Tohru Koike

Phosphate-binding Tag, a New Tool to Visualize Phosphorylated Proteins

We introduce two methods for the visualization of phosphorylated proteins using alkoxide-bridged dinuclear metal (i.e. Zn2+ or Mn2+) complexes as novel phosphate-binding tag (Phos-tag) molecules. Both Zn2+- and Mn2+-Phos-tag molecules preferentially capture phosphomonoester dianions bound to Ser, Thr, and Tyr residues. One method is based on an ECL system using biotin-pendant Zn2+-Phos-tag and horseradish peroxidase-conjugated streptavidin. We demonstrate the electroblotting analyses of protein phosphorylation status by the phosphate-selective ECL signals. Another method is based on the mobility shift of phosphorylated proteins in SDS-PAGE with polyacrylamide-bound Mn2+-Phos-tag. Phosphorylated proteins in the gel are visualized as slower migration bands compared with corresponding dephosphorylated proteins. We demonstrate the kinase and phosphatase assays by phosphate affinity electrophoresis (Mn2+-Phos-tag SDS-PAGE).

Recent development of zinc-fluorophores

Zinc-fluorophores have recently been attracting much interest in biological and environmental applications. How to detect selectively trace Zn2+ with efficient signal transduction is the central problem. Following carbonic anhydrase-based biosensors with fluorescent aromatic sulfonamides, a chemosensor, Zinquin, is now extensively used to study the role of intracellular Zn2+ in cellular biology. New types of zinc-fluorophores, zinc-finger peptides attached with fluorescent dyes and a dansylamide-pendant macrocyclic polyamine, have been developed in 1996. The principles, properties and limitations of these are discussed.

Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE.

We provide a standard phosphate-affinity SDS-PAGE (Mn2+–Phos-tag SDS-PAGE) protocol, in which Phos-tag is used to analyze large phosphoproteins with molecular masses of more than 200 kDa. A previous protocol required a long electrophoresis time of 12 h for separation of phosphoisotypes of large proteins (∼150 kDa). This protocol, which uses a 3% (wt/vol) polyacrylamide gel strengthened with 0.5% (wt/vol) agarose, permits the separation of protein phosphoisotypes larger than 200 kDa within 2 h. In subsequent immunoblotting, phosphoisotypes of high-molecular-mass proteins, such as mammalian target of rapamycin (289 kDa), ataxia telangiectasia-mutated kinase (350 kDa) and p53-binding protein 1 (213 kDa), can be clearly detected as up-shifted migration bands on the improved Mn2+–Phos-tag SDS-PAGE gel. The procedure from the beginning of gel preparation to the end of electrophoresis requires about 4 h in this protocol.

FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway

In response to DNA damage or replication fork stress, the Fanconi anemia pathway is activated, leading to monoubiquitination of FANCD2 and FANCI and their colocalization in foci. Here we show that, in the chicken DT40 cell system, multiple alanine-substitution mutations in six conserved and clustered Ser/Thr-Gln motifs of FANCI largely abrogate monoubiquitination and focus formation of both FANCI and FANCD2, resulting in loss of DNA repair function. Conversely, FANCI carrying phosphomimic mutations on the same six residues induces constitutive monoubiquitination and focus formation of FANCI and FANCD2, and protects against cell killing and chromosome breakage by DNA interstrand cross-linking agents. We propose that the multiple phosphorylation of FANCI serves as a molecular switch in activation of the Fanconi anemia pathway. Mutational analysis of putative phosphorylation sites in human FANCI indicates that this switch is evolutionarily conserved.

A macrocyclic zinc(II) fluorophore as a detector of apoptosis

Our originally designed dansylamidoethylcyclen 4 as a biomimetic Zn2+-selective fluorophore has been demonstrated to be a good detector of the apoptosis (induced by an anticancer agent, etoposide, and H2O2) in cancer cells such as HeLa and HL60 cells. The macrocyclic Zn2+ ligand 4 (mostly as a deprotonated form) is cell-permeable to show weak fluorescence (emission at 550 nm), which forms a strong fluorescent 1:1 Zn2+ complex 5 (emission at 530 nm) when Zn2+ is incorporated into the cells by a zinc(II) ionophore pyrithione. Thus formed, Zn2+ complex 5 is cell-impermeable and remains intact over a few hours. When apoptosis in HeLa or HL60 cells is artificially induced, 4 selectively and strongly stains apoptotic cells only at early stages, which was verified by using the conventional apoptosis detection probe annexin V-Cy3. Detection of the apoptotic cells by 4 was perhaps due to significantly increased free Zn2+ flux at early stages of apoptosis. Apoptotic detection by 4 has been compared with a presently available Zn2+ fluorophore, Zinquin 1. We present that 4 has advantages in detection of apoptosis over annexin V-Cy3 and Zinquin 1.

