JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M408018200 on July 27, 2004

J. Biol. Chem., Vol. 279, Issue 41, 43019-43026, October 8, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/41/43019    most recent
M408018200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yan, Q.
Right arrow Articles by Conaway, J. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yan, Q.
Right arrow Articles by Conaway, J. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification of Elongin C and Skp1 Sequences That Determine Cullin Selection*

Qin Yan{ddagger}§, Takumi Kamura||, Yong Cai{ddagger}, Jingji Jin{ddagger}, Mircea Ivan**, Arcady Mushegian{ddagger}, Ronald C. Conaway{ddagger}{ddagger}{ddagger}, and Joan Weliky Conaway{ddagger}§{ddagger}{ddagger}§§

From the {ddagger}Stowers Institute for Medical Research, Kansas City, Missouri 64110, the §Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, the Howard Hughes Medical Institute, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, the ||Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan, the **Department of Medicine, Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts 02111, and the {ddagger}{ddagger}Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, Kansas 66160

Received for publication, July 15, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) complexes belong to families of structurally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit, which is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that activates ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzyme Ubc5. In SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits, which are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that activates ubiquitylation of target proteins, in most cases by the E2 Cdc34. In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases. Our findings provide evidence that E2 selection by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate recognition subunit but by yet to be elucidated features of these macromolecular complexes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The von Hippel-Lindau (VHL)1 tumor suppressor complex is the founding member of the family of Elongin BC-containing E3 ubiquitin ligases, which are composed of a substrate recognition subunit, Elongins B and C, a member of the Cullin family of proteins (either Cul2 or Cul5), and the RING finger protein Rbx1 (also known as ROC1 or Hrt1) (1–4). The VHL protein is one of a large family of proteins that bind Elongins B and C through a conserved BC box motif, which is a 10-amino-acid degenerate motif of sequence (A,P,S,T)LXXXCXXX(A,-I,L,V) (5–9). In the context of the VHL ubiquitin ligase, the VHL protein serves as the substrate recognition subunit and is linked by the adaptor protein Elongin C to a heterodimeric Cul2/Rbx1 module that functions as a potent activator of ubiquitylation of target proteins by an E2 ubiquitin-conjugating enzyme. Elongin B, a ubiquitin-like protein, associates with the complex through interactions with Elongin C and appears to stabilize the binding of Elongin C to VHL.

The best characterized substrates of the VHL ubiquitin ligase are the HIF{alpha} family of DNA binding transcription factors (1–3, 10, 11), which function together with the constitutively expressed aryl hydrocarbon nuclear translocator to regulate expression of hypoxia-inducible genes when cells are starved for oxygen (12). Cellular HIF{alpha} protein levels are tightly controlled by the ubiquitin-dependent proteolytic system (13, 14). In cells grown in a plentiful supply of oxygen, HIF{alpha} family members are hydroxylated at critical proline residues within their oxygen-dependent degradation domains (15–20). Upon VHL binding to their hydroxylated oxygen-dependent degradation domains, HIF{alpha} family members are rapidly ubiquitylated by the VHL ubiquitin ligase complex and degraded by the proteasome. In cells grown in hypoxic conditions, HIF{alpha} family members are not hydroxylated. Accordingly, under hypoxic conditions, the VHL ubiquitin ligase complex cannot bind HIF{alpha}s and promote their ubiquitylation and degradation, so cellular levels of these transcription factors rise, leading to activation of hypoxically regulated genes.

The large family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases share striking structural similarities with the VHL ubiquitin ligase. SCF ubiquitin ligases are composed of one of many F-box proteins, the Elongin C-like protein Skp1, Cullin family member Cul1 (called Cdc53 in the Saccharomyces cerevisiae), and Rbx1 (4, 21–30). In the context of SCF ubiquitin ligases, F-box proteins serve as substrate recognition subunits and are linked by the adaptor protein Skp1 to a Cul1/Rbx1 module that activates an E2 to ubiquitylate target proteins.

Many substrates of SCF ubiquitin ligases have been characterized, and ubiquitylation of most, but not all, depends upon the E2 ubiquitin-conjugating enzyme Cdc34 (30–34). In contrast, at least in vitro, VHL ubiquitin ligase-dependent ubiquitylation of HIF{alpha}s depends on members of the Ubc5 family of E2s (2, 3).

Our laboratory is currently engaged in biochemical studies to define the functions of subunits of VHL and SCF ubiquitin ligases. In this report, we investigate the functions of the Elongin C and Skp1 adaptor proteins in reconstitution of active VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, by exploiting altered specificity Elongin C-Skp1 and Cdc4-VHL chimeric proteins, we provide insights into the determinants of E2 selectivity of VHL and SCF ubiquitin ligases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—Anti-VHL monoclonal antibody (Ig32) was purchased from BD PharMingen. Anti-Myc (9E10) and anti-HA (12CA5) monoclonal antibodies were obtained from Roche Applied Science. Anti-HSV monoclonal antibody was from Novagen. Anti-FLAG monoclonal antibody (M2) was purchased from Sigma. Anti-Cul2 and anti-Elongin C monoclonal antibodies were obtained from BD Transduction Laboratories. Anti-Cul2 (CT2) rabbit polyclonal antibodies were from Zymed Laboratories Inc.. Anti-HIF1{alpha} monoclonal antibody (H1{alpha}67) was purchased from Novus Biologicals. Anti-protein C monoclonal antibody (HPC4) (35) was provided by C. Esmon (Oklahoma Medical Research Foundation) and is commercially available as anti-protein C from Roche Applied Science. Anti-Elongin B polyclonal antibody was prepared as described (36).

Expression of Recombinant Proteins in Sf21 Insect Cells—Wild type human Elongin C and Elongin C mutants containing N-terminal epitope tags recognized by the HPC4 monoclonal antibody were subcloned into pBacPAK8. Recombinant baculoviruses were generated with the BacPAK baculovirus expression system (Clontech). Baculoviruses encoding human Cul1 and Cul2 containing N-terminal HA epitope tags were described previously (7). Baculoviruses encoding human Cul2, human VHL, human VHL containing an N-terminal His6 tag, human Elongin B and mouse Rbx1 containing N-terminal Myc epitope tags, and human HIF1{alpha} containing N-terminal His6 and HPC4 tags were described previously (3). Baculoviruses encoding S. cerevisiae Cdc53 and mouse Rbx1 containing N-terminal His6 and Myc tags were described previously (4).

