HUWE1 | GeneID:10075 | Homo sapiens
[ ] NCBI Entrez Gene
|Gene ID||10075||Official Symbol||HUWE1|
|Synonyms||ARF-BP1; HECTH9; HSPC272; Ib772; KIAA0312; LASU1; MULE; UREB1|
|Full Name||HECT, UBA and WWE domain containing 1|
|Description||HECT, UBA and WWE domain containing 1|
|Also Known As||ARF binding protein 1; BJ-HCC-24 tumor antigen; HECT domain protein LASU1; Mcl-1 ubiquitin ligase E3; OTTHUMP00000023356; OTTHUMP00000061860|
Orthologs and Paralogs
|GeneID:480931||HUWE1||XP_538052.2||Canis lupus familiaris|
[ ] Monoclonal and Polyclonal Antibodies
|1||abcam||ab70161||ARFBP1 antibody (ab70161); Rabbit polyclonal to ARFBP1|
|2||abcam||ab65153||ARFBP1 antibody (ab65153); Rabbit polyclonal to ARFBP1|
|3||acris||AP07305PU-N||HUWE1 / UREB1; antibody Ab|
|4||scbt||HUWE1||HUWE1 Antibody / HUWE1 Antibodies;|
|5||sigma||HPA002548||Anti-HUWE1 antibody produced in rabbit ;|
|GO:0016881||Function||acid-amino acid ligase activity|
|GO:0004842||Function||ubiquitin-protein ligase activity|
|GO:0019941||Process||modification-dependent protein catabolic process|
|GO:0006464||Process||protein modification process|
MicroRNA and Targets
[ ] MicroRNA Sequences and Transcript Targets from miRBase at Sanger
|RNA Target||miRNA #||mat miRNA||Mature miRNA Sequence|
AAC62492 AAF28950 AAH02602 AAH18750 AAH38184 AAH63505 AAI07577 AAV90838 AAX24125 AAY98258 BAA20771 BAC06833 BAD96887 BAG57767 CAB82393 CAG33094 CAI39578 CAI39579 CAI42351 CAI42352 CAI42353 CAI42354 CAI42644 CAI42654 CAI42656 CAJ47378 EAW93160 EAW93161 EAW93162 EAW93163 EAW93164 EAW93165 EAW93166 NP_113584 Q3B7K0 Q53FW4 Q5H924 Q5H935 Q5H962 Q5H963 Q7Z6Z7 Q8IWJ0 Q9NXW1
Mutations and SNPs
- All SNPs in GeneID:10075 / HUWE1 Gene
The mutation data was obtained from the Sanger Institute Catalogue Of Somatic Mutations In Cancer web site, http://www.sanger.ac.uk/cosmic Bamford et al (2004) The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website. Br J Cancer, 91,355-358.
|Mutation (top 10)||Total Observations|
|Primary Site / Histology (Top 10)||Mutations (sites * observations)|
|breast / carcinoma||2|
Chemicals and Drugs
[ ] Comparative Toxicogenomics Database from MDI Biological Lab
Curated [chemical–gene interactions|chemical–disease|gene–disease] data were retrieved from the Comparative Toxicogenomics Database (CTD), Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. World Wide Web (URL: http://ctd.mdibl.org/). [Jan. 2009].
|Chemical and Interaction|
Gene and Diseases
Curated [chemical–gene interactions|chemical–disease|gene–disease] data were retrieved from the Comparative Toxicogenomics Database (CTD), Mount Desert Island Biological Laboratory, Salisbury Cove, Maine. World Wide Web (URL: http://ctd.mdibl.org/). [Jan. 2009].
