pVHL modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development

Abstract

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is the cause of the familial VHL disease and most sporadic renal clear-cell carcinomas (RCC). pVHL has been shown to play a role in the destruction of hypoxia-inducible factor alpha (HIF-alpha) subunits via ubiquitin-mediated proteolysis and in the regulation of fibronectin matrix assembly. Although most disease-causing pVHL mutations hinder the regulation of the HIF pathway, every disease-causing pVHL mutant tested to date has failed to promote the assembly of the fibronectin matrix, underscoring its potential importance in VHL disease. Here, we report that a ubiquitin-like molecule called NEDD8 covalently modifies pVHL. A nonneddylateable pVHL mutant, while retaining its ability to ubiquitylate HIF, failed to bind to and promote the assembly of the fibronectin matrix. Expression of the neddylation-defective pVHL in RCC cells, while restoring the regulation of HIF, failed to promote the differentiated morphology in a three-dimensional growth assay and was insufficient to suppress the formation of tumors in SCID mice. These results suggest that NEDD8 modification of pVHL plays an important role in fibronectin matrix assembly and that in the absence of such regulation, an intact HIF pathway is insufficient to prevent VHL-associated tumorigenesis.

FIG. 1.

pVHL is conjugated by NEDD8. (A and B) U2OS cells were transfected with plasmids encoding HA-pVHL, T7-NEDD8, and T7-elongin C or with empty vector (MOCK). The cells were lysed and immunoprecipitated with anti-HA or anti-T7 antibodies. Bound proteins were resolved by SDS-PAGE and immunoblotted with an anti-T7 (A) or anti-HA (B) antibody. (C) U2OS cells were transfected with plasmids encoding the indicated ubiquitin-like molecules or empty vector, with (left panel) or without (right panel) a plasmid encoding HA-pVHL. The cells were lysed and immunoprecipitated with anti-HA (left panel) or anti-T7 (right panel) antibody. Bound proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. (D) Endogenous association of pVHL and NEDD8. Prostatic PC-3 cancer cells were lysed and immunoprecipitated with an anti-pVHL antibody. Bound proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was cut into three sections, and each section was immunoblotted with the indicated antibody. *, uncharacterized protein; IP, immunoprecipitated; IB, immunoblotted.

FIG. 2.

NEDD8 conjugation of pVHL is dependent on lysine 159. (A and B) U2OS cells were transfected with plasmids encoding T7-NEDD8 and the indicated HA-pVHL. The cells were lysed and immunoprecipitated with an anti-HA antibody. Bound proteins were resolved by SDS-PAGE and immunoblotted with an anti-HA antibody. *, uncharacterized modification of pVHL; IP, immunoprecipitated; IB, immunoblotted.

FIG. 3.

pVHL(RRR) ubiquitylates HIF-α subunits and regulates the expression of HIF-targeted GLUT1. (A) 786-O cells stably transfected with plasmids encoding pVHL(WT), pVHL(C162F), or pVHL(RRR) or empty plasmid (MOCK) were radiolabeled with [³⁵S]methionine, lysed, and immunoprecipitated with an anti-HA antibody. Bound proteins were resolved by SDS-PAGE and visualized by autoradiography. (B) The indicated 786-O stable cell lines were lysed and immunoprecipitated with an anti-HA antibody. The bound proteins were resolved by SDS-PAGE and immunoblotted with an anti-Cul2 antibody (upper panel) or an anti-HA antibody (lower panel). (C) In vitro ubiquitylation assay was performed where plasmids encoding pVHL(WT), pVHL(C162F), or pVHL(RRR) were in vitro translated and mixed with ³⁵S-labeled Gal4-HA-tagged HIF-α ODD. The reaction mixture was immunoprecipitated with an anti-Gal4 antibody, and the bound proteins were resolved by SDS-PAGE and visualized by autoradiography. (D) Equal amounts of the whole-cell extracts prepared from the indicated 786-O stable subclones were separated on SDS-PAGE and immunoblotted with an anti-GLUT1 (upper panel) or an anti-HA (lower panel) antibody. IP, immunoprecipitated; AR, autoradiography.

FIG. 4.

pVHL(RRR) shows reduced binding to fibronectin, and RCC cells expressing pVHL(RRR) show decreased fibronectin deposition and assembly. (A) The indicated 786-O subclones were radiolabeled with [³⁵S]methionine. Cells were lysed and immunoprecipitated with an anti-HA antibody. The bound proteins were resolved with SDS-PAGE and visualized by autoradiography. (B) The amount of fibronectin deposited by the indicated 786-O subclones was measured by ELISA at 405 nm. (C) Fibronectin immunofluorescence was performed on 786-MOCK, 786-WT, and 786-RRR subclones by using rabbit anti-fibronectin-rhodamine staining. IP, immunoprecipitated; OD, optical density; AR, autoradiography.

FIG. 5.

Neddylation of pVHL affects the growth of RCC cells in a three-dimensional growth assay. Digital images (×50) of the indicated and representative 786-O subclones grown as spheroids in semisolid media were captured on day 3. Two 786-MOCK, two 786-WT, and four 786-RRR subclones were tested.

FIG. 6.

pVHL(RRR) fails to suppress tumor formation in SCID mouse xenograft assay. (A) Tumor-take in representative mice injected with 786-O cells stably expressing either MOCK, pVHL(WT), or pVHL(RRR). The left hind leg was shaved and the tumor was resected (shown on the right). N/A, not applicable since no tumors were found on mice injected with 786-pVHL(WT) subclones; bar, 1 cm. (B) A graph of tumor growth over time in mice injected with the indicated 786-O stable subclones. One 786-MOCK, two 786-WT, and three 786-RRR subclones were tested, and four mice were injected per subclone. The total numbers of mice that grew tumors are indicated on the right.

FIG. 7.

VHL type 2C-causing pVHL(K159E) mutant is capable of NEDD8 conjugation and forms an active E3 ligase but fails to bind fibronectin. (A) U2OS cells were transfected with plasmids encoding T7-NEDD8 and the indicated HA-pVHL. The cells were lysed and immunoprecipitated with an anti-HA antibody. Bound proteins were resolved by SDS-PAGE and immunoblotted with an anti-Cul2 antibody (upper panel) or an anti-HA antibody (lower panel). (B) In vitro ubiquitylation assay was performed where plasmids encoding the indicated HA-pVHL were in vitro translated and mixed with ³⁵S-labeled Gal4-HA-HIF-α ODD. The reaction mixture was immunoprecipitated with an anti-Gal4 antibody, and the bound proteins were resolved by SDS-PAGE and visualized by autoradiography. (C) Equal amounts of the whole-cell extracts prepared from the indicated 786-O stable subclones were separated with SDS-PAGE and immunoblotted with an anti-GLUT1 (upper panel) or an anti-HA (lower panel) antibody. (D) The indicated 786-O subclones were radiolabeled with [³⁵S]methionine. Cells were lysed and immunoprecipitated with an anti-HA antibody. The bound proteins were resolved with SDS-PAGE and visualized by autoradiography. IP, immunoprecipitated; AR, autoradiography.