Sf21 cells were cultured at 27 °C in Sf-900 II SFM with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Plates containing 1 x 107 Sf21 cells were infected with the recombinant baculoviruses indicated in the text and figures. Sixty hours after infections, cells were lysed in 1 ml/plate of ice-cold buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 1 mM dithiothreitol, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin.

Immunoprecipitations and Western Blotting—Approximately 100 µg of Sf21 cell lysates was incubated at 4 °C for 2 h with either 10 µl of protein G-Sepharose and 2 µg of HPC4 antibody or 10 µl of protein A-Sepharose and 2 µg of the antibodies indicated in the figures. Sepharose beads were washed three times in buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 1 mM dithiothreitol, and 0.5% (v/v) Triton X-100. Immunoprecipitated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Hybond P membranes (Amersham Biosciences). Membranes were incubated at 4 °C overnight with the indicated antibodies in buffer containing 40 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 3% (w/v) nonfat dry milk and then with peroxidase-conjugated secondary antibodies (Sigma). Proteins were visualized on film following treatment of membranes with Supersignal West Pico, Dura, or Femto chemiluminescence reagent (Pierce). In some experiments, proteins were visualized with a Storm gel and blot imaging system following treatment of membranes with ECL plus Western blotting reagents (Amersham Biosciences). Alternatively, membranes were incubated at 4 °C overnight with the indicated antibodies in Odyssey blocking solution (LI-COR Biosciences) and then with the appropriate Alexa Fluor 680 (Molecular Probes) or IRDye 800 (Rockland) secondary antibodies. In this case, proteins were visualized and quantitated with the Odyssey infrared imaging system (LI-COR Biosciences).

Purification of Recombinant VHL and Cullin/Rbx1 Complexes from Sf21 Cell Lysates—Plates containing 1 x 107 Sf21 cells were coinfected with the recombinant baculoviruses indicated in the text and figures. Sixty hours after infections, cells were collected by centrifugation and resuspended in 1 ml/plate of ice-cold buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin A, 5 µg/ml aprotinin, and 40 mM imidazole (pH 7.9). Cells were lysed by French press (1-inch piston; 16,000 p.s.i. of pressure; American Instrument Company). Following centrifugation at 10,000 x g for 20 min at 4 °C, the resulting supernatant was mixed with 1 ml of Ni+2-agarose pre-equilibrated in buffer containing 40 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, and 40 mM imidazole (pH 7.9). The slurry was incubated at 4 °C for 2 h with gentle mixing, washed three times with the same buffer, and packed into a 0.8 cm-diameter column. The column was eluted stepwise with buffer containing 40 mM Hepes-NaOH (pH 7.9), 50 mM NaCl, 10% (v/v) glycerol, and 300 mM imidazole (pH 7.9). VBC-Cullin/Rbx1 complexes were reconstituted by mixing Ni+2-agarose-purified VHL-Elongin BC complex (VBC) and Cullin/Rbx1 together and incubating them for 1 h at 4 °C. For the experiment in Fig. 5 (see below), VBC-Cul2/Rbx1 complex was further purified by TSK DEAE-NPR HPLC. Ni+2-agarose fractions were dialyzed against buffer containing 40 mM Tris-HCl (pH 7.9), 100 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, and 10% (v/v) glycerol. Following centrifugation at 10,000 x g for 20 min at 4 °C, the resulting supernatant was applied to a TSK DEAE-NPR HPLC column (4.6 x 35 mm; Toso-Haas) preequilibrated in the same buffer. The column was eluted at 0.2 ml/min with a 3-ml linear gradient from 100 to 500 mM KCl in the same buffer, and 0.1-ml fractions were collected. For the experiment of Fig. 7 (see below), complexes containing Cdc4-VHL, Elongins B and C, and Cul2/Rbx1 were purified by the same procedure.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 5.
Reconstitution of VHL ubiquitin ligase with isolated VBC and Cul2/Rbx1 complexes. VBC-Cul2/Rbx1, VBC, and Cullin/Rbx1 complexes were prepared as described under "Experimental Procedures." EGLN1-treated HIF1{alpha} was assayed as described under "Experimental Procedures" in ubiquitylation reactions containing ~800 ng of hUbc5a in the absence (lane 1) or presence (lane 2) of purified VBC-Cul2/Rbx1 complex or a mixture of purified VBC and Cul2/Rbx1 that had been preincubated for 60 min at 4 °C (lane 3). Reaction mixtures were subjected to 8% SDS-polyacrylamide gel electrophoresis. Proteins in gels were transferred to Hybond P and visualized as described under "Experimental Procedures" by Western blotting with anti-HIF1{alpha} antibody.

 



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 7.
Ubiquitylation of phosphorylated Sic1 by an Elongin BC- and Cul2-containing ubiquitin ligase by hUbc5a. A, the Cdc4-VHL chimera contains residues 341–779 of Cdc4 fused to the complete VHL open reading frame. The Cdc4 F-box is indicated by the black box, the Cdc4 WD repeats are indicated by the dark gray boxes, and the VHL BC box is indicated by the white box. WT, wild type. B, ubiquitin ligase complexes containing either Cdc4 or Cdc4-VHL as F-box protein were expressed in insect cells and purified as described. ElobB, Elongin B; ElobC, Elongin C. C, purified Cdc4-VHL-BC-Cul2/Rbx1 complexes were assayed for their abilities to support ubiquitylation of phosphorylated HPC4-Sic1 in the presence of ~100 ng of hUbc5a or yCdc34. Reaction products were analyzed by Western blotting (WB) with HPC4 monoclonal antibody and visualized on film. D, purified Cdc4-VHL-BC-Cul2/Rbx1 or VBC-Cul2/Rbx1 complexes were assayed as in panel C for their abilities to support ubiquitylation of phosphorylated HPC4-Sic1.

 
Preparation of Hydroxylated HIF1{alpha}—Sf21 cells were infected with baculoviruses encoding human HIF1{alpha} containing N-terminal His6 and HPC4 tags. HIF1{alpha} was purified from Sf21 cell lysates by Ni+2-agarose chromatography as described previously (3). Approximately 5 µg of purified HIF1{alpha} was incubated at room temperature for 2 h with 200 µl of TNT wheat germ extract translation system (Promega) programmed with a pcDNA3 expression vector encoding the EGLN1 prolyl hydroxylase (20) and containing 100 µM FeCl2, 2 mM sodium ascorbate, and 5 mM {alpha}-ketoglutarate. Prolyl hydroxylated HIF1{alpha} was then purified from the reaction mixture by Ni+2-agarose chromatography (3).

