scholarly journals Functional analysis of the nuclear LIM domain interactor NLI.

1997 ◽  
Vol 17 (10) ◽  
pp. 5688-5698 ◽  
Author(s):  
L W Jurata ◽  
G N Gill
Keyword(s):  
Transcriptional Activation ◽  
Negative Regulator ◽  
Homeodomain Protein ◽  
Lim Domain ◽  
Transcriptional Responses ◽  
Amino Terminal ◽  
High Affinity ◽  
Lim Domains ◽  
Lim Homeodomain

LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

1998 ◽  
Vol 9 (7) ◽  
pp. 1803-1816 ◽  
Author(s):  
Michael C. Brown ◽  
Joseph A. Perrotta ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Binding Sites ◽  
Protein Localization ◽  
Model System ◽  
Lim Domain ◽  
Amino Terminal ◽  
Lim Domains ◽  
Inside Out

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.

Regulation of Apterous activity inDrosophilawing development

Development ◽  
2001 ◽  
Vol 128 (22) ◽  
pp. 4615-4622 ◽  
Author(s):  
Ulrich Weihe ◽  
Marco Milán ◽  
Stephen M. Cohen
Keyword(s):  
Complex Formation ◽  
Wing Development ◽  
Homeodomain Protein ◽  
Activity Levels ◽  
Present Evidence ◽  
Drosophila Wing ◽  
Lim Domains ◽  
Regulation Of Activity ◽  
Lim Homeodomain

Apterous is a LIM-homeodomain protein that confers dorsal compartment identity in Drosophila wing development. Apterous activity requires formation of a complex with a co-factor, Chip/dLDB. Apterous activity is regulated during wing development by dLMO, which competes with Apterous for complex formation. Here, we present evidence that complex formation between Apterous, Chip and DNA stabilizes Apterous protein in vivo. We also report that a difference in the ability of Chip to bind the LIM domains of Apterous and dLMO contributes to regulation of activity levels in vivo.

trans activation of gene expression by v-myb

1990 ◽  
Vol 10 (5) ◽  
pp. 2285-2293 ◽  
Author(s):  
C E Ibanez ◽  
J S Lipsick
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Vp16 Fusion ◽  
Acute Myelomonocytic Leukemia ◽  
Simplex Virus

The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation.

scholarly journals Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

2020 ◽  
Vol 117 (41) ◽  
pp. 25532-25542 ◽  
Author(s):  
Jonathan D. Winkelman ◽  
Caitlin A. Anderson ◽  
Cristian Suarez ◽  
David R. Kovar ◽  
Margaret L. Gardel
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Strain Sensing ◽  
Lim Domain ◽  
Lim Domains ◽  
Mechanical Homeostasis ◽  
Functionally Diverse

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

scholarly journals trans activation of gene expression by v-myb.

1990 ◽  
Vol 10 (5) ◽  
pp. 2285-2293 ◽  
Author(s):  
C E Ibanez ◽  
J S Lipsick
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Vp16 Fusion ◽  
Acute Myelomonocytic Leukemia ◽  
Simplex Virus

The v-myb oncogene causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its product, p48v-myb, is a short-lived nuclear protein which binds DNA. We demonstrate that p48v-myb can function as a trans activator of gene expression in transient DNA transfection assays. trans activation requires the highly conserved amino-terminal DNA-binding domain and the less highly conserved carboxyl-terminal domain of p48v-myb, both of which are required for transformation. Multiple copies of a consensus sequence for DNA binding by p48v-myb inserted upstream of a herpes simplex virus thymidine kinase promoter are strongly stimulatory for transcriptional activation by a v-myb-VP16 fusion protein but not by p48v-myb itself, suggesting that the binding of p48v-myb to DNA may not be sufficient for trans activation.

scholarly journals Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Frank C. Holstege ◽  
Richard A. Young ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Negative Regulator ◽  
Preinitiation Complex ◽  
Positive Role ◽  
Pol Ii ◽  
Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

scholarly journals p300-Mediated Tax Transactivation from Recombinant Chromatin: Histone Tail Deletion Mimics Coactivator Function

