scholarly journals Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation

2012 ◽  
Vol 23 (3) ◽  
pp. 412-422 ◽  
Author(s):  
Wenyu Liu ◽  
Felipe H. Santiago-Tirado ◽  
Anthony Bretscher
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Spindle Orientation ◽  
Bud Neck ◽  
Negative Effect ◽  
The Right ◽  
Actin Cables ◽  
And Function

Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1–333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334–821) covers the Diaphanous inhibitory–dimerization–coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1–formin homology 2–COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.

scholarly journals Heterozygous Orthodenticle Homeobox 2 Mutations Are Associated with Variable Pituitary Phenotype

2010 ◽  
Vol 31 (1) ◽  
pp. 133-133
Author(s):  
Sumito Dateki ◽  
Kitaro Kosaka ◽  
Kosei Hasegawa ◽  
Hiroyuki Tanaka ◽  
Noriyuki Azuma ◽  
...  
Keyword(s):  
Target Genes ◽  
Japanese Patients ◽  
Dominant Negative ◽  
Pituitary Function ◽  
Hormone Deficiency ◽  
Dominant Negative Effect ◽  
Pituitary Hormone ◽  
Positive Role ◽  
Negative Effect ◽  
And Function

ABSTRACT Context Although recent studies have suggested a positive role of OTX2 in pituitary as well as ocular development and function, detailed pituitary phenotypes in OTX2 mutations and OTX2 target genes for pituitary function other than HESX1 and POU1F1 remain to be determined. Objective We aimed to examine such unresolved issues. Subjects We studied 94 Japanese patients with various ocular or pituitary abnormalities. Results We identified heterozygous p.K74fsX103 in case 1, p.A72fsX86 in case 2, p.G188X in two unrelated cases (3 and 4), and a 2,860,561-bp microdeletion involving OTX2 in case 5. Clinical studies revealed isolated GH deficiency in cases 1 and 5; combined pituitary hormone deficiency in case 3; abnormal pituitary structures in cases 1, 3, and 5; and apparently normal pituitary function in cases 2 and 4, together with ocular anomalies in cases 1-5. The wild-type Orthodenticle homeobox 2 (OTX2) protein transactivated the GNRH1 promoter as well as the HESX1, POU1F1, and IRBP (interstitial retinoid-binding protein) promoters, whereas the p.K74fsX103-OTX2 and p.A72fsX86-OTX2 proteins had no transactivation functions and the p.G188X-OTX2 protein had reduced (∼50%) transactivation functions for the four promoters, with no dominant-negative effect. cDNA screening identified positive OTX2 expression in the hypothalamus. Conclusions The results imply that OTX2 mutations are associated with variable pituitary phenotype, with no genotype-phenotype correlations, and that OTX2 can transactivate GNRH1 as well as HESX1 and POU1F1.

scholarly journals Heterozygous Orthodenticle Homeobox 2 Mutations Are Associated with Variable Pituitary Phenotype

2010 ◽  
Vol 95 (2) ◽  
pp. 756-764 ◽  
Author(s):  
Sumito Dateki ◽  
Kitaro Kosaka ◽  
Kosei Hasegawa ◽  
Hiroyuki Tanaka ◽  
Noriyuki Azuma ◽  
...  
Keyword(s):  
Target Genes ◽  
Japanese Patients ◽  
Dominant Negative ◽  
Pituitary Function ◽  
Hormone Deficiency ◽  
Dominant Negative Effect ◽  
Pituitary Hormone ◽  
Positive Role ◽  
Negative Effect ◽  
And Function

Abstract Context: Although recent studies have suggested a positive role of OTX2 in pituitary as well as ocular development and function, detailed pituitary phenotypes in OTX2 mutations and OTX2 target genes for pituitary function other than HESX1 and POU1F1 remain to be determined. Objective: We aimed to examine such unresolved issues. Subjects: We studied 94 Japanese patients with various ocular or pituitary abnormalities. Results: We identified heterozygous p.K74fsX103 in case 1, p.A72fsX86 in case 2, p.G188X in two unrelated cases (3 and 4), and a 2,860,561-bp microdeletion involving OTX2 in case 5. Clinical studies revealed isolated GH deficiency in cases 1 and 5; combined pituitary hormone deficiency in case 3; abnormal pituitary structures in cases 1, 3, and 5; and apparently normal pituitary function in cases 2 and 4, together with ocular anomalies in cases 1–5. The wild-type Orthodenticle homeobox 2 (OTX2) protein transactivated the GNRH1 promoter as well as the HESX1, POU1F1, and IRBP (interstitial retinoid-binding protein) promoters, whereas the p.K74fsX103-OTX2 and p.A72fsX86-OTX2 proteins had no transactivation functions and the p.G188X-OTX2 protein had reduced (∼50%) transactivation functions for the four promoters, with no dominant-negative effect. cDNA screening identified positive OTX2 expression in the hypothalamus. Conclusions: The results imply that OTX2 mutations are associated with variable pituitary phenotype, with no genotype-phenotype correlations, and that OTX2 can transactivate GNRH1 as well as HESX1 and POU1F1.