Assays of HIF1{alpha} and Sic1 Ubiquitylation in Vitro—Human Ubc5a (hUbc5a), human Cdc34 (hCdc34), and S. cerevisiae Cdc34 (yCdc34), all containing N-terminal His6 tags, S. cerevisiae Uba1 (yUba1) containing N-terminal Myc and His6 tags, and mammalian GST·ubiquitin were prepared as described (3). Reconstituted VBC-Cullin/Rbx1 complexes or aliquots of TSK DEAE-NPR HPLC-purified VBC-Cul2/Rbx1 or VHL-Cdc4BC-Cul2/Rbx1 complexes were incubated with 50 ng of yUba1, 500 ng of GST·Ub, 20 ng of purified prolyl hydroxylated HIF1{alpha} or phosphorylated Sic1 (23), and varying amounts of the E2 ubiquitin-conjugating enzymes hUbc5a, hUbc3, or yCdc34 as indicated in the text and figures in 10 µl of ubiquitylation reaction buffer containing 40 mM Hepes-NaOH (pH 7.9), 60 mM potassium acetate, 2 mM dithiothreitol, 6 mM MgCl2, 0.5 mM EDTA (pH 8.0), 10% (v/v) glycerol, and 1.5 mM ATP. Reaction mixtures were incubated for 1 h at 25 °C, fractionated by SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with anti-HIF1{alpha} antibodies.

Assay of E2-Ubiquitin Thioester Bond Formation—hUbc5a, hUbc3, or yCdc34 was incubated with 50 ng of yUba1 and 100 ng of His-T7-Xpress-ubiquitin in 10 µl of 40 mM Hepes-NaOH (pH 7.9), 60 mM potassium acetate, 2 mM dithiothreitol, 6 mM MgCl2, 0.5 mM EDTA (pH 8.0), 10% (v/v) glycerol, and 1.5 mM ATP. Reaction mixtures were incubated for 1 h at 25 °C and then stopped with 15 µl of a buffer containing 60 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, and either 30 mM dithiothreitol (reducing) or 6 M urea (nonreducing). Samples were incubated at 100 °C for 3 min (reducing) or 37 °C for 15 min (nonreducing), subjected to SDS-polyacrylamide gel electrophoresis, and analyzed by Western blotting with anti-T7 antibodies.

Computational Methods—Multiple sequence alignments were made using the MACAW program (37). Homology modeling and loop rebuilding were done on the Swiss-Model server (38). Automated docking was done using the GRAMM program (39). Fig. 8 (see below) was generated with the PyMOL package (DeLano Scientific, LLC; www.pymol.org).



View larger version (46K):
[in this window]
[in a new window]
  		FIG. 8.
Model of EloC-Cul1 interface. The pink ribbon indicates the fragment of Cul1 (residues 16–55 and 112–149 of PDB entry 1LDK [PDB] , chain A) that contributes to the interface. Elongin C is shown with rebuilt loop 3 from wild type Elongin C (blue) or Skp1 (magenta). Elongin C {alpha}-helices are shown in red, {beta}-strands are shown in yellow, and the rebuilt loop 3 from wild type Elongin C is shown in blue whereas that from Skp1 (corresponding to the M1 mutant) is shown in magenta. Side chains predicted to be involved in EloC-Cul1 interactions are shown in green.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
As illustrated in Fig. 1, the Elongin C and Skp1 proteins share significant amino acid sequence similarity and belong to a protein superfamily with shared three-dimensional structure. Elongin C and Skp1 function as adaptors that recruit heterodimeric Cul2/Rbx1 and Cul1/Rbx1 modules, respectively, into VHL and SCF ubiquitin ligase complexes. In the case of the VHL ubiquitin ligase, Elongin C recruits a Cul2/Rbx1 module to the complex by interacting specifically with a BC box in the VHL protein (Ref. 40 and references therein) and with an N-terminal Cul2 region (41). In the case of SCF ubiquitin ligase complexes, Skp1 functions by an apparently similar mechanism as Elongin C to recruit a Cul1/Rbx1 module to the complex by interacting specifically with an F-box in F-box proteins and with an N-terminal Cul1 region (42, 43).



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 1.
Alignment of Elongin C (EloC) and Skp1 amino acid sequences. Human Elongin C and selected representatives of the Skp1 family were downloaded from the NCBI Conserved Domain Data base (www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=smart00512) and realigned using Gibbs sampling. Unique identifiers of each sequence in GenBankTM or PDB databases and distances from the N termini of the proteins are shown to the left of each sequence. Segments missing from the three-dimensional structures (see "Results") are underlined. Sequences from Skp1 that were used to replace Elongin C sequences in C M1 and C M2 mutants are shown in magenta. The green font with red shading indicates the residues that are predicted to be within 3 Å of a Cullin mollecule and are likely to make direct contacts. Yellow shading indicates conserved bulky hydrophobic residues (Ile, Leu, Val, Met, Phe, Tyr, and Trp), blue type indicates conserved residues with small side chains (Ala, Gly, and Ser), and green shading indicates other conserved residues (including a group of positively charge side chains Lys, Arg, and His and acidic/amine side chains Asp, Glu, Asn, and Gln). In the secondary structure lines, s stands for a strand, and h stands for a helix.

 
To begin to study the relationship between the roles of Elongin C and Skp1 in reconstitution of their respective ubiquitin ligases, we sought to identify Elongin C sequences required for its interaction with Cul2 and for its recruitment of the Cul2/Rbx1 module into the VHL complex. To accomplish this, we constructed baculoviruses encoding a systematic series of Elongin C deletion mutants and analyzed the abilities of these Elongin C mutants to support the assembly of VBC and of the complete VBC-Cul2/Rbx1 ubiquitin ligase in Sf21 insect cells. Sf21 cells were coinfected with baculoviruses encoding VHL, Elongin B, Cul2, Rbx1, and wild type or mutant Elongin C with an N-terminal epitope tag recognized by the HPC4 monoclonal antibody. Elongin C-containing complexes were immunoprecipitated with HPC4 monoclonal antibodies and analyzed by Western blotting for the presence of individual subunits of the VHL complex. As shown in Fig. 2A, recruitment of Cul2 to the VHL complex was strongly dependent on both Elongins B and C. Elongin C was quite sensitive to deletions. Only three of the mutants could assemble similarly to wild type Elongin C into the VBC complex; two of these, {Delta}41–50 and {Delta}51–60, were defective in recruitment of Cul2 into the VHL ubiquitin ligase complex (Fig. 2B). These results suggested that Elongin C sequences between residues 41 and 60 are not crucial for VBC assembly but are important for Elongin C interaction with Cul2 and recruitment of Cul2 into the VHL ubiquitin ligase complex. Notably, some residues within the corresponding region of Skp1 appear to contribute to formation of the Skp1-Cul1 interface (43).



