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Basic Region (basic + region)
Selected AbstractsBasic region of residues 228,231 of protein kinase CK1, is involved in its interaction with axin: Binding to axin does not affect the kinase activity,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Pablo Sobrado Abstract Protein kinase CK1, also known as casein kinase 1, participates in the phosphorylation of ,-catenin, which regulates the functioning of the Wnt signaling cascade involved in embryogenesis and carcinogenesis. ,-catenin phosphorylation occurs in a multiprotein complex assembled on the scaffold protein axin. The interaction of CK1, from Danio rerio with mouse-axin has been studied using a pull-down assay that uses fragments of axin fused to glutathione S transferase, which is bound to glutathione sepharose beads. The results indicate that the three lysines present in the basic region of residues 228,231 of CK1, are necessary for the binding of CK1 to axin. Lysine 231 is particularly important in this interaction. In order to define the relevance of the axin-CK1, interaction, the effect of the presence of axin on the phosphorylating activity of CK1, was tested. It is also evident that the region of axin downstream of residues 503,562 is required for CK1, interaction. The binding of CK1, to axin fragment 292,681 does not facilitate the phosphorylation of ,-catenin despite the fact that this axin fragment can also bind ,-catenin. Binding of CK1, to axin is not required for the phosphorylation of axin itself and, likewise, axin does not affect the kinetic parameters of the CK1, towards casein or a specific peptide substrate. © 2004 Wiley-Liss, Inc. [source] Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulationFEBS JOURNAL, Issue 22 2004Interaction with BMAL The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature419, 841,844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loop-helix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein,protein interaction with BMAL1 and DNA binding to the E-box. [source] Basic region of residues 228,231 of protein kinase CK1, is involved in its interaction with axin: Binding to axin does not affect the kinase activity,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2005Pablo Sobrado Abstract Protein kinase CK1, also known as casein kinase 1, participates in the phosphorylation of ,-catenin, which regulates the functioning of the Wnt signaling cascade involved in embryogenesis and carcinogenesis. ,-catenin phosphorylation occurs in a multiprotein complex assembled on the scaffold protein axin. The interaction of CK1, from Danio rerio with mouse-axin has been studied using a pull-down assay that uses fragments of axin fused to glutathione S transferase, which is bound to glutathione sepharose beads. The results indicate that the three lysines present in the basic region of residues 228,231 of CK1, are necessary for the binding of CK1 to axin. Lysine 231 is particularly important in this interaction. In order to define the relevance of the axin-CK1, interaction, the effect of the presence of axin on the phosphorylating activity of CK1, was tested. It is also evident that the region of axin downstream of residues 503,562 is required for CK1, interaction. The binding of CK1, to axin fragment 292,681 does not facilitate the phosphorylation of ,-catenin despite the fact that this axin fragment can also bind ,-catenin. Binding of CK1, to axin is not required for the phosphorylation of axin itself and, likewise, axin does not affect the kinetic parameters of the CK1, towards casein or a specific peptide substrate. © 2004 Wiley-Liss, Inc. [source] Identification of structural and molecular determinants of the tyrosine-kinase Wzc and implications in capsular polysaccharide exportMOLECULAR MICROBIOLOGY, Issue 5 2010Emmanuelle Bechet Summary Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX2RX2R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery. [source] Two-dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brainPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2010Ciara A. McManus Abstract We describe a 2-DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6,11 IPG strips using paper-bridge loading and on 12% SDS-PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in-house proteomics data analysis platform, Proline. The 2-DE reference map is available via the UCD 2-DE Proteome Database (http://proteomics-portal.ucd.ie:8082) and can also be accessed via the WORLD-2DPAGE Portal (http://www.expasy.ch/world-2dpage/). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018,10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2-DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia. [source] Over-expression of TGA5, which encodes a bZIP transcription factor that interacts with NIM1/NPR1, confers SAR-independent resistance in Arabidopsis thaliana to Peronospora parasiticaTHE PLANT JOURNAL, Issue 2 2002Han Suk Kim Summary The Arabidopsis thaliana NIM1/NPR1 gene product is required for induction of systemic acquired resistance (SAR) by pathogens, salicylic acid (SA) or synthetic SA analogs. We identified, in a yeast two-hybrid screen, two NIM1/NPR1 interacting proteins, TGA2 and TGA5, which belong to the basic region, leucine zipper (bZIP) family of transcription factors. Both TGA2 and TGA5 strongly interact with NIM1/NPR1 in yeast and in vitro, and recognize the as-1 cis element found within the promoter of several pathogenesis-related genes, such as PR-1. To determine the role TGA2 and TGA5 may play in NIM1/NPR1-mediated disease resistance, we introduced sense and antisense versions of both genes into transgenic Arabidopsis plants. Characterization of TGA2 transgenic plants revealed that inhibition or overexpression of TGA2 does not significantly affect PR-1 expression or induction of SAR after pathogen infection or INA treatment. Surprisingly, all TGA5 -antisense transgenic plants produced showed increased accumulation of TGA5 transcripts compared with untransformed control plants, while the TGA5 -sense lines showed no significant increase in TGA5 mRNA levels. Interestingly, the high level of TGA5 mRNA in the antisense lines was accompanied by significant resistance to a highly virulent isolate of the oomycete pathogen Peronospora parasitica. Further, resistance was not coupled to accumulation of products from the SAR-linked PR-1 gene following inoculation with P. parasitica or treatment with INA, indicating that these plants express a robust, PR-1 -independent resistance mechanism. Resistance was retained when a TGA5 -accumulating line was combined genetically with a nim1-1 mutation or nahG (salicylate hydroxylase) transgene, indicating that resistance in these plants is due to an SA and SAR-independent mechanism. [source] Sequence-specific recognition of DNA by hydrophobic, alanine-rich mutants of the basic region/leucine zipper motif investigated by fluorescence anisotropyBIOPOLYMERS, Issue 1 2002Gregory H. Bird Abstract We generated minimalist proteins capable of sequence-specific, high-affinity binding of DNA to probe how proteins are used and can be used to recognize DNA. In order to quantify binding affinities and specificities in our protein,DNA system, we used fluorescence anisotropy to measure in situ the thermodynamics of binding of alanine-rich mutants of the GCN4 basic region/leucine zipper (bZIP) domain to DNA duplexes containing target sites AP-1 (5,-TGACTCA-3,) or ATF/CREB (5,-TGACGTCA-3,). We simplified the ,-helical bZIP molecular recognition scaffold by alanine substitution: 4A, 11A, and 18A contain four, eleven, and eighteen alanine mutations in their DNA-binding basic regions, respectively. DNase I footprinting analysis demonstrates that all bZIP mutants retain the sequence-specific DNA-binding function of native GCN4 bZIP. Titration of fluorescein-labeled oligonucleotide duplexes with increasing amounts of protein yielded low nanomolar dissociation constants for all bZIP mutants in complex with the AP-1 and ATF/CREB sites: binding to the nonspecific control duplex was > 1000-fold weaker. Remarkably, the most heavily mutated protein 18A, containing 24 alanines in its 27-residue basic region, still binds AP-1 and ATF/CREB with dissociation constants of 15 and 7.8 nM, respectively. Similarly, wild-type bZIP binds these sites with Kd values of 9.1 and 14 nM. 11A also displays low nanomolar dissociation constants for AP-1 and ATF/CREB, while 4A binds these sites with , 10-fold weaker Kd values. Thus, both DNA-binding specificity and affinity are maintained in all our bZIP derivatives. This Ala-rich scaffold may be useful in design and synthesis of small ,-helical proteins with desired DNA-recognition properties capable of serving as therapeutics targeting transcription. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 10,20, 2002 [source] Crystallization and preliminary X-ray diffraction analysis of GCIP/HHM transcriptional regulatorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Azusa Seto GCIP/HHM is a human nuclear protein that is implicated in regulation of cell proliferation. Its primary structure contains helix,loop,helix and leucine-zipper motifs but lacks a DNA-binding basic region. Native and selenomethionine-derivatized (SeMet) crystals of full-length GCIP/HHM were obtained using the hanging-drop vapour-diffusion method. The crystals were greatly improved by adding tris(2-carboxyethyl)phosphine as a reducing reagent and diffracted to 3.5,Å resolution. Preliminary phase calculations using the data set obtained from the SeMet crystal suggested that the crystal belonged to space group P3221 and contained one molecule per asymmetric unit. Structure determination by the multiple-wavelength anomalous dispersion method using the SeMet crystals is in progress. [source] | |||||||||||||