scholarly journals Regulated Disruption of Inositol 1,4,5-Trisphosphate Signaling in Caenorhabditis elegans Reveals New Functions in Feeding and Embryogenesis

2002 ◽  
Vol 13 (4) ◽  
pp. 1329-1337 ◽  
Author(s):  
Denise S. Walker ◽  
Nicholas J.D. Gower ◽  
Sung Ly ◽  
Gemma L. Bradley ◽  
Howard A. Baylis
Keyword(s):  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Animal Cells ◽  
Specific Expression ◽  
C Elegans ◽  
Wide Range ◽  
Inositol 1,4,5 Trisphosphate ◽  
Negative Effect ◽  
Pharyngeal Pumping

Inositol 1,4,5-trisphosphate (IP3) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP3 is to regulate release of Ca2+ from intracellular stores through IP3 receptors (IP3Rs). We describe a system for the transient disruption of IP3 signaling in the model organismCaenorhabditis elegans. The IP3 binding domain of the C. elegans IP3R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP3 binding domain acting as an IP3“sponge.” Disruption of IP3 signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP3-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP3 signaling. RNA-mediated interference studies and analysis of itr-1mutants show that this process is also IP3R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP3 signaling through the IP3R and thus determine that IP3-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.

Dominant Negative Form of PAX5 Is Generated by Multimerization of Its DNA Binding Domain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 25-25
Author(s):  
Norihiko Kawamata ◽  
Mario Pennella ◽  
Jennifer Woo ◽  
Arnold Berk ◽  
H. Phillip Koeffler
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Binding Affinity ◽  
Alpha Helix ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
Dna Binding Affinity

Abstract Abstract 25 We have previously cloned a number of fusion genes involving PAX5 in acute lymphoblastic leukemia (ALL) (Kawamata N. et al. PNAS, 2008). All of these fusion products exerted a dominant negative effect over the wild-type PAX5. One of these fusion PAX5 proteins, PAX5-C20orf112, was generated by the fusion between the DNA binding domain of PAX5 (PAX5DB) and the C-terminal end of C20orf112. To find the mechanism of the dominant negative effect of the PAX5-C20 fusion, we performed Fluorescence Recovery After Photobleaching (FRAP) assay using PAX5-C20 and PAX5wt constructs connected with Yellow Fluorescence Proteins (YFP). Results showed extremely strong DNA binding affinity of PAX5-C20 compared to PAX5wt. FRAP experiments using deletion mutants of PAX5-C20 showed that both the DNA binding domain and C-terminal alpha-helix region of C20 were indispensable for this strong binding to DNA. Fluorescence Resonance Energy Transfer (FRET) assay, Bi-molecule Fluorescence Complementation (BiFC) assay, and co-immunoprecipitation assay showed that C-terminal end of C20 containing an alpha-helix region encodes a homo-multimerization domain. To confirm that homo-multimerization of PAX5DB increases DNA binding affinity, PAX5DB was fused to the inducible dimerization motif of FKBP (PAX5DB-FK). PAX5DB-FK increased its DNA binding affinity with addition of FKBP ligand inducing homo-dimerization. We also fused PAX5DB to homo-dimerization of MAX (bHLH domain), or tetramerization domain of TP53. FRAP assays showed that homo-dimerization increased its DNA binding activity, and homo-tetramerization further increased its DNA binding and its dominant negative effect over PAX5wt. PAX5-ETV6, also a common fusion protein in ALL, exerts a dominant negative effect over PAX5wt. The ETV6 region of this fusion protein has a multimerization (SAM) domain and the PAX5DB-ETV6SAM mutant protein also showed a dominant negative effect and strong binding to DNA. Importantly, in further studies, co-expression of PAX5-C20 and the YFP-C20-alpha-helix-region diminished the strong DNA binding and the dominant negative activity of the fusion protein. Our data show that multimerization of the DNA binding domain of PAX5 induces strong DNA binding activity, leading to its dominant negative effect over the wild type transcription factor. We believe this represents a new paradigm explaining how a number of fusion genes containing a DB motif from one protein and a multimerization motif from the other partner, can behave in a dominant negative fashion. These observations suggest that peptides/ small molecules inhibiting the multimerization of these oncogenic fusion transcription factors can be promising reagents for treating cancers. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Unravelling the molecular basis of the dominant negative effect of myosin XI tails on P-bodies

