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1.  pVHL Mediates K63-Linked Ubiquitination of nCLU 
PLoS ONE  2012;7(4):e35848.
pVHL, product of von Hippel-Lindau (VHL) tumor suppressor gene, functions as the substrate recognition component of an E3-ubiquitin ligase that targets proteins for ubiquitination and proteasomal degradation. Hypoxia-inducible factor α (HIFα) is the well-known substrate of pVHL. Besides HIFα, pVHL also binds to many other proteins and has multiple functions. In this manuscript, we report that the nuclear clusterin (nCLU) is a target of pVHL. We found that pVHL had a direct interaction with nCLU. nCLU bound to pVHL at pVHL's β domain, the site for recognition of substrate, indicating that nCLU might be a substrate of pVHL. Interestingly, pVHL bound to nCLU but did not lead to nCLU destruction. Further studies indicated that pVHL mediated K63-linked ubiquitination of nCLU and promoted nCLU nuclear translocation. In summary, our results disclose a novel function of pVHL that mediates K63-linked ubiquitination and identify nCLU as a new target of pVHL.
doi:10.1371/journal.pone.0035848
PMCID: PMC3332038  PMID: 22532874
2.  The VHL tumor suppressor and HIF: insights from genetic studies in mice 
Cell death and differentiation  2008;15(4):650-659.
The von Hippel–Lindau tumor suppressor gene product, pVHL, functions as the substrate recognition component of an E3-ubiquitin ligase, which targets the oxygen-sensitive α-subunit of hypoxia-inducible factor (HIF) for rapid proteasomal degradation under normoxic conditions and as such plays a central role in molecular oxygen sensing. Mutations in pVHL can be found in familial and sporadic clear cell carcinomas of the kidney, hemangioblastomas of the retina and central nervous system, and pheochromocytomas, underscoring its gatekeeper function in the pathogenesis of these tumors. Tissue-specific gene targeting of VHL in mice has demonstrated that efficient execution of pVHL-mediated HIF proteolysis under normoxia is fundamentally important for survival, proliferation, differentiation and normal physiology of many cell types, and has provided novel insights into the biological function of individual HIF transcription factors. In this review, we discuss the role of HIF in the development of the VHL phenotype.
doi:10.1038/sj.cdd.4402313
PMCID: PMC3799983  PMID: 18219317
von Hippel–indau (VHL) tumor suppressor; hypoxia-inducible factor (HIF); conditional knockout mice; renal cell cancer; hemangiomas; vascular tumors
3.  Cell-Type-Specific Regulation of Degradation of Hypoxia-Inducible Factor 1α: Role of Subcellular Compartmentalization 
Molecular and Cellular Biology  2006;26(12):4628-4641.
The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that mediates adaptive cellular responses to decreased oxygen availability (hypoxia). At normoxia, HIF-1α is targeted by the von Hippel-Lindau tumor suppressor protein (pVHL) for degradation by the ubiquitin-proteasome pathway. In the present study we have observed distinct cell-type-specific differences in the ability of various tested pVHL-interacting subfragments to stabilize HIF-1α and unmask its function at normoxia. These properties correlated with differences in subcellular compartmentalization and degradation of HIF-1α. We observed that the absence or presence of nuclear localization or export signals differently affected the ability of a minimal HIF-1α peptide spanning residues 559 to 573 of mouse HIF-1α to stabilize endogenous HIFα and induce HIF-driven reporter gene activity in two different cell types (primary mouse endothelial and HepG2 hepatoma cells). Degradation of HIF-1α occurred mainly in the cytoplasm of HepG2 cells, whereas it occurs with equal efficiency in nuclear and cytoplasmic compartments of primary endothelial cells. Consistent with these observations, green fluorescent protein-HIF-1α is differently distributed during hypoxia and reoxygenation in hepatoma and endothelial cells. Consequently, we propose that differential compartmentalization of degradation of HIF-1α and the subcellular distribution of HIF-1α may account for cell-type-specific differences in stabilizing HIF-1α protein levels under hypoxic conditions.
doi:10.1128/MCB.02236-05
PMCID: PMC1489139  PMID: 16738327
4.  The VHL Tumor Suppressor: Master Regulator of HIF 
Current pharmaceutical design  2009;15(33):3895-3903.
