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Originally published In Press as doi:10.1074/jbc.M312301200 on January 5, 2004

J. Biol. Chem., Vol. 279, Issue 12, 11074-11080, March 19, 2004
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Stability of Homologue of Slimb F-box Protein Is Regulated by Availability of Its Substrate*

Ying Li{ddagger}, Stefan Gazdoiu§, Zhen-Qiang Pan§, and Serge Y. Fuchs{ddagger}

From the {ddagger}Department of Animal Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and §The Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, November 10, 2003 , and in revised form, December 31, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The homologue of Slimb (HOS) F-box protein is a receptor of the Skp1-Cullin1-F-box protein (SCFHOS) E3 ubiquitin ligase, which mediates ubiquitination and degradation of {beta}-catenin and the inhibitor of NF{kappa}B, I{kappa}B. We found that HOS itself is an unstable protein that undergoes ubiquitination and degradation in a 26 S proteasome-dependent manner. A HOS mutant lacking the F-box that is deficient in binding to the core SCF components underwent ubiquitination less efficiently and was more stable than the wild type protein. Furthermore, ubiquitination and degradation of HOS was impaired in ts41 cells, in which the activities of Cullin-based ligases were decreased because the NEDD8 pathway was abrogated. Whereas HOS was directly ubiquitinated within the SCFHOS complex in vitro, the addition of phosphorylated I{kappa}B{alpha} inhibited this ubiquitination. Increasing cellular levels of HOS substrate (phosphorylated I{kappa}B{alpha}) by activating I{kappa}B kinase inhibited HOS ubiquitination and led to stabilization of HOS, indicating that interaction between HOS and its substrate might protect HOS from proteolysis. Taken together, our data suggest that proteolysis of HOS depends on its interaction with active components of the SCF complex and that HOS stability is regulated by a bound substrate. These findings may define a mechanism for maintaining activities of specific SCF complexes based on availability of a particular substrate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
E31 ubiquitin protein ligases determine specificity and timing of the ubiquitin-dependent proteolysis that plays a key role in regulating the activities of many proteins. Both recognition of a substrate and catalysis of ubiquitin transfer are the major functions of ubiquitin ligases that can be conferred by different domains of a single protein (e.g. Mdm2 and Nedd4) or different proteins within a complex enzyme (e.g. SCF, CBCVHL, APC (anaphase-promoting complex), etc.; for review, see Ref. 1).

The modular features of complex ubiquitin ligases offer the biological advantages of expanded substrate specificity and rapid switching between various substrates to satisfy cell demand for ligase activity toward a particular substrate. However, an important mechanism regulating disassembly of existing complexes and assembly of new ones must be in place to allow rapid switching. Ubiquitination and degradation of ligase receptors that actually recognize specific substrates may enable the core ligase components to recruit different types of receptors to mediate such a mechanism.

F-box proteins serve as receptors for the SCF (Skp1-Cul1-F-box protein) E3 ligases family, whose members ubiquitinate specifically phosphorylated substrates and play a key role in the regulation of the cell cycle, signal transduction and transcriptional activation (for review, see Ref. 2). F-box proteins contain WD40- or leucine-rich repeat domains that interact with phosphorylated substrates as well as an F-box motif, which is required for binding to Skp1 (3). Skp1 tethers F-box proteins to the core ligase complex consisting of Cdc53/Cullin1 and Roc1/Rbx1, which, in turn, recruits an E2 ubiquitin-conjugating enzyme and mediates substrate ubiquitination (for review, see Ref. 2).

Previous data show that F-box proteins undergo ubiquitination and 26S proteasome-dependent degradation in yeast and mammalian cells (4–8). Ubiquitination and degradation of F-box proteins as a mechanism underlying reassembly of SCF complexes that allows rapid switching of F-box protein-determined substrate specificity was shown to be essential for cell growth (4), although other mechanisms regulating SCF assembly are likely to exist (9).

In yeast, genetic evidence indicates that ubiquitination and degradation of F-box proteins requires all the core SCF components. Given that the integrity of the F-box motif is also essential for degradation of F-box proteins, it has been suggested that their ubiquitination occurs within the SCF complex in an autocatalytic manner (4, 5). This hypothesis has been confirmed by biochemical analysis of Cdc4p ubiquitination (10). Furthermore, it has been demonstrated that mammalian F-box protein Skp2 undergoes ubiquitination in vitro within the SCF complex (8). In addition, other E3 ligases (such as APC (anaphase-promoting complex) or other SCFs) have been shown to be involved in regulating degradation of the F-box proteins, including Tome1 and Emi1 (11–13).