View larger version (83K):
[in this window]
[in a new window]
  		FIG. 2.
Identification of Elongin C (Elo C) mutations that reduce binding of Cul2 to the VHL-Elongin BC complex. A, Sf21 cells were coinfected with baculoviruses encoding the proteins indicated in the figure. Cell lysates and HPC4 immunoprecipitates (IP) were subjected to 9 or 13% SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting as described under "Experimental Procedures." Proteins detected in Western blots are indicated to the left of the figure. Myc-tagged Rbx1 was detected with anti-Myc antibody, and HPC4-tagged Elongin C was detected with HPC4 antibody. Elo B, Elongin B. B, Sf21 cells were coinfected with baculoviruses encoding the proteins indicated in the figure. Cell lysates and HPC4 immunoprecipitates were subjected to 9 or 13% SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting as described under "Experimental Procedures." Proteins detected in Western blots are indicated to the left of the figure. Myc-tagged Rbx1 was detected with anti-Myc antibody, and HPC4-tagged Elongin C was detected with HPC4 antibody. Visualization of Western blots was accomplished with a Storm gel and blot imaging system as described under "Experimental Procedures." wt, wild type.

 
To investigate further the role of Elongin C sequences 41–60 in recruitment of Cul2 into the VHL ubiquitin ligase, we carried out domain swaps between Elongin C and Skp1 in an effort to identify an Elongin C-Skp1 chimeric protein capable of recruiting a Cul1/Rbx1 module into the VHL complex. In one mutant, designated M2, Elongin C residues 40–47 were replaced with Skp1 residues 25–32. These stretches of sequence in Elongin C and Skp1 fall largely within conserved helix 2 (Fig. 1) (40, 44). A second Elongin C-Skp1 chimeric protein, designated M1, was created by replacing Elongin C residues 47–57 with Skp1 residues 32–42. Elongin C residues 47–57 form a loop (loop 3) between helix 2 and strand 3 in human Elongin C; loop 3 was poorly resolved in the Elongin C x-ray structures (40, 45). The corresponding sequences from Skp1 fall within a protease-sensitive region that is also proposed to form an unstructured loop between helix 2 and strand 3 and that was partially deleted from the recombinant Skp1 molecules used to crystallize SCFF-boxSKP2 and Skp1-Skp2 for structural studies (43, 44). The detailed structure of this region in both Skp1 and Elongin C remains elusive, and the sequence of loop 3 is not well conserved.

As shown in Fig. 3, VBC complexes containing M1 and M2 Elongin C mutants were both capable of assembling into VHL complexes with Cul2/Rbx1, although less efficiently than VBC complexes containing wild type Elongin C. In contrast, only complexes containing the Elongin C M1 mutant, in which the Elongin C flexible loop residues were replaced with the corresponding residues from Skp1, were capable of assembling into VHL complexes with Cul1/Rbx1. Taken together, these results argue that Elongin C sequences between amino acids 47 and 57 and Skp1 sequences between amino acids 32 and 42 are important for determining the specificity with which these adaptor proteins interact with their respective Cullins and for recruitment of Cullin/Rbx1 modules into their respective E3 ubiquitin ligase complexes.



View larger version (68K):
[in this window]
[in a new window]
  		FIG. 3.
Characterization of an Elongin C-Skp1 chimeric protein capable of recruiting a Cul1/Rbx1 module to the VHL-Elongin BC complex. Sf21 cells were coinfected with baculoviruses encoding the proteins indicated in the figure. Cell lysates and anti-HPC4 immunoprecipitates (IP) were subjected to 9 or 13% SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting as described under "Experimental Procedures." Proteins detected in Western blots are indicated to the left of the figure. HA-tagged Cullin proteins were detected with anti-HA antibody, Myc-tagged Rbx1 was detected with anti-Myc antibody, and HPC4-tagged Elongin C (Elo C) was detected with HPC4 antibody. Elo B, Elongin B; WT, wild type Elongin C; M1, Elongin C mutant M1; M2, Elongin C mutant M2.

 
Evidence from previous studies indicated that HIF1{alpha} ubiquitylation by the wild type VHL complex is carried out specifically by the E2 ubiquitin-conjugating enzyme Ubc5 (2, 3), whereas ubiquitylation of SCF target proteins is in most cases carried out by the E2 Cdc34 (30–34). The E2 ubiquitinconjugating enzyme is believed to be recruited to these ubiquitin ligases by interactions with the RING finger protein Rbx1. In the SCFF-boxSKP2 complex, Rbx1 interacts with other subunits of the complex only through the C-terminal domain of the long, rigid Cul1 molecule, whereas Skp1 and its associated substrate-binding F-box protein subunit are bound to the Cul1 N-terminal domain nearly 100 Å away (43). For these reasons, we considered the possibilities that (i) the Cullin protein might play a significant role in determining E2 selectivity and (ii) HIF1{alpha} ubiquitylation by the VHL ubiquitin ligase complex containing the altered specificity Elongin C M1 mutant and the Cul1/Rbx1 module might depend on Cdc34 rather than Ubc5. To address these possibilities and to confirm the functionality of the altered specificity Elongin C M1 mutant, we tested the ability of the VHL ubiquitin ligase containing either wild type Elongin C and Cul2/Rbx1 or the Elongin C M1 mutant and Cul1/Rbx1 to activate HIF1{alpha} ubiquitylation in reactions carried out with the E2 ubiquitin-conjugating enzymes human Ubc5a (hUbc5a), human Cdc34 (hCdc34), or yeast Cdc34 (yCdc34).

The recombinant E2 ubiquitin-conjugating enzymes hUbc5a, hCdc34, and yCdc34 used in these experiments were purified to near homogeneity from Escherichia coli (Fig. 4A) and assayed for their abilities to form E2-ubiquitin thioester conjugates in the presence of an E1 ubiquitin-activating enzyme, GST·ubiquitin, and ATP. As shown in the experiment in Fig. 4B, all three enzyme preparations were active in this assay, although the maximum level of E2-ubiquitin thioester conjugate detected with hCdc34 was lower than with yCdc34 or hUbc5a. In addition, hUbc5a supported VHL ubiquitin ligase-dependent ubiquitylation of prolyl-hydroxylated HIF1{alpha}, and both yCdc34 and hCdc34 supported SCFCdc4-dependent ubiquitylation of phosphorylated Sic1 (Fig. 4, C and D). We note that hUbc5a could also support SCFCdc4-dependent ubiquitylation of phosphorylated Sic1, albeit at a much reduced level when compared with yCdc34 and hCdc34.



