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.     

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.     

Priming-dependent phosphorylation and regulation of the tumor suppressor pVHL by glycogen synthase kinase 3

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.     

Deubiquitylase OTUD6B stabilizes the mutated pVHL and suppresses cell migration in clear cell renal cell carcinoma

Von Hippel-Lindau (VHL) is an important tumor suppressor, and its inactivation is a hallmark of inherited VHL disease and most sporadic clear cell renal cell carcinoma (ccRCC). VHL protein (pVHL) with missense point mutations are unstable and degraded by the proteasome because of the disruption of elongin binding. Deubiquitylase ovarian tumor domain-containing 6B (OTUD6B) had been documented to couple pVHL and elongin B to form stable VHL - elonginB - elonginC complex, which protects pVHL from degradation. However, whether OTUD6B governs the stability of pVHL wild type and the missense mutants in ccRCC remains largely elusive. Here, we reported that low OTUD6B level predicted poorer survival in ccRCC patients with VHL missense mutation, but not frameshift deletion and nonsense mutation. OTUD6B is able to interact with wild type pVHL and tumor-derived pVHL missense mutants, except for pVHL I151T, and decrease their ubiquitylation and proteasomal degradation in ccRCC cells. Functionally, we revealed that OTUD6B depletion enhanced cell migration and HIF-2α level in ccRCC cells in a pVHL dependent manner. In addition, OTUD6B depletion reduced the inhibitory effects of ectopic pVHL missense mutants on cell migration and HIF-2α level, except for pVHL I151T. Thus, we speculated that I151 residue might be one of key sites of pVHL binding to OTUD6B. These results suggested that OTUD6B is an important regulator for the stability of pVHL missense mutants, which provides a potential therapeutic strategy for ccRCC with VHL mutations.Subject terms: Ubiquitylation, Renal cell carcinoma     

pVHL and GSK3beta are components of a primary cilium-maintenance signalling network

Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3beta are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3beta alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3beta is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3beta function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.     

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.     

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.     

Identification of a deubiquitinating enzyme subfamily as substrates of the von Hippel-Lindau tumor suppressor

The VHL protein (pVHL) is a component of an E3 ubiquitin ligase complex which is involved in the ubiquitination and degradation of the alpha subunits of HIF (hypoxia-inducible factor) in the presence of oxygen. However, it is of considerable interest to identify pVHL substrates other than HIF. In our previous studies, we have shown that VDU1 (pVHL-interacting deubiquitinating enzyme-1) can be ubiquitinated for rapid degradation in a pVHL-dependent manner. In this report we show that another uncharacterized deubiquitinating enzyme, named VDU2 (pVHL-interacting deubiquitinating enzyme-2), is a substrate of pVHL. Based on human and mouse cDNA sequences, VDU1 and VDU2 are identical in approximately 59% of the amino acids with strong homology in the N-terminus and C-terminus and a weaker similarity in the middle region. VDU2 contains the signature motifs of the ubiquitin-specific processing protease family and possesses deubiquitinating activity. Like VDU1, VDU2 interacts with pVHL beta-domain and these two proteins can compete with each other to bind to pVHL. Finally, we demonstrate that VDU2 can also be ubiquitinated and degraded in a pVHL-dependent manner. Based on their amino acid sequence homology and functional interaction with pVHL, VDU1 and VDU2 define a subfamily of ubiquitin specific processing proteases. Since deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes, VDU1 and VDU2 could be important substrates of pVHL E3 ligase complex.     

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.