Recent structural studies demonstrate that F-box proteins are positioned far apart from the core ligase components within the SCF E3 complexes, which exhibit a fairly rigid conformation with an ~59 Å distance between the substrate/F-box interacting determinants and E2 ubiquitin-conjugating enzyme recruited by the core ligase components (14–17). Such a distance between an F-box protein and E2 does not explain direct ubiquitination of an F-box protein within the SCF complex unless additional factors enabling ubiquitination are involved. Indeed, it has been recently proposed that a release of activated E2 from the core ligase domains toward the lysine residues within the substrates must occur (18). Whether in the absence of a substrate an F-box protein ubiquitination requires such release remains to be determined.

Two opposite models could be envisioned to address a major question regarding a potential role for substrate availability in regulating the F-box protein stability. One of these models favors co-ubiquitination of an F-box protein together with a bound substrate, predicting that, in the absence of substrate, stabilization of an F-box protein may occur. Another model proposes that a substrate would shield an F-box protein from ubiquitination by core components of the SCF ligase (2). Experimental evidence supporting either of these models has not been reported.

In this study we sought to investigate the role of SCF ligase and the availability of a substrate in regulating the stability of the HOS F-box protein. HOS (also termed {beta}-TrCP2/Fbw1b) serves as a receptor of the mammalian SCFHOS E3 ubiquitin ligase to mediate ubiquitination and degradation of phosphorylated I{kappa}B and {beta}-catenin and regulate NF-{kappa}B and {beta}-catenin-signaling pathways (19). Mechanisms determining the abundance of HOS play an important role in regulating these signaling cascades and in turn in modulating cell differentiation and malignant transformation (20, 21).

We show that the integrity of the F-box of HOS and the activity of Cullin-based ligases are essential for ubiquitination and degradation of HOS. HOS is ubiquitinated within the SCFHOS complex in vitro, and the addition of phosphorylated I{kappa}B inhibits this process. Moreover, stimuli that increase the levels of phosphorylated I{kappa}B in cells stabilize HOS and increase its levels. These data provide an insight into the potential contribution of mechanisms regulating HOS stability in satisfying the cellular demands for continuous activity of the SCF-HOS E3 ubiquitin ligase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs—The pCDNA3-based construct for expression of HA-tagged HOS was previously described (19). HOS cDNA was subcloned into pEF-HA vector (a gift from E. Spanopoulou), and PCR-mediated mutagenesis was used to delete the cDNA sequence encoding HOS F-box (amino acids 117–169). Vectors for mammalian expression of FLAG-tagged I{kappa}B{alpha} proteins (22), Cullin1, Skp1, and Roc1 (23), as well as constitutively active I{kappa}B kinase {beta} (IKK{beta}S177E,S181E) (24) and His- or HA-tagged ubiquitin (25) were described elsewhere. Plasmid for bacterial co-expression of Cullin 1 was prepared by PCR amplification from the pCDNA-FLAG-Cul1 template (with primers 5'-CATATGGATTACAAGGATGACGACGATAAGATGTCG-3' and 5'-CATATGTTAAGCCAAGTAACTGTAGGTGTCCTT-3'). The resulting PCR product was digested with NdeI and cloned into the pGEX4T3/pET15b-(GST-HA-Roc1) vector (26) via the pET15b-derived multiple cloning site, creating pGEX4T3/pET15b-(GST-HA-Roc1)-(FLAG-Cul1). To construct pET3a-Skp1, human Skp1 was amplified by PCR using pcDNA-hSkp1 vector as a template (with primers 5'-CGCCATATGCCTTCAATTAAGTTGCAGAG-3' and 5'-CGCGGATCCTCACTTCTCTTCACACCACTG-3'). The resulting PCR fragment was digested with NdeI and BamHI and cloned into the pET3a vector.

Tissue Culture and Transfections—Human 293T human embryo kidney cells and normal human fibroblasts (TIG, a gift from H. Tahara) were grown in Dulbecco's modified Eagle's medium in the presence of 10% fetal bovine serum and antibiotics at 37 °C and 5% CO2. Hamster CHO-K1 and ts41 cells (a gift from R. Neve) were grown in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, antibiotics, and Fungizone (0.625 µg/ml, Invitrogen) at 34 °C and 5% CO2. These cells were shifted to 40 °C 24 h before harvesting where indicated. Transfections were performed using the calcium phosphate procedure or LipofectAMINE Plus (Invitrogen) at 24–48 h before harvesting. For cell treatment, actinomycin D, sodium salicylate, and cycloheximide (Sigma) as well as neocarzinostatin (NCS, Kayaku Co. Tokyo, Japan) were purchased and used at the indicated concentrations.