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 4.
Characterization of E2 ubiquitin-conjugating enzymes used in this study. A, yCdc34, hCdc34, and hUbc5a, expressed in and purified from E. coli, were analyzed by SDS-polyacrylamide gel electrophoresis and visualized with Coomassie Brilliant Blue. B, ability of yCdc34, hCdc34, and hUbc5a to form thioester bonds. The indicated amounts of each E2 were incubated for 1 h at 25 °C with 50 ng of yeast Uba1 and 100 ng of His-T7-Xpress-ubiquitin in 10-µl reactions containing 40 mM Hepes-NaOH (pH 7.9), 60 mM potassium acetate, 2 mM dithiothreitol, 6 mM MgCl2, 0.5 mM EDTA (pH 8.0), 10% (v/v) glycerol, and 1.5 mM ATP. Reactions were stopped with 15 µl of buffer containing either 30 mM dithiothreitol (reducing) or 6 M urea (nonreducing). Reaction products were subjected to SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting (WB) with anti-T7 antibodies as described under "Experimental Procedures." Reaction products were visualized using the LI-COR Odyssey infrared imager as described under "Experimental Procedures." C, hUbc5a supports VBC-Cul2/Rbx1-dependent ubiquitylation of hydroxylated HIF1{alpha}. As described under "Experimental Procedures," purified His-HPC4-HIF1{alpha} was treated with wheat germ extract programmed with or without HA-EGLN1 prolyl hydroxylase, purified by Ni+2-agarose chromatography, and assayed in ubiquitylation reactions containing 500 ng of hUbc5a in the presence or absence of the purified VBC-Cul2/Rbx1 complex. Reaction mixtures were subjected to 8% SDS-polyacrylamide gel electrophoresis and analyzed as described under "Experimental Procedures" by Western blotting with HPC4 monoclonal antibody. D, yCdc34 and hCdc34 support SCFCdc4-dependent ubiquitylation of phosphorylated Sic1 (circled P indicates phosphorylation). Lane 1, phosphorylated HPC4-Sic1. Lanes 2–9, yCdc34 (60 ng), hCdc34 (110 ng), and hUbc5a (50 ng) were assayed for their abilities to support ubiquitylation of phosphorylated HPC4-Sic1 in the absence (lanes 2–5) or presence (lanes 6–9) of SCFCdc4, which had been immunopurified with anti-FLAG antibody from lysates of baculovirus-infected Sf21 cells expressing Cdc53, HSV-Cdc4, Myc-Rbx1, and FLAG-Skp1 as described (4). Reaction products were fractionated by SDS-polyacrylamide gel electrophoresis and analyzed as described under "Experimental Procedures" by Western blotting with HPC4 antibody. The band marked with an asterisk is a nonspecific band derived from FLAG immunoprecipitation.

 
In preliminary experiments, we found that it was difficult to reproducibly and in good yield prepare recombinant VHL complexes containing stoichiometric amounts of Cul2/Rbx1 or Cul1/Rbx1 by coexpression of VHL, Elongins B and C, Rbx1, and Cul2 or Cul1 in insect cells (data not shown). To reconstitute VHL ubiquitin ligase complexes, we therefore mixed recombinant heterotrimeric VBC with recombinant Cullin/Rbx1 complexes. Confirming the validity of this approach, hydroxylated HIF1{alpha} could be ubiquitylated with comparable efficiency by recombinant VHL complexes that had been expressed in and purified intact from insect cells or by VHL complexes reconstituted with a mixture of VBC and Cul2/Rbx1 complexes, which had been expressed and purified separately (Fig. 5).

As shown in Fig. 6, both wild type VHL complex and VHL complex containing the altered specificity Elongin C M1 mutant were capable of supporting HIF1{alpha} ubiquitylation. As expected, the VHL complex containing wild type Elongin C was much more active in HIF1{alpha} ubiquitylation in the presence of Cul2/Rbx1 than Cul1/Rbx1. VHL complex containing the Elongin C M1 mutant was active in HIF1{alpha} ubiquitylation in the presence of Cul1/Rbx1 but exhibited little activity in the presence of Cul2/Rbx1, although it could bind Cul2/Rbx1. Remarkably, HIF1{alpha} ubiquitylation was dependent on the E2 Ubc5a in all cases, despite previous studies indicating that ubiquitylation of target proteins by most SCF ubiquitin ligases containing the Cul1/Rbx1 module is carried out by the E2 Cdc34. Taken together, these findings demonstrated that VBC-dependent HIF1{alpha} ubiquitylation can be activated by a Cul1/Rbx1 module. In addition, they support the idea that the Cullin/Rbx1 module is not solely responsible for determining the E2 selectivity of Cullin-containing ubiquitin ligases, although the E2 is believed to dock on the Cullin/Rbx1 module at the C terminus of Cullin proteins, a relatively large distance away from other subunits of the ubiquitin ligase complex.



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 6.
Cul1-dependent ubiquitylation of HIF1{alpha} by hUbc5a. A, VBC complexes containing either wild type (WT) or M1 mutant Elongin C were preincubated for 60 min at 4 °C alone or in the presence of Cul1/Rbx1 or Cul2/Rbx1. The resulting mixtures were assayed for their abilities to support ubiquitylation of prolyl-hydroxylated HIF1{alpha} in the presence of 0.5 µg of hUbc5a or 5.4 µg of hCdc34. Reaction products were analyzed by Western blotting with anti-HIF1{alpha} antibody and visualized on film. B, VBC complexes containing either wild type or M1 mutant Elongin C were preincubated for 60 min at 4 °C in the presence of Cul1/Rbx1 or Cul2/Rbx1. The resulting mixtures were assayed for their abilities to support ubiquitylation of prolyl-hydroxylated HIF1{alpha} in the presence of the indicated amounts of yCdc34, hCdc34, or hUbc5a. Reaction products were analyzed by Western blotting with anti-HIF1{alpha} antibody and visualized using an Odyssey infrared imaging system (LI-COR Biosciences).

