Publications by authors named "Tohru Koike"

80 Publications

History of Phos-tag technology for phosphoproteomics.

J Proteomics 2021 Nov 21:104432. Epub 2021 Nov 21.

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan.

Phos-tag is a functional molecule that selectively captures a phosphate monoester dianion in neutral aqueous solutions. The affinity of Phos-tag for phosphate monoester dianions is more than 10,000 times greater than that for other anions present in living organisms, such as carboxylic acid anions. We have developed and applied useful techniques for phosphoproteomics based on Phos-tag. This review describes the history of Phos-tag development and outlines three main technologies that have been put to practical use. The first is a technique to separate and concentrate phosphopeptides and phosphoproteins using a Phos-tag derivative with a hydrophilic chromatography carrier (Phos-tag polymer beads). The second is a technology to detect phosphopeptides and phosphoproteins on various arrays using Phos-tag biotin. The third is a technique to separate and detect phosphoproteins by electrophoresis using Phos-tag acrylamide. We hope that these three technologies will make a significant contribution to phosphoproteomics and, ultimately, to life science research. SIGNIFICANCE: The authors found that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)-amino]propan-2-olato acted as a novel phosphate-binding tag nanomolecule, Phos-tag, in an aqueous solution under near physiological conditions. The metal complex having a vacancy on two metal ions is suitable for the access of a phosphomonoester dianion (R-OPO) as a bridging ligand. A dinuclear zinc(II) complex (Zn-Phos-tag) strongly binds to a p-nitrophenyl phosphate dianion (K = 2.5 × 10 M) at a neutral pH. The anion selectivity indexes against SO, CHCOO, Cl, and the bisphenyl phosphate monoanion at 25 °C are 5.2 × 10, 1.6 × 10, 8.0 × 10, and > 2 × 10, respectively. We have been involved in developing technologies by using the Phos-tag molecule and its derivatives to permit the analysis of phosphorylated biomolecules. To date, Phos-tag technology has contributed to the development of several procedures for phosphoproteomics, including a phosphate-affinity chromatography technique for the separation and enrichment of phosphopeptides and phosphoproteins, a wide variety of microarray/on-chip techniques for the detection of protein phosphorylation, and a phosphate-affinity electrophoresis technique for the detection of shifts in the mobilities of phosphoproteins. In this review article, the authors introduce the impact of Phos-tag-based technological advances for phosphoproteomics.
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http://dx.doi.org/10.1016/j.jprot.2021.104432DOI Listing
November 2021

Recent advances in the Phos-tag technique focused on the analysis of phosphoproteins in a bacterial two-component system.

J Proteomics 2021 Nov 20:104429. Epub 2021 Nov 20.

Department of Human Nutrition, Faculty of Human Sciences, Hiroshima Bunkyo University, Kabehigashi 1-2-1, Asakita-ku, Hiroshima 731-0295, Japan. Electronic address:

In a bacterial two-component system (TCS), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator (RR). The His- and Asp-bound phosphate groups are extremely unstable under acidic conditions easily to be hydrolyzed within a few hours. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation states in the TCS. Here, we describe that Phos-tag technique is suitable for the quantitative analysis of His- and Asp-phosphorylated proteins. The dynamics of the His-Asp phosphorelay of recombinant TCS derived from Escherichia coli, was examined by Phos-tag SDS-PAGE or Phos-tag fluorescent dye gel staining. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of HK and RR in the presence of ATP or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from HK to RR in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag fluorescent dye gel staining. Consequently, Phos-tag technique provides a simple and convenient approach for screening of HK inhibitors that have potential as new antimicrobial agents. SIGNIFICANCE: Bacterial cells have unique phosphotransfer signaling mechanisms known as two-component systems (TCSs) that permit the organism to sense and respond to various environmental conditions. Each system consists of a histidine kinase (HK) and a response regulator (RR). A typical HK contains an invariant His residue that is autophosphorylated in an ATP-dependent manner. A typical RR has a conserved Asp residue that can acquire a phosphoryl group from its cognate HK. In general, TCS has this type of a His-Asp phosphorelay scheme. Because TCS is also involved in the virulence of pathogens, it is potential targets for novel antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in the bacterial TCS. We believe that our Phos-tag technique provides a simple and convenient approach for drug discovery targeting the bacterial TCS.
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http://dx.doi.org/10.1016/j.jprot.2021.104429DOI Listing
November 2021

Characterization of Phosphorylation Status and Kinase Activity of Src Family Kinases Expressed in Cell-Based and Cell-Free Protein Expression Systems.

