Drosophila Topors Is a RING Finger-containing Protein That Functions as a Ubiquitin-protein Isopeptide Ligase for the Hairy Basic Helix-Loop-Helix Repressor Protein

Secombe, Julie; Parkhurst, Susan M.

doi:10.1074/jbc.m310097200

Cited by 17 publications

(22 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Precedent for such a dual-activity protein is provided by the conserved RING-finger protein, Topors. Topors binds DNA topoisomerase I [50] and p53 [51,52] and it displays both Ub E3 ligase [53,54] and SUMO ligase activity [55]. Topors Ub ligase activity is dependent on its RING-finger domain, while its SUMO ligase activity is not [55].…”

Section: Discussionmentioning

confidence: 99%

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

et al. 2007

View full text Add to dashboard Cite

The yeast genes SLX5 and SLX8 were identified based on their requirement for viability in the absence of the Sgs1 DNA helicase. Loss of these genes results in genome instability, nibbled colonies, and other phenotypes associated with defects in sumoylation. The Slx5 and Slx8 proteins form a stable complex and each subunit contains a single RING-finger domain at its C-terminus. To determine the physiological function of the Slx5-8 complex, we explored its interaction with the SUMO pathway. Curing 2μ circle from the mutants suppressed their nibbled colony phenotype and partially improved their growth rate, but did not affect their sensitivity to hydroxyurea. The increase in sumoylation observed in slx5Δ and slx8Δ mutants was found to be dependent on the Siz1 SUMO ligase. Physical interactions between the Slx5-8 complex and both Ubc9 and Smt3 were identified and characterized. Using in vitro reactions, we show that Slx5, Slx8, or the Slx5-8 complex stimulates the formation of SUMO chains and the sumoylation of a test substrate. Interestingly, a functional RING-finger domain is not required for this stimulation in vitro. These biochemical data demonstrate for the first time that the Slx5-8 complex is capable of interacting directly with the SUMO pathway.

show abstract

Section: Discussionmentioning

confidence: 99%

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

et al. 2007

View full text Add to dashboard Cite

show abstract

“…This localization was consistent with recombination mapping of nbl to the region between pr and px. Additional complementation tests revealed that Df(dtopors AA ), a small deletion that removes dtopors and the adjacent gene prod (Secombe and Parkhurst 2004), failed to complement the nuclear and chromosome segregation defects of nbl. Furthermore, an insertional mutation in dtopors, P(Bac{WH}Topors f05115 , failed to complement nbl, whereas an insertional mutation in prod, P{lacW}prod k08810 , fully complemented.…”

Section: Mapping Nbl Mutationsmentioning

confidence: 99%

“…In addition to the EMS-induced alleles, we made use of an existing deletion, Df(dtopors AA ) (Secombe and Parkhurst 2004), and a piggyBac insertion allele, dtopors f05115 . Conceptual translation of the piggyBac insertion allele sequence predicts that it encode a protein truncated after amino acid 47 and therefore is likely a null allele.…”

Section: Mutations In Dtopors Disrupt Segregation Of All Chromosomes mentioning

confidence: 99%

“…Coverslips were removed after freezing in liquid nitrogen and tissues fixed 10 min in cold methanol. Primary antibodies were used at the following dilutions in phosphate buffered saline (PBS) + 1.0% bovine serum albumin, fraction V (Fisher Scientific): rabbit anti-GFP 1:1000 (Invitrogen), mouse anti-dTopors, 1:1000 (Secombe and Parkhurst 2004), mouse anti-b-tubulin 1:500 (E7, Iowa Hybridoma Bank), mouse anti-g-tubulin 1:2000 (GTU-88, Sigma Scientific), mouse anti-lamin Dm0 1:10 (LC28), and mouse antilamin C (ADL67) 1:100 (Iowa Hybridoma Bank), rabbit anti-pH 3 (Sigma 06570) 1:500, anti-centrosomin 1:500 (Li et al 1998), anti-dTACC 1:2000 (Gergely et al 2000), anti-Aurora A 1:1000 (Berdnik and Knoblich 2002), and anti-HP1 (CA19) 1:500 (Iowa Hybridoma Bank). Secondary antibodies used were Alexa Fluor 546 goat anti-mouse, 1:1000 (Molecular Probes) and Alexa Fluor 488 goat antirabbit (Molecular Probes).…”

Section: Indirect Immunofluorescence and Confocal Microscopymentioning

confidence: 99%

See 1 more Smart Citation

Nuclear Structure and Chromosome Segregation inDrosophilaMale Meiosis Depend on the Ubiquitin Ligase dTopors