 
We next investigated the possibility that the identity of the target protein might influence E2 selectivity in our assays. In these experiments, we took advantage of a Cdc4-VHL chimeric substrate recognition subunit (46). The S. cerevisiae F-box protein Cdc4 interacts through its WD repeat domain with the S. cerevisiae Cdk inhibitor Sic1 and recruits it for ubiquitylation by the SCFCdc4 ubiquitin ligase. The Cdc4-VHL chimeric protein is composed of the entire 213-amino-acid human VHL protein fused to the C terminus of a Cdc4 deletion mutant containing residues 341–779, which includes the Cdc4 WD repeat domain but lacks the Cdc4 F-box (Fig. 7A). The Cdc4-VHL chimera binds stably to the Elongin BC complex through the VHL BC box but does not interact with Skp1 since it lacks an intact F-box (46).

The SCFCdc4 ubiquitin ligase targets phosphorylated Sic1 for ubiquitylation by the E2 ubiquitin-conjugating enzyme Cdc34 (21–23, 47). Cdc4 binds phosphorylated Sic1 via its WD repeat domain and recruits it to the SCFCdc4 complex for ubiquitylation by Cdc34. To determine whether the Cdc4-VHL chimeric protein can assemble into an E3 ubiquitin ligase containing Elongins B and C and a Cul2/Rbx1 module, Sf21 insect cells were coinfected with baculoviruses encoding the Cdc4-VHL chimera containing an N-terminal His6 tag, Elongins B and C, Cul2, and Rbx1. The Cdc4-VHL complex was purified from cell lysates by Ni2+-agarose chromatography followed by TSK DEAE 5-PW HPLC. As shown in the Coomassie-stained SDS-polyacrylamide gel of Fig. 7B, the Cdc4-VHL chimera could be purified as part of a multiprotein complex with roughly stoichiometric amounts of Elongins B and C, Cul2, and Rbx1. As shown in Fig. 7C, Sic1 ubiquitylation by the Cdc4-VHL ubiquitin ligase complex was strongly dependent on the E2 ubiquitin-conjugating enzyme Ubc5 rather than Cdc34. Sic1 ubiquitylation was dependent on the Cdc4 portion of the Cdc4-VHL chimeric protein since the purified VHL complex did not support Sic1 ubiquitylation in the presence of either Ubc5 (Fig. 7D) or Cdc34 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we investigate the functions of the Elongin C and Skp1 proteins in reconstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 structural elements responsible for selective interaction with their cognate Cullin/Rbx1 modules. In addition, using altered specificity Elongin C and F-box protein mutants, we investigate models for the mechanism underlying E2 selection by VHL and SCF ubiquitin ligases.

Our findings suggest that the ~11 amino acid loop 3 between helix 2 and strand 3 in the conserved three-dimensional structure of Elongin C and Skp1 (Fig. 1) plays an important role in Cullin selection by the VBC complex. Wild type Elongin C preferentially binds to Cul2. However, when loop 3 is replaced by its counterpart from Cul1-binding Skp1, the chimeric Elongin C M1 mutant can bind both Cul1 and Cul2, and it requires Cul1 for efficient HIF1{alpha} ubiquitylation.

Little is known about loop 3 structure and function. In the available structures of Elongin C (Protein Data Bank (PDB) accession numbers 1VCB [PDB] and 1LM8 [PDB] ), this region is disordered (40, 45), and in Skp1 (PDB accession number 1LDK [PDB] ), it appeared to interfere with crystallization and had to be deleted (43, 44). In an effort to gain better understanding of the function of loop 3, we rebuilt both the wild type loop and its replacement in Elongin C M1, using the corresponding sequences from either Elongin C or Skp1 with the isolated C chain of 1LM8 [PDB] as the template. The resulting model was docked onto Cul1 (the A chain of 1LDK [PDB] ) by global energy minimization or was homology-modeled onto the Skp1 structure (the C chain of 1LDK [PDB] ). The results obtained using both approaches are very similar and suggest that loop 3 might not be involved in a direct interaction with the Cullin protein (Fig. 8 and data not shown). In contrast with numerous direct, mainly hydrophobic interactions between helices that are conserved between Skp1 and Elongin C and between Cul1 and Cul2, respectively, all atoms in the predicted loop 3 structure in Elongin C M1 are separated by at least 6 Å from any atoms in Cul1, and this distance is even larger for the predicted loop 3 structure in wild type Elongin C.

What might be the molecular mechanism by which loop 3 affects the specificity with which Elongin C interacts with Cullin proteins? There are several possibilities that could not be addressed by our approximate structural model. First, loop 3 might adopt a folded or bent conformation in the process of Elongin C-Cullin interaction, which perhaps could produce additional contact(s) between Cul1 and the Elongin C M1 mutant but not between Cul1 and wild type Elongin C. Second, the ~6-Å distance between Elongin C M1 loop 3 and Cul1 could be bridged by a water molecule, thus producing additional Elongin C M1-specific interactions. Finally, we note that our model predicts that loop 3 of Elongin C M1 would contribute an extra hydrogen bond to interact with a lysine residue in the middle of strand 1 (data not shown), perhaps inducing a change in the packing of the structural core of the Elongin C M1 mutant. Experimental structure-function studies are needed to determine whether any of these possibilities contribute to the specificity of protein-protein interactions that we observed; what is clear is that the mechanism of loop 3-dependent Cullin selection is subtle.

By exploiting the altered specificity Elongin C M1 mutant, we have investigated the importance of the Cullin/Rbx1 module in E2 selection by the VHL ubiquitin ligase complex. Our observation that hUbc5a and not Cdc34 is the preferred E2 for ubiquitylation of prolyl-hydroxylated HIF1{alpha} directed by a Cul1-containing VHL ubiquitin ligase suggests that the Cullin/Rbx1 module is not solely responsible for determining the E2 selectivity of substrate-specific ubiquitylation. In addition, our observation that hUbc5a is the preferred E2 for ubiquitylation of phosphorylated Sic1 directed by an Elongin BC-based ubiquitin ligase containing a chimeric Cdc4-VHL substrate recognition subunit suggests that the target protein is not solely responsible for determining the E2 selectivity of the reaction. It could be argued that Sic1 presented to the E2 by the Cdc4-VHL ubiquitin ligase is not the same substrate as Sic1 presented to the E2 by SCFCdc4 since the position and orientation of Sic1 relative to the E2 could be different in the two cases. For several reasons, however, we do not favor this possibility. Comparison of the VHL-Elongin BC (1VBC [PDB] ) (40) and Skp1-Cdc4 (1NEX [PDB] ) (49) structures suggests that Sic1 could be positioned similarly in the two cases. The space occupied by the Cdc4 F-box and the F-box binding helices of Skp1 in the Skp1-Cdc4 structure is approximately the same as that occupied by VHL in the VHL-Elongin BC structure. Although the Cdc4 F-box is located N-terminal to the Sic1-binding WD repeat domain, and VHL is located C-terminal to the WD repeat in the Cdc4-VHL chimera, the N and C termini of the WD repeat domain emerge from the same face of the {beta}-propeller, suggesting that the Sic1 binding site is likely to be oriented similarly in SCFCdc4 and in the Cdc4-VHL ubiquitin ligase. Furthermore, high affinity binding of Sic1 to Cdc4 is due to cooperative interactions between any of several spatially separated, low affinity phosphobinding sites on Sic1 to a single site on Cdc4, suggesting that Sic1 can bind productively to Cdc4 in any of several orientations (48, 49).


