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Dimerization Domain (dimerization + domain)
Selected AbstractsMechanism of activation of the double-stranded-RNA-dependent protein kinase, PKRFEBS JOURNAL, Issue 13 2001Role of dimerization, cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2) An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the , subunit of eIF2. Binding of viral dsRNAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein,protein interactions and that trans -autophosphorylation is the final step in the mechanism of activation of PKR. [source] Nuclear import and DNA-binding activity of RFX1FEBS JOURNAL, Issue 10 2001Evidence for an autoinhibitory mechanism RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent ,acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC. [source] A BCR,JAK2 fusion gene as the result of a t(9;22)(p24;q11.2) translocation in a patient with a clinically typical chronic myeloid leukemiaGENES, CHROMOSOMES AND CANCER, Issue 3 2005Frank Griesinger Chronic myeloid leukemia (CML) is characterized by the presence of a t(9;22)(q34;q11.2), which leads to the well-known BCR,ABL1 fusion protein. We describe a patient who was diagnosed clinically with a typical CML but on cytogenetic analysis was found to have a t(9;22)(p24;q11.2). Chromosomal fluorescence in situ hybridization showed that the BCR gene locus spanned the breakpoint at band 22q11.2 but that the ABL1 gene was not rearranged. By means of a candidate gene approach, the JAK2 gene, at 9p24, was identified as the fusion partner of BCR in this case. The BCR,JAK2 fusion protein contains the coiled-coil dimerization domain of BCR and the protein tyrosine kinase domain (JH1) of JAK2. The patient's disease did not respond to Imatinib, and this unresponsiveness was most likely a result of the BCR,JAK2 fusion protein. © 2005 Wiley-Liss, Inc. [source] Novel germline BRCA1 and BRCA2 mutations in breast and breast/ovarian cancer families from the Czech Republic ,,HUMAN MUTATION, Issue 6 2001Eva Machackova Abstract Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high-risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non-radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high-risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761-3762delGA and 2616-2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073-5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA-binding protein. © 2000 Wiley-Liss, Inc. [source] Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responsesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Manchao Zhang Abstract The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin. J. Cell. Biochem. 103: 162,181, 2008. © 2007 Wiley-Liss, Inc. [source] Secretion of proteins with dimerization capacity by the haemolysin type I transport system of Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2004Sofía Fraile Summary The tolerance of the haemolysin transport system (Hly) for exporting dimeric protein substrates to the supernatants of Escherichia coli cultures was examined. A strong dimerization domain (i.e. an amphipathic ,-helix capable of forming a leucine zipper in the yeast transcription factor GCN4) was inserted into an epitope-tagged version of the 23 kDa C-terminal secretion signal of haemolysin (EHlyA). The zipper-containing polypeptide (ZEHlyA) was effectively secreted by E. coli cells carrying the HlyBD transporter and accumulated in the culture media as a stable dimer as determined by gel filtration chromatography. In vivo protein cross-linking experiments and coexpression with a secretion-deficient derivative of ZEHlyA indicated that leucine zipper-dependent dimerization occurs following secretion. To test whether dimerization allows the correct folding of the secreted polypeptide, immunoglobulin VHH -domains obtained from camel antibodies were fused to EHlyA and ZEHlyA. Functional dimerization of the ZEHlyA hybrid was anticipated to increase the apparent binding affinity (i.e. avidity) of the VHH moiety, thus becoming an excellent reporter of correct protein folding and dimerization. Both VHH -EHlyA and VHH -ZEHlyA hybrids were quantitatively secreted and found in the extracellular medium as active monomers and dimers respectively. When compared with their monomeric counterparts, the dimeric VHH -ZEHlyA molecules showed superior binding properties to their cognate antigen, with a 10-fold increase in their avidity. These data reveal a non-anticipated permissiveness of the Hly type I transport machinery for the secretion of substrates with dimerization capacity. [source] Molecular cloning and characterization of Bombyx mori CREB gene,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 1 2009Hongsheng Song Abstract The cAMP response element binding protein (CREB), as one of the best characterized stimulus-induced transcription factors, plays critical roles in activating transcription of target genes in response to a variety of environmental stimuli. To characterize this important molecule in the silkworm, Bombyx mori, we cloned a full-length cDNA of CREB gene from B. mori brains by using RACE-PCR. The sequence of B. mori CREB (named BmCREB1) gene contains a 88,bp 5, UTR, a 783,bp open reading frame (ORF) encoding 261 amino acids and a 348,bp 3, UTR. The deduced BmCREB amino acid sequence has 56.7% and 37.2% homology with CREB from Apis mellifera carnica and Drosophila melanogaster, respectively. The primary structure of the deduced BmCREB1 protein contains a kinase-inducible domain (KID) and a basic region/leucine zipper (bZIP) dimerization domain which exisits in all CREB family members. Genomic analysis showed there are 9 exons and 5 introns in B. mori CREB genome sequences. We identified three different isoforms of BmCREB (BmCREB1, BmCREB2 and BmCREB3) through alternative splicing in C terminal. In addition, the expression of BmCREB in different developmental stages was investigated