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Rbx1, a Component of the VHL Tumor Suppressor Complex and SCF Ubiquitin Ligase

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Science  23 Apr 1999:
Vol. 284, Issue 5414, pp. 657-661
DOI: 10.1126/science.284.5414.657

Abstract

The von Hippel–Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.

The VHL tumor suppressor gene on chromosome 3p25.5 is mutated in most sporadic clear cell renal carcinomas and in VHL disease, an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors (1). The VHL protein is expressed in most tissues and cell types and appears to perform multiple functions, including repression of hypoxia-inducible genes (2), regulation of p27 protein stability (3), and regulation of fibronectin matrix assembly (4). VHL is found in a multiprotein complex with Elongin B, which is ubiquitin-like, and Elongin C and CUL2, which share sequence similarity with the Skp1 and Cdc53 components of the SCF ubiquitin ligase (5, 6). The Elongin BC complex interacts with a short BC-box motif in VHL and bridges its interaction with CUL2 (6). A large fraction of VHL mutations alter the BC-box and disrupt the VHL complex (1, 4,6).

To investigate VHL function, we purified the endogenous VHL complex from rat liver (Fig. 1) (7). Greater than 90% of the detectable VHL protein copurified with CUL2, Elongins B and C, and a polypeptide of ∼16 kD (Fig. 1, B and C). The identities of the VHL, CUL2, and Elongin B and C polypeptides were confirmed by immunoblotting, peptide sequencing, or both. Ion trap mass spectrometry (8) of the ∼16-kD protein revealed that it was a previously undescribed RING-H2 fingerlike protein, which we designate RING-box protein Rbx1 (Fig. 1D). Rbx1 is highly homologous to Drosophila melanogaster open reading frame (ORF) 115C2.11, Caenorhabditis elegans ORF ZK287.5, and Saccharomyces cerevisiae ORF YOL133w. In addition, Rbx1 is similar in sequence to S. cerevisiae anaphase-promoting complex subunit APC11 (9).

Figure 1

Copurification of the VHL complex with Rbx1. (A) Purification of the VHL complex. P-cell, phosphocellulose P11. (B) Cochromatography of Rbx1 with the VHL complex. Samples of column fractions from the MonoQ column were subjected to 12% SDS-PAGE, and proteins were detected by silver staining. VHL, von Hippel–Lindau protein; CUL2, CUL2 protein; EloB, Elongin B; EloC, Elongin C. (C) SDS-polyacrylamide (5 to 20%) gel of sample used for peptide sequencing. (D) Alignment of predicted Rbx1 protein sequences from human, mouse,D. melanogaster (DROS), C. elegans (ELEGANS), andS. cerevisiae (SACCH) with Rbx2 from human and C. elegans and APC11 from S. cerevisiae. The alignment was generated with the MACAW program (30). Black shading indicates sequence identity, gray shading sequence similarity. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

We next studied Rbx1 interactions with VHL, CUL2, and the Elongin BC complex. Sf21 insect cells were coinfected with various combinations of baculoviruses encoding MYC-Rbx1, FLAG-VHL, hemagglutinin A (HA)–CUL2, HPC4–Elongin B, and herpes simplex virus (HSV)–Elongin C, and complexes were immunoprecipitated from infected cell lysates with antibodies to the epitope (10, 11). Rbx1 assembled into a complex with VHL, CUL2, and Elongins B and C when all five proteins were coexpressed (Fig. 2A, lanes 5 and 9). In addition, Rbx1 assembled with VHL and Elongins B and C independent of coexpressed CUL2 (lanes 6 and 10) and with CUL2 and Elongins B and C independent of coexpressed VHL (lane 11). Suggesting that Rbx1 interacts independently with VHL, CUL2, and the Elongin BC complex, Rbx1-VHL, Rbx1-CUL2, and Rbx1–Elongin BC complexes could be isolated from lysates of cells not expressing other VHL complex components (Fig. 2B). Consistent with these results, complexes containing Rbx1, Elongin BC, and VHL or Rbx1 and Elongin BC could be reconstituted in vitro with bacterially expressed proteins (Fig. 2C) (12).

