Ubiquitination of a Novel Deubiquitinating Enzyme Requires Direct Binding to von Hippel-Lindau Tumor Suppressor Protein

Abstract

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 twohybrid 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 ␤-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 ubiquitinproteasome degradation pathway.

The von Hippel-Lindau Tumor Suppressor Stabilizes Novel Plant Homeodomain Protein Jade-1

“…Jade-1 also interacted convincingly with human VHL aa 55-143 (3ϩ) as well as mouse VHL (2ϩ) in yeast. VDU1 (10) was also recovered in the screen, supporting the validity of this approach for identifying bona fide VHL interactors. Additional confirmed positive interactors in yeast include four other novel genes, 17 known genes encoding a wide range of proteins, and 14 known genes encoding chaperones or chaperone-like proteins, which are common false-positives.…”

“…VHL binds and promotes ubiquitination of hypoxia-inducible transcription factors HIF-1␣ and HIF-2␣ (7), protein kinase C (PKC) lambda (8), heterogeneous nuclear ribonucleoprotein A2 (9), and VHLinteracting deubiquitinating enzyme-1 (VDU1) (10). VHL inhibits transcription elongation (11)(12)(13)(14), mRNA stability (9,(15)(16)(17), Sp1-related promoter activity (18,19), and PKC activity (8,20,21).…”

von Hippel–Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination

“…Importantly, binding of pVHL to the full-size Rpb1 requires hydroxylation of proline-1465 within Rpb1 and phosphorylation of the CTD. To date, the proteins that pVHL targets for ubiquitination include, in addition to HIF-␣s, a subfamily of deubiquitinating enzymes (43,44) and activated atypical protein kinase C (45).…”

The VHL tumor suppressor and HIF: insights from genetic studies in mice

“…51 Furthermore, pVHL has been shown to regulate microtubule stability and cilia maintenance [52][53][54][55] and controls the activity of plant homeodomain protein Jade-1, 56,57 and atypical protein kinase C isoforms. [58][59][60][61][62] Other pVHL targets include a KRAB-A domain protein, VHLak, repressing HIF transcriptional activity, 63 de-ubiquitinating enzymes, 64 the large subunit of RNA polymerase II 65 and the RNA-binding protein hnRNP A2. 66 How these HIF-independent pVHL functions and recently discovered protein interactions exactly contribute to the initiation and progression of VHL-associated tumorigenesis is unclear and requires further investigation.…”