Abstract
The von Hippel-Lindau tumor suppressor (pVHL) targets hydroxylated alpha-subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated proteasomal destruction through direct interaction with the hydroxyproline binding pocket in its beta-domain. Although disruption of this process may contribute to VHL-associated tumor predisposition by up-regulation of HIF target genes, genetic and biochemical analyses support the existence of additional functions, including a role in the assembly of extracellular matrix. In an attempt to delineate these pathways, we searched for novel pVHL-binding proteins. Here we report a direct, hydroxylation-dependent interaction with alpha-chains of collagen IV. Interaction with pVHL was also observed with fibrillar collagen chains, but not the folded collagen triple helix. The interaction was suppressed by a wide range of tumor-associated mutations, including those that do not disturb the regulation of HIF, supporting a role in HIF-independent tumor suppressor functions.
Highlights
PVHL was observed with fibrillar collagen chains, but not the folded collagen triple helix
cell renal carcinomas (CCRC) cell line expressing VHLHA (RCC4-VHLHA), several co-immunoprecipitating species were identified by MS/MS
A similar pVHL binding species has previously been visualized by 35S labeling in RCC4 and 786-O cells [9, 10, 19, 21] and, in 786-O cells, assigned as fibronectin [12]
Summary
⬃220-kDa protein identified as ␣1- and ␣2-chains of collagen IV has an asterisk. Nonspecific bands are marked CA630 with “Complete” Protease ns. C, autoradiograph of 35S-labeled inhibitor (Roche) and 25 M proteins resolved by 8 –16% gradient SDS-PAGE. Lanes 7–12 display proteins re-immunoprecipitated by the indicated “Input” 8 g of cell extract was used, antibodies after HA peptide elution of anti-HA IP (lanes 4 and 5). Immunoprecipitates were washed five times with Igetumor-associated mutations in a manner that is concordant pal buffer and either analyzed directly or subjected to glycine elution and trichloroacetic acid precipitation Plasmids, Transfections, and 35S Labeling—HA-tagged at 95 °C), and SDS-PAGE. For HIF-1␣, V5 (PK tag), collagen wild type or mutant pVHL plasmids, empty vector (pcDNA3), I, and collagen IV immunoblotting, samples were first and the stably transfected RCC4 sublines have been described resolved by 7.5% SDS-PAGE. Human embryonic kidney 293T cells were grown under tubulin immunoblotting 15% SDS-PAGE was used. MS/MS spectra were matched to the SwissProt data base using the Mascot algorithm (Matrix Science)
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