Hypoxia-inducible factors (HIFs) are heterodimeric oxygen-sensitive basic helix-loop-helix transcription factors that play central roles in cellular adaptation to low oxygen environments. The von-Hippel Lindau tumor suppressor (pVHL) is the substrate recognition component of an E3 ubiquitin ligase and functions as a master regulator of HIF activity by targeting the hydroxylated HIF-alpha subunit for ubiquitylation and rapid proteasomal degradation under normoxic conditions. Mutations in pVHL can be found in familial and sporadic hemangioblastomas, clear cell carcinomas of the kidney, pheochromocytomas and inherited forms of erythrocytosis, illustrating the importance of disrupted molecular oxygen sensing in the pathogenesis of these diseases. Tissue-specific gene targeting of pVHL in mice has demonstrated that efficient execution of HIF proteolysis is critically important for normal tissue physiology, and has provided novel insights into the functional consequences of HIF activation on the cellular and tissue level. Here we focus on the contribution of individual HIF transcription factors to the development of VHL phenotypes and discuss how the pVHL/HIF axis could be exploited pharmacologically.
PMCID: PMC3622710  PMID: 19671042
von Hippel-Lindau (VHL) tumor suppressor; hypoxia-inducible factor (HIF); renal cell cancer; hemangioblastoma; erythropoietin; anemia; metabolism; kidney cysts; mouse model
5.  Design and Analysis of a Petri Net Model of the Von Hippel-Lindau (VHL) Tumor Suppressor Interaction Network 
PLoS ONE  2014;9(6):e96986.
Von Hippel-Lindau (VHL) syndrome is a hereditary condition predisposing to the development of different cancer forms, related to germline inactivation of the homonymous tumor suppressor pVHL. The best characterized function of pVHL is the ubiquitination dependent degradation of Hypoxia Inducible Factor (HIF) via the proteasome. It is also involved in several cellular pathways acting as a molecular hub and interacting with more than 200 different proteins. Molecular details of pVHL plasticity remain in large part unknown. Here, we present a novel manually curated Petri Net (PN) model of the main pVHL functional pathways. The model was built using functional information derived from the literature. It includes all major pVHL functions and is able to credibly reproduce VHL syndrome at the molecular level. The reliability of the PN model also allowed in silico knockout experiments, driven by previous model analysis. Interestingly, PN analysis suggests that the variability of different VHL manifestations is correlated with the concomitant inactivation of different metabolic pathways.
doi:10.1371/journal.pone.0096986
PMCID: PMC4041725  PMID: 24886840
6.  Inhibition of HIF2α Is Sufficient to Suppress pVHL-Defective Tumor Growth 
PLoS Biology  2003;1(3):e83.
Biallelic inactivation of the von Hippel–Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the α subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2α variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2α with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.
Specific downregulation of the transcription factor HIF2α is sufficient to suppress tumor formation by cells lacking the functional tumor suppressor (pVHL), demonstrating that tumor suppression by pVHL is linked to regulation of HIF target genes
doi:10.1371/journal.pbio.0000083
PMCID: PMC300692  PMID: 14691554
7.  The VHL tumor suppressor in development and disease: Functional studies in mice by conditional gene targeting 
The von Hippel–Lindau tumor suppressor pVHL plays a critical role in the pathogenesis of familial and sporadic clear cell carcinomas of the kidney and hemangioblastomas of the retina and central nervous system. pVHL targets the oxygen sensitive alpha subunit of hypoxia-inducible factor (HIF) for proteasomal degradation, thus providing a direct link between tumorigenesis and molecular pathways critical for cellular adaptation to hypoxia. Cell type specific gene targeting of VHL in mice has demonstrated that proper pVHL mediated HIF proteolysis is fundamentally important for survival, proliferation and differentiation of many cell types and furthermore, that inactivation of pVHL may, unexpectedly, inhibit tumor growth under certain conditions. Mouse knock out studies have provided novel mechanistic insights into VHL associated tumorigenesis and established a central role for HIF in the development of the VHL phenotype.
doi:10.1016/j.semcdb.2005.03.006
PMCID: PMC3787877  PMID: 15908240
von Hippel–Lindau (VHL) tumor suppressor; Hypoxia-inducible factor (HIF); Conditional knock out mice; Renal cell cancer; Hemangioma
8.  Nitric Oxide Impairs Normoxic Degradation of HIF-1α by Inhibition of Prolyl Hydroxylases 
Molecular Biology of the Cell  2003;14(8):3470-3481.
