Article

Anglesio, M.S. et al. Differential expression of a novel ankyrin containing E3 ubiquitin-protein ligase, Hace1, in sporadic Wilms' tumor versus normal kidney. Hum. Mol. Genet. 13, 2061-2074

Department of Pathology, British Columbia Research Institute for Children's and Women's Health, University of British Columbia, Vancouver, BC, V5Z 4H4, Canada.
Human Molecular Genetics (Impact Factor: 6.68). 10/2004; 13(18):2061-74. DOI: 10.1093/hmg/ddh215
Source: PubMed

ABSTRACT We have analyzed the chromosome 6q21 breakpoint of a non-constitutional t(6;15)(q21;q21) rearrangement in sporadic Wilms' tumor. This identified a novel gene encoding a protein with six N-terminal ankyrin repeats linked to a C-terminal HECT ubiquitin-protein ligase domain. We therefore designated this gene HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1). HACE1 is widely expressed in human tissues, including mature and fetal kidney. We show that Hace1 protein possesses intrinsic ubiquitin ligase activity, utilizes UbcH7 as a candidate partner E2 enzyme and localizes predominantly to the endoplasmic reticulum. Although the HACE1 locus was not directly interrupted by the translocation in the index Wilms' case, its expression was markedly lower in tumor tissue compared with adjacent normal kidney. Moreover, HACE1 expression was virtually undetectable in the SK-NEP-1 Wilms' tumor cell line and in four of five additional primary Wilms' tumor cases compared with patient-matched normal kidney. We found no evidence of HACE1 mutations or deletions, but hypermethylation of two upstream CpG islands correlates with low HACE1 expression in tumor samples. Our findings implicate Hace1 as a novel ubiquitin-protein ligase and demonstrate that its expression is very low in primary Wilms' tumors.