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant R37 GM041628. 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. Tel.: 816-926-4091; Fax: 816-926-2091; E-mail: jlc{at}stowers-institute.org.

1 The abbreviations used are: VHL, von Hippel-Lindau; VBC, the VHL-Elongin BC complex; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; SCF, Skp1-Cul1/Cdc53-F-box; Ub, ubiquitin; h, human; E1, Ub-activating enzyme; HIF, hypoxia-inducible factor; HA, hemagglutinin; PDB, Protein Data Bank. Back


   ACKNOWLEDGMENTS
 
We thank Dr. William Kaelin (Dana-Farber Cancer Institute) for the EGLN1 expression vector and Dr. Ning Zheng (University of Washington School of Medicine) for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cockman, M. E., Masson, N., Mole, D. R., Jaakkola, P., Chang, G. W., Clifford, S. C., Maher, E. R., Pugh, C. W., Ratcliffe, P. J., and Maxwell, P. H. (2000) J. Biol. Chem. 275, 25733-25741[Abstract/Free Full Text]
  2. Iwai, K., Yamanaka, K., Kamura, T., Minato, N., Conaway, R. C., Conaway, J. W., Klausner, R. D., and Pause, A. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12436-12441[Abstract/Free Full Text]
  3. Kamura, T., Sato, S., Iwai, K., Czyzyk-Krezeska, M. F., Conaway, R. C., and Conaway, J. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10430-10435[Abstract/Free Full Text]
  4. Kamura, T., Koepp, D. M., Conrad, M. N., Skowyra, D., Moreland, R. J., Iliopoulos, O., Lane, W. S., Kaelin, W. G., Elledge, S. J., Conaway, R. C., Harper, J. W., and Conaway, J. W. (1999) Science 284, 657-661[Abstract/Free Full Text]
  5. Kamura, T., Sato, S., Haque, D., Liu, L., Kaelin, W. G., Conaway, R. C., and Conaway, J. W. (1998) Genes Dev. 12, 3872-3881[Abstract/Free Full Text]
  6. Zhang, J. G., Farley, A., Nicholson, S. E., Willson, T. A., Zugaro, L. M., Simpson, R. J., Moritz, R. L., Cary, D., Richardson, R., Hausmann, G., Kile, B. J., Kent, S. B., Alexander, W. S., Metcalf, D., Hilton, D. J., Nicola, N. A., and Baca, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2071-2076[Abstract/Free Full Text]
  7. Kamura, T., Burian, D., Yan, Q., Schmidt, S. L., Lane, W. S., Querido, E., Branton, P. E., Shilatifard, A., Conaway, R. C., and Conaway, J. W. (2001) J. Biol. Chem. 276, 29748-29753[Abstract/Free Full Text]
  8. Kibel, A., Iliopoulos, O., DeCaprio, J. A., and Kaelin, W. G. (1995) Science 269, 1444-1446[Abstract/Free Full Text]
  9. Aso, T., Haque, D., Barstead, R. J., Conaway, R. C., and Conaway, J. W. (1996) EMBO J. 15, 5557-5566[Medline] [Order article via Infotrieve]
  10. Maxwell, P. H., Wiggener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., and Ratcliffe, P. J. (1999) Nature 399, 271-275[CrossRef][Medline] [Order article via Infotrieve]
  11. Lisztwan, J., Imbert, G., Wirbelauer, C., Gstaiger, M., and Krek, W. (1999) Genes Dev. 13, 1822-1833[Abstract/Free Full Text]
  12. Semenza, G. L. (2000) Annu. Rev. Cell Dev. Biol. 15, 551-578[CrossRef]
  13. Huang, L. E., Gu, J., Schau, M., and Bunn, H. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7987-7992[Abstract/Free Full Text]
  14. Kallio, P. J., Wilson, W. J., O'Brien, S., Makino, Y., and Poellinger, L. (1999) J. Biol. Chem. 274, 6519-6525[Abstract/Free Full Text]
  15. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, J. M., Lane, W. S., and Kaelin, W. G. (2001) Science 292, 464-468[Abstract/Free Full Text]
  16. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., Griegsheim, A. V., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468-472[Abstract/Free Full Text]
  17. Yu, F., White, S. B., Zhao, Q., and Lee, F. S. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9630-9635[Abstract/Free Full Text]
  18. Bruick, R. K., and McKnight, S. L. (2001) Science 294, 1337-1340[Abstract/Free Full Text]
  19. Epstein, A. C., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O'Rourke, J., Mole, D. R., Mukherji, M., Metzen, E., Wilson, M. I., Dhanda, A., Tian, Y. M., Masson, N., Hamilton, D. L., Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh, C. W., Schofield, C. J., and Ratcliffe, P. (2001) Cell 107, 43-54[CrossRef][Medline] [Order article via Infotrieve]
  20. Ivan, M., Haberberger, T., Gervasi, D. C., Michelson, K. S., Gunzler, V., Kondo, K., Yang, H., Sorokina, I., Conaway, R. C., Conaway, J. W., and Kaelin, W. G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 13459-13464[Abstract/Free Full Text]
  21. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J. W., and Elledge, S. J. (1996) Cell 86, 263-274[CrossRef][Medline] [Order article via Infotrieve]
  22. Feldman, R. M., Correll, C. C., Kaplan, K. B., and Deshaies, R. J. (1997) Cell 91, 221-230[CrossRef][Medline] [Order article via Infotrieve]
  23. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J., and Harper, J. W. (1997) Cell 91, 209-219[CrossRef][Medline] [Order article via Infotrieve]
  24. Lyapina, S. A., Correll, C. C., Kipreos, E. T., and Deshaies, R. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7451-7456[Abstract/Free Full Text]