Antibodies and Immunological Techniques—Antibodies against HA (Roche Applied Science), {alpha}-tubulin and FLAG M2 (Sigma), and I{kappa}B{alpha} (phosphoserine 32-specific, Santa Cruz) were purchased. Antibody against HOS (HOS-C) was previously described (27). Secondary antibodies conjugated with horseradish peroxidase (Amersham Biosciences) were purchased. Immunoprecipitation and immunoblotting analyses were described elsewhere (19). Degradation of HOS proteins was assessed by pulse-chase or CHX chase analyses. For pulse-chase analysis, 293T cells grown in 100-mm dishes and transfected with HOS-HA plasmid were starved in medium lacking methionines and cysteine (Invitrogen), metabolically labeled with a [35S]methionine/[35S]cysteine mixture (PerkinElmer Life Sciences), and chased with complete medium supplemented with unlabeled methionine and cysteine (2 mM). Cells from a 1/3 sector of each plate were harvested at each time point of the chase, lysed in radioimmune precipitation assay buffer (phosphate-buffered saline containing 0.1% of SDS, 1% of Triton X-100, and 0.5% of sodium deoxycholate and supplemented with inhibitors of proteases and phosphatases) and HOS proteins were immunoprecipitated with HA antibody, separated on SDS-PAGE, and analyzed by autoradiography. For CHX chase analysis, unlabeled cells from a 1/4 sector of each transfected plate were harvested at each time point after adding CHX (60 µg/ml) to the medium, lysed in radioimmune precipitation assay buffer, separated on SDS-PAGE, and analyzed by immunoblotting with HA antibody.

Ubiquitination Assays—In vivo ubiquitination assays of recombinant HOS were carried out as described elsewhere (25). For detection of ubiquitination of endogenous HOS proteins, cells were transfected with HA-tagged ubiquitin vector and treated with MG132 (40 µM for 1 h). Cells were harvested in a boiling solution of SDS (1% in Tris-buffered saline) and further disrupted by sonication. Lysates were diluted 10-fold with Triton X-100 solution (1% in Tris-buffered saline), incubated with protein A beads for 1 h, and centrifuged. Supernatants were analyzed using immunoprecipitation with HOS antibody and immunoblotting with HA and HOS antibodies. For an in vitro assay Skp1·FLAG-Cul1·GST-HA-Roc1 complex (SCR) was co-expressed in bacterial cells. To this end BL21pJY2 cells were transformed with both pGEX4T3/pET15b-(GST-HA-Roc1)-(FLAG-Cul1) and pET3a-Skp1. Freshly grown bacteria were induced by isopropyl-{beta}-D-thiogalactopyranoside, and the expressed proteins were co-purified by glutathione affinity columns (Amersham Biosciences) as described previously (26). Bacterial lysates (200 µl) yielded ~10 pmol of the SCR complex (as shown in Fig. 4A) by Coomassie staining. The SCR complex, derived from 50 µl of bacterial extracts and immobilized to glutathione beads, was assayed for ubiquitin ligase activity as previously described (28). Briefly, this complex was incubated with 32P-labeled ubiquitin (1 µg), E1 (20 pmol), E2 (Cdc34 or UbcH5c, 100 pmol), and ATP (2 mM) in the presence of ubiquitination buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM NaF, 10 nM okadaic acid) in a 20-µl total volume at 37 °C for 60 min. Formation of ubiquitin chains was analyzed by autoradiography. To prepare mammalian SCR plasmids for expression of FLAG-tagged Cullin1, Roc1, and Skp1 were transfected into 293T cells, and the entire complex was purified with immobilized FLAG antibody (M2, Sigma).



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  		FIG. 4.
Ubiquitination of HOS within the SCFHOS complex in vitro; an inhibitory effect of a substrate. A, characterization of bacterially expressed and purified core ligase complex containing GST-tagged Roc1, Skp1, and FLAG-tagged Cullin1 proteins (SCR). The purified complex was separated on SDS-PAGE and analyzed using Coomassie staining (left panel). The ability of this immobilized SCR complex to bind in vitro translated and [35S]methionine-labeled HOS (middle panel) and to conjugate 32P-labeled ubiquitin (Ub) into polyubiquitin chains (right panel) was analyzed by means of autoradiography. wt, wild type. B, in vitro ubiquitination of in vitro translated and 35S-labeled HOS pre-bound to an immobilized Skp1·FLAG·Cullin1/GST·Roc1 (SCR) complex and incubated with ATP and ubiquitin in the presence or absence of E1 and E2 (Cdc34 or UbcH5c) as indicated. An autoradiograph of 35S-labeled proteins separated on SDS-PAGE is depicted. C, in vitro ubiquitination of in vitro translated and 35S-labeled HOS pre-bound to an immobilized Skp1·FLAG·Cullin1·Roc1 complex (expressed in 293T cells and purified with FLAG antibody) and incubated with ATP and ubiquitin in the presence or absence of E1 and E2 (Cdc34) as well as GST-I{kappa}B{alpha} proteins in either phosphorylated (by IKK{beta}; PI{kappa}B) or non-phosphorylated (I{kappa}B) forms as indicated. An autoradiograph of 35S-labeled proteins separated on SDS-PAGE is depicted (upper panel). Aliquots of these reactions were also analyzed by immunoblotting (IB) with antibodies against GST (middle panel) to detect GST-I{kappa}B{alpha} proteins and FLAG (lower panel) to detect Cullin1.

