Abstract
von Hippel-Lindau (VHL) disease is a rare familial cancer predisposition syndrome caused by a loss or mutation in a single gene,VHL, but it exhibits a wide phenotypic variability that can be categorized into distinct subtypes. The phenotypic variability has been largely argued to be attributable to the extent of deregulation of the α subunit of hypoxia-inducible factor α, a well established target of VHL E3 ubiquitin ligase, ECV (Elongins/Cul2/VHL). Here, we show that erythropoietin receptor (EPOR) is hydroxylated on proline 419 and 426 via prolyl hydroxylase 3. EPOR hydroxylation is required for binding to the β domain of VHL and polyubiquitylation via ECV, leading to increased EPOR turnover. In addition, several type-specific VHL disease-causing mutants, including those that have retained proper binding and regulation of hypoxia-inducible factor α, showed a severe defect in binding prolyl hydroxylated EPOR peptides. These results identify EPOR as the secondbona fidehydroxylation-dependent substrate of VHL that potentially influences oxygen homeostasis and contributes to the complex genotype-phenotype correlation in VHL disease.
Highlights
APRIL 1, 2016 VOLUME 291 NUMBER 14 without renal cell carcinoma (RCC); type 2B, characterized by PHEO and HAB with RCC; type 2C, characterized by exclusive development of PHEO; and type 3, which is characterized by the development of polycythemia without an increased cancer predisposition [2]
Truncation mutations in EPO receptor (EPOR) that lead to a failure in negative regulation or JAK2 mutations that cause increased activity are often found in primary form of polycythemia, which are instigated by an exaggerated response to EPO stimulus (24 –26)
Oxygen tension was inversely correlated to total EPOR level, whereas the level of total JAK2 remained unchanged (Fig. 1C)
Summary
Cells—786-O, HEK293A, and HEK293T cells (American Type Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% heatinactivated fetal bovine serum (Wisent) at 37 °C in a humidified atmosphere with 5% CO2. 786-O lines stably expressing HAVHL or empty plasmid were previously described [3, 4]. ␥2A cells were a kind gift from Dr George Stark (Cleveland Clinic) and were maintained in DMEM supplemented with 10% heatinactivated fetal bovine serum, 1 mM sodium pyruvate, and 0.4 mg/ml G418 (Sigma). Binding assays using biotinylated peptides were performed as previously described [37] with the following modifications: methionine was not radiolabeled, so immunoblotting was used to detect rabbit reticulocyte in vitro translated (Promega) proteins; EBC buffer was supplemented with 1 mM DTT; washes were conducted in NETN containing 300 mM NaCl instead of 100 mM NaCl. In Vitro Hydroxylation—In vitro hydroxylation assays were performed essentially as previously described [37]. In Vitro Differentiation of UT-7 Cells—The cells were washed extensively in plain ␣-MEM and starved overnight in ␣-MEM supplemented with 20% fetal bovine serum and puromycin and without GM-CSF. Cells were washed in PBS twice and lysed by vortexing in 3 volumes of lysis solution H (50 mM Tris, pH 7, 25 mM KCl, 5 mM MgCl2, 1 mM -mercaptoethanol, 0.3% Triton X-100) similar to as previously described [38] and centrifuged on a tabletop centrifuge at 4 °C for 10 min at maximum speed. Statistical analysis was performed using GraphPad Prism 5.0 software
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