- [ ] Stankiewicz AR, et al. (2009) "Regulation of heat-induced apoptosis by Mcl-1 degradation and its inhibition by Hsp70." Cell Death Differ. 16(4):638-647. PMID:19148187
- [ ] Horikawa Y, et al. (2008) "Single nucleotide polymorphisms of microRNA machinery genes modify the risk of renal cell carcinoma." Clin Cancer Res. 14(23):7956-7962. PMID:19047128
- [ ] Herold S, et al. (2008) "Miz1 and HectH9 regulate the stability of the checkpoint protein, TopBP1." EMBO J. 27(21):2851-2861. PMID:18923429
- [ ] Barbe L, et al. (2008) "Toward a confocal subcellular atlas of the human proteome." Mol Cell Proteomics. 7(3):499-508. PMID:18029348
- [ ] Zhao X, et al. (2008) "The HECT-domain ubiquitin ligase Huwe1 controls neural differentiation and proliferation by destabilizing the N-Myc oncoprotein." Nat Cell Biol. 10(6):643-653. PMID:18488021
- [ ] Froyen G, et al. (2008) "Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation." Am J Hum Genet. 82(2):432-443. PMID:18252223
- [ ] Hall JR, et al. (2007) "Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage." Mol Biol Cell. 18(9):3340-3350. PMID:17567951
- [ ] Olsen JV, et al. (2006) "Global, in vivo, and site-specific phosphorylation dynamics in signaling networks." Cell. 127(3):635-648. PMID:17081983
- [ ] Beausoleil SA, et al. (2006) "A probability-based approach for high-throughput protein phosphorylation analysis and site localization." Nat Biotechnol. 24(10):1285-1292. PMID:16964243
- [ ] Ross MT, et al. (2005) "The DNA sequence of the human X chromosome." Nature. 434(7031):325-337. PMID:15772651
- [ ] Yoon SY, et al. (2005) "Over-expression of human UREB1 in colorectal cancer: HECT domain of human UREB1 inhibits the activity of tumor suppressor p53 protein." Biochem Biophys Res Commun. 326(1):7-17. PMID:15567145
- [ ] Liu Z, et al. (2005) "Characterization of E3Histone, a novel testis ubiquitin protein ligase which ubiquitinates histones." Mol Cell Biol. 25(7):2819-2831. PMID:15767685
- [ ] Chen D, et al. (2005) "ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor." Cell. 121(7):1071-1083. PMID:15989956
- [ ] Zhong Q, et al. (2005) "Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis." Cell. 121(7):1085-1095. PMID:15989957
- [ ] Warr MR, et al. (2005) "BH3-ligand regulates access of MCL-1 to its E3 ligase." FEBS Lett. 579(25):5603-5608. PMID:16213503
- [ ] Adhikary S, et al. (2005) "The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation." Cell. 123(3):409-421. PMID:16269333
- [ ] Ota T, et al. (2004) "Complete sequencing and characterization of 21,243 full-length human cDNAs." Nat Genet. 36(1):40-45. PMID:14702039
- [ ] Gerhard DS, et al. (2004) "The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC)." Genome Res. 14(10B):2121-2127. PMID:15489334
- [ ] Beausoleil SA, et al. (2004) "Large-scale characterization of HeLa cell nuclear phosphoproteins." Proc Natl Acad Sci U S A. 101(33):12130-12135. PMID:15302935
- [ ] Strausberg RL, et al. (2002) "Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences." Proc Natl Acad Sci U S A. 99(26):16899-16903. PMID:12477932
- [ ] Zhang QH, et al. (2000) "Cloning and functional analysis of cDNAs with open reading frames for 300 previously undefined genes expressed in CD34+ hematopoietic stem/progenitor cells." Genome Res. 10(10):1546-1560. PMID:11042152
- [ ] Nagase T, et al. (2000) "Prediction of the coding sequences of unidentified human genes. XVIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro." DNA Res. 7(4):273-281. PMID:10997877
- [ ] Jackson PK, et al. (2000) "The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases." Trends Cell Biol. 10(10):429-439. PMID:10998601
- [ ] Suzuki Y, et al. (1997) "Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library." Gene. 200(1-2):149-156. PMID:9373149
- [ ] Nagase T, et al. (1997) "Prediction of the coding sequences of unidentified human genes. VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro." DNA Res. 4(2):141-150. PMID:9205841
- [ ] Gu J, et al. (1995) "UREB1, a tyrosine phosphorylated nuclear protein, inhibits p53 transactivation." Oncogene. 11(10):2175-2178. PMID:7478539
- [ ] Maruyama K, et al. (1994) "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides." Gene. 138(1-2):171-174. PMID:8125298
- [ ] Gu J, et al. (1994) "Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstream initiator-like sequence in the promoter of the preprodynorphin gene." Brain Res Mol Brain Res. 24(1-4):77-88. PMID:7968380
Cellular stress eliminates irreversibly damaged cells by initiating the intrinsic death pathway. Cell stress is sensed by pro- and antiapoptotic members of the Bcl-2 protein family, which regulate the release of apoptogenic factors, such as cytochrome c, from mitochondria. Exposure of cells to hyperthermia results in the activation of the proapoptotic Bcl-2 family protein Bax, which plays an essential role in cytochrome c release. Heat directly affects Bax activity in vitro; however, antiapoptotic Bcl-2 family proteins, such as Bcl-xL, can suppress this activation, suggesting that a second heat-sensitive step must be breached before apoptosis ensues in cells exposed to hyperthermia. Here we show that heat shock causes the loss of Mcl-1 protein. Depletion of Noxa by short hairpin RNA protected cells from hyperthermia by preventing Mcl-1 degradation. Heat shock caused the dissociation of Noxa from Mcl-1, which allowed binding of the BH3-containing ubiquitin ligase Mule followed by Mcl-1 ubiquitination and degradation. Overexpression of Hsp70, which prevents heat-induced Bax activation, stabilized Mcl-1 protein levels in heat-shocked cells. This resulted from reduced Mule binding and ubiquitination as well as enhanced Mcl-1 expression compared with cells without Hsp70. Our results demonstrate that loss of Mcl-1 is a critical heat-sensitive step leading to Bax activation that is controlled by Hsp70.
