To look for pVHL-binding proteins, 786-O [VHL (−/−)], renal carcinoma cells that were stably transfected with a plasmid encoding HA-tagged pVHL (Fig. A, lanes 3 and 4) or the backbone expression plasmid (Fig. A, lanes 1 and 2) were metabolically labelled with [35
S]methionine, lysed, and immunoprecipitated with an anti-HA antibody (Fig. A, lanes 2 and 4) or a control antibody (Fig. A, lanes 1 and 3) under stringent conditions. Bound proteins were detected by autoradiography (Fig. A) or immunoblot analysis (Fig. C). pVHL coimmunoprecipitated with cellular proteins with molecular masses of ~200, 70, 18, and 15 kDa (compare Fig. A, lane 4, to lanes 2 and 3). These proteins were also detected in monoclonal and polyclonal anti-VHL immunoprecipitates but not in isotype-matched control immunoprecipitates (data not shown). The 18- and 15-kDa proteins were shown previously to be elongins B and C, respectively (6
). The characterization of p200 will be described elsewhere.
FIG. 1 Coimmunoprecipitation of Cul2 with ectopically produced pVHL. (A and C) VHL (−/−) renal carcinoma cells stably transfected with a plasmid encoding HA-tagged pVHL (lanes 3 and 4) or with the backbone expression plasmid (lanes 1 and 2) were (more ...)
p70 was purified by preparative anti-HA immunoelectrophoresis and subjected to microsequence analysis following tryptic digestion. Three peptide sequences that were present in the conceptual open reading frame of the human Cul2
gene were obtained (23
). A full-length human Cul2 cDNA clone was isolated by screening a cDNA library with the available partial human Cul2 cDNA clone and was translated in vitro. The major Cul2 in vitro translation product comigrated with p70 (Fig. A, compare lanes 4 and 5), and the partial proteolytic peptide maps of these two proteins generated with α-chymotrypsin (Fig. B) and Staphylococcus aureus
V8 protease (data not shown) were identical. In some experiments, translation of Cul2, whether in vitro or in vivo, also gave rise to a second band that migrated more slowly in SDS-polyacrylamide gels (see, for example, Fig. C and ). The nature of this second band is currently under investigation. Finally, both p70 and the Cul2 in vitro translation product were specifically recognized by a polyclonal anti-Cul2 antibody in Western blot assays (Fig. C). Thus, pVHL, at least when overproduced, can bind to human Cul2. While these experiments were in progress, the same conclusion was reported by Pause and coworkers (38
FIG. 4 Cul2 binds to elongin C in the absence of pVHL. VHL (−/−) renal carcinoma cells stably transfected with a plasmid encoding HA-tagged pVHL (lanes 3 and 4), T7 epitope-tagged elongin C (lanes 5 and 6), or the backbone expression plasmid (more ...)
To ask whether pVHL and Cul2 might associate under physiological conditions, 293 human embryonic kidney cells containing endogenous, wild-type pVHL (18
) were immunoprecipitated with anti-VHL antibody or a control antibody (Fig. A). The stable transfectants described above were analyzed in parallel. As expected, anti-VHL but not control 293 cell immunoprecipitates contained wild-type pVHL, as determined by anti-VHL immunoblot analysis (Fig. A, compare lanes 5 and 6). Two species of pVHL (pVHL30
) were detected in these and other cells (data not shown). pVHL19
results from translation initiation at an internal methionine codon (17a
). In addition, anti-Cul2 immunoblot analysis confirmed the presence of Cul2 specifically in the anti-VHL immunoprecipitate (compare Fig. A, lane 5, to lanes 4 and 6). The available anti-Cul2 sera do not immunoprecipitate Cul2 efficiently from mammalian cell extracts (data not shown). Thus, the reciprocal experiment, in which an anti-Cul2 immunoprecipitate would be analyzed for the presence of pVHL, cannot yet be performed.