Download full-text

Full-text

Available from: Poul H B Sorensen, Sep 02, 2015
0 Followers
 · 
175 Views
 · 
51 Downloads
  • Source
    • ".H.S.). 15. Anglesio MS, et al. (2004) "
    [Show abstract] [Hide abstract]
    ABSTRACT: Oxidative stress plays a key role in late onset diseases including cancer and neurodegenerative diseases such as Huntington disease. Therefore, uncovering regulators of the antioxidant stress responses is important for understanding the course of these diseases. Indeed, the nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of the cellular antioxidative stress response, is deregulated in both cancer and neurodegeneration. Similar to NRF2, the tumor suppressor Homologous to the E6-AP Carboxyl Terminus (HECT) domain and Ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays a protective role against stress-induced tumorigenesis in mice, but its roles in the antioxidative stress response or its involvement in neurodegeneration have not been investigated. To this end we examined Hace1 WT and KO mice and found that Hace1 KO animals exhibited increased oxidative stress in brain and that the antioxidative stress response was impaired. Moreover, HACE1 was found to be essential for optimal NRF2 activation in cells challenged with oxidative stress, as HACE1 depletion resulted in reduced NRF2 activity, stability, and protein synthesis, leading to lower tolerance against oxidative stress triggers. Strikingly, we found a reduction of HACE1 levels in the striatum of Huntington disease patients, implicating HACE1 in the pathology of Huntington disease. Moreover, ectopic expression of HACE1 in striatal neuronal progenitor cells provided protection against mutant Huntingtin-induced redox imbalance and hypersensitivity to oxidative stress, by augmenting NRF2 functions. These findings reveal that the tumor suppressor HACE1 plays a role in the NRF2 antioxidative stress response pathway and in neurodegeneration.
    Proceedings of the National Academy of Sciences 02/2014; 111(8). DOI:10.1073/pnas.1314421111 · 9.81 Impact Factor
  • Source
    • "The effect of HACE1 on trafficking of transiently expressed b 2 AR was then determined by ELISA. Transient expression of Myc-HACE1 and Myc-HACE1-C876S (a mutant with deficient E3-ligase activity) (Anglesio et al., 2004; Tang et al., 2011; Zhang et al., 2007) increased cell surface expression of b 2 AR by roughly 25%, indicating that the catalytic activity of the enzyme is not required for this effect (Fig. 1B). Time-course analyses established that HACE1 significantly decreased the apparent agonist-induced internalization of the b 2 AR, whereas HACE1-C876S had virtually no effect (Fig. 1C). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We and others have shown that trafficking of G protein-coupled receptors is regulated by Rab GTPases. Cargo-mediated regulation of vesicular transport has received great attention lately. Rab GTPases, forming the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Rab GTPases are well-recognized targets of human diseases but their regulation and the mechanisms connecting them to cargo proteins are still poorly understood. Herein, we show by overexpression/depletion studies that HACE1, a HECT domain-containing ubiquitin ligase, promotes the recycling of the β2-adrenergic receptor (β2AR), a prototypical G protein-coupled receptor, through a Rab11a-dependent mechanism. Interestingly, the β2AR in conjunction with HACE1 triggered ubiquitination of Rab11a, as observed by Western blot analysis. LC-MS/MS experiments determined that Rab11a is ubiquitnatied on Lys(145). A Rab11a-K145R mutant failed to undergo β2AR/HACE1-induced ubiquitination and inhibited the HACE1-mediated recycling of the β2AR. Rab11a, but not Rab11a-K145R, was activated by β2AR/HACE1 indicating that ubiquitination of Lys(145) is involved in Rab11a activation. β2AR/HACE1 co-expression also potentiated ubiquitination of Rab6a and Rab8a, but not of other Rab GTPases that were tested. We report a novel regulatory mechanism of Rab GTPases by their ubiquitination with associated functional effects demonstrated on Rab11a. This partakes into a new pathway whereby a cargo protein, like a G protein-coupled receptor, can regulate its own trafficking by inducing the ubiquitination and activation of a Rab GTPase.
    Journal of Cell Science 11/2013; 127(1). DOI:10.1242/jcs.132944 · 5.33 Impact Factor
  • Source
    • "BRCA1 interacts with many proteins to serve its function in the cell. Protein kinases have been shown to be critical in BRCA1-phosyphorylation, where they are involved in activation or deactivation of the BRCA1 protein function including its stability, protein-interactions and sub-cellular location [34]–[36], its regulation of DNA repair [37]–[40] and its transcriptional activity [41]–[43]. The phosphorylation pattern of BRCA2 is less well known but it is shown to be essential in the regulation of BRCA2-mediated DNA recombination repair [44], [45]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mutations in BRCA1 and BRCA2 are responsible for a large proportion of breast-ovarian cancer families. Protein-truncating mutations have been effectively used in the clinical management of familial breast cancer due to their deleterious impact on protein function. However, the majority of missense variants identified throughout the genes continue to pose an obstacle for predictive informative testing due to low frequency and lack of information on how they affect BRCA1/2 function. Phosphorylation of BRCA1 and BRCA2 play an important role in their function as regulators of DNA repair, transcription and cell cycle in response to DNA damage but whether missense variants of uncertain significance (VUS) are able to disrupt this important process is not known. Here we employed a novel approach using NetworKIN which predicts in vivo kinase-substrate relationship, and evolutionary conservation algorithms SIFT, PolyPhen and Align-GVGD. We evaluated whether 191 BRCA1 and 43 BRCA2 VUS from the Breast Cancer Information Core (BIC) database can functionally alter the consensus phosphorylation motifs and abolish kinase recognition and binding to sites known to be phosphorylated in vivo. Our results show that 13.09% (25/191) BRCA1 and 13.95% (6/43) BRCA2 VUS altered the phosphorylation of BRCA1 and BRCA2. We highlight six BRCA1 (K309T, S632N, S1143F, Q1144H, Q1281P, S1542C) and three BRCA2 (S196I, T207A, P3292L) VUS as potentially clinically significant. These occurred rarely (n<2 in BIC), mutated evolutionarily conserved residues and abolished kinase binding to motifs established in the literature involved in DNA repair, cell cycle regulation, transcription or response to DNA damage. Additionally in vivo phosphorylation sites identified via through-put methods are also affected by VUS and are attractive targets for studying their biological and functional significance. We propose that rare VUS affecting phosphorylation may be a novel and important mechanism for which BRCA1 and BRCA2 functions are disrupted in breast cancer.
    PLoS ONE 05/2013; 8(5):e62468. DOI:10.1371/journal.pone.0062468 · 3.23 Impact Factor
Show more