Analytical Biochemistry 360 (2007) 160–162

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ANALYTICALBIOCHEMISTRY

Notes & Tips

Separation of a phosphorylated histidine protein using phosphate aYnity polyacrylamide gel electrophoresis

Seiji Yamada a,¤, Hiro Nakamura a,b, Eiji Kinoshita c, Emiko Kinoshita-Kikuta c, Tohru Koike c, Yoshitsugu Shiro a

a Biometal Science Laboratory, RIKEN SPring-8 Center, Harima Institute, Kouto 1-1-1, Sayo, Hyogo 679-5148, Japanb Yokohama City University International Graduate School of Arts and Sciences, Tsurumi, Yokohama, Kanagawa 230-0045, Japan

c Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan

Received 19 September 2006Available online 25 October 2006

It is important to visualize phosphorylated proteins inevaluating diverse signal transductions in biological systems.Kinoshita and coworkers recently reported the separation ofphosphorylated proteins from nonphosphorylated proteinsusing a novel phosphate aYnity sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)1 technique[1]. The aYnity gel copolymerized with a phosphate-bindingtag molecule, Phos-tag, a dinuclear Mn2+ complex with anacrylamide-pendant Phos-tag ligand (commercially availableat www.phos-tag.com). The principle of this method is as fol-lows: phosphorylated proteins move slower in SDS–PAGEthan do the corresponding nonphosphorylated moleculesbecause the phospho-group interacts with the Mn2+ Phos-tagligand in the gel.

This novel technique was used successfully to detect someproteins containing phosphorylated Ser, Thr, and Tyr resi-dues (O-phosphates) [1]. In addition, because the standardfree energy of the N–P bond of N-phosphorylated His islarge (¡42 kJ/mol), the phosphorylated His has the potentialto serve as an intermediate in phosphotransfer reactions toother amino acids [2]. Therefore, in addition to Ser/Thr andTyr kinases, His kinases (HKs) play a crucial role as a sensorapparatus in signal transduction, the two-component regula-

* Corresponding author. Fax: +81 791 58 2818.E-mail address: seijiy@spring8.or.jp (S. Yamada).

1 Abbreviations used: SDS–PAGE, sodium dodecyl sulfate–polyacryl-amide gel electrophoresis; acrylamide-pendant Phos-tag ligand, N-(5-(2-acryloylaminoethylcarbamoyl)pyridin-2-ylmethyl)-N,N�,N�-tris(pyridine-2-yl-methyl)1,3-diaminopropan-2-ol; Mn2+ Phos-tag, dinuclear Mn2+

complex with Phos-tag ligand; HK, histidine kinase; DTT, dithiothreitol;BPB, bromophenol blue; EDTA, ethylenediaminetetraacetic acid; ThkA,T. maritima HK-TM1359 (PIR ID: H72262); CBB, Coomassie brilliantblue.

0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2006.10.005

tory system, in prokaryotes, fungi, and plants [3]. In responseto intra- and extracellular signals (e.g., nutrients, light, oxy-gen), HK promotes autophosphorylation at a conserved Hisresidue using ATP, and the phosphoryl group subsequentlyis transferred to a unique Asp residue in a response regulatorprotein. The detection of phosphorylated HK is very impor-tant in terms of measuring the signal response by the two-component regulatory system. Here, we Wrst demonstrate thevisualization of a phosphorylated His protein in the HKreaction using Mn2+ Phos-tag SDS–PAGE.

A recombinant His kinase �ThkA, a deletion mutant(residue 408–755) of HK-TM1359 (PIR ID: H72262)from the hyperthermophile Thermotoga maritima, waspuriWed according to a method described previously [4].The reaction premixture (total volume 0.20 ml) for theautophosphorylation assay was composed of 50 mMTris–HCl (pH 7.5), 50 mM KCl, 20 �M MgCl2, and5.6 �M �ThkA. The reaction was initiated by the addi-tion of 1/9 volume of 2.0 mM ATP at room temperature.After 0.5, 1, 2, 4, or 6 min, 6.0 �l of the reaction mixturewas mixed with 10 �l of a stop solution composed of0.21 M Tris–HCl (pH 6.8), 13% (w/v) SDS, 32% (v/v) glyc-erol, 0.33 M dithiothreitol (DTT), 0.05% (w/v) bromophe-nol blue (BPB), and 1.0 mM ethylenediaminetetraaceticacid (EDTA) at 70 °C.

SDS–PAGE was performed using a 1-mm thick, 8.5-cmwide, 9.5-cm long gel at 120 V and room temperatureaccording to a method described previously [1]. The gelconsisted of 1.8 ml of a stacking gel (3.0% [w/v] acrylamide,0.12 M Tris–HCl [pH 6.8], and 0.20 % [w/v] SDS) and 6.3 mlof a separating gel copolymerized with Phos-tag (7.5% [w/v]acrylamide, 0.37 M Tris–HCl [pH 8.8], 0.20% [w/v] SDS,0.10 mM acrylamide-pendant Phos-tag, and 0.20 mM

