The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the β domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.     

Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein

The von Hippel-Lindau tumor suppressor protein (pVHL) has emerged as a key factor in cellular responses to oxygen availability, being required for the oxygen-dependent proteolysis of alpha subunits of hypoxia inducible factor-1 (HIF). Mutations in VHL cause a hereditary cancer syndrome associated with dysregulated angiogenesis, and up-regulation of hypoxia inducible genes. Here we investigate the mechanisms underlying these processes and show that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-alpha ubiquitylation activity which is complemented by exogenous pVHL. This defect was specific for HIF-alpha among a range of substrates tested. Furthermore, HIF-alpha subunits were the only pVHL-associated proteasomal substrates identified by comparison of metabolically labeled anti-pVHL immunoprecipitates from proteosomally inhibited cells and normal cells. Analysis of pVHL/HIF-alpha interactions defined short sequences of conserved residues within the internal transactivation domains of HIF-alpha molecules sufficient for recognition by pVHL. In contrast, while full-length pVHL and the p19 variant interact with HIF-alpha, the association was abrogated by further N-terminal and C-terminal truncations. The interaction was also disrupted by tumor-associated mutations in the beta-domain of pVHL and loss of interaction was associated with defective HIF-alpha ubiquitylation and regulation, defining a mechanism by which these mutations generate a constitutively hypoxic pattern of gene expression promoting angiogenesis. The findings indicate that pVHL regulates HIF-alpha proteolysis by acting as the recognition component of a ubiquitin ligase complex, and support a model in which its beta domain interacts with short recognition sequences in HIF-alpha subunits.     

The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery

The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.     

The von Hippel-Lindau tumor suppressor stabilizes novel plant homeodomain protein Jade-1

The von Hippel-Lindau disease gene (VHL) is the causative gene for most adult renal cancers. However, the mechanism by which VHL protein functions as a renal tumor suppressor remains largely unknown. To identify low occupancy VHL protein partners with potential relevance to renal cancer, we screened a human kidney library against human VHL p30 using a yeast two-hybrid approach. Jade-1 (gene for Apoptosis and Differentiation in Epithelia) encodes a previously uncharacterized 64-kDa protein that interacts strongly with VHL protein and is most highly expressed in kidney. Jade-1 protein is short-lived and contains a candidate destabilizing (PEST) motif and plant homeodomains that are not required for the VHL interaction. Jade-1 is abundant in proximal tubule cells, which are clear-cell renal cancer precursors, and expression increases with differentiation. Jade-1 is expressed in cytoplasm and the nucleus diffusely and in speckles, where it partly colocalizes with VHL. VHL reintroduction into renal cancer cells increases endogenous Jade-1 protein abundance up to 10-fold. Furthermore, VHL increases Jade-1 protein half-life up to 3-fold. Thus, direct protein stabilization is identified as a new VHL function. Moreover, Jade-1 protein represents a novel candidate regulatory factor in VHL-mediated renal tumor suppression.     

The von Hippel-Lindau tumor suppressor protein mediates ubiquitination of activated atypical protein kinase C

The von Hippel-Lindau tumor-suppressor protein (pVHL) forms a protein complex (VCB-Cul2) with elongin C, elongin B, Cul-2, and Rbx1, which functions as a ubiquitin-protein ligase (E3). The alpha-subunits of the hypoxia-inducible factors have been identified as targets for the VCB-Cul2 ubiquitin ligase. However, a variety of cellular defects caused by the depletion of pVHL cannot be explained solely by the ubiquitin-mediated degradation of hypoxia-inducible factor-alpha. We show here that a member of the atypical protein kinase C (PKC) group, PKClambda, is ubiquitinated by the pVHL-containing E3 enzyme. An active PKClambda mutant is ubiquitinated more extensively than wild-type PKClambda in HEK293 cells, and the ubiquitination is further enhanced by the overexpression of pVHL. The activation of wild-type PKClambda by serum stimulation of cells enhances the ubiquitination of the protein, supporting the notion that active PKClambda is preferentially ubiquitinated by VCB-Cul2 ubiquitin ligase. Furthermore, we show that PKClambda can be ubiquitinated in vitro in a cell-free ubiquitination assay using purified recombinant components including VCB-Cul2. Given the known function of aPKC in the regulation of cell polarity and cell growth, PKClambda may be a target of pVHL in its function as a tumor suppressor.     

