The von Hippel-Lindau tumour suppressor interacts with microtubules through kinesin-2

Synthesis and maintenance of primary cilia are regulated by the von Hippel-Lindau (VHL) tumour suppressor protein. Recent studies indicate that this regulation is linked to microtubule-dependent functions of pVHL such as orienting microtubule growth and increasing plus-end microtubule stability, however little is known how this occurs. We have identified the kinesin-2 motor complex, known to regulate cilia, as a novel and endogenous pVHL binding partner. The interaction with kinesin-2 facilitates pVHL binding to microtubules. These data suggest that microtubule-dependent functions of pVHL are influenced by kinesin-2.     

The VHL Tumor Suppressor: Riding Tandem with GSK3β in Primary Cilium Maintenance

Amongst other clinical manifestations, patients with the von Hippel-Lindau (VHL) cancer syndrome are predisposed to develop kidney cysts, which are considered to be precursor lesions of clear cell renal cell carcinoma (ccRCC). Recent evidence has highlighted an unexpected function of the VHL tumour suppressor protein (pVHL) in maintaining the structural integrity of the primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled proliferation of kidney epithelial cells and cyst formation. Intriguingly, this function of pVHL is directly linked to its capacity to regulate the microtubule cytoskeleton independent of its well-characterized role in the degradation of hypoxia inducible factor alpha (HIFα) subunits. However, loss of pVHL alone does not suffice for a cell to lose the primary cilium. Other pathways need to be additionally inactivated, including one involving glycogen synthase kinase 3 beta (GSK3β). These new findings draw attention to a primary cilium-maintenance network as new territory for pVHL tumour suppressive activity and have implications for understanding the development of kidney pathology in the setting of VHL disease.     

Mobility of the von Hippel-Lindau tumour suppressor protein is regulated by kinesin-2

The von Hippel-Lindau tumour suppressor protein (pVHL) participates in many cellular processes including oxygen sensing, microtubule stability and primary cilia regulation. Recently, we identified ATP-dependent motor complex kinesin-2 to endogenously bind the full-length variant of VHL (pVHL30) in primary kidney cells, and mediate its association to microtubules. Here we show that pVHL also endogenously binds the neuronal kinesin-2 complex, which slightly differs from renal kinesin-2. To investigate the role of kinesin-2 in pVHL mobility, we performed fluorescence recovery after photobleaching (FRAP) experiments in neuroblastoma cells. We observe that pVHL30 is a highly mobile cytoplasmic protein, which becomes an immobile centrosomal protein after ATP-depletion in living cells. This response to ATP-depletion is independent of GSK3beta-dependent phosphorylation of pVHL30. Furthermore, VHL variant alleles with reduced binding to kinesin-2 fail to respond to ATP-depletion. Accordingly, interfering with pVHL30-KIF3A interaction by either overexpressing a dominant negative construct or by reducing endogenous cellular levels of KIF3A by RNAi abolishes pVHL's response to ATP-depletion. From these data we suggest that mobility of a subcellular pool of pVHL is regulated by the ATP-dependent kinesin-2 motor. Kinesin-2 driven mobility of cytoplasmic pVHL might enable pVHL to function as a tumour suppressor.     

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.     

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.     

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.     

von Hippel-Lindau病的分子机制研究

VHL病(Von Hippel-Lindau disease)是一种遗传性肿瘤综合征,由VHL抑癌基因突变引起.研究表明,VHL蛋白在体内具有多种功能,VHL基因突变形式和部位的差异所造成的VHL蛋白功能增加、减少或缺失可能是导致肿瘤不同表型的重要原因.     

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.     