Hypoxia inducible factor-1 (HIF-1) is the master regulator of metabolic adaptation to hypoxia. It is appreciated that HIF-1α accumulation is achieved under normoxic conditions by e.g., nitric oxide. We determined molecular mechanisms of HIF-1α accumulation under the impact of S-nitrosoglutathione (GSNO). In human embryonic kidney cells GSNO provoked nuclear accumulation of HIF-1α. This appeared unrelated to gene transcription and protein translation, thus pointing to inhibition of HIF-1α degradation. Indeed, GSNO as well as the hypoxia mimic CoCl2 decreased ubiquitination of HIF-1α and GSNO-induced HIF-1α failed to coimmunoprecipitate with pVHL (von Hippel Lindau protein). Considering that HIF-1α-pVHL interactions require prolyl hydroxylation of HIF-1α, we went on to demonstrate inhibition of HIF-1α prolyl hydroxylases (PHDs) by GSNO. In vitro HIF-1α-pVHL interactions revealed that GSNO dose-dependently inhibits PHD activity but not the interaction of a synthetic peptide resembling the hydroxylated oxygen-dependent degradation domain of HIF-1α with pVHL. We conclude that GSNO-attenuated prolyl hydroxylase activity accounts for HIF-1α accumulation under conditions of NO formation during normoxia and that PHD activity is subject to regulation by NO.
doi:10.1091/mbc.E02-12-0791
PMCID: PMC181582  PMID: 12925778
9.  HIF-2α downregulation in the absence of functional VHL is not sufficient for renal cell differentiation 
Background
Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary as well as sporadic clear cell renal carcinomas. The product of the VHL gene, pVHL, acts to target hypoxia-inducible factor alpha (HIF-α) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2α in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2α toward cellular pVHL activities.
Results
Knockdown of HIF-2α resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2α regulation. However, cellular morphological changes and downregulation of integrins α5 and β1, which were seen upon pVHL replacement, were not faithfully phenocopied by HIF-2α reduction. Moreover, fibronectin deposition and expression of renal cell differentiation markers were observed in cells containing replaced pVHL, but not in HIF-2α knockdown cells, indicating that these pVHL functions may occur independently of HIF-2α downregulation.
Conclusion
These results indicate that HIF-2α regulation is not sufficient for pVHL-induced renal cell differentiation. We hypothesize that in addition to HIF-2α dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.
doi:10.1186/1475-2867-7-13
PMCID: PMC1919349  PMID: 17598890
10.  pVHL Modification by NEDD8 Is Required for Fibronectin Matrix Assembly and Suppression of Tumor Development 
Molecular and Cellular Biology  2004;24(8):3251-3261.
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 α (HIF-α) 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.
doi:10.1128/MCB.24.8.3251-3261.2004
PMCID: PMC381603  PMID: 15060148
11.  Renal Cyst Development in Mice with Conditional Inactivation of the von Hippel-Lindau Tumor Suppressor 
Cancer research  2006;66(5):2576-2583.
