The von Hippel-Lindau tumour suppressor protein: new perspectives.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of the VHL tumour suppressor gene. The VHL gene product, pVHL, forms multiprotein complexes that contain elongin B, elongin C and Cul-2, and negatively regulates hypoxia-inducible mRNAs. pVHL is suspected to play a role in ubiquitination given the similarity of elongin C and Cul-2 with Skp1 and Cdc53, respectively. pVHL can also interact with fibronectin and is required for the assembly of a fibronectin matrix. Finally, pVHL, at least indirectly, plays a role in the ability of cells to exit the cell cycle. Thus, pVHL is a tumour suppressor protein that regulates angiogenesis, extracellular matrix formation and the cell cycle.     

VHL Type 2B Mutations Retain VBC Complex Form and Function

Background

von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1α and HIF-2α), resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease.

Methodology/Principal Findings

This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1α.

Conclusions

Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition for cancer development in VHL disease.     

The Positive Regulation of p53 by the Tumor Suppressor VHL

The ubiquitin-mediated degradation of hypoxia-inducible factor-α (HIF-α) by a von Hippel-Lindau tumor suppressor protein (pVHL) is mechanistically responsible for controlling gene expression due to oxygen availability. Germline mutations in the VHL gene cause dysregulation of HIF and induce an autosomal dominant cancer syndrome referred to as VHL disease. However, it is unclear whether HIF accumulation caused by VHL mutations is sufficient for tumorigenesis. Recently, we found that pVHL directly associates and positively regulates the tumor suppressor p53 by inhibiting Mdm2-mediated ubiquitination, and by subsequently recruiting p53-modifying enzymes. Moreover, VHL-deleted RCC cells showed attenuated apoptosis or abnormal cell-cycle arrest upon DNA damage, but became normal when pVHL was restored. Thus, pVHL appears to play a pivotal role in tumor suppression by participating actively as a component of p53 transactivation complex during DNA damage response.     

Effects of point mutations in pVHL on the binding of HIF-1α

The von Hippel-Lindau tumor suppressor protein (pVHL) has an essential role in the regulation of the hypoxia response pathway in animal cells. Under normoxic conditions, the hypoxia-inducible factor (HIF) undergoes trans-4-prolyl hydroxylation and is subsequently recognised by the β-domain of pVHL, leading to the ubiquitination and degradation of HIF. Mutations of pVHL alter the binding of HIF. A subset of relevant clinically observed mutations to pVHL are thought to cause weaker binding of HIF-1α and are associated with cancer and cardiovascular diseases. Here, we present computational studies analyzing the interaction of HIF with mutant forms of pVHL, describing at atomic detail the local structural reorganization caused by substitution of certain residues of pVHL. The results reveal that the canonical configuration in the wild-type system is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the h-bond network in the binding site disrupts HIF binding. Although the experimentally observed ordering of binding energies for mutants of Tyr98 is reproduced, our examination of a broader range of mutations does not support the hypothesis of a correlation between the degree of disruption of the pVHL/HIF-1α interaction caused by a mutation and the phenotype with which the mutation is associated. We suggest that disruption of the binding interaction is one of many factors behind the manifestation of VHL disease.     

Expression of p53 in renal carcinoma cells is independent of pVHL

Renal clear-cell carcinoma (RCC) is the predominant form of kidney cancer and is highly refractory to conventional anti-cancer therapies. The status of p53 tumor suppressor gene has been correlated with the efficacy of radio- and chemotherapies, where presence of mutant p53 is associated with reduced responsiveness to treatment. However, p53 itself is rarely mutated in RCC, rather suggesting that the p53 pathway might be compromized in RCC cells. In support of this notion, the transactivation property of normal p53 was shown to be repressed in various transformed kidney epithelial cells via an unknown dominant-negative mechanism. Mutation of the von Hippel-Lindau (VHL) gene causes familial VHL disease, which includes predisposition to RCC. Moreover, biallelic inactivation of VHL has been observed in the vast majority of sporadic RCC. Recently, the expression of pVHL in RCC cells was demonstrated to elevate the expression of p53 by inducing the binding of RNA-stabilizing protein HuR to the 3'untranslated region of p53 mRNA. Contrary to this finding, we report here that the reconstitution of a variety of VHL(-/-) RCC lines including 786-O, RCC4, and A498 or non-RCC cells with wild-type pVHL does not influence the expression of p53 and fails to induce p53-responsive gene p21CIP1/WAF1 or p53-responsive reporters. These results suggest that the expression of p53 in RCC cells is independent of pVHL.     

NEDD8 acts as a 'molecular switch' defining the functional selectivity of VHL

The von Hippel-Lindau (VHL) tumour suppressor protein is important in the E3 ubiquitin ligase ECV (Elongin B/C-CUL2-VHL)-mediated destruction of hypoxia-inducible factor and the promotion of fibronectin (FN) extracellular matrix assembly. Although the precise molecular mechanism controlling the selectivity of VHL function remains unknown, a failure in either process is associated with oncogenic progression. Here, we show that VHL performs its FN-associated function independently of the ECV complex, highlighting the autonomy of these pathways. Furthermore, we show that NEDD8, a ubiquitin-like molecule, acts as a 'molecular switch' in which its covalent conjugation to VHL prohibits the engagement of the scaffold component CUL2 and, concomitantly, activates the association with FN. These findings provide the first mechanistic step in defining the functional selectivity of VHL and explain a previously unrecognized function of NEDD8.     

Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is characterized by the development of multiple vascular tumors and is caused by inactivation of the von Hippel-Lindau protein (pVHL). Here we show that pVHL, through its beta-domain, binds directly to hypoxia-inducible factor (HIF), thereby targeting HIF for ubiquitination in an alpha-domain-dependent manner. This is the first function to be ascribed to the pVHL beta-domain. Furthermore, we provide the first direct evidence that pVHL has a function analogous to that of an F-box protein, namely, to recruit substrates to a ubiquitination machine. These results strengthen the link between overaccumulation of HIF and development of VHL disease.     

Diverse effects of mutations in exon II of the von Hippel-Lindau (VHL) tumor suppressor gene on the interaction of pVHL with the cytosolic chaperonin and pVHL-dependent ubiquitin ligase activity

We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1alpha (HIF-1alpha), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.     

Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

Tumor suppression by the von Hippel-Lindau protein requires phosphorylation of the acidic domain

The tumor suppressor function of the von Hippel-Lindau protein (pVHL) has previously been linked to its role in regulating hypoxia-inducible factor levels. However, VHL gene mutations suggest a hypoxia-inducible factor-independent function for the N-terminal acidic domain in tumor suppression. Here, we report that phosphorylation of the N-terminal acidic domain of pVHL by casein kinase-2 is essential for its tumor suppressor function. This post-translational modification did not affect the levels of hypoxia-inducible factor; however, it did change the binding of pVHL to another known binding partner, fibronectin. Cells expressing phospho-defective mutants caused improper fibronectin matrix deposition and demonstrated retarded tumor formation in mice. We propose that phosphorylation of the acidic domain plays a role in the regulation of proper fibronectin matrix deposition and that this may be relevant for the development of VHL-associated malignancies.