by using quantitative real-time PCR in both diapause and non-diapause type of B. mori bivoltine race (Dazao). BmCREB transcripts showed two peaks in embryonic stage and pupal stage in both types of bivoltine race. However, consistently higher expression of BmCREB was found throughout the developmental stages in the diapause type than in the non-diapause type. These results suggest that BmCREB is involved in the processs of diapause induced by environmental factors. © 2009 Wiley Periodicals, Inc. [source] Structures of three diphtheria toxin repressor (DtxR) variants with decreased repressor activityACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2001Ehmke Pohl The diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae regulates the expression of the gene on corynebacteriophages that encodes diphtheria toxin (DT). Other genes regulated by DtxR include those that encode proteins involved in siderophore-mediated iron uptake. DtxR requires activation by divalent metals and holo-DtxR is a dimeric regulator with two distinct metal-binding sites per three-domain monomer. At site 1, three side chains and a sulfate or phosphate anion are involved in metal coordination. In the DtxR,DNA complex this anion is replaced by the side chain of Glu170 provided by the third domain of the repressor. At site 2 the metal ion is coordinated exclusively by constituents of the polypeptide chain. In this paper, five crystal structures of three DtxR variants focusing on residues Glu20, Arg80 and Cys102 are reported. The resolution of these structures ranges from 2.3 to 2.8,Å. The side chain of Glu20 provided by the DNA-binding domain forms a salt bridge to Arg80, which in turn interacts with the anion. Replacing either of the salt-bridge partners with an alanine reduces repressor activity substantially and it has been inferred that the salt bridge could possibly control the wedge angle between the DNA-binding domain and the dimerization domain, thereby modulating repressor activity. Cys102 is a key residue of metal site 2 and its substitution into a serine abolishes repressor activity. The crystal structures of Zn-Glu20Ala-DtxR, Zn-Arg80Ala-DtxR, Cd-Cys102Ser-DtxR and apo-Cys102Ser-DtxR in two related space groups reveal that none of these substitutions leads to dramatic rearrangements of the DtxR fold. However, the five crystal structures presented here show significant local changes and a considerable degree of flexibility of the DNA-binding domain with respect to the dimerization domain. Furthermore, all five structures deviate significantly from the structure in the DtxR,DNA complex with respect to overall domain orientation. These results confirm the importance of the hinge motion for repressor activity. Since the third domain has often been invisible in previous crystal structures of DtxR, it is also noteworthy that the SH3-like domain could be traced in four of the five crystal structures. [source] Crystallization and preliminary X-ray diffraction analysis of the dimerization domain of the tumour suppressor ING4ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Simone Culurgioni Inhibitor of growth protein 4 (ING4) belongs to the ING family of tumour suppressors and is involved in chromatin remodelling, in growth arrest and, in cooperation with p53, in senescence and apoptosis. Whereas the structure and histone H3-binding properties of the C-terminal PHD domains of the ING proteins are known, no structural information is available for the N-terminal domains. This domain contains a putative oligomerization site rich in helical structure in the ING2,5 members of the family. The N-terminal domain of ING4 was overexpressed in Escherichia coli and purified to homogeneity. Crystallization experiments yielded crystals that were suitable for high-resolution X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 129.7, b = 188.3, c = 62.7,Å. The self-rotation function and the Matthews coefficient suggested the presence of three protein dimers per asymmetric unit. The crystals diffracted to a resolution of 2.3,Å using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF). [source] Crystallographic characterization of a multidomain histidine protein kinase from an essential two-component regulatory systemACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Haiyan Zhao YycGF is a highly conserved two-component signal transduction system that is specific to low-G+C Gram-positive bacteria, including many important human pathogens. It has been recognized as a crucial regulatory system for cell-wall metabolism. YycG, the histidine protein kinase of this system, is a multidomain transmembrane protein. The truncated cytoplasmic portion of YycG from Bacillus subtilis encompassing the PAS domain, the dimerization domain and the catalytic domain was expressed, purified and crystallized. X-ray data were collected to 2.8,Å resolution with a completeness of 98.2% and an overall Rmerge of 5.6%. The crystals belonged to space group P61 or P65, with unit-cell parameters a = 135.0, c = 133.0,Å. The selenomethionine-substituted version of the protein was crystallized and X-ray data were collected to 3.6,Å resolution for subsequent MAD phasing. [source] The structure of NMB1585, a MarR-family regulator from Neisseria meningitidisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Charles E. Nichols The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1,Å. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix,turn,helix (wHtH) domain connected to an ,-helical dimerization domain. The spacing of the recognition helices of the wHtH domain indicates that NMB1585 is pre-configured for DNA binding, with a putative inducer pocket that is largely occluded by the side chains of two aromatic residues (Tyr29 and Trp53). NMB1585 was shown to bind to its own promoter region in a gel-shift assay, indicating that the protein acts as an auto-repressor. [source] | |||||||||||||