  25. Patton, E. E., Willems, A. R., Sa, D., Kuras, L., Thomas, D., Craig, K. L., and Tyers, M. (1998) Genes Dev. 12, 692-705[Abstract/Free Full Text]
  26. Skowyra, D., Koepp, D. M., Kamura, T., Conrad, M. N., Conaway, R. C., Conaway, J. W., Elledge, S. J., and Harper, J. W. (1999) Science 284, 662-665[Abstract/Free Full Text]
  27. Ohta, T., Michel, J. J., Schottelius, A. J., and Xiong, Y. (1999) Mol. Cell 3, 535-541[CrossRef][Medline] [Order article via Infotrieve]
  28. Tan, P., Fuchs, S. Y., Chen, A., Wu, K., Gomez, C., Ronai, Z., and Pan, Z. Q. (1999) Mol. Cell 3, 527-533[CrossRef][Medline] [Order article via Infotrieve]
  29. Seol, J. H., Feldman, R. M., Zachariae, W., Shevchenko, A., Correll, C. C., Lyapina, S. A., Chi, Y., Galova, M., Claypool, J., Sandmeyer, S., Nasmyth, K., and Deshaies, R. J. (1999) Genes Dev. 13, 1614-1626[Abstract/Free Full Text]
  30. Deshaies, R. J. (1999) Annu. Rev. Cell Dev. Biol. 15, 435-467[CrossRef][Medline] [Order article via Infotrieve]
  31. Pickart, C. (2001) Annu. Rev. Biochem. 70, 503-533[CrossRef][Medline] [Order article via Infotrieve]
  32. Yaron, A., Hatzubai, A., Davis, M., Lavon, I., Amit, S., Manning, A. M., Andersen, J. S., Mann, M., Mercurio, F., and Ben-Neriah, Y. (1998) Nature 396, 590-594[CrossRef][Medline] [Order article via Infotrieve]
  33. Oh, K. J., Kalinina, A., Wang, J., Nakayama, K., Nakayama, K. I., and Bagchi, S. (2004) J. Virol. 78, 5338-5346[Abstract/Free Full Text]
  34. Strack, P., Caligiuri, M., Pelletier, M., Boisclair, M., Theodoras, A., Beer-Romero, P., Glass, S., Parsons, T., Copeland, R. A., Auger, K. R., Benfield, P., Brizuela, L., and Rolfe, M. (2000) Oncogene 19, 3529-3536[CrossRef][Medline] [Order article via Infotrieve]
  35. Stearns, D. J., Kurosawa, S., Sims, P. J., Esmon, N. L., and Esmon, C. T. (1988) J. Biol. Chem. 263, 826-832[Abstract/Free Full Text]
  36. Garrett, K. P., Aso, T., Bradsher, J. N., Foundling, S. I., Lane, W. S., Conaway, R. C., and Conaway, J. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7172-7176[Abstract/Free Full Text]
  37. Schuler, G. D., Altschul, S. F., and Lipman, D. J. (1991) Proteins Struct. Funct. Genet. 9, 180-190[CrossRef][Medline] [Order article via Infotrieve]
  38. Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003) Nucleic Acids Res. 31, 3381-3385[Abstract/Free Full Text]
  39. Katchalski-Katzir, E., Shariv, I., Eisenstein, M., Friesem, A. A., Alflalo, C., and Vakser, I. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2195-2199[Abstract/Free Full Text]
  40. Stebbins, C. E., Kaelin, W. G., and Pavletich, N. P. (1999) Science 284, 455-461[Abstract/Free Full Text]
  41. Pause, A., Peterson, B., Schaffar, G., Stearman, R., and Klausner, R. D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9533-9538[Abstract/Free Full Text]
  42. Wu, K., Fuchs, S. Y., Chen, A., Tan, P., Gomez, C., Ronai, Z., and Pan, Z. Q. (2000) Mol. Cell. Biol. 20, 1382-1393[Abstract/Free Full Text]
  43. Zheng, N., Schulman, B. A., Song, L., Miller, J. J., Jeffrey, P. D., Wang, P., Chu, C., Koepp, D. M., Elledge, S. J., Pagano, M., Conaway, R. C., Conaway, J. W., Harper, J. W., and Pavletich, N. P. (2002) Nature 416, 703-709[CrossRef][Medline] [Order article via Infotrieve]
  44. Schulman, B. A., Carrano, A. c., Jeffrey, P. D., Bowen, Z., Kinnucan, E. R. E., Finnin, M. S., Elledge, S. J., Harper, J. W., Pagano, M., and Pavletich, N. P. (2000) Nature 408, 381-386[CrossRef][Medline] [Order article via Infotrieve]
  45. Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., and Pavletich, N. P. (2002) Science 296, 1886-1889[Abstract/Free Full Text]
  46. Kamura, T., Brower, C. S., Conaway, R. C., and Conaway, J. W. (2002) J. Biol. Chem. 277, 30388-30393[Abstract/Free Full Text]
  47. Schwob, E., Boehm, T., Mendenhall, M. D., and Nasmyth, K. (1994) Cell 79, 233-244[CrossRef][Medline] [Order article via Infotrieve]
  48. Nash, P., Tang, X., Orlicky, S., Chen, Q., Gertler, F. B., Mendenhall, M. D., Sicheri, F., Pawson, T., and Tyers, M. (2001) Nature 414, 514-521[CrossRef][Medline] [Order article via Infotrieve]
  49. Orlicky, S., Tang, X., Willems, A., Tyers, M., and Sicheri, F. (2003) Cell 112, 243-256[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 FASEB J.Home page
G. Zhou, L. A. Dada, N. S. Chandel, K. Iwai, E. Lecuona, A. Ciechanover, and J. I. Sznajder
Hypoxia-mediated Na-K-ATPase degradation requires von Hippel Lindau protein
FASEB J, May 1, 2008; 22(5): 1335 - 1342.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
J. C. Aon, R. J. Caimi, A. H. Taylor, Q. Lu, F. Oluboyede, J. Dally, M. D. Kessler, J. J. Kerrigan, T. S. Lewis, L. A. Wysocki, et al.
Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli
Appl. Envir. Microbiol., February 15, 2008; 74(4): 950 - 958.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/41/43019    most recent
M408018200v1
Right arrow Alert me when this article is cited