Figure 2

Reconstitution of Rbx1-containing complexes. (A) Rbx1 forms complexes with VHL and CUL2 in the presence of Elongin BC. Lysates from Sf21 cells expressing the indicated viruses were immunoprecipitated with anti-FLAG or anti-MYC. Immunoprecipitated proteins in (A) to (D) were detected by immunoblotting. (B) Interaction of Rbx1 with Elongin BC, VHL, and CUL2. Lysates from Sf21 cells expressing the indicated viruses were immunoprecipitated with anti-HPC4, anti-FLAG, or anti-MYC. (C) In vitro binding of recombinant Rbx1, VHL, and Elongin BC. Proteins expressed in and purified from E. coli were mixed together in the combinations indicated, renatured by dilution and dialysis, and immunoprecipitated with anti-HPC4. (D) Skp1-independent association of mRbx1 with CUL1. Lysates from Hi5 cells expressing the indicated viruses were immunoprecipitated with anti-HA or anti-MYC.

CUL2 is a member of the Cullin protein family that includes CUL1 and its S. cerevisiae homolog Cdc53 (13). CUL1/Cdc53 proteins are components of SCF (Skp1–Cdc53–F-box protein) complexes, the E3 ubiquitin ligases that target diverse proteins for ubiquitin-mediated proteolysis (14–18). In SCF complexes, CUL1 is linked to one of a number of F-box proteins through an adapter protein, Skp1. F-box proteins interact with ubiquitination substrates through COOH-terminal protein-protein interaction domains and with Skp1 through the F-box motif (14, 18). The finding that Rbx1 interacts with CUL2 led us to test whether it might also interact with CUL1. As shown in Fig. 2D, Rbx1 bound to CUL1 in insect cells, and this interaction was independent of Skp1. Rbx1 did not associate with Skp1 directly.

SCF complexes are best understood in S. cerevisiae, where they have been implicated in multiple phosphorylation-dependent proteolysis pathways, including destruction of the cyclin-dependent kinase inhibitor Sic1 by SCFCdc4, a process required for the G1 to S transition (14–16, 18,19). We therefore examined whether Rbx1 is involved in SCF function in S. cerevisiae. An S. cerevisiaestrain lacking the RBX1 gene was constructed by replacing the complete coding sequence (ORF YOL133w) with the HIS3gene (20, 21). Sporulation and tetrad dissection showed 2:0 segregation for viability, indicating thatRBX1 is an essential gene (21). Inviable spores produced microcolonies of 10 to 20 cells, many of which were abnormally elongated or contained multiple, abnormally shaped buds.Saccharomyces cerevisiae strains containing mutations in genes encoding the SCF components Cdc53, Skp1, Cdc4, and Cdc34 exhibit a similar morphology (14).

The viability defect of the rbx1 deletion strain was rescued by expression of either MYC-tagged mammalian Rbx1 (mRbx1) or a mutant mRbx1 (M4), in which putative RING finger cysteines at positions 53 and 56 were replaced with serines, but not by expression of a mutant mRbx1 (M3), in which putative ring finger cysteines 42 and 45 were replaced by serines. When expressed in either the rbx1deletion strain or in a wild-type background, MYC-tagged mRbx1 interacted with endogenous Cdc53 (Fig. 3A). Interaction of wild-type and mutant mRbx1 proteins with Cdc53 correlated with their abilities to rescue the deletionphenotype: Substantially more Cdc53 was coimmunoprecipitated with M4 than with M3 (Fig. 3A) (22). Consistent with these in vivo interactions, S. cerevisiae Rbx1 associated with Cdc53 (Fig. 4B) and assembled into SCFCdc4 complexes (Fig. 4A) when coexpressed in insect cells. Efficient assembly into SCF complexes required the presence of Cdc53 (Fig. 4A, lane 4). Rbx1 also associated with Cdc4 in the absence of Cdc53 and Skp1 (Fig. 4A, lane 5), an interaction that was reduced in the presence of Skp1 (Fig. 4A, lane 4). This interaction may be analogous to that between Rbx1 and VHL (Fig. 2B).

