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Yeast Two-hybrid System (yeast + two-hybrid_system)
Selected AbstractsKi-1/57 interacts with PRMT1 and is a substrate for arginine methylationFEBS JOURNAL, Issue 17 2006Dario O. Passos The human 57 kDa Ki-1 antigen (Ki-1/57) is a cytoplasmic and nuclear protein, associated with Ser/Thr protein kinase activity, and phosphorylated at the serine and threonine residues upon cellular activation. We have shown that Ki-1/57 interacts with chromo-helicase DNA-binding domain protein 3 and with the adaptor/signaling protein receptor of activated kinase 1 in the nucleus. Among the identified proteins that interacted with Ki-1/57 in a yeast two-hybrid system was the protein arginine-methyltransferase-1 (PRMT1). Most interestingly, when PRMT1 was used as bait in a yeast two-hybrid system we were able to identify Ki-1/57 as prey among 14 other interacting proteins, the majority of which are involved in RNA metabolism or in the regulation of transcription. We found that Ki-1/57 and its putative paralog CGI-55 have two conserved Gly/Arg-rich motif clusters (RGG/RXR box, where X is any amino acid) that may be substrates for arginine-methylation by PRMT1. We observed that all Ki-1/57 protein fragments containing RGG/RXR box clusters interact with PRMT1 and are targets for methylation in vitro. Furthermore, we found that Ki-1/57 is a target for methylation in vivo. Using immunofluorescence experiments we observed that treatment of HeLa cells with an inhibitor of methylation, adenosine-2,,3,-dialdehyde (Adox), led to a reduction in the cytoplasmic immunostaining of Ki-1/57, whereas its paralog CGI-55 was partially redistributed from the nucleus to the cytoplasm upon Adox treatment. In summary, our data show that the yeast two-hybrid assay is an effective system for identifying novel PRMT arginine-methylation substrates and may be successfully applied to other members of the growing family of PRMTs. [source] Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracleFEBS JOURNAL, Issue 13 2002Markus Lezzi The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development. [source] CK2,tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogasterFEBS JOURNAL, Issue 5 2002Alla I. Kalmykova An earlier described CK2,tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory ,,subunit of casein kinase 2. Western-analysis using anti-CK2,tes Ig revealed CK2,tes protein in Drosophila testes extract. Expression of a CK2,tes,,-galactosidase fusion protein driven by the CK2,tes promoter was found in transgenic flies at postmitotic stages of spermatogenesis. Examination of biochemical characteristics of a recombinant CK2,tes protein expressed in Escherichia coli revealed properties similar to those of CK2,: (a) CK2,tes protein stimulates CK2, catalytic activity toward synthetic peptide; (b) it inhibits phosphorylation of calmodulin and mediates stimulation of CK2, by polylysine; (c) it is able to form (CK2,tes)2 dimers, as well as (CK2,)2(CK2,tes)2 tetramers. Using the yeast two-hybrid system and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2,tes and CK2,. Northern-analysis has shown that another regulatory (,,) subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two tissue-specific regulatory subunits of CK2 which might serve to provide CK2 substrate recognition during spermatogenesis. [source] Uncoupling proteins 2 and 3 interact with members of the 14.3.3 familyFEBS JOURNAL, Issue 9 2000Benoit Pierrat Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms ,, ,, , were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the , isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 , could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria. [source] Identification of tudor repeat associator with PCTAIRE 2 (Trap)FEBS JOURNAL, Issue 7 2000A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons. [source] The stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hiraeFEMS MICROBIOLOGY LETTERS, Issue 1 2010Jivko V. Stoyanov Abstract Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu+ to the CopY repressor, thereby releasing its bound zinc and abolishing repressor,DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis. [source] Detection of a homotetrameric structure and protein,protein interactions of Paracoccidioides brasiliensis formamidase lead to new functional insightsFEMS YEAST RESEARCH, Issue 1 2010Clayton Luiz Borges Abstract Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role in fungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was overexpressed in bacteria and a polyclonal antibody to this protein was produced. We identified a 180-kDa protein species reactive to the antibody produced in mice against the P. brasiliensis recombinant purified formamidase of 45 kDa. The 180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide mass fingerprinting using MS. The identical mass spectra generated by the 180 and the 45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formamidase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights into formamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism. [source] Ternary complex formation of EphA4, FGFR and FRS2, plays an important role in the proliferation of embryonic neural stem/progenitor cellsGENES TO CELLS, Issue 3 2010Takahiro Sawada EphA4 belongs to a superfamily of receptor tyrosine kinases and interacts with several molecules including fibroblast growth factor receptors (FGFRs) as we reported earlier. Several receptor tyrosine kinases, FGFRs, Trks, Alk and Ret, are currently known to transduce a signal through a docking protein, fibroblast growth factor receptor substrate 2, (FRS2,). However, nothing has been reported about the interaction of FRS2, with EphA4. Using the yeast two-hybrid system and the in vitro binding and kinase assays, we found that the mid-kinase region of EphA4 directly interacts with the FRS2, PTB domain upon tyrosine phosphorylation of the EphA4 juxtamembrane (JM) domain and EphA4 directly phosphorylates FRS2,. We also found that the FRS2, PTB domain and the amino-terminal region of EphA4 bind to the amino- and carboxy-terminal regions of the FGFR JM domain, respectively, suggesting that FRS2, and EphA4 interact with FGFR simultaneously. Furthermore, a kinase-dead EphA4 mutant that constitutively binds to FGFR functions as a dominant-negative molecule for signaling through both EphA4 and FGFR, and so does the truncated FRS2, lacking multiple tyrosine phosphorylation sites. These dominant-negative mutants similarly inhibit the ligand-dependent proliferation of the mouse embryonic neural stem/progenitor cells. These results suggest the formation of a ternary complex comprising EphA4, FGFR and FRS2,. The signaling complex appears to integrate the input from FGFR and EphA4, and release the output signal through FRS2,. [source] A yeast two-hybrid system using Sp17 identified Ropporin as a novel cancer,testis antigen in hematologic malignanciesINTERNATIONAL JOURNAL OF CANCER, Issue 7 2007Zhanfei Li Abstract Since most intracellular proteins are expressed with their ligands, ligands of cancer,testis (CT) antigens may also be CT in their distribution. Applying Sperm protein 17 (Sp17) as the bait in a yeast 2-hybrid system of a testicular cDNA library, 17 interacting clones were isolated and all encoded Ropporin, a spermatogenic cell-specific protein that serves as an anchoring protein for the A-kinase anchoring protein, AKAP110. Ropporin showed a very restricted normal tissue gene expression, detected only in testis and fetal liver. Ropporin mRNA could also be detected in tumor cells from patients with multiple myeloma, chronic lymphocytic leukemia and acute myeloid leukemia. Interestingly, expression of Sp17 did not necessarily predict for the expression of Ropporin suggesting that their coexpression in these tumor cells was random rather than coordinated. Ropporin gene expression in tumor cells is associated with the presence of high titer IgG antibodies against Ropporin, suggesting the in vivo translation of the mRNA into protein and the immunogenicity of the protein to the autologous hosts. Using a CT antigen as the bait in a yeast 2-hybrid system may, therefore, identify novel tumor antigen. Our results also suggest that Ropporin is a novel CT antigen in hematologic malignancies. © 2007 Wiley-Liss, Inc. [source] Focal Adhesion Kinase pp125FAK Interacts With the Large Conductance Calcium-Activated hSlo Potassium Channel in Human Osteoblasts: Potential Role in Mechanotransduction,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2003Roger Rezzonico Abstract Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. Introduction: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. Methods: Interaction of FAK with the C terminus of the hSlo ,-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. Results: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. Conclusions: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts. [source] Identification