32-P antibody (top panel) or immunoprecipitated (IP alpha -Myc) with myc antibody-agarose conjugates (13). Precipitated proteins were probed with either the alpha -Ikappa Balpha -S32-P antibody (middle panel) or the 10B monoclonal antibody (alpha -Ikappa Balpha , bottom panel) that recognizes both unmodified and phosphorylated Ikappa Balpha (13). The migration pattern of anti-myc-precipitated (IP alpha -Myc) Ikappa Balpha is compared with that of Ikappa Balpha in total cytoplasmic extracts (control HeLa cell extracts, purchased from New England Biolabs) of untreated (-) or TNF-treated (+) HeLa cells detected by either alpha -Ikappa Balpha -S32-P or alpha -Ikappa Balpha antibodies (right, panel A). B, beta TrCP proteins fail to co-precipitate with a S32A/S36A phosphorylation-deficient Ikappa Balpha mutant. 293 cells were co-transfected with pcDNA3-beta TrCP or -beta TrCPDelta F DNA vectors and either SV5-tagged wild type (lanes 1-6) or SV5-tagged S32A/S36A phosphorylation-deficient mutant Ikappa Balpha proteins (lanes 7-10) deficient in cell activation-induced serine phosphorylation (11). A pcDNA3 plasmid expressing beta -galactosidase was used as a control (lanes 1 and 2). Cytoplasmic proteins were precipitated by anti-myc antibodies and probed (WB) with alpha -Ikappa Balpha -S32-P or alpha -Ikappa Balpha antibodies indicated under the gels. Migration of either tagged (SV5-Ikappa Balpha ) or endogenous (End-Ikappa Balpha ) Ikappa Balpha is indicated. Long and short exposures using the ECL kit are shown. C, interaction of beta TrCP with wild type but not with the phosphorylation-deficient S32A/S36A Ikappa Balpha in the two-hybrid system. beta TrCP proteins or control Ras protein were fused to the Escherichia coli LexA binding domain. Ikappa Balpha proteins or control Raf protein were fused to the Gal4 activation domain. The yeast reporter strain L40 expressing the indicated hybrid protein pairs was analyzed for histidine auxotrophy and beta -galactosidase expression. D, wild type Ikappa Balpha is constitutively phosphorylated in yeast. L40 strain yeast colonies expressing the Gal4 activation domain fused to Raf, Ikappa Balpha wild type, or Ikappa Balpha S32A/S36A were lysed, and protein extracts were blotted onto nitrocellulose. Left, detection with alpha -Ikappa Balpha antibody. Right, detection with alpha -Ikappa Balpha -S32-P antibody.

To confirm the selective association of phosphorylated Ikappa Balpha and beta TrCP, we performed experiments using a S32A/S36A mutant of Ikappa Balpha lacking the capacity to be phosphorylated by cell activation signals promoting NF-kappa B activation (3). The presence of a 15-amino acid SV5 carboxyl terminus tag allows distinction of endogenous from transiently expressed wild type or S32A/S36A Ikappa Balpha . Despite expression of similar amounts of wild type or S32A/S36A-SV5 tagged proteins (data not shown), only the endogenous and wild type SV5-tagged Ikappa Balpha (Fig. 3B, lanes 4-6), but not the SV5-S32A/S36A phosphorylation-deficient mutant (Fig. 3B, lanes 7-10) were able to associate with either beta TrCP or beta TrCPDelta F. Expression of either wild type or phosphorylation-deficient tagged Ikappa Balpha proteins from SFV was consistently detected in more than 90% of cells (data not shown). The high infection efficiency of this system allows us to conclude that co-precipitation of the endogenous, but not the S32A/S36A-SV5 Ikappa Balpha (Fig. 3B, lanes 7-10), reflects the incapacity of the phosphorylation-deficient mutant to compete for binding to Ikappa Balpha .

Further evidence of Ikappa Balpha interaction with beta TrCP was provided by the yeast two-hybrid system (carried out as described previously (37)). We observed that beta TrCP fused to the LexA DNA binding domain (LexA-beta TrCP) associates specifically with Ikappa Balpha fused to the Gal4 activation domain (Gal4AD-Ikappa Balpha ), as detected by histidine auxotrophy or beta -galactosidase expression (Fig. 3C). The interaction between Ikappa Balpha and beta TrCP is likely accounted for by the existence in yeast of phosphorylated Ikappa Balpha as shown by the recognition of the Gal4AD-Ikappa Balpha hybrid by the antibody that specifically reacts with Ser32-Ser36 phosphorylated Ikappa Balpha (Fig. 3D). This hypothesis is reinforced by the fact that the Gal4AD-Ikappa Balpha S32A/S36A mutant, which is not recognized by the anti-phosphoserine Ikappa Balpha antibody (Fig. 3D), did not associate with LexA-beta TrCP (Fig. 3C).

