Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

Accepted Manuscript Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay Yasunori Sugiyama, Sho Yamashita, Yuuki Uezato, Yukako Senga, Syouichi Katayama, Naoki Goshima, Yasushi Shigeri, Noriyuki Sueyoshi, Isamu Kameshita PII:

S0003-2697(16)30258-5

DOI:

10.1016/j.ab.2016.08.020

Reference:

YABIO 12485

To appear in:

Analytical Biochemistry

Received Date: 21 June 2016 Revised Date:

18 August 2016

Accepted Date: 22 August 2016

Please cite this article as: Y. Sugiyama, S. Yamashita, Y. Uezato, Y. Senga, S. Katayama, N. Goshima, Y. Shigeri, N. Sueyoshi, I. Kameshita, Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.08.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Analytical Biochemistry

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Enzymatic assays and analyses

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

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Short title: P-TandeMBP for PPase assay

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Yasunori Sugiyamaa,*, Sho Yamashitaa, Yuuki Uezatoa, Yukako Sengaa,b, Syouichi Katayamaa, Naoki Goshimac, Yasushi Shigerid, Noriyuki Sueyoshia and Isamu Kameshitaa

a

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa 761-0795,

b

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Japan

Biomedical Research Institute, National Institute of Advanced Industrial Science and

Technology (AIST), Ibaraki 305-8566, Japan Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced

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Industrial Science and Technology (AIST), Tokyo 135-0064, Japan Health Research Institute, National Institute of Advanced Industrial Science and Technology

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(AIST), Osaka 563-8577, Japan

*Corresponding author Department of Life Sciences, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kagawa 761-0795, Japan Tel.: +81-87-891-3116

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Fax: +81-87-891-3021 E-mail address: [email protected] (Y. Sugiyama)

Abbreviations used: CaMKIδ, Ca2+/calmodulin-dependent protein kinase Iδ; CK1δ, casein

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kinase 1δ; DCLK, doublecortin-like protein kinase; DYRK1A, dual-specificity tyrosine phosphorylation-regulated kinase 1A; ERK2, extracellular signal-regulated kinase 2; IPTG,

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isopropyl β-D-thiogalactoside; λPPase, lambda protein phosphatase; MBP, myelin basic protein; MEK, mitogen-activated protein kinase kinase; MUP, 4-methylumbelliferyl

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phosphate; Pi, orthophosphoric acid; PKA, cAMP-dependent protein kinase; P-TandeMBP,

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phosphorylated TandeMBP; TCA, trichloroacetic acid.

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Abstract To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in

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tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture

(Ca2+/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular

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signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein

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kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G,

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PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the

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detection of protein phosphatase activities.

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Keywords: phosphatase assay; protein phosphatase; protein substrate; myelin basic protein; malachite green assay.

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Various biological phenomena, such as proliferation, development, differentiation, and apoptosis are regulated by protein phosphorylation [1]. Phosphorylation regulates the function, localization, and binding specificity of target proteins [2]. Intracellular phosphorylation

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signaling is constructed on the basis of the balance between phosphorylation by protein kinases and dephosphorylation by protein phosphatases. Many tools to detect a wide variety of protein kinase activities have been developed, whereas only a few tools exist to detect various

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protein phosphatase activities. Therefore, there is a demand for new tools to analyze the activities of protein phosphatases.

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Although chromogenic substrates, such as p-nitrophenyl phosphate [3, 4], fluorogenic substrates, such as 6,8-difluoro-4-methylumbelliferyl phosphate and 4-methylumbelliferyl phosphate (MUP) [5, 6], and phosphopeptide substrates [7] have been developed to detect protein phosphatase activities, these compounds are not protein substrates. However,

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preexisting phosphorylated protein substrates [8] have often been used for detecting protein phosphatase activities, but their use is limited owing to the specificity of protein phosphatases. Recently, we developed TandeMBP, in which two different mouse myelin basic protein

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(MBP) isoforms were fused in tandem as a substrate for in vitro assays of various protein kinases [9]. TandeMBP was phosphorylated more efficiently by various Ser/Thr protein

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kinases, such as casein kinase 1δ (CK1δ), doublecortin-like protein kinase (DCLK), Ca2+/calmodulin-dependent protein kinase Iδ (CaMKIδ), dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), extracellular signal-regulated kinase 2 (ERK2), cAMP-dependent protein kinase (PKA) and PKL01 than other MBP isoforms [9]. Therefore, we hypothesized that phosphorylated TandeMBP (P-TandeMBP) would be a unique substrate for a wide variety of protein phosphatases. In this study, we established a method to prepare P-TandeMBP efficiently and confirmed

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that P-TandeMBP was a better substrate for various protein phosphatases than a chemical compound and a peptide substrate.