 
For assessment of in vitro HOS ubiquitination, in vitro translated and [35S]methionine-labeled HOS was captured on immobilized SCR, washed with A-500 buffer (25 mM Tris-HCl, pH7.4, 0.5 M NaCl, 0.25% Nonidet P-40, 10% glycerol), re-equilibrated with ubiquitination buffer, and subjected to the ubiquitination reaction with recombinant E1 and E2 as indicated above. Phosphorylated by IKK{beta} (as described earlier (28)) or non-phosphorylated GST-I{kappa}B{alpha}, proteins (25 pmol) were added before the start of the reaction as indicated. Products of the reactions were separated on SDS-PAGE and analyzed by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HOS Is Ubiquitinated in Cells and Undergoes Proteasome-dependent Degradation—We sought to investigate whether HOS undergoes ubiquitination and degradation in cells. 293T cells were co-transfected with HA-tagged HOS and hexahistidine-tagged ubiquitin and harvested. Immunoblotting analysis of ubiquitinated proteins purified on nickel resins under denaturing conditions (25) revealed slower migrating HA-reactive species as a smear at the top of the gel. These species were not observed when either HA-HOS of his-ubiquitin plasmids were omitted from transfection (Fig. 1A). These data indicate that HOS is covalently conjugated with ubiquitin in vivo under these experimental conditions.



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  		FIG. 1.
HOS is ubiquitinated and degraded in a proteasome-dependent manner. A, immunoblotting analysis of HA-tagged HOS proteins from 293T cells (co-transfected with Ub-his vector as indicated) in whole cell extracts (w) or eluates of proteins purified on nickel nitrilotriacetic acid-agarose (n) was carried out with HA antibody. Positions of HOS-HA proteins and ubiquitinated (Ub) HOS-HA species are indicated. B, pulse-chase analysis of HA-HOS protein expressed in 293T cells, which were treated with a vehicle (Me2SO (DMSO)) or an inhibitor of 26 S proteasome (MG132, 40 µM) at the beginning of the chase. An autoradiograph of HA-HOS proteins immunoprecipitated with HA antibody and separated on SDS-PAGE is shown. M depicts material immunoprecipitated from mock-transfected and metabolically labeled cells. C, immunoblotting (IB) analysis of endogenous HOS immunoprecipitated (IP) from 293T cells transfected with HA-tagged ubiquitin and treated with MG132 (40 µM for 1 h) as indicated was carried out with HA (upper panel) or HOS (lower panel) antibody. Positions of HOS protein and ubiquitinated HOS species are indicated. Ig, heavy chain of immunoglobulins. Vec, vector.

 
Previous findings demonstrate that treatment of cells with inhibitors of 26 S proteasome results in accumulation of recombinant HOS (19). We assessed the effect of proteasome inhibition on the rate of HOS proteolysis by pulse-chase analysis. Recombinant HOS was used in these experiments because the low levels of HOS in cells precluded analyzing degradation of the endogenous protein. HOS rapidly degraded in 293T cells, and treating cells with proteasome inhibitors including MG132 (Fig. 1B) or lactacystin (data not shown) delayed this degradation.

We next sought to investigate the effects of proteasome inhibitors on the levels of endogenous HOS. We immunoprecipitated endogenous HOS under stringent conditions from cells transfected with HA-tagged ubiquitin and treated or not with MG132. Analysis of HOS immunoprecipitates revealed that treatment with MG132 led to accumulation of HOS levels (Fig. 1C, lower panel) as well as of levels of slowly migrating HA-reactive species, indicative of ubiquitinated endogenous HOS (Fig. 1C, upper panel). In all, these data suggest that HOS is an unstable protein that undergoes ubiquitination and proteasome-dependent proteolysis.

Integrity of the F-box Motif Is Required for Efficient Ubiquitination and Proteolysis of HOS—Ubiquitination and degradation of yeast F-box proteins depend on the integrity of their F-box motifs (4, 5). We found that deletion of the F-box in HOS noticeably decreases, but does not entirely abolish this ubiquitination (Fig. 2A). This result suggests that more than one mode of regulation of HOS ubiquitination (F-box-dependent and F-box-independent) may be operative. Cycloheximide chase analysis showed that degradation of wild type HOS protein (Fig. 2B) occurs at a rate comparable with that observed in 35S-labeling pulse-chase experiments (Fig. 1B). Under these conditions degradation of HOS{Delta}F mutant was substantially slower than that of HOSwt (Fig. 2B). These findings show that the efficiency of HOS ubiquitination and degradation depends on the integrity of the HOS F-box motif and suggest that, as in the case of yeast F-box proteins, the association of HOS with Skp1 within the SCF complex may play a role in regulating HOS stability.