of a novel protein from glial cells based on its ability to interact with NF-,B subunitsrJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2003Thersa Sweet Abstract Nuclear factor ,B (NF-,B) represents a family of inducible DNA-binding transcription factors whose activity is critical for expression of the HIV-1 genome in a broad range of cells. In addition to its interaction with the ,B DNA sequence, the association of NF-,B subunits with other cellular proteins plays an important role in stimulation of HIV-1 gene transcription in astrocytic cells. Here, we utilized a yeast two-hybrid system to screen a cDNA library from a human astrocytic cell line and were able to isolate a partial cDNA belonging to a gene with an open reading frame of 1,871 amino acid residues which binds to both the p50 and p65 subunits of NF-,B. This gene, named NF-,B-binding protein (NFBP) is located on chromosome 10q24.2-25.1 and hybridized to a single transcript of nearly 6 kb in size. It is localized to the nucleus, specifically the nucleolus of cells. Extensive computer analysis was performed with the sequence of the full length NFBP and significant homology was found between NFBP, and yeast and mouse proteins. A discussion of the potential roles of NFBP in normal and viral infected cells is included. © 2003 Wiley-Liss, Inc. [source] Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cellsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Hiroaki Mitsuhashi Abstract SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S -transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro. Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation. [source] Calsenilin interacts with transcriptional co-repressor C-terminal binding protein(s)JOURNAL OF NEUROCHEMISTRY, Issue 4 2006Nikhat F. Zaidi Abstract Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence antagonist modulator (DREAM) is a neuronal calcium-binding protein that has been shown to have multiple functions in the cell, including the regulation of presenilin processing, repression of transcription and modulation of A-type potassium channels. To gain a better understanding of the precise role of calsenilin in specific cellular compartments, an interactor hunt for proteins that bind to the N-terminal domain of calsenilin was carried out. Using a yeast two-hybrid system and co-immunoprecipitation studies, we have identified the transcriptional co-repressor C-terminal binding protein (CtBP)2 as an interactor for calsenilin and have shown that the two proteins can interact in vivo. In co-immunoprecipitation studies, calsenilin also interacted with CtBP1, a CtBP2 homolog. Our data also showed a calsenilin-dependent increase in c-fos protein levels in CtBP knockout fibroblasts, suggesting that CtBP may modulate the transcriptional repression of c-fos by calsenilin. Furthermore, the finding that histone deacetylase protein and activity were associated with the calsenilin,CtBP immunocomplex suggests a mechanism by which calsenilin,CtBP may act to repress transcription. Finally, we demonstrated that calsenilin and CtBP are present in synaptic vesicles and can interact in vivo. [source] The zinc-finger protein ZFR is critical for Staufen 2 isoform specific nucleocytoplasmic shuttling in neuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2006George Elvira Abstract In mammalian neurons, transport and translation of mRNA to individual potentiated synapses is believed to occur via a heterogeneous population of RNA granules. To identify components of Staufen2-containing granules, we used the yeast two-hybrid system. A mouse fetal cDNA library was screened with the N-terminal fragment of Staufen2 as bait. ZFR, a three zinc finger protein, was identified as an interacting protein. Confocal microscopy showed that ZFR, although mainly nuclear, was also found in the somatodendritic compartment of primary hippocampal neurons where it localized as granule-like structures. Co-localization with Staufen2 was observed in several granules. Biochemical analyses (immunoprecipitation, cell fractionation) further confirmed the ZFR/Staufen2 association. ZFR was shown to interact with at least the Staufen262 isoform, but not with Staufen1. ZFR also co-fractionated with ribosomes and Staufen259 and Staufen252 in a sucrose gradient. Interestingly, knockdown expression of ZFR through RNA interference in neurons