Notes & Tips / Anal. Biochem. 360 (2007) 160–162 161

MnCl2). The electrophoresis running buVer consisted of25 mM Tris, 192 mM glycine, and 0.10% (w/v) SDS. To cali-brate the amount of protein, 8, 16, 24, 32, and 40 pmol of�ThkA, which had been treated with the stop solution inthe same manner as shown above, were loaded onto the gel(calibration lanes in Fig. 1A). Each of the assayed samples(16 �l, total 30 pmol of �ThkA) was loaded onto the samegel (assay lanes in Fig. 1A). After the electrophoresis, thegels were soaked in a bleaching solution (30% [v/v] metha-nol and 10% [v/v] acetic acid) for 10 min. The gels werestained in a Coomassie brilliant blue (CBB) solution pre-pared by adding one tablet of PhastGel Blue R (GEHealthcare) to 400 ml of bleaching solution for 30 min andthen washing with the bleaching solution until the back-ground of the gel was completely decolored. The gel imagewas taken as a TIFF Wle with a common scanner for per-sonal computer (CanoScan 8000F, Canon). The intensity ofthe protein bands in the gel was measured with the one-dimensional gel analyze tool in the Image J program (freeshareware available at http://rsb.info.nih.gov/ij). An autora-diography assay was performed with the same conditionsfor kinase reaction and gel electrophoresis as mentionedabove except using [�-32P]-ATP (0.09 MBq/nmol). Theradioactivity of the protein bands was measured using aphosphorimaging plate scanner (BAS2500, Fuji Film).

As shown in the calibration lanes of Fig. 1A, the ATP-untreated (nonphosphorylated) �ThkA showed a single

band, whereas the ATP-treated �ThkA gave two migrationbands in the assay lanes. The lower bands appeared at thesame position (relative mobility, RfD0.34) as that for thenonphosphorylated HK, where RfD1 is the mobility of BPB.The upper bands at RfD0.29 in the assay lanes are assignedto phosphorylated �ThkA because the position is consistentwith that observed by 32P autoradiography (Fig. 1A). ThediVerence in mobility between the upper and lower bandsshows that the phosphorylated �ThkA was preferentiallycaptured by the Mn2+ Phos-tag in the gel. This behavior ofthe phosphorylated HK is comparable to that of phosphory-lated proteins containing phospho-Ser, phospho-Thr, andphospho-Tyr residues in a previous report [1].

The gel image of the calibration lanes in Fig. 1A gave a lin-ear relationship between the band intensity and the amount of�ThkA (Fig. 1B). The amount of the phosphorylated andnonphosphorylated proteins could then be estimated on thebasis of the upper and lower bands in the assay lanes ofFig. 1A and were plotted against reaction time in Fig. 1C. Theamount of phosphorylated �ThkA produced increased time-dependently, consistent with the result from 32P radioisotopemethod, whereas the total amount of nonphosphorylated andphosphorylated �ThkA in each lane was nearly constant.This technique can be quantitatively useful in assays of HKreactions in the two-component regulatory system.

In summary, Mn2+ Phos-tag can be useful for capturingphosphorylated His (N-phosphate), as well as O-phosphates,

Fig. 1. Mn2+ Phos-tag SDS–PAGE of �ThkA. (A) Autophosphorylation assay of �ThkA in a polyacrylamide gel (7.5% [w/v] acrylamide and 100 �MMn2+ Phos-tag). The nonphosphorylated �ThkA (calibration lanes: 8, 16, 24, 32, and 40 pmol) and ATP-treated �ThkA (assay lanes: total 30 pmol) wereloaded onto the same gel, as shown in the left panel. Autoradiography under the same conditions using 32P is shown in the right panel. Open and closedarrowheads indicate nonphosphorylated and phosphorylated �ThkA, respectively. (B) Linear relationship between band intensity (in AU) and theamount of �ThkA (in pmol). Each plot is the mean value of triplicate experiments. The solid line is the linear Wtting of the data. (C) Autophosphorylationactivity of �ThkA. Open circles with solid line, phosphorylated �ThkA; squares with dotted line, nonphosphorylated �ThkA; triangles, sum of nonphos-phorylated and phosphorylated �ThkA; closed circles with dashed line, phosphorylated �ThkA determined using 32P. Each symbol is the mean value oftriplicate experiments.

162 Notes & Tips / Anal. Biochem. 360 (2007) 160–162

in protein samples. We believe that phosphoproteomics onHK would progress greatly by the eVective and quantitativeseparation of phosphorylated HK using Mn2+ Phos-tagSDS–PAGE.

Acknowledgments

This work was supported, in part, by the Special Post-doctoral Researchers Program in RIKEN (to S.Y.), aGrant-in-Aid for ScientiWc Research (B) (15390013) fromJSPS (to E.K. and T.K.), a Grant-in-Aid for Young Scien-tists (B) (17790034) from MEXT (to E.K.), and a Grant-in-Aid for Young Scientists (B) (18790120) from MEXT(to E.K.-K.).

References

[1] E. Kinoshita, E. Kinoshita-Kikuta, K. Takiyama, T. Koike, Phosphate-binding tag: A new tool to visualize phosphorylated proteins, Mol.Cell. Proteomics 5 (2006) 749–757.

[2] Y.A. Kosinsky, P.E. Volynsky, P. Lagant, G. Vergoten, E. Suzuki, A.S.Arseniev, R.G. Efremov, Development of the force Weld parameters forphosphoimidazole and phosphohistidine, J. Comput. Chem. 25 (2004)1313–1321.

[3] A.H. West, A.M. Stock, Histidine kinases and response regulator pro-teins in two-component signaling systems, Trends Biochem. Sci. 26(2001) 369–376.

[4] S. Yamada, S. Akiyama, H. Sugimoto, H. Kumita, K. Ito, T. Fujisawa,H. Nakamura, Y. Shiro, The signaling pathway in histidine kinase andthe response regulator complex revealed by X-ray crystallography andsolution scattering, J. Mol. Biol. 362 (2006) 123–139.