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  		FIG. 2.
The F-box motif of HOS is required for efficient ubiquitination and degradation. A, immunoblotting analysis of HA-tagged HOS proteins from 293T cells (co-transfected with Ub-his vector as indicated) in whole cell extracts (lower panel) or eluates of ubiquitinated proteins purified on nickel nitrilotriacetic acid (Ni-NTA)-agarose (upper panel) was carried out with HA antibody. Positions of HOS-HA proteins and ubiquitinated HOS-HA species are indicated. B, CHX chase analysis of degradation of HA-HOS proteins (wild type and {Delta}F mutant) expressed in 293T cells. Levels of proteins at the indicated time points after adding CHX to cells (at 60 µg/ml) were analyzed by immunoblotting with HA antibody. NS, nonspecific band; M, material from mock-transfected cells.

 
HOS Degradation Depends on the Activity of the NEDD8 Pathway—The ability of SCF E3 ligases to ubiquitinate their substrates is enhanced by covalent modification of Cullin proteins with ubiquitin-like protein NEDD8 (26, 29–31). Conjugation of NEDD8 to Cullin proteins requires adequate activity of NEDD8-activating E1 enzyme. In temperature-sensitive hamster ts41 fibroblast cells, the APP-BP1 subunit of this enzyme is inactivated when cells are incubated at a non-permissive temperatures (40 °C) (32), enabling use of these cells as a convenient model for studies of Cullin-dependent protein degradation (33). When expressed in ts41 grown at permissive temperature (34 °C), HOS underwent rapid degradation as measured by CHX chase (Fig. 3A). However, incubation of ts41 cells at 40 °C noticeably delayed proteolysis of HOS.



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  		FIG. 3.
Degradation of HOS depends on NEDD8 pathway. A, Chinese hamster ovarian (CHO) and ts41 hamster cells grown at 34 °C were transfected with HA-HOS and 24 h later incubated at the indicated temperatures for another 24 h. Degradation of HOS was assessed using CHX, as described in the legend to Fig. 2B. B, hamster ts41 cells were transfected with a HA-ubiquitin construct as indicated and incubated at the indicated temperatures before treatment with MG132 (40 µM for 1 h) and harvesting. Immunoblotting (IB) analysis of endogenous HOS immunoprecipitated (IP) from these cells was carried out with HA (upper panel) or HOS (lower panel) antibody. Positions of HOS protein and ubiquitinated HOS species are indicated. Ig, heavy chain of immunoglobulins; NS, nonspecific band; M, material from mock-transfected cells.

 
To exclude the possibility that inhibition of HOS degradation is a general characteristic of hamster cells exposed to 40 °C, we carried out a similar experiment in Chinese hamster ovarian cells, which harbor wild type APP-BP1 (33). In this cell line, HOS degraded at 34 °C (although less efficiently than in ts41 cells, most likely because of the genetic differences between epithelial Chinese hamster ovarian cells and ts41 fibroblasts). However, HOS degradation was accelerated in Chinese hamster ovarian cells grown at 40 °C (Fig. 3A).

We further focused our analysis at the levels of endogenous HOS in ts41 cells. Experiments with immunoprecipitation of HOS from ts41 cells transfected with HA-tagged ubiquitin revealed that incubation of cells at 40 °C results in accumulation of endogenous HOS as well as in decrease in the extent of HOS ubiquitination (Fig. 3B). Altogether, these findings suggest that activity of NEDD8 pathway is important for HOS ubiquitination and proteolysis and, thus, are consistent with a role of SCF activities in regulating the rate of HOS degradation.