Biomolecules 2021 10 2;11(10). Epub 2021 Oct 2.

Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

The production of heterologous proteins is an important procedure for biologists in basic and applied sciences. A variety of cell-based and cell-free protein expression systems are available to achieve this. The expression system must be selected carefully, especially for target proteins that require post-translational modifications. In this study, human Src family kinases were prepared using six different protein expression systems: 293 human embryonic kidney cells, and cell-free expression systems derived from rabbit reticulocytes, wheat germ, insect cells, or . The phosphorylation status of each kinase was analyzed by Phos-tag SDS-PAGE. The kinase activities were also investigated. In the eukaryotic systems, multiple phosphorylated forms of the expressed kinases were observed. In the rabbit reticulocyte lysate system and 293 cells, differences in phosphorylation status between the wild-type and kinase-dead mutants were observed. Whether the expressed kinase was active depended on the properties of both the kinase and each expression system. In the prokaryotic systems, Src and Hck were expressed in autophosphorylated active forms. Clear differences in post-translational phosphorylation among the protein expression systems were revealed. These results provide useful information for preparing functional proteins regulated by phosphorylation.
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http://dx.doi.org/10.3390/biom11101448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8533471PMC
October 2021

An assay of human tyrosine protein kinase ABL activity using an protein expression system.

Biotechniques 2021 04 6;70(4):209-217. Epub 2021 Apr 6.

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 7348553, Japan.

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in . Tyrosine phosphorylation of the substrate in was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Relative to wild-type ABL, kinase activity was comparable in the H396P mutant, reduced in both Y253F and E255K mutants and undetectable in T315I and M351T mutants. These comparative results demonstrated that the phosphorylation states of the mutants correlated with their activity. The bacterial co-expression system permits rapid production of tyrosine kinase variants and provides a simple approach for examining their structure-activity relationships.
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http://dx.doi.org/10.2144/btn-2020-0154DOI Listing
April 2021

Phos-Tag Fluorescent Gel Staining for the Quantitative Detection of His- and Asp-Phosphorylated Proteins.

Methods Mol Biol 2021 ;2261:73-78

Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

We describe a standard protocol for phosphate-affinity fluorescent gel staining that uses a fluorophore-labeled dizinc(II) complex of a derivative of the phosphate-binding tag molecule Phos-tag to detect His- and Asp-phosphorylated proteins separated by SDS-PAGE. The procedure permits the quantitative monitoring of phosphorylated histidine kinases (His-phosphoproteins) and their cognate phosphorylated response regulators (Asp-phosphoproteins) in bacterial two-component signaling transduction systems. The total time required for each gel staining operation is about 2 h at room temperature.
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http://dx.doi.org/10.1007/978-1-0716-1186-9_6DOI Listing
March 2021

Determining Protein Phosphorylation Status Using Antibody Arrays and Phos-Tag Biotin.

Methods Mol Biol 2021 ;2237:217-224

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

We describe here a standard protocol for determining the phosphorylation status of protein multiplexes using antibody arrays and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer (Phos-tag Biotin). The procedure is based on an antibody microarray technique used in conjunction with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. By using this procedure, we have demonstrated the quantitative detection of the entire phosphorylation status of a target protein involved in intracellular signaling.
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http://dx.doi.org/10.1007/978-1-0716-1064-0_18DOI Listing
March 2021

Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic.

Sci Rep 2020 10 1;10(1):16273. Epub 2020 Oct 1.

Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.
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http://dx.doi.org/10.1038/s41598-020-73248-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531007PMC
October 2020

Phos-tag-based micropipette-tip method for analysis of phosphomonoester-type impurities in synthetic oligonucleotides.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 May 30;1151:122198. Epub 2020 May 30.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address:

Various chromatographic techniques, combined with mass spectrometry, have been developed for the analysis of impurities in oligonucleotide drugs, but those methods have generally been less focused on possible phosphomonoester-type compounds. Here, we introduce a simple method for separating terminally phosphorylated impurities from parent oligonucleotides by using a phosphate-affinity micropipette tip (Phos-tag tip). All steps for the phosphate-affinity separation (binding, washing, and elution) are conducted in aqueous buffers at neutral pH. The entire separation protocol requires less than 30 min per sample. In practical examples, we demonstrated that phosphorylated impurities in natural-type and chemically modified oligonucleotides can be efficiently separated by the Phos-tag tip method and subsequently characterized by using ion-pairing reversed-phase liquid chromatography mass spectrometry (IP-RPLC-MS). Thus, a combination of the Phos-tag tip method and IP-RPLC-MS is useful for characterizing and identifying phosphomonoester-type impurities in oligonucleotide drugs.
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http://dx.doi.org/10.1016/j.jchromb.2020.122198DOI Listing
May 2020

An immuno-dot blot assay for screening histidine kinase inhibitors.

Anal Biochem 2020 07 30;600:113765. Epub 2020 Apr 30.

Department of Functional Molecular Science, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.
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http://dx.doi.org/10.1016/j.ab.2020.113765DOI Listing
July 2020

A strategy to identify protein-N-myristoylation-dependent phosphorylation reactions of cellular proteins by using Phos-tag SDS-PAGE.

PLoS One 2019 21;14(11):e0225510. Epub 2019 Nov 21.

Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi, Japan.

To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin β1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225510PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872159PMC
March 2020

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes.

Electrophoresis 2019 11 16;40(22):3005-3013. Epub 2019 Sep 16.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.
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http://dx.doi.org/10.1002/elps.201900261DOI Listing
November 2019

Increase in constitutively active MEK1 species by introduction of MEK1 mutations identified in cancers.

Biochim Biophys Acta Proteins Proteom 2019 01 9;1867(1):62-70. Epub 2018 May 9.

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan.

The kinase MEK1 is an essential component of the mitogen-activated protein kinase cascades. Somatic mutations that have been identified in the MEK1-coding gene generally enhance kinase activity. Consequently, MEK1 has attracted much interest as a target for cancer therapy to block the aberrant activity. By using Phos-tag affinity electrophoresis, we found that the introduction of mutations detected in certain sporadic cancers or in MEK-inhibitor-resistant cancer cells produced constitutively active MEK1 species containing phosphorylated Ser-218 and Ser-222 residues; it also enhanced the constitutive activity of the kinase. Phosphorylation profiling of the mutants in the presence of inhibitors of RAF/MEK demonstrated that several mutations conferred resistance to multiple inhibitors as a result of an increase in the quantity of active MEK1 species containing the two phosphorylated Ser-218 and Ser-222 residues. Phos-tag-based phosphorylation profiling of MEK1 can therefore provide clinical insights into characteristics of individual mutations in the MEK1-coding gene.
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http://dx.doi.org/10.1016/j.bbapap.2018.05.004DOI Listing
January 2019

A method for profiling the phosphorylation state of tyrosine protein kinases.

Biochim Biophys Acta Proteins Proteom 2019 01 9;1867(1):71-75. Epub 2018 May 9.

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan. Electronic address:

Protein kinases are known to be implicated in various biological phenomena and diseases through their involvement in protein phosphorylation. Therefore, analysis of the activity of protein kinases by examination of their phosphorylation state is important to elucidate their mechanisms. However, a method for analyzing the phosphorylation state of entire protein kinases in cells is not established. In the present study, we developed a new profiling method to analyze the expression and phosphorylation state of protein kinases using a Multi-PK antibody and Phos-tag 2D-PAGE. When HL-60 cells were differentiated into macrophage-like cells induced by 12-O-tetradecanoylphorbol-13-acetate, we observed significant changes in the expression and phosphorylation state of immunoreactive spots by this method. These results show that tyrosine kinase expression levels and phosphorylation state are changed by differentiation. Taken together, the developed method will be a useful tool for analysis of intracellular tyrosine protein kinases.
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http://dx.doi.org/10.1016/j.bbapap.2018.05.003DOI Listing
January 2019

A simple method for determining the ligand affinity toward a zinc-enzyme model by using a TAMRA/TAMRA interaction.