Figure 3

Rbx1 function in yeast. (A) Binding of Rbx1 to endogenous yeast Cdc53 correlates with function. (Upper panel) Phenotypes of rbx1Δ cells expressing wild-type or mutant mRbx1. (Lower panel) Lysates from cells expressing wild-type and mutant mammalian MYC-Rbx1 proteins in the rbx1deletion strain (deleted) or in the parental strain MCY453 (wild type) were immunoprecipitated with anti-MYC and immunoblotted with anti-MYC or anti-Cdc53. (B) Sic1 accumulates in Rbx1-depleted cells. rbx1Δ/pGAL-mrbx1(M4) cells were grown to an absorbance at 600 nm of 1 in galactose-containing medium and then shifted into glucose medium. Cells were harvested after 8 hours of growth in glucose, and cell lysates were analyzed by immunoblotting with anti-MYC and two different anti-Sic1 antibodies. (C) Morphological changes associated with Rbx1-depletion.rbx1Δ/pGAL-mrbx1(M4) cells were grown in galactose (gal) or for 8 hours after glucose shift (glu) before fixation and staining. Nuclear morphology was visualized by DAPI (4′,6′-diamidino-2-phenylindole) staining. (Left) Differential interference contrast; (right) DAPI. (D) Sic1 ubiquitination is defective in extracts from rbx1-1 cells. Ubiquitination assays were performed in fractionated yeast lysates (F250) with Cln1/Cdc28-phosphorylated Sic1 as substrate (15, 31). Reactions were supplemented with 500 nM bacterial Cdc34, 200 nM human E1, 20 μM ubiquitin, 5 mM ATP, an ATP-regenerating system, 50 μM LLnL, 1 μM okadaic acid, and protease inhibitors (5 μg/ml each of pepstatin, leupeptin, and aprotinin. FLAG-Skp1–Cdc4 immune complexes (100 ng of Cdc4 per 25 μg of yeast extract) were added to the yeast lysates before addition of substrate to increase activity toward Sic1. Assays were performed with 25, 50, or 75 μg of yeast proteins.

Figure 4

Rbx1 assembles with SCFCdc4complexes and activates ubiquitination of phosphorylated Sic1 in vitro. (A) Rbx1 associates with SCFCdc4complexes and Cdc4 in insect cells. Lysates from insect cells expressing the indicated proteins were immunoprecipitated with anti-MYC to immunoprecipitate MYC-Rbx1 or anti-FLAG to immunoprecipitate FLAG-Cdc4. Washed immune complexes (middle and lower panels) and crude lysates (top panel) were separated by SDS-PAGE and immunoblotted. (B) Rbx1 interacts with Cdc53. MYC-Rbx1 was immunoprecipitated from insect cells in the presence or absence of Cdc53 and immunoblots probed for both proteins. (C) Rbx1 stimulates SCFCdc4-mediated Sic1 ubiquitination in vitro. Baculoviruses expressing FLAG-Skp1, MYC-Cdc53, and Cdc4 were coinfected into insect cells in the presence or absence of viruses expressing mouse or yeast Rbx1. SCF complexes from 4 × 106 cells were immunoprecipitated with either anti-FLAG (lanes 8 to 13) or anti-MYC (lanes 1 to 6) (10 μl) and immune complexes used for Sic1 ubiquitination reactions (15). Reaction mixtures were separated by SDS-PAGE and Sic1-ubiquitin conjugates visualized with anti-Sic1. Blots were stripped and reprobed with anti-Cdc53 and anti-Cdc4 to control for protein levels. The asterisk (*) indicates the position of a cross-reacting band in some batches of anti-Sic1. (D) The indicated quantities of MYC-Rbx1 baculovirus were coinfected into 2 × 106 insect cells with 100 μl each of Skp1, FLAG-Cdc4, and Cdc53 viruses (∼108particles per milliliter). Anti-FLAG immune complexes were used for Sic1 ubiquitination reactions as described in (C). Comparable amounts of Skp1, Cdc53, and FLAG-Cdc4 were present in each reaction as determined by immunoblotting of stripped blots (lower panels), and assembly of Rbx1 with the SCF complex was near maximal, with 100 μl of virus (∼108 particles per milliliter) (lane 6). In addition, the levels of MYC-Rbx1 in lysates used for immunoprecipitation were determined by immunoblotting.

To address the role of Rbx1 in Sic1 ubiquitination, we introduced MYC-tagged wild-type or M4 mRbx1 into the rbx1 deletion strain on a high copy number plasmid under control of the GAL1,10 promoter. When cells carrying the plasmid were shifted from galactose to glucose medium, Rbx1 protein was depleted, and the fraction of cells exhibiting the elongated bud morphology increased substantially (Fig 3, B and C). Cells expressing M4 stopped growing within a few hours of the glucose shift, whereas cells expressing wild-type Rbx1 continued to grow slowly, presumably owing to the presence of residual Rbx1. Consistent with the hypothesis that M4 cells arrest because they cannot ubiquitinate and destroy Sic1, the M4 cells accumulated Sic1 protein when shifted into glucose (Fig 3B).