PURPOSE: MicroRNAs (miRNA) are a class of small noncoding RNA molecules that have been implicated in a wide variety of basic cellular functions through posttranscriptional regulations on their target genes. Compelling evidence has shown that miRNAs are involved in cancer initiation and progression. We hypothesized that genetic variations of the miRNA machinery genes could be associated with the risk of renal cell carcinoma. EXPERIMENTAL DESIGN: We genotyped 40 single nucleotide polymorphisms (SNP) from 11 miRNA processing genes (DROSHA, DGCR8, XPO5, RAN, DICER1, TARBP2, AGO1, AGO2, GEMIN3, GEMIN4, HIWI) and 15 miRNA genes in 279 Caucasian patients with renal cell carcinoma and 278 matched controls. RESULTS: We found that two SNPs in the GEMIN4 gene were significantly associated with altered renal cell carcinoma risks. The variant-containing genotypes of Asn929Asp and Cys1033Arg exhibited significantly reduced risks, with odds ratios (OR) of 0.67 [95% confidence interval (95% CI), 0.47-0.96] and 0.68 (95% CI, 0.47-0.98), respectively. Haplotype analysis showed that a common haplotype of GEMIN4 was associated with a significant reduction in the risk of renal cell carcinoma (OR, 0.66; 95% CI, 0.45-0.97). We also conducted a combined unfavorable genotype analysis including five promising SNPs showing at least a borderline significant risk association. Compared with the low-risk reference group with one unfavorable genotype, the median-risk and high-risk groups exhibited a 1.55-fold (95% CI, 0.96-2.50) and a 2.49-fold (95% CI, 1.58-3.91) increased risk of renal cell carcinoma, respectively (P for trend < 0.001). CONCLUSIONS: Our results suggested that genetic polymorphisms of the miRNA-machinery genes may affect renal cell carcinoma susceptibility individually and jointly.
The Myc-associated zinc-finger protein, Miz1, activates transcription of the p21cip1 gene in response to UV irradiation. Miz1 associates with topoisomerase II binding protein1 (TopBP1), an essential activator of the Atr kinase. We show here that Miz1 is required for the recruitment of a fraction of TopBP1 to chromatin, for the protection of TopBP1 from proteasomal degradation and for Atr-dependent signal transduction. TopBP1 that is not bound to chromatin is degraded by the HectH9 (Mule, ARF-BP1 and HUWE1) ubiquitin ligase. Myc antagonizes the binding of TopBP1 to Miz1; as a result, expression of Myc leads to dissociation of TopBP1 from chromatin, reduces the amount of total TopBP1 and attenuates Atr-dependent signal transduction. Our data show that Miz1 and Myc affect the activity of the Atr checkpoint through their effect on TopBP1 chromatin association and stability.
Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
Development of the nervous system requires that timely withdrawal from the cell cycle be coupled with initiation of differentiation. Ubiquitin-mediated degradation of the N-Myc oncoprotein in neural stem/progenitor cells is thought to trigger the arrest of proliferation and begin differentiation. Here we report that the HECT-domain ubiquitin ligase Huwe1 ubiquitinates the N-Myc oncoprotein through Lys 48-mediated linkages and targets it for destruction by the proteasome. This process is physiologically implemented by embryonic stem (ES) cells differentiating along the neuronal lineage and in the mouse brain during development. Genetic and RNA interference-mediated inactivation of the Huwe1 gene impedes N-Myc degradation, prevents exit from the cell cycle by opposing the expression of Cdk inhibitors and blocks differentiation through persistent inhibition of early and late markers of neuronal differentiation. Silencing of N-myc in cells lacking Huwe1 restores neural differentiation of ES cells and rescues cell-cycle exit and differentiation of the mouse cortex, demonstrating that Huwe1 restrains proliferation and enables neuronal differentiation by mediating the degradation of N-Myc. These findings indicate that Huwe1 links destruction of N-Myc to the quiescent state that complements differentiation in the neural tissue.
Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.
The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.
Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.
The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
Many fundamental processes, including oncogenesis, have implicated HECT domain proteins with ubiquitin ligase activity. The protein human upstream regulatory element binding protein 1 (hUREB1) is a HECT domain protein whose function is not defined yet. Here, we investigate the function of hUREB1 as a ubiquitin-protein ligase in human colorectal cells. Ectopic expression of the HECT domain of hUREB1 reduces the protein level and transcriptional activity of the p53 tumor suppressor, which is abrogated by the deletion in the HECT domain or point mutations in the essential residues of the HECT domain. The ubiquitination and destabilization of p53 is observed in cells treated with the protease inhibitor MG132, implying that the HECT domain of hUREB1 suppresses the transcriptional activity of p53 through a ubiquitin-dependent degradation pathway. Based on the results of Northern blot analysis, RT-PCR, and immunohistochemical analyses, the over-expression of hUREB1 is associated with colorectal carcinoma. Moreover, protein levels of hUREB1 and p53 were inversely correlated. These findings suggest that hUREB1 can function, at least in part, as a negative regulator of p53 during the colorectal carcinoma progression through the ubiquitination pathway mediated by the HECT domain.
During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.
Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.
The elimination of Mcl-1, an anti-apoptotic Bcl-2 family member, is an early and required step for DNA damage-induced apoptosis. The degradation of Mcl-1 can be blocked by proteasome inhibitors, suggesting a role for the ubiquitin proteasome pathway in apoptosis. Here, we demonstrate that Mcl-1 is ubiquinated at five lysines. Biochemical fractionation of cell extracts allowed us to identify a 482 kDa HECT-domain-containing ubiquitin ligase named Mule (Mcl-1 ubiquitin ligase E3) that is both required and sufficient for the polyubiquitination of Mcl-1. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) domain that allows Mule to specifically interact with Mcl-1. Elimination of Mule expression by RNA interference stabilizes Mcl-1 protein, resulting in an attenuation of the apoptosis induced by DNA-damage agents. Thus, Mule is a unique BH3-containing E3 ubiquitin ligase apical to Bcl-2 family proteins during DNA damage-induced apoptosis.
A genome wide search for new BH3-containing Bcl-2 family members was conducted using position weight matrices (PWM) and identified a large (480kDa), novel BH3-only protein, originally called LASU1 (now also known as Ureb-1, E3(histone), ARF-BP1, and Mule). We demonstrated that LASU1 is an E3 ligase that ubiquitinated Mcl-1 in vitro and was required for its proteasome-dependent degradation in HeLa cells. Of note, the BH3 domain of LASU1 interacted with Mcl-1 but not with Bcl-2 or Bcl-Xl. A competing BH3-ligand derived from Bim interacted with Mcl-1 and prevented its interaction with LASU1 in HeLa cells, causing elevation of the steady-state levels of Mcl-1. This suggests that the unliganded form of Mcl-1 is sensitive to LASU1-mediated degradation of Mcl-1.
The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked polyubiquitin chain. Miz1 inhibits this ubiquitination. HectH9-mediated ubiquitination of Myc is required for transactivation of multiple target genes, recruitment of the coactivator p300, and induction of cell proliferation by Myc. HectH9 is overexpressed in multiple human tumors and is essential for proliferation of a subset of tumor cells. Our results suggest that site-specific ubiquitination regulates the switch between an activating and a repressive state of the Myc protein, and they suggest a strategy to interfere with Myc function in vivo.
As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.
The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.
Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.
The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http:mgc.nci.nih.gov).
Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58-752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon-intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3' UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation.