The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth

Cilia are specialized organelles that play an important role in several biological processes, including mechanosensation, photoperception, and osmosignaling. Mutations in proteins localized to cilia have been implicated in a growing number of human diseases. In this study, we demonstrate that the von Hippel-Lindau (VHL) protein (pVHL) is a ciliary protein that controls ciliogenesis in kidney cells. Knockdown of pVHL impeded the formation of cilia in mouse inner medullary collecting duct 3 kidney cells, whereas the expression of pVHL in VHL-negative renal cancer cells rescued the ciliogenesis defect. Using green fluorescent protein-tagged end-binding protein 1 to label microtubule plus ends, we found that pVHL does not affect the microtubule growth rate but is needed to orient the growth of microtubules toward the cell periphery, a prerequisite for the formation of cilia. Furthermore, pVHL interacts with the Par3-Par6-atypical PKC complex, suggesting a mechanism for linking polarity pathways to microtubule capture and ciliogenesis.     

The von Hippel-Lindau tumor suppressor protein promotes c-Cbl-independent poly-ubiquitylation and degradation of the activated EGFR

Somatic mutations or reduced expression of the von Hippel-Lindau (VHL) tumor suppressor occurs in the majority of the clear cell renal cell carcinoma (ccRCC) and is a causal factor for the pathogenesis of ccRCC. pVHL was reported to suppress the oncogenic activity of Epidermal Growth Factor Receptor (EGFR) by reducing the expression of the EGFR agonist TGF-α and by reducing the translation efficiency of EGFR itself. Furthermore, it was reported that pVHL down-regulates activated EGFR by promoting efficient lysosomal degradation of the receptor. These modes of negative regulation of EGFR by pVHL were dependent on Hypoxia Inducible Factor (HIF). In this study, we report that HIF was not the only factor stabilizing the activated EGFR in VHL-deficient ccRCC cells. Down-regulation of endogenous HIF in these cells had little effect on the turnover rates of the activated EGFR. Furthermore, neither pretreatment with lysosomal inhibitors pretreatment nor down-regulation of c-Cbl, a major E3 ubiquitin ligase that targets the activated EGFR for lysosomal degradation, significantly increased the stabilities of EGFR in VHL-expressing ccRCC cells. In contrast, pretreatment with proteasomal inhibitors extended EGFR lifetime and led to similar EGFR half-lives in VHL-expressing and VHL-deficient ccRCC cells. Down-regulation of c-Cbl in VHL-deficient ccRCC cells revealed that the c-Cbl and pVHL collaborated to down-regulate the activated EGFR. Finally, we found that pVHL promoted the poly-ubiquitylation of the activated EGFR, and this function was c-Cbl-independent. Thus these results indicate that pVHL limits EGFR signaling by promoting c-Cbl-independent poly-ubiquitylation of the activated receptor, which likely results in its degradation by proteasome.     

Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.     

The von Hippel-Lindau tumor suppressor protein: new insights into oxygen sensing and cancer

The von Hippel-Lindau tumor suppressor protein (pVHL) is the substrate-recognition module of an E3 ubiquitin ligase that targets the alpha subunits of hypoxia-inducible factor (HIF) for degradation in the presence of oxygen. Recognition of HIF by pVHL is linked to enzymatic hydroxylation of conserved prolyl residues in the HIF alpha subunits by members of the EGLN family. Dysregulation of HIF-target genes such as vascular endothelial growth factor and transforming growth factor alpha has been implicated in the pathogenesis of renal cell carcinomas and of hemangioblastomas, both of which frequently lack pVHL function.     

Ubiquitination of a novel deubiquitinating enzyme requires direct binding to von Hippel-Lindau tumor suppressor protein.