Dalton Trans 2018 Feb;47(6):1841-1848

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

Thiolate coordination to zinc(ii) ions occurs widely in such functional biomolecules as zinc enzymes or zinc finger proteins. Here, we introduce a simple method for determining the affinity of ligands toward the zinc-enzyme active-center model tetramethylrhodamine (TAMRA)-labeled 1,4,7,10-tetraazacyclododecane (cyclen)-zinc(ii) complex (TAMRA-ZnL). The 1 : 1 complexation of TAMRA-labeled cysteine (TAMRA-Cys) with TAMRA-ZnL (each at 2.5 μM), in which the TAMRA moieties approach one another closely, induces remarkable changes in the visible absorption and fluorescence spectra at pH 7.4 and 25 °C. The 1 : 1 complex formation constant (K = [thiolate-bound zinc(ii) complex]/[uncomplexed TAMRA-ZnL][uncomplexed TAMRA-Cys], M) was determined to be 10 M from a Job's plot of the absorbances at 552 nm. By a ligand-competition method with the 1 : 1 complexation equilibrium, analogous K values for thiol-containing ligands, such as N-acetyl-l-cysteine, l-glutathione, and N-acetyl-l-cysteinamide, were evaluated to have similar values of about 10 M. As a result of the ligand affinities to TAMRA-ZnL, nonlabeled zinc(ii)-cyclen induced remarkable stabilization of the reduced form of l-glutathione and a cysteine-containing enolase peptide to aerial oxidation in aqueous solution at pH 7.4 and 25 °C.
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http://dx.doi.org/10.1039/c7dt04364cDOI Listing
February 2018

TAMRA/TAMRA Fluorescence Quenching Systems for the Activity Assay of Alkaline Phosphatase.

Sensors (Basel) 2017 Aug 15;17(8). Epub 2017 Aug 15.

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

We introduce two types of fluorescence-quenching assay for alkaline phosphatases (APs) by using a carboxytetramethyl-rhodamine (TAMRA)-labeled phosphate-binding tag molecule (TAMRA-Phos-tag). In the first assay, TAMRA-labeled -phosphorylethanolamine (TAMRA-PEA) was used as an artificial AP-substrate. TAMRA-Phos-tag specifically captured TAMRA-PEA to form a 1:1 complex at pH 7.4; the intensity of the fluorescence peak of the complex at 580 nm (λ = 523 nm) was significantly reduced to 32% of the average value for the two individual components as a result of the mutual approach of the TAMRA moieties. As TAMRA-PEA was dephosphorylated by AP, the resulting TAMRA-labeled ethanolamine dissociated and the fluorescence increased in a manner dependent on the AP dose and the time. In the second assay, pyrophosphate (PP), a natural AP-substrate, was used as a bridging ligand to form a dimeric TAMRA-Phos-tag complex. The dimerization reduced the fluorescence intensity to 49% of that in the absence of PP. As pyrophosphate was hydrolyzed to two orthophosphate moieties by AP, the 580-nm fluorescence recovered in a time-dependent manner. By examining the initial slope of this time-dependent fluorescence recovery, we succeeded in evaluating the 50% inhibitory concentrations of orthovanadate toward two AP isozymes under near-physiological conditions.
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http://dx.doi.org/10.3390/s17081877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579763PMC
August 2017

A Phos-tag-based micropipette-tip method for rapid and selective enrichment of phosphopeptides.