FIG. 2 Copurification of Cul2 and endogenous pVHL. (A) 293 [VHL (+/+)] human embryonic kidney cells (lanes 5 and 6) and VHL (−/−) renal carcinoma cells stably transfected with a plasmid encoding HA-tagged pVHL (more ...)
In addition, a stable multiprotein complex containing endogenous pVHL, Cul2, and elongins B and C could be extensively purified from rat liver cytosolic extracts according to the procedure outlined in Fig. B. Analysis of column fractions from the final two steps in the purification of this complex revealed close cochromatographic elution of pVHL with Cul2 and elongin B (Fig. C and D) as well as elongin C (data not shown). The coimmunoprecipitation and chromatographic data, taken together, suggest that pVHL-Cul2 complexes exist naturally in cells.
The minimal region of pVHL capable of binding to elongins B and C, at least in vitro, corresponds to residues 157 to 172 (22
). To map the region of pVHL required for binding to Cul2, 786-O subclones that stably produced N-terminal HA epitope-tagged pVHL mutants were metabolically labelled with [35
S]methionine, lysed, and immunoprecipitated with an anti-HA antibody under stringent conditions (Fig. A). Comparable recovery of each of the pVHL mutants was confirmed by anti-HA immunoblot analysis (Fig. A, middle panel). Elongins B and C were detected by autoradiography (lower panel), and Cul2 was detected by anti-Cul2 immunoblot analysis (upper panel). In keeping with the earlier in vitro mapping studies, pVHL(1-197) and pVHL(94-213) retained the ability to bind to elongins B and C, whereas pVHL(1-167) did not. pVHL(1-187), surprisingly, did not stably associate with elongins B and C, suggesting that residues C terminal to pVHL residue 172 contribute to elongin B and C binding in vivo. The ability of pVHL to bind to Cul2 mirrored its ability to bind to elongins B and C in these assays (compare upper and lower panels in Fig. A).
FIG. 3 Binding of Cul2 and elongins to pVHL mutants. (A) Deletion mutants; (B) missense mutants. VHL (−/−) renal carcinoma cells ectopically producing the indicated HA-tagged pVHL species were metabolically labelled with 35S, lysed, and immunoprecipitated (more ...)
pVHL(157-172), a synthetic peptide, blocks the binding of pVHL to elongins B and C in vitro (22
). Scanning mutagenesis of this peptide, in which single amino acid residues were converted to either alanine or a residue corresponding to a tumor-derived mutation, suggested that T157, L158, and C162 were likely to form critical contacts between pVHL and the elongins (4a
). We therefore generated stable 786-O renal carcinoma subclones that ectopically produced HA-tagged versions of pVHL(L158S), pVHL(C162F), or pVHL(157-162NAAIRS). The former two correspond to naturally occuring VHL gene mutations (48
). The latter replaces pVHL residues 157 to 162 with the sequence NAAIRS, which is believed to be highly flexible based on its appearance in both alpha-helical and beta-sheet structures (34
). These three mutants were, as expected, unable to bind to elongins B and C (Fig. B and data not shown). In addition, none of these mutants bound to Cul2.
pVHL binding to elongins B and C appeared, based on these studies, to be necessary and sufficient for binding to Cul2, raising the possibility that Cul2 can bind to elongins B and C in the absence of pVHL. To test this hypothesis, 786-O cells that stably produced a T7 epitope-tagged version of elongin C were immunoprecipitated with an anti-T7 antibody or a control antibody (Fig. ). In parallel, 786-O cells that stably produced HA-pVHL were immunoprecipitated with an anti-HA antibody or a control antibody. Bound proteins were detected by anti-Cul2 (Fig. , upper panel), anti-HA (middle panel), or anti-T7 (lower panel) immunoblot analysis. Cul2 coimmunoprecipitated with elongin C (compare Fig. , lane 5, to lanes 4 and 6). As 786-O cells lack endogenous, wild-type pVHL, this result suggests that elongin C can, at least indirectly, bind to Cul2 in the absence of pVHL.