2002 ◽  
Vol 22 (1) ◽  
pp. 127-137 ◽  
Author(s):  
Sara A. Georges ◽  
W. Lee Kraus ◽  
Karolin Luger ◽  
Jennifer K. Nyborg ◽  
Paul J. Laybourn
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Leukemia Virus ◽  
Acetyl Coa ◽  
Histone Tails ◽  
Cell Leukemia Virus Type ◽  
Cellular Transcription Factor ◽  
Amino Terminal ◽  
Human T Cell

ABSTRACT Efficient transcription of the human T-cell leukemia virus type 1 (HTLV-1) genome requires Tax, a virally encoded oncogenic transcription factor, in complex with the cellular transcription factor CREB and the coactivators p300/CBP. To examine Tax transactivation in vitro, we used a chromatin assembly system that included recombinant core histones. The addition of Tax, CREB, and p300 to the HTLV-1 promoter assembled into chromatin activated transcription several hundredfold. Chromatin templates selectively lacking amino-terminal histone tails demonstrated enhanced transcriptional activation by Tax and CREB, with significantly reduced dependence on p300 and acetyl coenzyme A (acetyl-CoA). Interestingly, Tax/CREB activation from the tailless chromatin templates retained a substantial requirement for acetyl-CoA, indicating a role for acetyl-CoA beyond histone acetylation. These data indicate that during Tax transcriptional activation, the amino-terminal histone tails are the major targets of p300 and that tail deletion and acetylation are functionally equivalent.

scholarly journals LIM Protein KyoT2 Negatively Regulates Transcription by Association with the RBP-J DNA-Binding Protein

1998 ◽  
Vol 18 (1) ◽  
pp. 644-654 ◽  
Author(s):  
Yoshihito Taniguchi ◽  
Takahisa Furukawa ◽  
Tin Tun ◽  
Hua Han ◽  
Tasuku Honjo
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Negative Regulator ◽  
Nuclear Antigen ◽  
Differential Splicing ◽  
Barr Virus ◽  
Lim Domains ◽  
C Terminus

ABSTRACT The RBP-J/Su(H) DNA-binding protein plays a key role in transcriptional regulation by targeting Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors to specific promoters. Using the yeast two-hybrid system, we isolated a LIM-only protein, KyoT, which physically interacts with RBP-J. Differential splicing gave rise to two transcripts of theKyoT gene, KyoT1 and KyoT2, that encoded proteins with four and two LIM domains, respectively. With differential splicing resulting in deletion of an exon, KyoT2 lacked two LIM domains from the C terminus and had a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoT1 had a negligible level of interaction with RBP-J. Strong expression of KyoT mRNAs was detected in skeletal muscle and lung, with a predominance of KyoT1 mRNA. When expressed in F9 embryonal carcinoma cells, KyoT1 and KyoT2 were localized in the cytoplasm and the nucleus, respectively. The binding site of KyoT2 on RBP-J overlaps those of EBNA2 and Notch1 but is distinct from that of Hairless, the negative regulator of RBP-J-mediated transcription in Drosophila. KyoT2 but not KyoT1 repressed the RBP-J-mediated transcriptional activation by EBNA2 and Notch1 by competing with them for binding to RBP-J and by dislocating RBP-J from DNA. KyoT2 is a novel negative regulatory molecule for RBP-J-mediated transcription in mammalian systems.

scholarly journals Crystallization and diffraction of an Isl1–Ldb1 complex

2012 ◽  
Vol 68 (11) ◽  
pp. 1398-1401 ◽  
Author(s):  
Morgan S. Gadd ◽  
David A. Jacques ◽  
J. Mitchell Guss ◽  
Jacqueline M. Matthews
Keyword(s):  
Unit Cell ◽  
Homeodomain Protein ◽  
Unit Cell Parameters ◽  
Space Group ◽  
Cell Parameters ◽  
Lim Domains ◽  
Intramolecular Complex ◽  
Orthorhombic Crystals ◽  
Peptide Region ◽  
Lim Homeodomain

A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Isl1 tethered to a peptide region of Ldb1 has been engineered, purified and crystallized. The orthorhombic crystals belonged to space groupP2221, with unit-cell parametersa= 57.2,b= 56.7,c= 179.8 Å, and diffracted to 3.10 Å resolution.

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