Electrophoresis 2017 10 10;38(19):2447-2455. Epub 2017 Jul 10.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Phosphorylated peptides are attractive targets in the study of the phosphoproteome. Here, we introduce a simple and convenient micropipette-tip method for the separation of phosphorylated and nonphosphorylated peptides by using a phosphate-binding zinc(II) complex of 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag). A 200-μL micropipette tip containing 10 μL of swollen agarose beads functionalized with Phos-tag moieties was prepared. All steps in the phosphate-affinity separation (binding, washing, and elution) were conducted by using aqueous buffers at neutral pH values. The entire separation protocol required less than 30 min per sample. By means of three independent separation experiments, followed by mass spectrometric (MS) analyses, we identified 1,649 non-redundant phosphopeptides from the lysates of human embryonic kidney cells (the peptides sample derived from 25 μg proteins per an MS analysis). The average ratio of identified phosphopeptides to total peptides in the respective experiments was >90%, showing a high selectivity. Furthermore, the high correlation between the triplicate analyses was confirmed by scatter plots based on the normalized abundance of each peptide, as calculated by a label-free peptide relative quantification analysis in Progenesis QI. This micropipette-tip method would be thus used preferentially as an alternative to existing tools for the reliable enrichment of phosphopeptides.
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http://dx.doi.org/10.1002/elps.201700175DOI Listing
October 2017

Zn(II)-Phos-Tag SDS-PAGE for Separation and Detection of a DNA Damage-Related Signaling Large Phosphoprotein.

Methods Mol Biol 2017 ;1599:113-126

Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8553, Japan.

In this chapter, we provide a standard protocol for phosphate-affinity sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Zn-Phos-tag SDS-PAGE). This technique uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system, Tris [tris(hydroxymethyl)aminomethane], and acetic acid (Tris-AcOH), to detect shifts in the mobility of phosphorylated ataxia telangiectasia-mutated (ATM) kinase. This protocol, which employs a 3% (w/v) polyacrylamide gel strengthened with 0.5% (w/v) agarose, permits the separation of larger phosphoproteins with molecular masses in the order of 200 kDa over a period of approximately 4 h. Subsequently, multiple phosphorylated forms of high-molecular-mass ATM kinase (350 kDa) can be clearly detected via immunoblotting as multiple upshifted migration bands on the Zn-Phos-tag SDS-PAGE gel. The procedure described in this protocol requires a completion time of approximately 5 h from the beginning of gel preparation to the end of electrophoresis.
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http://dx.doi.org/10.1007/978-1-4939-6955-5_9DOI Listing
February 2018

Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration.

Electrophoresis 2017 04 16;38(8):1139-1146. Epub 2017 Feb 16.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

We describe two unique proteins, Escherichia coli ClpX and human histone H2A, that show extremely retarded migrations relative to their molecular weights in Phos-tag SDS-PAGE, despite being nonphosphorylated. Although ClpX separated into multiple migration bands in Phos-tag gels, the separation was not due to phosphorylation. The N-terminal 47-61 region of ClpX was responsible for producing multiple phosphorylation-independent structural variants, even under denaturing conditions, and some of these variants were detected as highly up-shifted bands. By systematic Ala-scanning mutation analysis in the N-47-61 region, we concluded that the Glu-51 or Glu-54 residue was responsible for the appearance of exaggerated mobility-shifting bands. Histone H2A showed a much slower migration in Phos-tag gels in comparison with other major histones having similar molecular weights, and we found that the Glu-62 or Glu-65 residue caused the retarded migration. In addition, Phos-tag SDS-PAGE permitted us to detect a shift in the mobility of the phosphorylated form of histone H2A from that of the nonphosphorylated one. This is the first report showing that exaggerated retardation in the migration of a certain protein in Phos-tag SDS-PAGE is induced by interactions between the Phos-tag molecule and the carboxylate group of a specific Glu residue on the target.
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http://dx.doi.org/10.1002/elps.201600520DOI Listing
April 2017

A novel thiol-affinity micropipette tip method using zinc(II)-cyclen-attached agarose beads for enrichment of cysteine-containing molecules.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Sep 28;1031:195-201. Epub 2016 Jul 28.