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0252327
Author(s):  
Lisa Stephan ◽  
Marc Jakoby ◽  
Arijit Das ◽  
Eva Koebke ◽  
Martin Hülskamp
Keyword(s):  
Intracellular Transport ◽  
Body Movement ◽  
Coiled Coil ◽  
Transport Processes ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cellular Functions ◽  
Directional Movement ◽  
Negative Effect ◽  
Myosin Xi

The directional movement and positioning of organelles and macromolecules is essential for regulating and maintaining cellular functions in eukaryotic cells. In plants, these processes are actin-based and driven by class XI myosins, which transport various cargos in a directed manner. As the analysis of myosin function is challenging due to high levels of redundancy, dominant negative acting truncated myosins have frequently been used to study intracellular transport processes. A comparison of the dominant negative effect of the coiled-coil domains and the GTD domains revealed a much stronger inhibition of P-body movement by the GTD domains. In addition, we show that the GTD domain does not inhibit P-body movement when driven by a hybrid myosin in which the GTD domain was replaced by DCP2. These data suggest that the dominant negative effect of myosin tails involves a competition of the GTD domains for cargo binding sites.

scholarly journals p53 Isoforms and Their Implications in Cancer

Cancers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 288 ◽  
Author(s):  
Maximilian Vieler ◽  
Suparna Sanyal
Keyword(s):  
Dna Binding ◽  
Tp53 Gene ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Evolutionary Significance ◽  
Dna Binding Domain ◽  
Negative Effect ◽  
P53 Isoforms ◽  
P53 Inactivation

In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the TP53 gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are ∆40p53, ∆133p53, and ∆160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered ‘responsive elements’ than p53. Furthermore, they may exert a ‘dominant negative effect’ or induce more aggressive cancer by the ‘gain of function’. One possible mechanism of p53 inactivation can be through tetramerization with the ∆133p53 and ∆160p53 isoforms—both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation—possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.

scholarly journals The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

1996 ◽  
Vol 16 (3) ◽  
pp. 1203-1211 ◽  
Author(s):  
K Takebayashi ◽  
K Chida ◽  
I Tsukamoto ◽  
E Morii ◽  
H Munakata ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
T Antigen ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Negative Effect

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

scholarly journals PSTPIP: A Tyrosine Phosphorylated Cleavage Furrow–associated Protein that Is a Substrate for a PEST Tyrosine Phosphatase

1997 ◽  
Vol 138 (4) ◽  
pp. 845-860 ◽  
Author(s):  
Susan Spencer ◽  
Donald Dowbenko ◽  
Jill Cheng ◽  
Wenlu Li ◽  
Jennifer Brush ◽  
...  
Keyword(s):  
Tyrosine Phosphatase ◽  
Coiled Coil ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cleavage Furrow ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Hematopoietic Stem ◽  
Coiled Coil Region ◽  
Negative Effect

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

scholarly journals Truncation of TRIM5 in the Feliformia Explains the Absence of Retroviral Restriction in Cells of the Domestic Cat

2009 ◽  
Vol 83 (16) ◽  
pp. 8270-8275 ◽  
Author(s):  
William A. McEwan ◽  
Torsten Schaller ◽  
Laura M. Ylinen ◽  
Margaret J. Hosie ◽  
Greg J. Towers ◽  
...  
Keyword(s):  
Mammalian Species ◽  
Ectopic Expression ◽  
Binding Domain ◽  
Dominant Negative ◽  
Domestic Cat ◽  
Dominant Negative Effect ◽  
Negative Effect ◽  
In Cells

ABSTRACT TRIM5α mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5α. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

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