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Materials and methods

Materials

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MUP and goat anti-mouse IgG, conjugated with horseradish peroxidase, were purchased from Sigma-Aldrich. [γ-32P]ATP (111 TBq/mmol) was obtained from PerkinElmer. An

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anti-His6 antibody was obtained from Wako Chemicals. A phosphopeptide substrate pp10, YGGMHRQET(p)VDC, and Erk2(179–189) peptide, TGFLT(p)EY(p)VATR, were synthesized and purified as described previously [7]. TandeMBP was prepared as described previously [9]. The constitutively active form of CaMKIδ was expressed and purified as

[11, 12].

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Protein kinase assay

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described previously [10]. Purified CK1δ and ERK2 were prepared as described previously

Phosphorylation of TandeMBP by various protein kinases was carried out essentially as

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described previously [9]. Phosphorylation of TandeMBP (200 ng) by CaMKIδ, CK1δ, ERK2 (20 ng), or a protein kinase mixture (CaMKIδ, CK1δ, and ERK2; 20 ng each) was carried out in a standard reaction mixture (10 µl) composed of 40 mM HEPES-NaOH (pH 8.0), 1 mM dithiothreitol, 0.1 mM EGTA, 5 mM Mg(CH3COO)2, and 100 µM [γ-32P]ATP. The reactions were started by the addition of the kinases, incubated at 30 °C for 30 min and stopped by the addition of 10 µl of 2× SDS-PAGE sample buffer. Phosphorylated proteins were subjected to SDS-PAGE and visualized by autoradiography. ERK2 was pre-activated by phosphorylation

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with mitogen-activated protein kinase kinase (MEK) before the protein kinase assay. Briefly, ERK2 (500 ng) was incubated in a reaction mixture (20 µl) composed of 40 mM HEPES-NaOH (pH 8.0), 1 mM dithiothreitol, 0.1 mM EGTA, 5 mM Mg(CH3COO)2, 100 µM

for 30 min and terminated by placing the tube on ice.

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ATP, and MEK (50 ng). The reaction was started by the addition of MEK, incubated at 30°C

The stoichiometry of phosphate incorporation into TandeMBP was determined as follows.

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TandeMBP was phosphorylated by various kinases using an aforementioned reaction mixture in a final volume of 200 µl. After incubation at 30°C for 2, 4, 6, 8, 10 and 12 h, a 10 µl aliquot

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of the mixture was withdrawn, spotted onto a 2 × 2 cm Whatman 3MM chromatography paper (Sigma-Aldrich), and immediately placed into 5% (w/v) trichloroacetic acid (TCA) containing 1% (w/v) sodium diphosphate. The 32P-phosphate incorporation into TandeMBP was

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measured according to the method of Corbin and Reimann [13].

Preparation of P-TandeMBP

TandeMBP (80 µg) and the protein kinase mixture (2 µg each) were incubated in a

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standard reaction mixture (40 ml) at 30°C for 10 h. After incubation, 1% (w/v) sodium deoxycholate (1 ml) was added. After incubation at 30°C for 10 min, 5 ml of 100% (w/v) TCA

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was added and placed on ice for 5 min. Proteins were precipitated by centrifugation at 20,000 g for 10 min at 4°C and the supernatant was carefully removed for elimination of ATP, orthophosphoric acid (Pi) and ADP. To the precipitate, 10 ml of cold acetone was added, mixed and centrifuged at 20,000 g for 10 min at 4°C. This washing step was repeated three times to remove remaining TCA in the precipitate. The precipitate was air-dried and dissolved in 80 µl of 0.2 N HCl by agitation for resolubilization of P-TandeMBP. The kinases were precipitated by centrifugation at 20,000g for 10 min at 4°C, and then the supernatant was

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harvested. The supernatant was neutralized with 2 M Tris and stored in aliquots at −80°C until used.

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Construction of plasmids

To generate the plasmids pET-hPPM1A, pET-hPPM1B, pET-hPPM1D, pET-hPPM1G, pET-hPPM1H, pET-hPPM1K and pET-hPPM1M, the primers listed in Table 1 were used for

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PCR with pDONR201-hPPM1A, pDONR201-hPPM1B, pDONR201-hPPM1D, pDONR201-hPPM1G, pDONR201-hPPM1H, pDONR201-hPPM1K, and

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pDONR201-hPPM1M [14] as templates, respectively. The PCR fragments were digested by restriction enzymes (Table 1), and ligated into pET-23a(+) (Novagen) to generate the plasmids pET-hPPM1A, pET-hPPM1B, pET-hPPM1D, pET-hPPM1G, pET-hPPM1H, pET-hPPM1K

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and pET-hPPM1M.