In our series of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 100 cDNA clones of unidentified genes, named KIAA1544 to KIAA1643, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here reached 4.6 kb and 2.8 kb (930 amino acid residues), respectively. By computer-assisted database search of the deduced amino acid sequences, 48 predicted gene products were classified into the five functional categories of proteins relating to cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. Homology search against the databases for proteins deduced from yeast, nematode and fly full genome sequences revealed only one gene (KIAA1630) was entirely conserved among human and these three organisms in the 100 genes reported here. Additionally, their chromosomal loci were determined by using human-rodent hybrid panels unless they were already assigned in the public databases. Furthermore, the expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.
Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.
In this series of projects of sequencing human cDNA clones which correspond to relatively long transcripts, we newly determined the entire sequences of 100 cDNA clones which were screened on the basis of the potentiality of coding for large proteins in vitro. The cDNA libraries used were the fractions with average insert sizes from 5.3 to 7.0 kb of the size-fractionated cDNA libraries from human brain. The randomly sampled clones were single-pass sequenced from both the ends to select clones that are not registered in the public database. Then their protein-coding potentialities were examined by an in vitro transcription/translation system, and the clones that generated proteins larger than 60 kDa were entirely sequenced. Each clone gave a distinct open reading frame (ORF), and the length of the ORF was roughly coincident with the approximate molecular mass of the in vitro product estimated from its mobility on SDS-polyacrylamide gel electrophoresis. The average size of the cDNA clones sequenced was 6.1 kb, and that of the ORFs corresponded to 1200 amino acid residues. By computer-assisted analysis of the sequences with DNA and protein-motif databases (GenBank and PROSITE databases), the functions of at least 73% of the gene products could be anticipated, and 88% of them (the products of 64 clones) were assigned to the functional categories of proteins relating to cell signaling/communication, nucleic acid managing, and cell structure/motility. The expression profiles in a variety of tissues and chromosomal locations of the sequenced clones have been determined. According to the expression spectra, approximately 11 genes appeared to be predominantly expressed in brain. Most of the remaining genes were categorized into one of the following classes: either the expression occurs in a limited number of tissues (31 genes) or the expression occurs ubiquitously in all but a few tissues (47 genes).
Tumor suppressor p53 is a transcription activator that upregulates target genes containing the p53 binding site. UREB1, a DNA binding protein that is tyrosine phosphorylated in vivo, shares a significant homology with the human papilloma virus E6 associated protein (E6-AP). E6-AP forms a ternary complex with E6 and p53 and participates in the ubiquitination of p53. Based on the homology with E6-AP, but taking into account the nuclear localization of UREB1 and its smaller size, the present study used a transient transfection system to examine whether UREB1 influenced p53-stimulated transcription. Co-transfection of a vector expressing wildtype UREB1 with one expressing p53 into H1299, a p53 negative cell line, resulted in a pronounced suppression of p53 transactivation. The inhibitory effect was significantly attenuated by mutation of a tyrosine residue in the consensus tyrosine phosphorylation sequence of UREB1. These data suggest that optimal suppression of p53 transactivation requires tyrosine phosphorylated UREB1 and that tyrosine phosphorylation and dephosphorylation processes may be involved in the regulation of p53 transactivation.
We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).
A 90 bp fragment prepared from the promoter region of the rat preprodynorphin gene formed a complex with rat brain nuclear extracts as assessed by gel mobility shift assays. An 8 base pair sequence, CACTCTCC, termed upstream regulatory element (URE), was identified within this fragment as a binding site by DNase 1 footprint analysis and gel mobility shift assays with synthetic oligonucleotides. The URE is a consensus sequence for a transcription initiator (Inr) element although in the preprodynorphin promoter it is located upstream at -208 and overlaps a region conserved between rat and human promoters. A unique 310 amino acid protein (UreB1) that specifically bound the URE was cloned from a rat brain cDNA library using the URE-containing oligonucleotide. Recombinantly expressed, affinity purified UreB1 protein retains specific binding to the URE oligonucleotide. UreB1 contains a tyrosine kinase phosphorylation consensus and binding is enhanced following phosphorylation with the p43v-abl tyrosine kinase. The UreB1 tyrosine phosphoprotein increases transcription in vitro, consistent with a positive transcriptional regulatory function. UreB1 transcripts are well expressed in subsets of neurons in multiple brain areas suggesting that, in addition to regulation of the preprodynorphin gene, it may have a more generalized role in gene transcription.