Ubiquitination of HOS within the SCF Complex in Vitro Is Impaired in the Presence of a Substrate—Previous studies of yeast F-box proteins and Skp2 (4, 5, 8) and our own data (Figs. 2 and 3) implicate SCF activities in HOS ubiquitination and degradation. These findings suggest that F-box proteins may undergo ubiquitination within the SCF complex in an autocatalytic manner. In addition, Cdc4p and Skp2 were efficiently ubiquitinated within the respective SCF complexes in vitro (8, 10). We sought to analyze HOS ubiquitination in vitro. Our previous data showed that in vitro translated HOS efficiently binds to bacterially expressed GST-Skp1 protein (34). We expressed and purified the core SCF complex (SCR) consisting of recombinant Skp1, FLAG-tagged Cullin1, and GST-tagged Roc1 and confirmed its ability to bind HOS but not HOS{Delta}F mutant and to conjugate ubiquitin in the presence of E1 and E2 (Cdc34) in an in vitro ubiquitin ligase reaction (Fig. 4A). Intriguingly, in vitro-translated HOS captured on the (SCR) complex immobilized on glutathione-Sepharose did not undergo ubiquitination in the presence of either Cdc34 (Fig. 4B, lanes 1–3) or UbcH5c (Fig. 4B, lanes 4–6). Ubiquitination of an in vitro translated HOS was also undetectable, either when SCR was immobilized on M2-agarose or when immunoprecipitated HOS (stringently washed to remove proteins from rabbit reticulocyte lysate) was incubated with SCR in solution (data not shown).

To exclude the possibility that in vitro translated HOS lacks modifications that may be required for its autocatalytic ubiquitination, we tested ubiquitination of HOS expressed in cells. Nevertheless, bacterially expressed SCR did not efficiently ubiquitinate HA-HOS immunopurified from 293T cells (data not shown). Because the complex lacks NEDD8 conjugation (Fig. 4A), which is required for its maximal activities (26) as well as for degradation of HOS in vivo (Fig. 3), we purified SCR from 293T cells in which Cullin1 undergoes appropriate NEDD8 modification (Refs. 26, 28, and 35) and Fig. 4C, lower panel). In vitro translated HOS immobilized on mammalian SCR underwent noticeable ubiquitination, which was dependent on the presence of E1 and Cdc34 (Fig. 4C, upper panel, lanes 1–3). Similar results were obtained when the entire recombinant SCFHOS complex was assembled in 293T cells after purification of this complex on an HA affinity column and elution with HA peptide (data not shown).

We next tested how HOS ubiquitination is affected by the presence of its substrate. The addition of non-phosphorylated GST-I{kappa}B{alpha} did not affect the extent of HOS ubiquitination, although in some (but not all) experiments it led to a moderate decrease in the levels of non-ubiquitinated HOS (Fig. 4C, lane 4). Interestingly, preincubation of SCR and HOS with recombinant GST-I{kappa}B{alpha} fusion protein, which was phosphorylated by IKK{beta}, strikingly decreased the extent of HOS ubiquitination in vitro (Fig. 4C; compare lane 5 versus lanes 3–4). Similar inhibition of HOS ubiquitination by its substrate was observed with the entire mammalian SCFHOS complex (data not shown). Analysis of the ubiquitination reactions with GST antibody showed that I{kappa}B{alpha} underwent ubiquitination under these experimental conditions (Fig. 4C, middle panel). These results collectively indicate that in the presence of phosphorylated substrate, HOS is protected from ubiquitination in vitro.

Activation of IKK Inhibits Ubiquitination and Degradation of HOS—We next sought to determine whether increasing the levels of a substrate (e.g. phospho-I{kappa}B) would affect HOS ubiquitination in cells. I{kappa}B is phosphorylated within the HOS recognition motif by IKK, which is induced by various inflammatory cytokines and stress stimuli (36). We chose to use a constitutively active IKK{beta}S177,181E mutant known to efficiently phosphorylate I{kappa}B in vivo (19, 24). Co-expression of this mutant with I{kappa}B{alpha}, His-tagged ubiquitin, and HA-tagged HOS led to a noticeable inhibition of HOS ubiquitination in 293T cells (Fig. 5A).



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  		FIG. 5.
Phosphorylated I{kappa}B protects HOS from ubiquitination and degradation. A, in vivo ubiquitination of HA-HOS is inhibited by co-expression of constitutively active IKK{beta}. Immunoblotting analysis of HA-tagged HOS proteins from 293T cells (co-transfected with Ub-his and FLAG-I{kappa}B{alpha}wt vectors as well as with IKK{beta}S177,181E as indicated) in whole cell extracts (lower panel) or in the eluates of ubiquitinated proteins purified on nickel nitrilotriacetic acid (Ni-NTA)-agarose (upper panel) was carried out with HA antibody. Positions of HOS-HA proteins and ubiquitinated (Ub) HOS-HA species are indicated. B, analysis of expression and phosphorylation of FLAG-tagged I{kappa}B{alpha} proteins expressed in 293T cells. Aliquots of lysates (corresponding to time 0 of the CHX chase shown in C) were analyzed by immunoblotting with FLAG (upper panel) or phosphor-specific I{kappa}B{alpha} antibody (lower panel) under conditions that did not allow detection of endogenous phosphorylated I{kappa}. Symbols below refer to the graph in panel C. C, CHX chase analysis of degradation of HA-HOS protein expressed in 293T cells, which were co-transfected with constitutively active IKK{beta} and FLAG-tagged I{kappa}B vectors as indicated. Levels of HOS proteins were analyzed by immunoblotting with HA antibody (upper panel). M, mock-transfected cells; NS, nonspecific band. Symbols on the right correspond to the levels of I{kappa}B proteins at time point 0 (shown in Fig. 5B) and to the graph (lower panel), which depicts the % of remaining HOS at various time points of the CHX chase.