relocated specifically the Staufen262, but not the Staufen259, isoform to the nucleus. Our results demonstrate that ZFR is a native component of Staufen2-containing granules and likely plays its role during early steps of RNA transport and localization. They also suggest that one of these roles may be linked to Staufen262 -containing RNA granule formation in the nucleus and/or to their nucleo-cytoplasmic shuttling. [source] Interaction of Rab31 and OCRL-1 in oligodendrocytes: Its role in transport of mannose 6-phosphate receptorsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2010A.G. Rodriguez-Gabin Abstract Rab31, a protein that we cloned from an oligodendrocyte cDNA library, is required for transport of mannose 6-phosphate receptors (MPRs) from the trans-Golgi network (TGN) to endosomes and for Golgi/TGN organization. Here we extend the knowledge of the mechanism of action of Rab31 by demonstrating its interaction with OCRL-1, a phosphatidylinositol 4,5-diphosphate 5-phosphatase (PI(4,5)P2 5-phosphatase) that regulates the levels of PI(4,5)P2 and PI(4)P, molecules involved in transport and Golgi/TGN organization. We show that Rab31 interacts with OCRL-1 in a yeast two-hybrid system, GST-Rab31 pull-down experiments, and coimmunoprecipitation of OCRL-1 using oligodendrocyte culture lysates. Rab31 and OCRL-1 colocalize in the TGN, post-TGN carriers, and endosomes. Cation-dependent MPR (CD-MPR) is sorted to OCRL-1-containing carriers, but CD63 and vesicular stomatitis virus G (VSVG) are not. siRNA-mediated depletion of endogenous Rab31 causes collapse of the TGN apparatus and markedly decreases the levels of OCRL-1 in the TGN and endosomes. Our observations indicate that the role of Rab31 in the Golgi/TGN structure and transport of MPRs depends on its capability to recruit OCRL-1 to domains of the TGN where the formation of carriers occurs. The importance of our observations is highlighted by the fact that mutation of OCRL-1 causes demyelination in humans. © 2009 Wiley-Liss, Inc. [source] The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoeaMOLECULAR MICROBIOLOGY, Issue 2 2006Nandi Simpson Summary Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentium,map. These results provide new insights into the mechanisms of EPEC diarrhoea. [source] Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneriMOLECULAR MICROBIOLOGY, Issue 4 2001Anne-Laure Page The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process. [source] A novel upstream regulator of WRKY53 transcription during leaf senescence in Arabidopsis thalianaPLANT BIOLOGY, Issue 2008Y. Miao Abstract Arabidopsis WRKY proteins comprise a family of zinc finger-type transcription factors involved in the regulation of gene expression during pathogen defence, wounding, trichome development and senescence. To better understand the regulatory role of the senescence-related WRKY53 factor, we identified upstream regulatory factors using the yeast one-hybrid system. Among others, we identified a DNA-binding protein with a so far unknown function that contains a transcriptional activation domain and a kinase domain with similarities to HPT kinases. In vitro studies revealed that this activation domain protein (AD protein) can phosphorylate itself and that phosphorylation increases its DNA-binding activity to the WRKY53 promoter region. Using the yeast two-hybrid system, an interaction with proteins that were previously shown to bind to the WRKY53 promoter was tested. The AD protein interacted with MEKK1. The interaction with MEKK1 was confirmed in vivo by bimolecular fluorescence complementation (BiFC); however, the AD protein was not phosphorylated by MEKK1 in vitro and vice versa. This indicates that there may be competition between WRKY53 and AD protein for binding of MEKK1 at the WRKY53 promoter. Overexpression and knockout of the respective gene resulted in changes in transcription levels of WRKY53, indicating that AD protein is a positive regulator of WRKY53 expression. Expression of the AD protein gene can be induced by hydrogen peroxide treatment and reduced by jasmonic acid treatment, as previously shown for WRKY53. [source] Characterization of an Arabidopsis thaliana Homologue of the Nuclear Export Receptor CAS by its Interaction with Importin,PLANT BIOLOGY, Issue 4 2002D. Haasen Abstract: We have previously described four genes encoding different Importin ,-like proteins from Arabidopsis thaliana. Here we describe the putative nuclear