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of the VHL gene. Recent studies suggest that VHL protein (pVHL) is a component of an E3 ubiquitin ligase, but the detailed biological function of pVHL remains to be determined. To further elucidate the biological functions of pVHL, we searched pVHL-interacting proteins using yeast two-hybrid screening. A novel protein named VHL-interacting deubiquitinating enzyme 1 (VDU1) was identified as being able to directly interact with pVHL in vitro and in vivo. We have determined the full-length cDNA of this enzyme, which includes two putative subtypes. Type I consists of 942 amino acids, and type II consists of 911 amino acids with predicted molecular masses of 107 and 103 kDa, respectively. We have also cloned a mouse homologue of this enzyme. Sequence analysis reveals that this protein is conserved between human and mouse and contains the signature motifs of the ubiquitin-specific processing protease family. Enzymatic function studies demonstrate its deubiquitinating activity. We have determined that the VDU1-interacting region in pVHL is located in its beta-domain, and several naturally occurring mutations located in this domain disrupt the interaction between pVHL and VDU1 protein. Co-immunoprecipitation demonstrates that VDU1 can be recruited into the pVHL-elongin C-elongin B complex. Finally, we demonstrate that VDU1 is able to be ubiquitinated via a pVHL-dependent pathway for proteasomal degradation, and VHL mutations that disrupt the interaction between VDU1 and pVHL abrogate the ubiquitination of VDU1. Our findings indicate that VDU1, a novel ubiquitin-specific processing protease, is a downstream target for ubiquitination and degradation by pVHL E3 ligase. Targeted degradation of VDU1 by pVHL could be crucial for regulating the ubiquitin-proteasome degradation pathway.     

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.     

Quantitative proteomics identifies the Myb-binding protein p160 as a novel target of the von Hippel-Lindau tumor suppressor

Background

The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of a ubiquitin ligase complex, which is best understood as a negative regulator of hypoxia inducible factor (HIF). VHL ubiquitinates and degrades the α subunits of HIF, and this is proposed to suppress tumorigenesis and tumor angiogenesis. However, several lines of evidence suggest that there are unidentified substrates or targets for VHL that play important roles in tumor suppression.

Methodology/Principal Findings

Employing quantitative proteomics, we developed an approach to systematically identify the substrates of ubiquitin ligases and using this method, we identified the Myb-binding protein p160 as a novel substrate of VHL.

Conclusions/Significance

A major barrier to understanding the functions of ubiquitin ligases has been the difficulty in pinpointing their ubiquitination substrates. The quantitative proteomics approach we devised for the identification of VHL substrates will be widely applicable to other ubiquitin ligases.     

Regulation of microtubule stability by the von Hippel-Lindau tumour suppressor protein pVHL

Von Hippel-Lindau (VHL) tumour suppressor gene inactivation is linked to the development of haemangioblastomas in the central nervous system and retina, often in association with other tumours, such as clear-cell carcinomas of the kidney and phaeochromocytomas. Here we show that the VHL protein (pVHL) is a microtubule-associated protein that can protect microtubules from depolymerization in vivo. Both the microtubule binding and stabilization functions of pVHL depend on amino acids 95-123 of pVHL, a mutational 'hot-spot' in VHL disease. From analysis of naturally occurring pVHL mutants, it seems that only point mutations such as pVHL(Y98H) and pVHL(Y112H) (that predispose to haemangioblastoma and phaeochromocytoma, but not to renal cell carcinoma) disrupt pVHL's microtubule-stabilizing function. Our data identify a role for pVHL in the regulation of microtubule dynamics and potentially provide a link between this function of pVHL and the pathogenesis of haemangioblastoma and phaeochromocytoma in the context of VHL disease.     

Ran-mediated nuclear export of the von Hippel-Lindau tumor suppressor protein occurs independently of its assembly with cullin-2

Inactivating mutations of the von Hippel-Lindau (VHL) tumor suppressor gene cause the VHL cancer syndrome and sporadic renal clear cell carcinoma. VHL engages in a nucleocytoplasmic shuttle, which is required for its function. Here, we pursue our investigation to identify mechanisms by which VHL-green fluorescent protein (VHL-GFP) is exported from the nucleus. We show that nuclear export of VHL-GFP in living cells requires ongoing RNA polymerase II activity, and is mediated by mechanisms that are temperature-sensitive and energy-dependent. In vitro nuclear export of VHL-GFP is inhibited by nuclear pore-specific lectins, requires ATP hydrolysis and polyadenylated mRNAs, and occurs with kinetics that are similar to those of proteins containing a nuclear export signal. Biochemical fractionation has revealed that nuclear export of VHL-GFP occurs by way of a Ran-dependent pathway. Size exclusion column chromatography and deletion mutant analysis suggest that VHL-GFP does not require assembly with one of its associated proteins, cullin-2, to engage in nuclear export. These results demonstrate that nuclear export of VHL-GFP is Ran-mediated and ATP hydrolysis-dependent. They also suggest that sequences outside the elongin C binding box may function as a nuclear export domain, potentially providing a novel role for this region of VHL frequently mutated in renal cell carcinoma.