 
To determine whether an increase in the levels of phosphorylated I{kappa}B in cells affects the rate of HOS degradation, we used forced expression of constitutively active IKK{beta} with expression of various FLAG-tagged I{kappa}B{alpha} proteins (Fig. 5B, upper panel). Co-expression of the wild type protein and I{kappa}B{alpha}K21,22R mutant, but not of I{kappa}B{alpha}S32,36A mutant, led to accumulation of very high levels of phosphorylated I{kappa}B{alpha}, detected by immunoblotting with phospho-specific antibody (Fig. 5B, lower panel).

Analysis of HOS degradation in cells treated with CHX showed that expression of constitutively active IKK{beta}S177,181E mutant resulted in noticeable stabilization of HOS (Fig. 5C). Co-expression of wild type I{kappa}B{alpha}, but not of IKK phosphorylation-resistant I{kappa}B{alpha}S32,36A mutant (24), led to further inhibition of HOS degradation. These data indicate that increasing levels of a phosphorylated substrate in cells protect HOS from degradation. Notably, co-expression of I{kappa}B{alpha}K21,22R mutant, which is deficient in IKK-mediated ubiquitination and, hence, inhibits activation of NF-{kappa}B (22), also resulted in robust stabilization of HOS (Fig. 5C). This result excludes the possibility that stabilization of HOS is indirectly mediated by the products of NF-{kappa}B target genes. In all, these data suggest that in the presence of a phosphorylated substrate HOS is protected from ubiquitination and subsequent degradation.

HOS Is Stabilized and Accumulated in Response to DNA-damaging Agents—To further confirm a role of phosphorylated I{kappa}B in regulating HOS degradation we investigated whether HOS stability is altered in cells in response to agents known to cause DNA damage and induce sustained activation of IKK (37–39). Treatment of 293T cells with either actinomycin D or NCS substantially delayed degradation of recombinant HOS (Fig. 6A) and led to accumulation of endogenous HOS (Fig. 6B). Similar observations were made in normal human fibroblasts (data not shown). Furthermore, pretreatment of cells with sodium salicylate, which is known to efficiently inhibit IKK activities (40), prevented an increase in the levels of endogenous HOS in response to actinomycin D and NCS (Fig. 6B). In addition, treatment of cells with NCS decreased the extent of ubiquitination of endogenous HOS, whereas the addition of sodium salicylate to the NCS-treated cells partially prevented this decrease (Fig. 6C). These results collectively indicate that HOS ubiquitination is inhibited and that HOS stability and levels are increased in cells under conditions of genotoxic stress in an IKK-dependent manner.



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  		FIG. 6.
HOS is stabilized in the cells treated with DNA-damaging agents. A, CHX chase analysis of degradation of HA-HOS protein expressed in 293T cells that had been pretreated with either actinomycin D (ActD, 10 µg/ml for 1 h before adding CHX) or NCS (100 ng/ml for 12 h before adding CHX). Levels of proteins were analyzed by immunoblotting with HA antibody. NS, nonspecific band. DMSO, dimethyl sulfoxide (Me2SO). B, accumulation of endogenous HOS in 293T cells treated with vehicle (Me2SO), actinomycin D, or NCS as described in panel A. Cells were also pretreated with sodium salicylate (10 mM for 24 h before harvesting) as indicated. HOS was analyzed by immunoblotting with HOS antibody (upper panel). Levels of {alpha}-tubulin ({alpha}-tub; lower panel) in the lysates were also analyzed by immunoblotting with respective antibody. C, cells were transfected with HA-tagged ubiquitin and treated with Me2SO or NCS (100 ng/ml for 12 h before harvesting as indicated), sodium salicylate (10 mM for 24 h before harvesting as indicated), and MG132 (40 µM for 1 h before harvesting). Endogenous HOS proteins were immunoprecipitated (IP) and analyzed by immunoblotting (IB) with HA (upper panel) or HOS (lower panel) antibody. Positions of HOS protein and ubiquitinated HOS species are indicated. Ig, heavy chain of immunoglobulins.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented here show that HOS stability is regulated via the ubiquitin-proteasome pathway (Fig. 1). Ubiquitination and degradation of HOS depends on the integrity of its F-box motif (Fig. 2) and the activity of Cullin-based E3 ligases (Fig. 3), and ubiquitination of HOS within the SCFHOS complex can be detected in vitro (Fig. 4). In the presence of a phosphorylated substrate, HOS is protected from ubiquitination and degradation (Figs. 4, 5), and treatment of cells with DNA-damaging agents inhibits HOS ubiquitination and leads to stabilization and accumulation of HOS (Fig. 6).