et al. 2011

View full text Add to dashboard Cite

In many organisms, homolog pairing and synapsis at meiotic prophase depend on interactions between chromosomes and the nuclear membrane. Male Drosophila lack synapsis, but nonetheless, their chromosomes closely associate with the nuclear periphery at prophase I. To explore the functional significance of this association, we characterize mutations in nuclear blebber (nbl), a gene required for both spermatocyte nuclear shape and meiotic chromosome transmission. We demonstrate that nbl corresponds to dtopors, the Drosophila homolog of the mammalian dual ubiquitin/small ubiquitin-related modifier (SUMO) ligase Topors. We show that mutations in dtopors cause abnormalities in lamin localizations, centriole separation, and prophase I chromatin condensation and also cause anaphase I bridges that likely result from unresolved homolog connections. Bridge formation does not require mod(mdg4) in meiosis, suggesting that bridges do not result from misregulation of the male homolog conjunction complex. At the ultrastructural level, we observe disruption of nuclear shape, an uneven perinuclear space, and excess membranous structures. We show that dTopors localizes to the nuclear lamina at prophase, and also transiently to intranuclear foci. As a role of dtopors at gypsy insulator has been reported, we also asked whether these new alleles affected expression of the gypsy-induced mutation ct 6 and found that it was unaltered in dtopors homozygotes. Our results indicate that dTopors is required for germline nuclear structure and meiotic chromosome segregation, but in contrast, is not necessary for gypsy insulator function. We suggest that dtopors plays a structural role in spermatocyte lamina that is critical for multiple aspects of meiotic chromosome transmission.A SSOCIATIONS between chromosomes and the nuclear envelope during meiotic prophase are a widely conserved phenomenon important for proper chromosome transmission. In many species, such interactions are required for bouquet formation, an arrangement in which telomeres cluster in association with the nuclear envelope to facilitate homolog pairing and synapsis (reviewed in Scherthan 2007). In Caenorhabditis elegans, analogous interactions between nuclear envelope proteins and chromosomes are mediated by zinc finger proteins that connect chromosomespecific pairing sites to integral nuclear membrane proteins ). These connections establish bridges across the nuclear envelope and allow for interactions between meiotic chromosomes and cytoskeletal actin. Chromosome movements dependent on these connections are important for homolog pairing (Sato et al. 2009).The association of meiotic chromosomes with the nuclear periphery is particularly striking in Drosophila males, in which paired homologs occupy discreet domains closely apposed to the nuclear membrane. The relevance of this organization to meiotic chromosome segregation in this organism, however, has not been explored. Drosophila males have an unconventional meiosis in which homologs pair but do not assemble synapto...

show abstract

“…Because TOPORS is a known E3 ubiquitin ligase for the transcription factors p53 (23) and Hairy (29), we sought to determine whether TOPORS can also ubiquitinate NKX3.1. In the presence of ubiquitin, E1, and E2 (UbcH5a) enzymes, TOPORS induced the appearance of higher molecular weight species of NKX3.1 (Fig.…”

Section: Topors Interacts With and Ubiquitinates Nkx31-tomentioning

confidence: 99%

Ubiquitination by TOPORS Regulates the Prostate Tumor Suppressor NKX3.1

Guan

Pungaliya

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

The NKX3.1 gene located at 8p21.2 encodes a homeodomaincontaining transcription factor that acts as a haploinsufficient tumor suppressor in prostate cancer. Diminished protein expression of NKX3.1 has been observed in prostate cancer precursors and carcinomas. TOPORS is a ubiquitously expressed E3 ubiquitin ligase that can ubiquitinate tumor suppressor p53. Here we report interaction between NKX3.1 and TOPORS. NKX3.1 can be ubiquitinated by TOPORS in vitro and in vivo, and overexpression of TOPORS leads to NKX3.1 proteasomal degradation in prostate cancer cells. Conversely, small interfering RNA-mediated knockdown of TOPORS leads to an increased steady-state level and prolonged half-life of NKX3.1. These data establish TOPORS as a negative regulator of NKX3.1 and implicate TOPORS in prostate cancer progression.Prostate cancer is the second leading cause of cancer deaths and the most frequently diagnosed malignancy in American men (1). Prostate carcinomas are considered to arise from cancer precursors including prostatic intraepithelial neoplasia (PIN) 2 and proliferative inflammatory atrophy (2). Molecular alterations, including hereditary and somatic gene mutations, gene deletions, gene amplification, chromosomal rearrangements, as well as epigenetic changes, have been implicated in prostate cancer initiation and progression (3).The NK class homeobox gene NKX3.1 has been studied extensively over the past decade for its roles in prostate development and carcinogenesis (4). The expression of the murine Nkx3.1 gene is androgen-dependent and is restricted largely to prostate epithelial cells in adults (5-7). Deletion of Nkx3.1 by gene targeting leads to prostate ductal morphological defects, as well as prostatic dysplasia and hyperplasia that resembles human PIN (8 -10). Interestingly, heterozygous Nkx3.1 mice also develop hyperplasia and PIN-like lesions (8). The human NKX3.1 gene maps to 8p21.2 within a region where loss of heterozygosity occurs in PIN and is common in prostate carcinomas; however, no mutations have been found in the coding region of the NKX3.1 allele remaining (11,12). In light of these observations, NKX3.1 has been proposed to function as a haploinsufficient tumor suppressor. In support of a dose-dependent growth regulatory function of NKX3.1, reduced but not complete loss of NKX3.1 protein expression is now well documented in most human prostate cancer samples in a manner inversely correlated with Gleason score (13) and also with disease progression (14). Diminished NKX3.1 expression is thought to be an early event in prostate carcinogenesis, and reduced NKX3.1 immunostaining was observed in most proliferative inflammatory atrophy and PIN lesions analyzed (13). The diminished NKX3.1 expression may be partly attributed to selective CpG methylation of the NKX3.1 promoter in some prostate cancer cases (15). Nevertheless, quantitative analyses of mRNA and protein levels of NKX3.1 in prostate carcinoma lesions revealed a lack of concordance between mRNA and protein levels (13). In particular, decre...

show abstract

Drosophila Topors Is a RING Finger-containing Protein That Functions as a Ubiquitin-protein Isopeptide Ligase for the Hairy Basic Helix-Loop-Helix Repressor Protein

Cited by 17 publications

References 41 publications

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

Nuclear Structure and Chromosome Segregation inDrosophilaMale Meiosis Depend on the Ubiquitin Ligase dTopors

Ubiquitination by TOPORS Regulates the Prostate Tumor Suppressor NKX3.1

Contact Info

Product

Resources

About