Identification of the RNA polymerase II subunit hsRPB7 as a novel target of the von Hippel-Lindau protein

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the hereditary VHL disease and sporadic clear cell renal cell carcinomas (CCRCC). VHL-associated tumors are highly vascularized, a characteristic associated with overproduction of vascular endothelial growth factor (VEGF). The VHL protein (pVHL) is a component of the ubiquitin ligase E3 complex, targeting substrate proteins for ubiquitylation and subsequent proteasomic degradation. Here, we report that the pVHL can directly bind to the human RNA polymerase II seventh subunit (hsRPB7) through its beta-domain, and naturally occurring beta-domain mutations can decrease the binding of pVHL to hsRPB7. Introducing wild-type pVHL into human kidney tumor cell lines carrying endogenous mutant non-functional pVHL facilitates ubiquitylation and proteasomal degradation of hsRPB7, and decreases its nuclear accumulation. pVHL can also suppress hsRPB7-induced VEGF promoter transactivation, mRNA expression and VEGF protein secretion. Together, our results suggest that hsRPB7 is a downstream target of the VHL ubiquitylating complex and pVHL may regulate angiogenesis by targeting hsRPB7 for degradation via the ubiquitylation pathway and preventing VEGF expression.     

Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network

The von Hippel-Lindau (VHL) tumor suppressor gene is a protein interaction hub, controlling numerous genes implicated in tumor progression. Here we focus on structural aspects of protein interactions for a list of 35 experimentally verified protein VHL (pVHL) interactors. Using structural information and computational analysis we have located three distinct interaction interfaces (A, B, and C). Interface B is the most versatile, recognizing a refined linear motif present in 17 otherwise non-related proteins. It has been possible to distinguish compatible and exclusive interactions by relating pVHL function to interaction interfaces and subcellular localization. A novel hypothesis is presented regarding the possible function of the N-terminus as an inhibitor of pVHL function.     

Comparative sequence analysis of the VHL tumor suppressor gene

Comparative genome analysis may provide novel insights into gene evolution and function. To investigate the von Hippel-Lindau (VHL) disease tumor suppressor gene, we sequenced the VHL gene in seven primate species. Comparative analysis was performed for human, primate, and rodent VHL genes and for a putative Caenorhabditis elegans VHL homologue identified by database analysis. The VHL gene has two translation initiation sites (at codons 1 and 54); however, the relative importance of the full-length translation product (pVHL30) and that translated from the second internal translation initiation site (pVHL19) is unclear. The N-terminal sequence of pVHL30 contains eight copies of a GXEEX acidic repeat motif in human and higher primates, but only three copies were present in the marmoset, and only one copy was present in rodent VHL genes. Evolutionary analysis suggested that the N-terminal repetitive sequence in pVHL30 was of less functional importance than those regions present in both pVHL30 and pVHL19. The VHL gene product is reported to form complexes with various proteins including elongin B, elongin C, VBP-1, fibronectin, Spl, CUL2, and HIF-1. Although most of the regions in pVHL that had been implicated in binding specific proteins demonstrated evolutionary conservation, the carboxy-terminal putative VBP-1 binding site was less well conserved, suggesting that VBP-1 binding may have less functional significance. Although an amino acid substitution (K171T) close to the pVHL elongin binding region was found in baboon, analysis of the structure of human pVHL suggested that this substitution would not interfere with pVHL/elongin C interaction. In general, there was a good correlation between the pVHL domains that demonstrated most evolutionary conservation and those that were most frequently mutated in tumors. Analysis of human/C. elegans conservation and human germline and somatic mutation patterns identified a highly conserved mutation cluster region between codons 74 and 90. However, this region is likely to be important for the structural integrity of pVHL rather than representing an additional protein binding domain.     

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.     

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.     

Multitasking by pVHL in tumour suppression

Functional inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product, pVHL, leads to cancer in humans. It is widely accepted that pVHL functions to destabilise hypoxia inducible factor alpha (HIFalpha) subunits, key effectors of the hypoxia signalling pathway. However, growing evidence indicates that tumour suppression by pVHL also involves the control of a wide variety of HIFalpha-independent processes including microtubule dynamics, primary cilium maintenance, cell proliferation, neuronal apoptosis, extracellular matrix deposition and responses to DNA damage. Moreover, it is becoming apparent that tumour initiation requires not only VHL mutation but also the alteration of additional cooperating cancer pathways. These studies are beginning to provide insights into the signalling networks involving pVHL that normally control diverse cellular processes and how disruption of these networks leads to tumour formation.     

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

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.