The von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins

Abstract

The inactivation of the von Hippel-Lindau (VHL) gene predisposes affected individuals to VHL syndrome and is an early genetic event associated with sporadic renal cell carcinoma and CNS hemangioblastomas. The VHL protein (pVHL) has been shown to form a stable complex with elongin B and elongin C, two factors that stabilize and activate the transcription elongation factor elongin A. Here, Hs-CUL-2, a member of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-elongin B-C (VBC) complex in vitro and in vivo. Nearly 70% of naturally occurring cancer-predisposing mutations of VHL disrupt this interaction. The pVHL-Hs-CUL-2 association is strictly dependent on the integrity of the trimeric VBC complex. Immunofluorescence studies show Hs-CUL-2 to be a cytosolic protein that can be translocated to the nucleus by pVHL. Recently it has been shown that a yeast Hs-CUL-2 homolog, Cdc53, is part of a ubiquitin protein ligase complex that targets cell cycle proteins for degradation by the ubiquitin proteolytic pathway. In Caenorhabditis elegans, a null mutation of another Hs-cul-2 homolog, Ce-cul-1, results in hyperplasia in all tissues and is required for cell cycle exit. Hence, Hs-cul-2 may be required for VHL function and, therefore, may be a candidate human tumor-suppressor gene.

Figure 1

Immunoprecipitation analysis of different recombinant pVHL complexes. pVHL complexes VHL-F, F-VHL, and F-VHL-157Δ were renatured in the presence or absence of recombinant elongin B-C followed by immunoprecipitation with M2 antibody and analysis on a SDS/14% polyacrylamide gel stained with Coomassie Blue (F, FLAG epitope). The bands at 55 and 30 kDa, present in all lanes, represent the heavy and light IgG chains, respectively.

Figure 2

Immunoaffinity purification of pVHL-associated proteins using recombinant pVHL complexes. Different pVHL complexes were incubated with a lysate of [³⁵S]methionine-labeled 786-0 RCC cells and precipitated with M2 resin, followed by analysis on a SDS/14% polyacrylamide gel and autoradiography.

Figure 3

pVHL associates with p76 in the cytosol of cells. Lanes 1–4 and 8, VBC complexes were incubated with lysates of [³⁵S]methionine-labeled 786-0 RCC cells and precipitated with M2 resin. The precipitates in lanes 3 and 4 were washed in the presence of 0.5% Sarkosyl or 1 M NaCl, respectively. Lanes 5–7, HeLa cells expressing either rat VHL-F, human VHL-F, or a R167W mutant VHL-F were labeled with [³⁵S]methionine and lysed, and pVHL was immunoprecipitated with M2 antibody. Lanes 9–12, HeLa cells expressing the rat VHL protein were labeled with [³⁵S]methionine and fractionated into a nuclear fraction (600 × g pellet), cytosolic fraction (100,000 × g supernatant), and a membrane fraction (100,000 × g pellet). Total lysate (lane 12) and subcellular fractions were solubilized in Triton X-100 lysis buffer and immunoprecipitated with M2 antibody. All reactions were analyzed on a split SDS/polyacrylamide gel (7% on the top/14% on the bottom) followed by autoradiography.

Figure 4

Amino acid sequence comparison between predicted full-length human CUL-2 and C. elegans CUL-2 proteins. Predicted sequences of the human Hs-CUL-2 (745 aa) and C. elegans Ce-CUL-2 (743 aa) are shown. Amino acids identical between human and C. elegans CUL-2 proteins are boxed.

Figure 5

(A) Coimmunoprecipitation of VBC with Hs-CUL-2 but not with Hs-CUL-1 in vitro. Various combinations of the cDNAs encoding human F-VHL, elongin B, elongin C, Hs-CUL-1-HA, and HS-CUL-2-HA were expressed in a coupled transcription–translation system in the presence of [³⁵S]methionine. The translation products were immunoprecipitated with either M2 or HA antibodies and analyzed on a SDS/14% polyacrylamide gel followed by autoradiography. (B) Coimmunoprecipitation of Hs-CUL-2 with wild-type (wt) or mutant VHL (R167W, 157Δ) in vitro. cDNAs encoding human wt F-VHL, R167W, and 157Δ mutant VHL, elongin B, elongin C, and Hs-CUL-2-HA were expressed in a coupled transcription–translation system in the presence of [³⁵S]methionine. The translation products were immunoprecipitated with M2 antibody and analyzed on a SDS/14% polyacrylamide gel, followed by autoradiography.

Figure 6

Colocalization of Hs-CUL-2 and pVHL-GFP in transfected cells. COS-7 cells were transiently transfected with cDNAs encoding Hs-CUL-2-HA or pVHL-GFP (wt) separately (A) or concurrently (B–E). (C–E) COS-7 cells were transfected with cDNAs encoding Hs-CUL-2-HA and mutant pVHL-GFP [Δ60 (C and D) and 157Δ (E)]. Cells were stained with the anti-HA antibody to detect Hs-CUL-2-HA or observed under fluorescein isothiocyanate illumination to detect pVHL-GFP.