export receptor for Importin ,. Using protein interaction assays in the yeast two-hybrid system, we characterized an Arabidopsis protein showing high similarity to human CAS, the nuclear export receptor for Importin ,. Arabidopsis CAS specifically bound to four different plant Importin , proteins but not to proteins containing leucine-rich nuclear export signals (NESs) that are recognized by Exportin 1 (XPO1/CRM1). Like all members of the Importin , family, Arabidopsis CAS also interacted with the regulatory GTPase Ran. Deletion of 15 amino acid residues from the amino terminus of CAS abolished binding of Importin ,, but did not influence the interaction with the GTPase Ran. We found two regions of Importin ,1 that profoundly influence the binding to CAS: the amino terminal Importin beta-binding (IBB) domain and the carboxy terminus. Whereas the IBB domain did not directly bind to CAS, but might rather affect the interaction through conformational changes within the Importin , protein, the carboxy terminal domain strongly interacted with CAS. [source] A UVB-hypersensitive mutant in Arabidopsis thaliana is defective in the DNA damage responseTHE PLANT JOURNAL, Issue 3 2009Ayako N. Sakamoto Summary To investigate UVB DNA damage response in higher plants, we used a genetic screen to isolate Arabidopsis thaliana mutants that are hypersensitive to UVB irradiation, and isolated a UVB-sensitive mutant, termed suv2 (for sensitive to UV 2) that also displayed hypersensitivity to ,-radiation and hydroxyurea. This phenotype is reminiscent of the Arabidopsis DNA damage-response mutant atr. The suv2 mutation was mapped to the bottom of chromosome 5, and contains an insertion in an unknown gene annotated as MRA19.1. RT-PCR analysis with specific primers to MRA19.1 detected a transcript consisting of 12 exons. The transcript is predicted to encode a 646 amino acid protein that contains a coiled-coil domain and two instances of predicted PIKK target sequences within the N-terminal region. Fusion proteins consisting of the predicted MRA19.1 and DNA-binding or activation domain of yeast transcription factor GAL4 interacted with each other in a yeast two-hybrid system, suggesting that the proteins form a homodimer. Expression of CYCB1;1:GUS gene, which encodes a labile cyclin:GUS fusion protein to monitor mitotic activity by GUS activity, was weaker in the suv2 plant after ,-irradiation than in the wild-type plants and was similar to that in the atr plants, suggesting that the suv2 mutant is defective in cell-cycle arrest in response to DNA damage. Overall, these results suggest that the gene disrupted in the suv2 mutant encodes an Arabidopsis homologue of the ATR-interacting protein ATRIP. [source] Interaction of proteins involved in ecdysone and juvenile hormone signal transduction,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Kavita Bitra Abstract Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] BIP, a BRAM-interacting protein involved in TGF-, signalling, regulates body length in Caenorhabditis elegansGENES TO CELLS, Issue 7 2001Katsura Sugawara Background The TGF-, superfamily has diverse biological activities and is involved in the early development of animals. We previously identified a novel family member, BMP receptor associated molecule (BRAM), which binds to the intracellular domain of BMP type IA receptor and is involved in the BMP signalling pathway. Results To identify novel molecules involved in TGF-, signalling pathways, we performed yeast two-hybrid screening using BRAM as bait. From a Xenopus cDNA library, we cloned a cDNA encoding 693 amino acids and containing the motif for an oxysterol binding protein (OSBP), which we designated BRAM interacting protein (BIP). We then isolated a BIP homologue from the Caenorhabditis elegans that encodes 733 amino acids and also contains the OSBP-like motif. Immunoprecipitation and Western blotting studies revealed that C. elegans BIP could interact with the C. elegans BRAM homologues BRA-1 and BRA-2. C. elegans BIP was expressed in pharyngeal muscle, hypodermis and several neuronal cells, an expression pattern overlaps with those of BRA-1 and BRA-2. Finally, we found that inhibition of BIP expression in C. elegans by double stranded RNA interference produces a Sma phenotype. Conclusions BIP was isolated using the yeast two-hybrid systems. BIP may function in the TGF-, pathway and regulate body length in C. elegans. [source] | |||||||||||||||||||||||||||||||