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan. Electronic address:

Cysteine-containing biomolecules are attractive targets in the study of thiol biology. Here we introduce a novel method for the selective enrichment of thiol-containing molecules using a thiol-capture zinc(II) complex of 1,4,7,10-tetraazacyclododecane (Zn(2+)-cyclen). Recognition of N-acetylcysteine amide by Zn(2+)-cyclen has been studied by potentiometric pH titration, revealing formation of a 1:1 thiolate-bound Zn(2+)-cyclen complex with a large thiolate-affinity constant of 10(6.2)M(-1) at 25°C and I=0.10M (NaCl). The Zn(2+)-bound thiolate anion is unexpectedly stable in aqueous solution at pH 7.8 under atmospheric conditions for a few days. These findings have contributed to the development of a convenient method for separation of thiol compounds by using a micropipette tip. A 200μL micropipette tip containing 10μL of hydrophilic cross-linked agarose beads attached to Zn(2+)-cyclen moieties was prepared. All steps for thiol-affinity separation (binding, washing, and eluting) are conducted using aqueous buffers at room temperature. The entire separation protocol requires less than 15min per sample. We demonstrate practical example separations of cysteine-containing molecules. This micropipette tip method would be used preferentially as an alternative to existing tools for reliable enrichment of thiol-containing molecules.
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http://dx.doi.org/10.1016/j.jchromb.2016.07.049DOI Listing
September 2016

A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1.

Proteomics 2016 07 8;16(13):1825-36. Epub 2016 Jun 8.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Japan.

MEK1, an essential component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor-specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK-733, PD98059, and U0126.
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http://dx.doi.org/10.1002/pmic.201500494DOI Listing
July 2016

Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and Phos-tag SDS-PAGE.

MethodsX 2015 19;2:469-74. Epub 2015 Nov 19.

Laboratory of Molecular Biology, Science Research Center, Kochi Medical School, Kochi 783-8505, Japan.

Protein kinase expression and activity play important roles in diverse cellular functions through regulation of phosphorylation signaling. The most commonly used tools for detecting the protein kinase are protein kinase-specific antibodies, and phosphorylation site-specific antibodies were used for detecting activated protein kinase. Using these antibodies, only one kinase was analyzed at a time, however, a method for analyzing the expression and activation of a panel of protein kinases in cells is not established. Therefore, we developed a combined method using Multi-PK antibody and Phos-tag SDS-PAGE for profiling the expression and phosphorylation state of intracellular protein kinases. Using the new method, changes in the expression and phosphorylation state of various protein kinases were detected in cells treated with anticancer agent which inhibit multiple tyrosine kinase activities. Therefore, the new method is a useful technique for analysis of intracellular protein kinases.•Multi-PK antibody recognizes a wide variety of protein kinases in various species.•Using Phos-tag SDS-PAGE, phosphorylated proteins are visualized as slower migration bands compared with corresponding non-phosphorylated proteins.•This combined method can be used for detecting changes in the expression and phosphorylation state of various intracellular protein kinases.
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http://dx.doi.org/10.1016/j.mex.2015.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4703585PMC
February 2016

Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE.

PLoS One 2016 1;11(2):e0148294. Epub 2016 Feb 1.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148294PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734776PMC
July 2016

Phosphopeptide Detection with Biotin-Labeled Phos-tag.

Methods Mol Biol 2016 ;1355:17-29

Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.

Protein kinases are widely considered to be invaluable target enzymes for drug discovery and for diagnosing diseases and assessing their prognosis. Effective analytical techniques for measuring the activities of cellular protein kinases are therefore required for studies in the field of phosphoproteomics. We have recently developed a highly sensitive microarray-based technique for tracing the activities of protein kinases. A series of peptides that are specific substrates of various protein kinases are immobilized on a glass slide and subjected to phosphorylation by cell lysates. The resulting phosphorylated forms of the various peptides are then selectively and simultaneously detected by using a phosphate-binding tag molecule, biotin-labeled Phos-tag, bound to horseradish peroxidase-conjugated streptavidin. Enhanced chemiluminescence signals can then be readily detected by using an automatic image analyzer. In this chapter, we describe a standard protocol for detecting phosphopeptides by biotin-labeled Phos-tag. We also describe a microarray system for high-throughput profiling of intracellular protein kinase activities. The Phos-tag-based method is expected to be useful in the rapid detection of the complex range of phosphorylation reactions involved in cellular signaling events, and it has potential applications in high-throughput screening of kinase activators or inhibitors.
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http://dx.doi.org/10.1007/978-1-4939-3049-4_2DOI Listing
August 2016

Functional Characterization of the Receiver Domain for Phosphorelay Control in Hybrid Sensor Kinases.

