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Robert S Adelstein - One of the best experts on this subject based on the ideXlab platform.
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identification of the Serine Residue phosphorylated by protein kinase c in vertebrate nonmuscle myosin heavy chains
Biochemistry, 1991Co-Authors: Mary Anne Conti, Robert S Adelstein, James R Sellers, Marshall ElzingaAbstract:Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the Serine Residue phosphorylated by protein kinase C, this Residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.
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phosphorylation of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells
Journal of Cell Science, 1991Co-Authors: Christine A Kelley, Sachiyo Kawamoto, Mary Anne Conti, Robert S AdelsteinAbstract:Summary In this article we summarize our recent experiments studying the phosphorylation of vertebrate myosin heavy chains by protein kinase C and casein kinase II. Protein kinase C phosphorylates vertebrate nonmuscle myosin heavy chains both in vitro and in intact cells. A single Serine Residue near the end of the helical portion of the myosin rod is the only site phosphorylated in a variety of vertebrate nonmuscle myosin heavy chains. There does not appear to be a site for protein kinase C phosphorylation in vertebrate smooth muscle myosin heavy chains. Casein kinase II phosphorylates a single Serine Residue located near the carboxyl terminus of the 204×10 3 M r , smooth muscle myosin heavy chain in vitro as well as in cultured smooth muscle cells. It does not phosphorylate the 200×10 3 M r smooth muscle myosin heavy chain. However, the site is present in vertebrate nonmuscle myosin heavy chains. The 204×10 3 M r myosin heavy chain of embryonic chicken gizzard smooth muscle is exceptional in not containing a site for casein kinase II phosphorylation.
Shunichi Nakamura - One of the best experts on this subject based on the ideXlab platform.
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protein kinase d mediated phosphorylation and nuclear export of sphingosine kinase 2
Journal of Biological Chemistry, 2007Co-Authors: Guo Ding, Hirofumi Sonoda, Taketoshi Kajimoto, Sravan K Goparaju, Saleem Jahangeer, Taro Okada, Shunichi NakamuraAbstract:Next Section Abstract Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a Serine Residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this Serine Residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.
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protein kinase d mediated phosphorylation and nuclear export of sphingosine kinase 2
Journal of Biological Chemistry, 2007Co-Authors: Guo Ding, Hirofumi Sonoda, Taketoshi Kajimoto, Sravan K Goparaju, Saleem Jahangeer, Taro Okada, Shunichi NakamuraAbstract:Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a Serine Residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this Serine Residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.
Shuying Wang - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of clostridium difficile sortase b reveals key Residues for catalytic activity and substrate specificity
Journal of Biological Chemistry, 2020Co-Authors: Chiayu Kang, Ihsiu Huang, Chichi Chou, Jyuncyuan Chang, Yuyuan Hsiao, Chenghsuan Cheng, Weijiun Tsai, Kaicheng Hsu, Shuying WangAbstract:Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key Residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved Serine Residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus. The Serine Residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic Residue nor a substrate-interacting Residue, based on the LC-MS/MS data and available structural models of SrtB–substrate complexes. Furthermore, we also demonstrated that Residues 163–168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key Residues with roles in catalysis and substrate specificity of Cd-SrtB.
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functional analysis of clostridium difficile sortase b reveals key Residues for catalytic activity and substrate specificity
Journal of Biological Chemistry, 2020Co-Authors: Chiayu Kang, Ihsiu Huang, Chichi Chou, Jyuncyuan Chang, Yuyuan Hsiao, Chenghsuan Cheng, Weijiun Tsai, Tsaiyu Wu, Shuying WangAbstract:Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key Residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved Serine Residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus The Serine Residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic Residue nor a substrate-interacting Residue, based on the LC-MS/MS data and available structural models of SrtB-substrate complexes. Furthermore, we also demonstrated that Residues 163-168 located on the beta6/beta7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key Residues with roles in catalysis and substrate specificity of Cd-SrtB.
Attila P Vegh - One of the best experts on this subject based on the ideXlab platform.
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hydrogen bonding in a model bacteriochlorophyll binding site drives assembly of light harvesting complex
Journal of Biological Chemistry, 2004Co-Authors: Lee G Kwa, Attila P Vegh, Brigitte Strohmann, Bruno Robert, Adela Garciamartin, Paula BraunAbstract:In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane Serine Residue at position -4 relative to the liganding histidine of the α-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the Residue at this position in the helix. A hydrogen bond between the hydroxy group of the Serine and the 131 keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the Serine Residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.
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identification of intramembrane hydrogen bonding between 13 1 keto group of bacteriochlorophyll and Serine Residue alpha27 in the lh2 light harvesting complex
Biochimica et Biophysica Acta, 2003Co-Authors: Paula Braun, Attila P Vegh, M Von Jan, Brigitte Strohmann, C N Hunter, Bruno Robert, Hugo ScheerAbstract:Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a Serine Residue of the membrane-spanning region of LH2 alpha-subunit, and the C-13(1) keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the beta-subunit. Replacement of the Serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13(1) keto carbonyl and a methyl hydrogen of the alanine replacing Serine(-4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13(1) not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 A in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa Residues suitable as hydrogen bond donors to their keto groups.
Paula Braun - One of the best experts on this subject based on the ideXlab platform.
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hydrogen bonding in a model bacteriochlorophyll binding site drives assembly of light harvesting complex
Journal of Biological Chemistry, 2004Co-Authors: Lee G Kwa, Attila P Vegh, Brigitte Strohmann, Bruno Robert, Adela Garciamartin, Paula BraunAbstract:In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane Serine Residue at position -4 relative to the liganding histidine of the α-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the Residue at this position in the helix. A hydrogen bond between the hydroxy group of the Serine and the 131 keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the Serine Residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.
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identification of intramembrane hydrogen bonding between 13 1 keto group of bacteriochlorophyll and Serine Residue alpha27 in the lh2 light harvesting complex
Biochimica et Biophysica Acta, 2003Co-Authors: Paula Braun, Attila P Vegh, M Von Jan, Brigitte Strohmann, C N Hunter, Bruno Robert, Hugo ScheerAbstract:Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a Serine Residue of the membrane-spanning region of LH2 alpha-subunit, and the C-13(1) keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the beta-subunit. Replacement of the Serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13(1) keto carbonyl and a methyl hydrogen of the alanine replacing Serine(-4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13(1) not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 A in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa Residues suitable as hydrogen bond donors to their keto groups.