A common property of temperature-sensitive mutations in the SCF components Skp1, Cdc53, Cdc4, and Cdc34 is that Sic1 overexpression is lethal at permissive temperatures (14, 19). Sic1 overexpression from the GAL1,10 promoter was lethal in cells expressing an RBX1 temperature-sensitive mutant, rbx1-1(23), but not in cells expressing wild-typeRBX1. Even on glucose, rbx1-1 cells grew more slowly in the presence of the SIC1 plasmid than did cells containing wild-type RBX1, suggesting that low levels of SIC1 expression are toxic in combination with the rbx1-1mutation. Moreover, extracts from RBX1 cells displayed Sic1 ubiquitination activity when supplemented with Cdc34, E1, and an adenosine triphosphate (ATP)–regenerating system, whereas extracts from rbx1-1 were deficient in Sic1 ubiquitination (Fig. 3D). These data indicate that Rbx1 is involved in SCFCdc4function and in Sic1 ubiquitination.

To test directly the role of Rbx1 in SCFCdc4function, we used an in vitro Sic1 ubiquitination assay dependent on Sic1 phosphorylation and the E2 Cdc34 (15,24). SCFCdc4 components were coexpressed in insect cells in the presence or absence of mammalian or yeast Rbx1, and complexes were purified by immunoprecipitation of MYC-tagged Cdc53 (MYC-Cdc53) or FLAG-tagged Skp1 (FLAG-Skp1) subunits. Complexes were supplemented with phosphorylated Sic1, Cdc34, E1 ubiquitin-activating enzyme, ATP, and glutathione S-transferase (GST)–UbRA before analysis of Sic1 conjugates by immunoblotting. GST-UbRA forms polyubiquitinated products poorly, so Sic1 conjugates are integrated into a ladder of bands differing by ∼35 kD, the size of GST-UbRA. Low but detectable amounts of Sic1-GST-UbRA conjugates were produced by the SCFCdc4 complex after a 60-min reaction (Fig. 4C, lanes 2 and 9). In the presence of Rbx1, accumulation of Sic1-GST-UbRA conjugates was markedly increased after 20 min (lanes 3, 5, 10, and 12), and substantial amounts of higher molecular mass conjugates accumulated after 60 min (lanes 4, 6, 11, and 13). In contrast to reactions lacking Rbx1, where <5% of Sic1 was conjugated (lane 9), >85% of Sic1 was converted to GST-UbRA conjugates in the presence of Rbx1 and FLAG-Skp1 complexes (lanes 11 and 13). To examine the extent of activation and the concentration dependence of Rbx1 activation, we purified SCFCdc4 complexes from insect cells coexpressing increasing levels of MYC-Rbx1 and then assayed for Sic1 ubiquitin-conjugating activity (Fig. 4D). In the absence of Rbx1, low levels of conjugates were observed (lane 1). Increased quantities of Rbx1 led to increased levels of ubiquitination, with the maximal extent of activation approaching 20-fold at the point where MYC-Rbx1 assembly into SCFCdc4 complexes was saturated and stoichiometric (lanes 6 to 9). This estimate represents a lower limit of the extent to which Rbx1 can increase the rate of accumulation of Sic1-GSTUbRAconjugates because a large fraction of the phosphorylated Sic1 substrate was depleted at the end of reactions (lane 9). Immunoblot analysis of these complexes revealed that the levels of Cdc53, Cdc4, and Skp1 were constant throughout the Rbx1 titration. It is likely that incorporation of small amounts of insect cell Rbx1 protein contributed to the previous observation of Sic1 ubiquitination activity by SCFCdc4 complexes in vitro (15,16).

Our findings have implications for the functions of both the VHL complex and Rbx1. First, our observation that Rbx1 is a component of both the VHL and SCFCdc4 complexes extends their marked structural similarity and raises the possibility that the VHL complex, and perhaps other Cullin complexes, may function as ubiquitin ligases for as yet unidentified target proteins. Such a function for the VHL complex could explain the remarkably pleiotropic phenotypes associated with VHL mutations. Second, our finding that Rbx1 is a component of the SCFCdc4 complex, together with the observations that Rbx1 is required for Cln1 ubiquitination by an SCFGrr1 complex (23), indicates that, like Cdc53/CUL1 and Skp1, Rbx1 functions as a common SCF subunit. Thus, it seems likely that Rbx1 regulates ubiquitination by SCF complexes containing additional F-box proteins, including the mammalian β-TRCP protein, which directs ubiquitination of the human immunodeficiency virus–1 coreceptor CD4 and the transcriptional regulators IκB and β-catenin (25–28).

  • * To whom correspondence should be addressed. E-mail: conawayj{at}omrf.ouhsc.edu

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