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Herein we describe three applications of label-free kinase profiling using a novel type of phosphate affinity polyacrylamide gel electrophoresis. The phosphate affinity site is a polyacrylamide-bound dinuclear Mn2+ complex that enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterpart. The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in phosphorylation status. The activity profiles of six kinds of kinases, glycogen synthase kinase-3β, cyclin-dependent kinase 5/p35, protein kinase A, mitogen-activated protein kinase (MAPK), casein kinase II, and calmodulin-dependent protein kinase II, were determined using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase demonstrated characteristic multiple electrophoresis migration bands up-shifted from the nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes in the epidermal growth factor-induced phosphorylation levels of Shc and MAPK in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK antibodies. The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. The inhibition profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was determined using the substrate Abltide-GST (a fusion protein consisting of a specific substrate peptide for Abl and glutathione S-transferase) and the approved drug Glivec (an ATP competitor). In the kinase assay, the slower migration band, monophosphorylated Abltide-GST, increased time-dependently, whereas the faster migration band, nonphosphorylated Abltide-GST, decreased. The dose-dependent inhibition of Glivec was determined by a change in the ratio of the faster and slower migration bands, which showed an IC50 value of 1.6 μm in the presence of 0.10 mm ATP.

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex (Zn2L3+) has been studied (L = alkoxide species of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol). Potentiometric pH titration study disclosed a 1 : 1 phenyl phosphate complexation with Zn2L3+ in aqueous solution. The dissociation constant (= [Zn2L3+][PhOPO3(2-)]/[Zn2L3+-PhOPO3(2-)]) is an extremely small value of 2.5 x 10(-8) mol dm(-3) at 25 degrees C with I = 0.10 (NaNO3). The X-ray crystal analysis of the dizinc(II) complex with p-nitrophenyl phosphate showed that the phosphate dianion binds as a bridging ligand to the two zinc(II) ions.

Shock-tube and modeling study of acetylene pyrolysis and oxidation

Abstract Pyrolysis and oxidation of acetylene were studied behind reflected shock waves in the temperature range 1100–2000 K at pressures of 1.1–2.6 atm analyzing the reacted gas mixtures. The progress of the oxidation reaction was also observed with real-time spectroscopy. Main products in the pyrolysis with H 2 or without H 2 were vinylacetylene, diacetylene, and triacetylene. In the oxidation with fuel-rich mixtures, it was found that CO was the main product, and C 1 and C 3 carbon species appeared. The pyrolysis and oxidation of acetylene were modeled using a kinetic reaction mechanism, including the most recent mechanism for formaldehyde and ketene oxidations. The present and earlier shock tube data were reproduced by a proposed mechanism with 103 reaction steps and 38 species. It was found that reactions C 2 H 2 + O 2 → CHO + CHO or C 2 H 2 + O 2 → CHCO + OH and C 2 H 2 + CH 2 → C 3 H 4 were important to predict our data in a wide range of mixtures from acetylene pyrolysis to acetylene-lean oxidation.

Intrinsic Properties of Zinc(II) Ion Pertinent To Zinc Enzymes

Publisher Summary This chapter discusses intrinsic properties of zinc(II) ion pertinent to zinc enzymes. Zinc(II) ion is a biologically essential element. The crystal structure of serine/threonine phosphatase-l revealed that it contains two metal ions M(1) and M(2) close to each other. Each of the metal ions is coordinated by five ligands. The distance between the two metal ions is 3.3 A, which is facilitated by Asp 92 and a water molecule (or OH - ion); each bridges the two ions. Both metal ions and two arginine residues together create the active site. Design of useful models is, therefore, a great help in elucidating the role of metals, functions of amino acid residues, and principles of multimetal functions. The metal ions, by stabilizing negative charges, can make a phosphomonoester more susceptible to nucleophilic attack, probably by the metal-bound H 2 O.

Separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE.

Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate‐affinity SDS‐PAGE. The phosphate‐affinity site is a polyacrylamide‐bound Phos‐tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c‐Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr‐394, Tyr‐197, or Tyr‐18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two‐component system. FixL is autophosphorylated at the His‐285 with ATP, and the phosphate group is transferred to the Asp‐54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well‐known isotype phosphorylated at the Asp‐54. The other is a novel isotype phosphorylated at the His‐84.