Inactivation of the von Hippel-Lindau tumor suppressor, pVHL, is associated with both hereditary and sporadic renal cysts and renal cell carcinoma, which are commonly thought to arise from the renal proximal tubule. pVHL regulates the protein stability of hypoxia-inducible factor (HIF)-α subunits and loss of pVHL function leads to HIF stabilization. The role of HIF in the development of VHL-associated renal lesions remains to be determined. To investigate the functional consequences of pVHL inactivation and the role of HIF signaling in renal epithelial cells, we used the phosphoenolpyruvate carboxykinase (PEPCK) promoter to generate transgenic mice in which Cre-recombinase is expressed in the renal proximal tubule and in hepatocytes. We found that conditional inactivation of VHL in PEPCK-Cre mutants resulted in renal cyst development that was associated with increased erythropoietin levels and polycythemia. Increased expression of the HIF target gene erythropoietin was limited to the liver, whereas expression of carbonic anhydrase 9 and multidrug resistance gene 1 was up-regulated in the renal cortex of mutant mice. Inactivation of the HIF-α binding partner, arylhydrocarbon receptor nuclear translocator (Arnt), but not Hif-1α, suppressed the development of renal cysts. Here, we present the first mouse model of VHL-associated renal disease that will provide a basis for further genetic studies to define the molecular events that are required for the progression of VHL-associated renal cysts to clear cell renal cell carcinoma.
doi:10.1158/0008-5472.CAN-05-3241
PMCID: PMC3514875  PMID: 16510575
12.  Hypoxia and cell cycle regulation of the von Hippel-Lindau tumor suppressor 
Oncogene  2010;30(1):21-31.
Inactivation of von Hippel-Lindau tumor suppressor protein (pVHL) is associated with von Hippel-Lindau disease, an inherited cancer syndrome, as well as the majority of patients with sporadic clear cell renal carcinoma (RCC). While the involvement of pVHL in oxygen sensing through targeting HIFα subunits to ubiquitin-dependent proteolysis has been well documented, less is known about pVHL regulation under both normoxic and hypoxic conditions. We found that pVHL levels decreased in hypoxia and that hypoxia-induced cell cycle arrest is associated with pVHL expression in RCC cells. pVHL levels fluctuate during the cell cycle, paralleling cyclin B1 levels, with decreased levels in mitosis and G1. pVHL contains consensus Destruction box sequences, and pVHL associates with Cdh1, an activator of the anaphase promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. We show that pVHL has a decreased half-life in G1, Cdh1 downregulation results in increased pVHL expression, while Cdh1 overexpression results in decreased pVHL expression. Taken together these results suggest that pVHL is a novel substrate of APC/CCdh1. Destruction box-independent pVHL degradation was also detected, indicating that other ubiquitin ligases are also activated for pVHL degradation.
doi:10.1038/onc.2010.395
PMCID: PMC2995849  PMID: 20802534
Von Hippel-Lindau protein; renal cell carcinoma; anaphase promoting complex/cyclosome; hypoxia; cell cycle
13.  Playing Tag with HIF: The VHL Story 
Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL is the cause of inherited VHL disease and is associated with sporadic kidney cancer. pVHL is found in a multiprotein complex with elongins B/C, Cul2, and Rbx1 forming an E3 ubiquitin ligase complex called VEC. This modular enzyme targets the α subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction. Consequently, tumour cells lacking functional pVHL overproduce the products of HIF-target genes such as vascular endothelial growth factor (VEGF), which promotes angiogenesis. This likely accounts for the hypervascular nature of VHL-associated neoplasms. Although pVHL has been linked to the cell-cycle, differentiation, and the regulation of extracellular matrix assembly, microenvironment pH, and tissue invasiveness, this review will focus on the recent insights into the molecular mechanisms governing the E3 ubiquitin ligase function of VEC.
doi:10.1155/S1110724302205057
PMCID: PMC161365  PMID: 12488577
14.  A yeast two-hybrid system reconstituting substrate recognition of the von Hippel-Lindau tumor suppressor protein 
Nucleic Acids Research  2007;35(21):e142.
The von Hippel-Lindau tumor suppressor protein (pVHL) is inactivated in the hereditary cancer syndrome von Hippel-Lindau disease and in the majority of sporadic renal carcinomas. pVHL is the substrate-binding subunit of the CBCVHL ubiquitin ligase complex that negatively regulates cell growth by promoting the degradation of hypoxia-inducible transcription factor subunits (HIF1/2α). Proteomics-based identification of novel pVHL substrates is hampered by their short half-life and low abundancy in mammalian cells. The usefulness of yeast two-hybrid (Y2H) approaches, on the other hand, has been limited by the failure of pVHL to adopt its native structure and by the absence of prolylhydroxylase activity critical for pVHL substrate recognition. Therefore, we modified the Y2H system to faithfully reconstitute the physical interaction between pVHL and its substrates. Our approach relies on the coexpression of pVHL with the cofactors Elongin B and Elongin C and with HIF1/2α prolylhydroxylases. In a proof-of-principle Y2H screen, we identified the known substrates HIF1/2α and new candidate substrates including diacylglycerol kinase iota, demonstrating that our strategy allows detection of stable interactions between pVHL and otherwise elusive cellular targets. Additional future applications may include structure/function analyses of pVHL-HIF1/2α binding and screens for therapeutically relevant compounds that either stabilize or disrupt this interaction.