Cul2 and elongin C share significant sequence similarity with Saccharomyces cerevisiae
proteins Cdc53 and Skp1, respectively (Fig. and ) (see Discussion) (2
). The potential significance of the elongin C similarity to Skp1 is underscored by the following observation. A peptide containing elongin C residues 18 to 50, produced as either a GST fusion protein (Fig. ) or a synthetic peptide (data not shown), was sufficient to bind to elongin B. An earlier study had shown that elongin C residues 21 to 30 were necessary for binding to elongin B (42
). Thus, the region of highest similarity between elongin C and Skp1, corresponding to elongin C residues 18 to 50, is a functional domain.
Similarity of human Cul2 to S. cerevisiae Cdc53 protein. Identical amino acid residues are shaded with black.
FIG. 6 The region of elongin C that is most similar to Skp1 is an elongin B-binding domain. (A) Similarity of elongin C to Skp1. Identical amino acid residues are shaded with black. Elongin C residues 18 to 50 are underlined. (B) Binding of radiolabelled elongin (more ...)
pVHL plays a critical role in the degradation of hypoxia-inducible mRNAs (11
). Consequently, cells lacking pVHL overproduce proteins such as VEGF and the Glut1 glucose transporter, which are both encoded by hypoxia-inducible mRNAs (11
). To determine whether this activity might be linked to the ability of pVHL to bind to elongins and Cul2, 786-O subclones producing the various pVHL mutants described above were tested for Glut1 protein production by steady-state anti-Glut1 immunoblot analysis (Fig. ). Three or more independent clones were tested for each pVHL mutant. In parallel, total RNA was harvested from representative clones and analyzed for Glut1 and VEGF mRNA abundance by Northern blot analysis (Fig. ). It was shown previously that the inhibition of VEGF mRNA accumulation by pVHL leads to a commensurate decrease in VEGF protein production (11
FIG. 7 Inhibition of Glut1 protein production by pVHL mutants. Whole-cell extracts (~50 μg of protein per lane, as determined by the Bradford method) prepared from VHL (−/−) renal carcinoma cells ectopically producing the indicated (more ...)
FIG. 8 Inhibition of hypoxia-inducible mRNA accumulation by pVHL mutants. Total RNA was isolated from VHL (−/−) renal carcinoma cells ectopically producing the indicated HA-tagged pVHL species and analyzed by Northern hybridization with radiolabelled (more ...)
Wild-type pVHL, as expected, inhibited Glut1 and VEGF expression, whereas pVHL(1-167) did not (Fig. and ). Similar results were obtained following the reintroduction of wild-type pVHL and pVHL(1-167) into A498 VHL (−/−) renal carcinoma cells (data not shown). pVHL(1-197), which did not bind to p200 (data not shown) but which retained the ability to bind to elongins B and C and Cul2 (Fig. A), also inhibited Glut1 and VEGF expression (Fig. and ). These results suggested that the binding of pVHL to elongins and Cul2 might be linked to its ability to regulate Glut1 and VEGF. In keeping with this view, all of the missense mutants that were unable to bind to elongins and Cul2 were likewise unable to inhibit Glut1 and VEGF mRNA accumulation. Some, but not all, clones producing pVHL(72-213) produced levels of Glut1 comparable to those produced by cells producing wild-type pVHL. The reason for this clonal variation is unclear (shown in Fig. and are data for clones in which Glut1 production was inhibited). Finally, pVHL(94-213), although able to bind to elongins and Cul2, was unable to inhibit Glut1 and VEGF expression (Fig. and ). The simplest interpretation of these data, summarized in Fig. , is that binding to elongins and Cul2 is necessary but not sufficient for pVHL to regulate hypoxia-inducible genes.
FIG. 9 Summary of elongin-Cul2 binding and Glut1 inhibition by pVHL mutants. For Glut1 inhibition, + indicates that all clones tested produced Glut1 at levels similar to those in cells producing wild-type (wt) pVHL, − indicates that all clones (more ...)