PLoS One 2015 7;10(7):e0132598. Epub 2015 Jul 7.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Hybrid sensor kinase, which contains a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, conveys signals to its cognate response regulator by means of a His-Asp-His-Asp phosphorelay. We examined the multistep phosphorelay of a recombinant EvgAS system in Escherichia coli and performed in vitro quantitative analyses of phosphorylation by using Phos-tag SDS-PAGE. Replacement of Asp in the receiver domain of EvgS by Ala markedly promoted phosphorylation at His in the HK domain compared with that in wild-type EvgS. Similar Ala-substituted mutants of other hybrid sensor kinases BarA and ArcB showed similar characteristics. In the presence of sufficient ATP, autophosphorylation of the HK domain in the mutant progressed efficiently with nearly pseudo-first-order kinetics until the phosphorylation ratio reached a plateau value of more than 95% within 60 min, and the value was maintained until 180 min. However, both wild-type EvgS and the Ala-substituted mutant of His in the HPt domain showed a phosphorylation ratio of less than 25%, which gradually decreased after 10 min. These results showed that the phosphorylation level is regulated negatively by the receiver domain. Furthermore, our in vivo assays confirmed the existence of a similar hyperphosphorylation reaction in the HK domain of the EvgS mutant in which the Asp residue was replaced with Ala, confirming the validity of the control mechanism proposed from profiling of phosphorylation in vitro [corrected].
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132598PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494823PMC
April 2016

Neutral Phosphate-Affinity SDS-PAGE system for profiling of protein phosphorylation.

Methods Mol Biol 2015 ;1295:323-54

Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.

In this chapter, we describe a standard protocol for phosphate-affinity SDS-PAGE that uses a dizinc(II) complex of the phosphate-binding molecule Phos-tag in conjunction with a neutral-pH gel system (Zn(2+-)Phos-tag SDS-PAGE) to detect shifts in the mobilities of phosphoproteins. A previous protocol for affinity electrophoresis that uses polyacrylamide-bound Mn(2+)-Phos-tag and Laemmli's buffer system under conditions of alkaline pH has limitations in separating certain phosphoproteins. The current protocol provides major improvements in separation and detection of various phosphorylated protein species. We here introduce two neutral-pH gel systems buffered with Bis-Tris-HCl and Tris-AcOH, respectively, for Zn(2+)-Phos-tag SDS-PAGE, and we also discuss their characteristics on the basis of comparative studies on phosphorylation profiling of proteins with a wide range of molecular masses. Each analytical procedure, from the beginning of gel preparation to the end of electrophoresis, requires 2.5-5 h with either buffer system.
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http://dx.doi.org/10.1007/978-1-4939-2550-6_24DOI Listing
December 2015

The Cutting Edge of Affinity Electrophoresis Technology.

Proteomes 2015 Mar 18;3(1):42-55. Epub 2015 Mar 18.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan.

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years.
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http://dx.doi.org/10.3390/proteomes3010042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302491PMC
March 2015

Advances in Phos-tag-based methodologies for separation and detection of the phosphoproteome.

Biochim Biophys Acta 2015 Jun 12;1854(6):601-8. Epub 2014 Oct 12.

Department of Functional Molecular Science, Institute of Biomedical & Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan.

This review article describes analytical techniques based on the phosphate-binding tag molecule "Phos-tag", which is an alkoxide-bridged dinuclear metal complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate, for studying the protein phosphorylome. The dinuclear zinc(II) complex forms a stable 1:1 complex with a phosphate monoester dianion in an aqueous solution under conditions of neutral pH. By using a series of functional Phos-tag derivatives, our group has developed novel techniques that are useful in studies on kinomics and phosphoproteomics. Among the derivatives, a series of biotinylated Phos-tag derivatives have been used as molecular tools in applications such as Western blotting for comprehensive detection of phosphorylated proteins and in highly sensitive peptide microarray-based techniques for the detection of kinase activities in biological samples. The review also gives an outline of phosphate affinity electrophoresis, in which immobilized Phos-tag molecules in a general polyacrylamide gel are used to separate proteins and detect differences in their phosphorylation status. This technique permits quantitative analyses of multiple phosphorylation statuses of individual cellular proteins and their time-dependent changes. Conventional mass spectrometry-based shotgun techniques used in phosphoproteomics detect the phosphorylation modification of proteins in peptide fragments, whereas the Phos-tag electrophoresis technique permits the direct analysis of the phosphorylation status of full-length proteins. The technique therefore provides a greater understanding of the detailed properties of particular proteins involved in specific physiological and pathological events. This article is part of a Special Issue entitled: Medical Proteomics.
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http://dx.doi.org/10.1016/j.bbapap.2014.10.004DOI Listing
June 2015