doi:10.1093/nar/gkm932
PMCID: PMC2175351  PMID: 17986458
15.  Hypoxia-inducible factor–2 (HIF-2) regulates hepatic erythropoietin in vivo 
Journal of Clinical Investigation  2007;117(4):1068-1077.
Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1α and/or Hif-2α (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel–Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1α. Here we show that conditional inactivation of Hif-2α in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2α but not Hif-1α and that the hypoxic induction of liver Epo in anemic adults was Hif-2α dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1α dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.
doi:10.1172/JCI30117
PMCID: PMC1838939  PMID: 17404621
16.  The von Hippel–Lindau Tumor Suppressor Protein Is Destabilized by Src: Implications for Tumor Angiogenesis and Progression 
Genes & cancer  2010;1(3):225-238.
The von Hippel–Lindau tumor suppressor protein (VHL), when mutated and inactivated, has been associated with renal and CNS cancer development. VHL normally plays an important role in targeting for degradation of the HIF-1α (hypoxia inducible factor-1α) transcription factor, a primary positive regulator of vascular endothelial growth factor (VEGF) production. In this report we demonstrate that VHL destabilization can be induced by Src kinase and may be involved in other cancers, including breast cancer. We have found that elevated Src can trigger a drastic reduction in VHL stability even under normoxic conditions, through phosphorylation of VHL tyrosine residue 185, leading to ubiquitination and proteasome-mediated degradation of VHL. The Src-induced degradation of VHL protein leads to increased HIF-1α levels and transcriptional activity and increased VEGF production. In this manner, Src regulation of VHL protein stability may play an important role in promoting VEGF expression, tumor angiogenesis, and cancer progression.
doi:10.1177/1947601910366719
PMCID: PMC3014987  PMID: 21212839 CAMSID: cams1625
VHL; tumor suppressor; stability; Src; proto-oncogene; oncogene
17.  The von Hippel–Lindau Tumor Suppressor Protein Is Destabilized by Src 
Genes & Cancer  2010;1(3):225-238.
The von Hippel–Lindau tumor suppressor protein (VHL), when mutated and inactivated, has been associated with renal and CNS cancer development. VHL normally plays an important role in targeting for degradation of the HIF-1α (hypoxia inducible factor-1α) transcription factor, a primary positive regulator of vascular endothelial growth factor (VEGF) production. In this report we demonstrate that VHL destabilization can be induced by Src kinase and may be involved in other cancers, including breast cancer. We have found that elevated Src can trigger a drastic reduction in VHL stability even under normoxic conditions, through phosphorylation of VHL tyrosine residue 185, leading to ubiquitination and proteasome-mediated degradation of VHL. The Src-induced degradation of VHL protein leads to increased HIF-1α levels and transcriptional activity and increased VEGF production. In this manner, Src regulation of VHL protein stability may play an important role in promoting VEGF expression, tumor angiogenesis, and cancer progression.
doi:10.1177/1947601910366719
PMCID: PMC3014987  PMID: 21212839
VHL; tumor suppressor; stability; Src; proto-oncogene; oncogene
18.  The von Hippel-Lindau Tumor Suppressor Protein Promotes c-Cbl-Independent Poly-Ubiquitylation and Degradation of the Activated EGFR 
PLoS ONE  2011;6(9):e23936.
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 lysomomal 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.
doi:10.1371/journal.pone.0023936
PMCID: PMC3174936  PMID: 21949687
19.  Calpain Mediates a von Hippel-Lindau Protein–independent Destruction of Hypoxia-inducible Factor-1α 
Molecular Biology of the Cell  2006;17(4):1549-1558.
Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1α requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1α under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1α expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2′-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1α protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1α degradation. Furthermore, chelating intracellular calcium attenuated HIF-1α destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1α even under normoxia. An active role of calpain in lowering HIF-1α amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1α that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1α destruction involving the calpain system.
doi:10.1091/mbc.E05-08-0770
PMCID: PMC1415322  PMID: 16421254
20.  A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1α (HIF-1α) does not impair Pro-564 hydroxylation 
Molecular Cancer  2003;2:31.
Background
The hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).
Results
Patients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.
Conclusion
The Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.
doi:10.1186/1476-4598-2-31
PMCID: PMC212228  PMID: 14521712
21.  Hypoxia-Inducible Factor Linked to Differential Kidney Cancer Risk Seen with Type 2A and Type 2B VHL Mutations▿ † 
Molecular and Cellular Biology  2007;27(15):5381-5392.
Clear cell carcinoma of the kidney is a major cause of mortality in patients with von Hippel-Lindau (VHL) disease, which is caused by germ line mutations that inactivate the VHL tumor suppressor gene. Biallelic VHL inactivation, due to mutations or hypermethylation, is also common in sporadic clear cell renal carcinomas. The VHL gene product, pVHL, is part of a ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF) for destruction under well-oxygenated conditions. All VHL mutations linked to classical VHL disease compromise this pVHL function although some missense mutations result in a low risk of kidney cancer (type 2A VHL disease) while others result in a high risk (type 2B VHL disease). We found that type 2A mutants were less defective than type 2B mutants when reintroduced into VHL−/− renal carcinoma cells with respect to HIF regulation. A stabilized version of HIF2α promoted tumor growth by VHL−/− cells engineered to produce type 2A mutants, while knock-down of HIF2α in cells producing type 2B mutants had the opposite effect. Therefore, quantitative differences with respect to HIF deregulation are sufficient to account for the differential risks of kidney cancer linked to VHL mutations.
doi:10.1128/MCB.00282-07
PMCID: PMC1952077  PMID: 17526729
22.  Proteostasis modulators prolong missense VHL protein activity and halt tumor progression 
Cell reports  2013;3(1):52-59.
SUMMARY
While missense mutations of von Hippel-Lindau disease (VHL) gene are the most common germline mutation underlying this heritable cancer syndrome, the mechanism of tumorigenesis is unknown. We found a quantitative reduction of missense mutant VHL protein (pVHL) in VHL-associated tumors associated with physiologic mRNA expression. While mutant pVHL is unstable and degraded contemporarily with translation, it retains its E3 ligase function, including hypoxia inducible factor degradation. The premature pVHL degradation is due to misfolding and imbalance of chaperonin binding. Histone deacetylase inhibitors (HDACis) can modulate this pathway by inhibiting the HDAC6-Hsp90 chaperone axis, stabilizing pVHL and restoring activity comparable to wild type protein in vitro and in animal models (786-O tumor xenografts). HDACi mediated stabilization of missense pVHL significantly attenuates the growth of 786-O rodent tumor model. These findings provide direct biologic insight into VHL-associated tumors and elucidate a new treatment paradigm for VHL.
doi:10.1016/j.celrep.2012.12.007
PMCID: PMC3563731  PMID: 23318261
23.  Estrogen receptor α is a novel target of the Von Hippel-Lindau protein and is responsible for the proliferation of VHL-deficient cells under hypoxic conditions 
Cell Cycle  2012;11(23):4462-4473.
