pVHL19 is a biologically active product of the von Hippel-Lindau gene arising from internal translation initiation

Abstract

The von Hippel-Lindau (VHL) gene encodes a protein consisting of 213 amino acid residues with an apparent molecular mass of 30 kDa (pVHL30). Here we show that cells also produce a VHL protein (pVHL19) that appears to arise as a result of internal translation from the second methionine within the VHL ORF. pVHL30 resides primarily in the cytosol, with less amounts found in the nucleus or associated with cell membranes. In contrast pVHL19, in biochemical fractionation experiments, is equally distributed between the nucleus and cytosol and is not found in association with membranes. pVHL19, like pVHL30, can bind to elongin B, elongin C, and Hs-Cul2 in coimmunoprecipitation assays and can inhibit the production of hypoxia-inducible proteins such as vascular endothelial growth factor (VEGF) and GLUT1 when reintroduced into renal carcinoma cells that lack a wild-type VHL allele. Thus, cells contain two biologically active VHL gene products.

Figure 1

Identification of pVHL₁₉ in vivo. (A) 786-O VHL(−/−) renal carcinoma cells were transfected with plasmid encoding pVHL₁₉ (lanes 3 and 4) or with the backbone expression plasmid (lines 1 and 2). The transfected cells and 293 human embryonic kidney cells (lines 5 and 6) were labeled with ³⁵S, lysed, and immunoprecipitated with control or IG32 monoclonal anti-VHL antibody as indicated. Bound proteins were detected by immunoblot analysis with affinity purified polyclonal anti-VHL antibody. (B) The ³⁵S-labeled pVHL₁₉ bands corresponding to lanes 4 and 5 of A were excised and digested with the indicated amounts of α-chymotryspin (micrograms) and V8 protease (nanograms). pVHL₁₉ translated in vitro was digested in parallel. Digestion products were resolved by SDS/PAGE and detected by autoradiography.

Figure 2

Pulse-chase analysis of pVHL. 293 human embryonic kidney cells were pulse radiolabeled with [³⁵S]methionine. Cell extracts were prepared at the indicated time points after chase with unlabeled methionine and immunoprecipitated with an anti-VHL mAb (IG32) under antibody excess conditions (lanes 1–6). In parallel, 293 cells were radiolabeled with [³⁵S]methionine under steady-state conditions and immunoprecipitated with control (lane 7) or anti-VHL(IG32) (lane 8) antibody. Bound proteins were resolved by SDS/PAGE and detected by fluorography. Positions of pVHL₃₀ and pVHL₁₉, as confirmed by anti-VHL immunoblots performed in parallel, are shown by arrows.

Figure 3

Subcellular localization of pVHL₁₉. pVHL was immunoprecipitated with preimmune (lane 1) or affinity purified anti-VHL serum (lanes 2–5) from 293 whole-cell extracts (W, lanes 1 and 2) or the indicated cellular fractions [nuclei (N) (lane 3), membranes (M) (lane 4), and cytosol (C) (lane 5)]. The insoluble pellets following extraction of 293 cells (W) (lane 6), 293 nuclei (N) (lane 10), 293 cell membranes (M) (lane 7), as well as pellets from the corresponding insoluble pellets from 786-O VHL(−/−) cells [(N) (lane 8) and (M) (lane 9)] were boiled in SDS-containing protein running buffer. Immunoprecipitated (lanes 1–5) and resolubilized (lanes 6–10) proteins were resolved by SDS/PAGE and detected by immunoblot analysis with R98 anti-VHL antibody. Bound antibody was detected colorimetrically. The identities of the higher molecular weight species in lanes 6, 7, and 10 are unknown.

Figure 4

pVHL₁₉-associated proteins. Stable 786-O renal carcinoma cell clones expressing HA-pVHL₃₀ (lanes 3 and 4), HA-pVHL₁₉ (lanes 5 and 6), or backbone vector transfectants (lanes 1 and 2) were labeled with [³⁵S]methionine, lysed, and immunoprecipitated with control or anti-HA mAb, as indicated. Bound proteins were detected by autoradiography.

Figure 5

Negative regulation of hypoxia-inducible proteins by pVHL₁₉. (A) Concentration of VEGF in the supernatant of the 786-O VHL(−/−) renal carcinoma cell clones ectopically producing HA-pVHL₃₀, HA-pVHL₁₉, or backbone vector transfectants (pRC) 1, 2 and 6 h after medium change. VEGF concentrations (pg/ml) were normalized to total cellular protein. (B) Whole-cell extracts (50 μg per lane) prepared from the above cells were resolved by SDS/PAGE and immunoblotted with anti-GLUT1 polyclonal (Upper) or anti-HA monoclonal (Lower) antibodies. Arrows indicate ectopically produced VHL proteins.