Interchanging different F-box proteins allows utilization of the core SCF ligase for ubiquitination of various phosphorylated substrates according to cellular needs. Ubiquitination and degradation of F-box proteins emerge as an important mechanism underlying rapid switching. It has been suggested that F-box proteins undergo ubiquitination within the SCF complex by its core ligase components in an autocatalytic manner (4, 5, 8). As in yeast F-box proteins and mammalian Skp2, the efficiency of HOS ubiquitination and degradation depends on the presence of the F-box and requires Cullin activities.

Furthermore, we observed ubiquitination of HOS within the SCF complex in vitro, albeit at limited efficiency (Fig. 4C). Whereas autocatalytic ubiquitination of Skp2 (8) and Cdc4p (10) within their respective SCF complexes was previously reported, other groups observed a less efficient ubiquitination of Cdc4p within the SCFCdc4 complex in vitro (18).2 Together with these data our findings give insight into the role of substrate in ubiquitination and degradation of F-box proteins. HOS ubiquitination and degradation are inhibited in the presence of phosphorylated I{kappa}B{alpha}. These results argue against a model of co-ubiquitination of an F-box protein together with a bound substrate and favor another model predicting that a bound substrate shields an F-box protein from ubiquitination (2). The latter model is not only less wasteful but also provides cells with an advantage of maintaining specific F-box protein-dependent SCF E3 activities based on cellular needs to eliminate a specific substrate as long as it is present.

It remains to be seen whether other F-box proteins are stabilized in the presence of their substrates, although some indirect evidence corroborates this speculation. Skp2 is rapidly degraded in quiescent cells and becomes stabilized in cells that re-start cycling (8). Intriguingly, such stabilization parallels the levels of specific phosphorylation of the SCFSkp2 substrate p27Kip1 by cyclin E-Cdk2 on threonine 187 (41), which enables the recognition of p27Kip1 by Skp2 followed by p27Kip1 ubiquitination (41, 42).

{beta}-TrCP1 protein (43), which also functions as an SCF E3 ligase receptor in ubiquitination of I{kappa}B and {beta}-catenin (for review, see Ref. 44), is stabilized via interaction with heterogeneous nuclear ribonucleoprotein U protein, which does not interact with HOS (45). Because {beta}-TrCP1 is closely related to HOS, it is conceivable that, in addition to heterogeneous nuclear ribonucleoprotein U effects, interaction of phosphorylated substrates with {beta}-TrCP1 may also contribute to stabilization of this F-box protein.

Activation of the NF-{kappa}B pathway, which depends on IKK activation (for review, see Ref. 36) and HOS/{beta}-TrCP1-mediated ubiquitination and degradation of I{kappa}B (19, 44), is a key mechanism underlying the ability of the cell to cope with stress (36, 46). HOS activities are required for survival of cells treated with DNA-damaging agents (47). Conversely, in response to DNA damage, inhibition of HOS ubiquitination and stabilization of HOS in the presence of phosphorylated I{kappa}B would be expected to preserve active SCFHOS E3 ubiquitin ligase to ubiquitinate I{kappa}B and activate NF-{kappa}B and, thus, may play an important role in cellular responses to prolonged stress.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA92900 (to S. Y. F.) and GM61051 (to Z.-Q. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Animal Biology, University of Pennsylvania, 3800 Spruce St., Rm. 161E, Philadelphia, PA 19104-6046. Tel.: 215-573-6949; Fax: 215-573-5188; E-mail: syfuchs{at}vet.upenn.edu.

1 The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme, HOS, homologue of Slimb; SCF, Skp1·Cullin1·Roc1·F-box protein complex; SCR, Skp1·Cullin1·Roc1 complex; GST, glutathione S-transferase; NCS, neocarzinostatin; CHX, cycloheximide; HA, hemagglutinin virus protein tag; {beta}-TrCP, {beta}-transducin repeats-containing protein; NF-{kappa}B, nuclear factor {kappa}B; I{kappa}B, inhibitor of NF-{kappa}B; IKK, I{kappa}B kinase. Back

2 R. Deshaies, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank D. Ballard, D. Bohmann, M. Karin, R. Neve, H. Tahara, and Y. Xiong for reagents, D. Skowyra and R. Deshaies for communicating results before publication, R. Deshaies and P. Zhou for critical comments and suggestions, and M. Meyer for assistance in editing the manuscript.



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