Tips on improving the efficiency of electrotransfer of target proteins from Phos-tag SDS-PAGE gel.

Proteomics 2014 Nov 18;14(21-22):2437-42. Epub 2014 Oct 18.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

The sensitivity of Western blotting analysis after Phos-tag SDS-PAGE is occasionally inferior to that after normal (Phos-tag-free) SDS-PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos-tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet-tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos-tag SDS-PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos-tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet-tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos-tag gels were similar to those from normal gels. In some cases involving the use of a Phos-tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.
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http://dx.doi.org/10.1002/pmic.201400380DOI Listing
November 2014

Simple enrichment of thiol-containing biomolecules by using zinc(II)-cyclen-functionalized magnetic beads.

J Sep Sci 2014 Jul 11;37(13):1601-9. Epub 2014 May 11.

Laboratory of Organic Medicinal Chemistry, Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University, Fukuyama, Japan.

A simple and efficient method based on magnetic-bead technology has been developed for the enrichment of thiol-containing biomolecules, such as l-glutathione and cysteine-containing peptides. The thiol-binding site on the bead is a mononuclear complex of zinc(II) with 1,4,7,10-tetraazacyclododecane (cyclen); this is linked to a hydrophilic cross-linked agarose coating on a particle that has a magnetic core. All steps for the thiol-affinity separation are conducted in aqueous buffers with 0.10 mL of the magnetic beads in a 1.5 mL microtube. The entire separation protocol for thiol-containing compounds, from addition to elution, requires less than one hour per sample, provided the buffers and the zinc(II)-cyclen-functionalized magnetic beads have been prepared in advance. The thiol-affinity magnetic beads are reusable at least 15 times without a decrease in their thiol-binding ability, and they are stable for six months at room temperature.
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http://dx.doi.org/10.1002/jssc.201400135DOI Listing
July 2014

Profiling of protein thiophosphorylation by Phos-tag affinity electrophoresis: evaluation of adenosine 5'-O-(3-thiotriphosphate) as a phosphoryl donor in protein kinase reactions.

Proteomics 2014 Mar 18;14(6):668-79. Epub 2014 Feb 18.

Department of Functional Molecular Science, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been widely used as a phosphoryl donor to trace protein kinase activities. However, the question remains whether particular kinases accept ATPγS as readily as they accept natural ATP. We investigated the characteristics of several kinase reactions in the presence of ATPγS by using Phos-tag affinity electrophoresis. The Phos-tag gel permitted quantitative analysis of thiophosphorylated proteins produced by kinase reactions in vitro and it identified differences in the efficiencies of utilization of ATPγS and ATP in these reactions. Using the method, we evaluated the utility of ATPγS as a phosphoryl donor in studies on bacterial two-component systems. Histidine kinases accepted ATPγS as readily as they accepted ATP in autophosphorylation reactions. However, downstream phosphotransfer reactions with ATPγS were markedly slower than the corresponding reactions with ATP. In an analysis of the sluggish thiophosphate transfer, we found that detergent-denatured thiophosphorylated histidine kinases gradually hydrolyzed at the P-N bond, even at neutral pH, during incubation for 24 h, whereas the native form of the thiophosphorylated enzymes were much more stable. Profiling of protein thiophosphorylation by using Phos-tag affinity electrophoresis might provide new insights into the characteristics of various types of kinase reactions with ATPγS.
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http://dx.doi.org/10.1002/pmic.201300533DOI Listing
March 2014
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