We have previously documented that beta TrCP is a component of a SCF complex involved in HIV-1 Vpu-mediated CD4 degradation. The WD domain of beta TrCP is responsible for the interaction with Vpu (37). Although beta TrCP is involved in both Vpu-mediated CD4 and Ikappa Balpha proteolysis, it should be stressed that important differences between the two degradation pathways exist. Indeed, if both Vpu and Ikappa Balpha can be phosphorylated at serine in DSGXXS motifs, phosphorylation of Vpu occurs constitutively (45-46) while that of Ikappa Balpha requires activation of cell signaling. Furthermore, and in contrast to Ikappa Balpha , Vpu has not yet been characterized as a substrate for ubiquitination or degradation by the proteasome. No human protein recognized as ubiquitination substrate by an SCF complex and undergoing degradation by the proteasome has been documented so far. Ikappa Balpha phosphorylated at critical serine residues represents the first example of this kind of substrate (8).

In the SCF complexes with E3-ubiquitin-protein ligase activity, the F-box component allows specific recognition of substrates (28-33). The existence of a large number of F-box-containing proteins revealed by genome sequencing and the combinatorial interactions of SCF components that belong to different protein families (Cullin, E2 ubiquitin-protein conjugating enzymes) suggest that the growing family of E3 ligases is composed of a large number of different SCF complexes. This diversity has likely hampered the identification of the E3 ligase responsible for ubiquitin conjugation of Ikappa Balpha required for Ikappa Balpha degradation and NF-kappa B activation. While precise characterization of the ubiquitin conjugating activity associated with SCFbeta TrCP is still missing, our findings provide evidence that beta TrCP is ultimately responsible for recognition of phosphorylated Ikappa Balpha by the SCF complex.

As shown for other F-box proteins in yeast, beta TrCP could target different substrates as well as Ikappa Balpha to the proteasome. This hypothesis is sustained by the recent discovery that, Slimb, the Drosophila homolog of beta TrCP, may be involved in an as yet unidentified step of regulation of the wingless and hedgehog pathways (47). However, it cannot be assumed from this that beta TrCP is a broad, universal adaptor for ubiquitinated substrates. Indeed, in the SCF complex, F-box proteins determine and restrict substrate recognition by the proteasome. Thus, the yeast F-box protein Cdc4 is able to selectively bind phosphorylated Sic1, but not phosphorylated Cln1 or Cln2, whereas Grr1, another yeast F-box protein, shows selective association with the latter substrates but not with Sic1-(30-32).

In conclusion, our findings characterize beta TrCP as a F-box protein, which selectively associates with Ser32-Ser36 phosphorylated, but not unmodified, Ikappa Balpha . beta TrCP represents the SCF adaptor which ultimately accounts for recognition of phosphorylated Ikappa Balpha by the ubiquitination machinery and allows targeting of the ubiquitinated inhibitor to the proteasome (see model in Fig. 4). While this manuscript was completed and sent for review, a report by Yaron et al. (48) was published that supports the involvement of beta TrCP in Ikappa Balpha degradation and NF-kappa B activation. Thus, beta TrCP can be considered as a new target for pharmacological intervention in the physiopathological processes regulated by NF-kappa B.


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Fig. 4.   A model for the role of beta TrCP in the proteolysis of Ikappa Balpha and NF-kappa B activation. Upon cell activation and Ikappa Balpha phosphorylation, beta TrCP interacts with Ser32-Ser36 phosphorylated Ikappa Balpha but not with unmodified Ikappa Balpha . In the SCF-beta TrCP complex, beta TrCP allows recognition of Ikappa Balpha and triggers the E3-dependent ubiquitination that ultimately marks Ikappa Balpha for the proteolytic attack by the 26 S proteasome. Degradation of Ikappa Balpha releases the transcription factor NF-kappa B, which localizes to the nucleus.


    ACKNOWLEDGEMENTS

We thank Michèle Bouloy and Agnès Billecocq of the Bunyaviridés research group at Institut Pasteur for their generous support to this project. S. Michelson is acknowledged for critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by the European Union Concerted Action BIOMED II (ROCIO II project), Agence Nationale de Recherche sur le SIDA, SIDACTION, and Association pour la Recherche sur le Cancer (France).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.

§ Supported by a fellowship of the German Academic Exchange Service (Program HSP III).

Dagger Dagger To whom correspondence should be addressed. Tel.: 33-1-44-41-25-65; E-mail: benarous{at}cochin.inserm.fr.

    ABBREVIATIONS

The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; SCF, Skp1-Cullin-F-box protein; ConA, concanavalin A; RSV, Rous sarcoma virus; SFV, Semliki forest virus; TNF, tumor necrosis factor; OKA, okadaic acid.

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