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1.  Role of SUMOylation in Full Antiestrogenicity 
Molecular and Cellular Biology  2012;32(19):3823-3837.
The selective estrogen receptor downregulator (SERD) fulvestrant can be used as second-line treatment for patients relapsing after treatment with tamoxifen, a selective estrogen receptor modulator (SERM). Unlike tamoxifen, SERDs are devoid of partial agonist activity. While the full antiestrogenicity of SERDs may result in part from their capacity to downregulate levels of estrogen receptor alpha (ERα) through proteasome-mediated degradation, SERDs are also fully antiestrogenic in the absence of increased receptor turnover in HepG2 cells. Here we report that SERDs induce the rapid and strong SUMOylation of ERα in ERα-positive and -negative cell lines, including HepG2 cells. Four sites of SUMOylation were identified by mass spectrometry analysis. In derivatives of the SERD ICI164,384, SUMOylation was dependent on the length of the side chain and correlated with full antiestrogenicity. Preventing SUMOylation by the overexpression of a SUMO-specific protease (SENP) deSUMOylase partially derepressed transcription in the presence of full antiestrogens in HepG2 cells without a corresponding increase in activity in the presence of agonists or of the SERM tamoxifen. Mutations increasing transcriptional activity in the presence of full antiestrogens reduced SUMOylation levels and suppressed stimulation by SENP1. Our results indicate that ERα SUMOylation contributes to full antiestrogenicity in the absence of accelerated receptor turnover.
PMCID: PMC3457522  PMID: 22826433
2.  Glycosyltransferase Function in Core 2-Type Protein O Glycosylation▿  
Molecular and Cellular Biology  2009;29(13):3770-3782.
Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes.
PMCID: PMC2698761  PMID: 19349303
3.  A Class III PDZ Binding Motif in the Myotilin and FATZ Families Binds Enigma Family Proteins: a Common Link for Z-Disc Myopathies▿  
Molecular and Cellular Biology  2008;29(3):822-834.
Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.
PMCID: PMC2630697  PMID: 19047374
4.  Targeting of the ETS Factor Gabpα Disrupts Neuromuscular Junction Synaptic Function▿ §  
Molecular and Cellular Biology  2007;27(9):3470-3480.
The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits δ and ɛ through the N-box promoter motif (5′-CCGGAA-3′), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor ɛ subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPα in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor ɛ subunit and increased expression of the γ subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.
PMCID: PMC1899955  PMID: 17325042
5.  The Ubiquitin-Conjugating Enzyme UBCH7 Acts as a Coactivator for Steroid Hormone Receptors 
Molecular and Cellular Biology  2004;24(19):8716-8726.
We investigated the role of the ubiquitin-conjugating enzyme UBCH7 in nuclear receptor transactivation. Using transient transfection assays, we demonstrated that UBCH7 modulates the transcriptional activity of progesterone receptor (PR) and glucocorticoid, androgen, and retinoic acid receptors in a hormone-dependent manner and that the ubiquitin conjugation activity of UBCH7 is required for its ability to potentiate transactivation by steroid hormone receptors (SHR). However, UBCH7 showed no significant effect on the transactivation functions of p53 and VP-16 activation domain. Depletion of endogenous UBCH7 protein by small interfering RNAs suggests that UBCH7 is required for the proper function of SHR. Furthermore, a chromatin immunoprecipitation assay demonstrated the hormone-dependent recruitment of UBCH7 onto estrogen receptor- and PR-responsive promoters. Additionally, we show that UBCH7 and E6-associated protein (E6-AP) synergistically enhance PR transactivation. We also demonstrate that UBCH7 interacts with steroid receptor coactivator 1 (SRC-1) and that UBCH7 coactivation function is dependent on SRC-1. Taken together, our results reveal the possible role of UBCH7 in steroid receptor transactivation and provide insights into the mechanism of action of UBCH7 in receptor function.
PMCID: PMC516762  PMID: 15367689
6.  The ETS Transcription Factor GABPα Is Essential for Early Embryogenesis 
Molecular and Cellular Biology  2004;24(13):5844-5849.
The ETS transcription factor complex GABP consists of the GABPα protein, containing an ETS DNA binding domain, and an unrelated GABPβ protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpα gene. Embryos homozygous for the null Gabpα allele die prior to implantation, consistent with the broad expression of Gabpα throughout embryogenesis and in embryonic stem cells. Gabpα+/− mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpα protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpα function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.
PMCID: PMC480913  PMID: 15199140
7.  Cell-Type-Dependent Activity of the Ubiquitous Transcription Factor USF in Cellular Proliferation and Transcriptional Activation 
Molecular and Cellular Biology  1999;19(2):1508-1517.
USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.
PMCID: PMC116079  PMID: 9891084
8.  Structure of the SAD mutation and the location of control sites at silent mating type genes in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1984;4(7):1278-1285.
The SAD mutation, an extra mating type cassette, has been shown to arise from an unequal mitotic crossover between the MAT and HMR loci, resulting in the formation of a hybrid cassette and a duplication of the MAT-HMR interval. The SAD cassette contains the "a" information and left-hand flanking regions from the parental HMRa cassette and the right-hand flanking sequences of the parental MAT cassette. This arrangement of flanking sequences causes a leaky but reproducible mating phenotype correlated with a low-level expression of the cassette as measured by RNA blotting. This weak expression is attributed to the loss of one flanking control site normally present at the silent HM storage loci.
PMCID: PMC368909  PMID: 6095058

Results 1-8 (8)