The Von Hippel-Lindau gene (VHL) is frequently deleted or mutated in human renal cell carcinoma (RCC) at the early stage. According to the well-established theory, pVHL acts as a tumor suppressor through its E3 ligase activity, which targets hypoxia-inducing factor-1α (HIF-1α). However, the elevated expression of HIF-1α did not promote cell proliferation, indicating that there would be another target, which could promote cell proliferation at the early cancer stage of RCC. In this study, we show that estrogen receptor-α (ER-α) is a novel proteasomal degradation target of the pVHL E3 ligase. Indeed, the overexpression of VHL suppresses exo- and endogenous ER-α expression, whereas si-pVHL can increase ER-α expression. The negative regulation of pVHL on ER-α expression is achieved by its E3 ligase activity. Thus, pVHL can promote the ER-α ubiquitinylation. In addition, we revealed that ER-α and HIF-1α are competitive substrates of pVHL. Thus, under normal conditions, ER-α overexpression can increase the transcription factor activity of HIF-1α. Under the hypoxic condition, where HIF-1α is not a suitable target of pVHL, ER-α is more rapidly degraded by pVHL. However, in VHL-deficient cells, the expression of ER-α and HIF-1α is retained, so that the hypoxic condition did not suppress cell proliferation obviously compared with cells that are expressing pVHL. Thus, blocking of ER-α using its inhibitor could suppress the proliferation of VHL-deficient cells as effectively as hypoxia-induced growth suppression. Considering our results, blocking of ER-α signaling in VHL-deficient cancer cells would be beneficial for cancer suppression. Indeed, we showed the anti-proliferative effect of Faslodex in VHL-deficient cells.
doi:10.4161/cc.22794
PMCID: PMC3552928  PMID: 23159849
RCC; VHL; ER-alpha; hypoxia; cell proliferation
24.  Hypoxia-Inducible Factor (HIF)-2 regulates Vascular Tumorigenesis in Mice 
Oncogene  2008;27(40):5354-5358.
The von Hippel-Lindau tumor suppressor pVHL regulates the stability of Hypoxia-Inducible Factors (HIF) -1 and –2, oxygen-sensitive basic helix-loop-helix transcription factors, which mediate the hypoxic induction of angiogenic growth factors such as vascular endothelial growth factor (VEGF). Loss of VHL function results in constitutive activation of HIF-1 and HIF-2 and is associated with the development of highly vascularized tumors in multiple organs. We have used a conditional gene targeting approach to investigate the relative contributions of HIF-1 and HIF-2 to VHL-associated vascular tumorigenesis in a mouse model of liver hemangiomas. Here we demonstrate genetically that conditional inactivation of HIF-2α suppressed the development of VHL-associated liver hemangiomas and that angiogenic gene expression in hepatocytes is predominantly regulated by HIF-2 and not by HIF-1. These findings suggest that HIF-2 is the dominant HIF in the pathogenesis of VHL-associated vascular tumors and that pharmacologic targeting of HIF-2 may be an effective strategy for their treatment.
doi:10.1038/onc.2008.160
PMCID: PMC2575082  PMID: 18490920
25.  Sequence Determinants in Hypoxia-inducible Factor-1α for Hydroxylation by the Prolyl Hydroxylases PHD1, PHD2, and PHD3 
The Journal of biological chemistry  2002;277(42):39792-39800.
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor induced by hypoxia. Under normoxic conditions, site-specific proline hydroxylation of the α subunits of HIF allows recognition by the von Hippel-Lindau tumor suppressor protein (VHL), a component of an E3 ubiquitin ligase complex that targets these subunits for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, this hydroxylation is inhibited, allowing the α subunits of HIF to escape VHL-mediated degradation. Three enzymes, prolyl hydroxylase domain-containing proteins 1, 2, and 3 (PHD1, -2, and -3; also known as HIF prolyl hydroxylase 3, 2, and 1, respectively), have recently been identified that catalyze proline hydroxylation of HIF α subunits. These enzymes hydroxylate specific prolines in HIF α subunits in the context of a strongly conserved LXXLAP sequence motif (where X indicates any amino acid and P indicates the hydroxylacceptor proline). We report here that PHD2 has the highest specific activity toward the primary hydroxylation site of HIF-1α. Furthermore, and unexpectedly, mutations can be tolerated at the −5, −2, and −1 positions (relative to proline) of the LXXLAP motif. Thus, these results provide evidence that the only obligatory residue for proline hydroxylation in HIF-1α is the hydroxylacceptor proline itself.
doi:10.1074/jbc.M206955200
PMCID: PMC1828037  PMID: 12181324

Results 1-25 (688247)