Protein expression and purification

Recombinant human PPM1F and rat PP5 were expressed in Escherichia coli BL21 (DE3)

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cells (Novagen) and purified as described previously [15, 16]. pET-hPPM1A, pET-hPPM1B, pET-hPPM1D, pET-hPPM1G, pET-hPPM1H, pET-hPPM1K and pET-hPPM1M were

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introduced into BL21 (DE3) pLysE cells (Novagen). The transformed bacteria were grown at 37°C to an A600 of 1.0, and then isopropyl β-D-thiogalactoside (IPTG) was added. After incubation, the bacteria were harvested by centrifugation (2,300 g for 10 min) and suspended in buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 40). The culture conditions are listed in Table 2. After sonication with a Sonifier model 450 (Branson) for 30 × 30 s with 30-s intervals on ice, cell debris were removed by centrifugation (20,000 g) at 4°C for 10 min, and the supernatant obtained was loaded onto a HiTrap Chelating HP column (GE

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Healthcare BioSciences) pre-equilibrated with buffer A. The column was washed with buffer A, buffer A containing 20 mM imidazole, buffer A containing 50 mM imidazole, and then the target protein was eluted with buffer A containing 200 mM imidazole. The purified fractions

Dephosphorylation of P-TandeMBP by λPPase

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2-mercaptoethanol) and stored in aliquots at −30°C until used.

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were pooled, dialyzed against buffer B (20 mM Tris-HCl, pH 7.5, 0.05% Tween 40 and 1 mM

P-TandeMBP was dephosphorylated by lambda protein phosphatase (λPPase) as described

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previously [17]. Dephosphorylation of P-TandeMBP was carried out at 30°C for 60 min in a standard reaction mixture (50 µl) composed of 40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MnCl2, 100 ng of P-TandeMBP, and 100 ng of λPPase, and the reaction was terminated by adding an equal volume of 2 × SDS-PAGE sample buffer and boiled for 90 s, and analyzed

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by SDS-PAGE followed by Western blotting with an anti-His6 antibody.

SDS-PAGE and Western blotting

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SDS-PAGE was carried out according to the method of Laemmli [18] in slab gels consisting of a 15% (w/v) acrylamide separating gel and a 3% (w/v) stacking gel. Western

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blotting was performed as described previously [19].

Gel filtration chromatography of mouse brain extract Mouse brain was homogenized with five volumes of buffer A and centrifuged at 20,000 g at 4°C for 30 min. The supernatant (2 ml) was loaded onto a HiLoad 16/60 Superdex 200 pg column (GE Healthcare Bio-Sciences) prewashed with buffer A. Chromatography was performed using a fast protein liquid chromatography system (BioLogic, Bio-Rad) with a flow

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rate of 1 ml/min, and 1.25-ml fractions were collected. The molecular-mass standard proteins used were β-amylase (200 kDa), transferrin (81 kDa), and ovalbumin (43 kDa).

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Protein phosphatase assay using phosphopeptide and P-TandeMBP as substrates

Protein phosphatase assay using phosphopeptide pp10 was carried out at 30°C for 15 min in a reaction mixture (50 µl) composed of 50 mM Tris-HCl (pH 8.0), 2 mM MnCl2, 0.1 mM

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EGTA, 0.01% Tween 20, 20 µM pp10 as a substrate, and 50 ng phosphatases or gel filtration fractions (5 µl). Using P-TandeMBP, the assay was carried out at 30°C for 15 min in a

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reaction mixture (50 µl) composed of 50 mM Tris-HCl (pH 8.0), 2 mM MnCl2, 0.1 mM EGTA, 0.01% Tween 20, P-TandeMBP (4 or 10 µg) as a substrate and 50 ng phosphatases or gel filtration fractions (1 µl). A value for Pi was calculated by comparison with the standard curve obtained by using phosphoric acid [20]. The assay for kinetic parameters of

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P-TandeMBP, pp10 and Erk2(179–189) peptide for PPM1A and PPM1G were carried out at 30°C for 6 or 15 min [7]. The experiments were performed three times, and the data were

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presented as means ± SE.

Protein phosphatase assay using MUP as a substrate

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The protein phosphatase assay using MUP was carried out at 30 °C for 15 min in a

reaction mixture (100 µl) composed of 50 ng phosphatases or gel filtration fractions (20 µl), 50 mM Tris-HCl (pH 8.0), 2 mM MnCl2, 0.1 mM EGTA, 1 mM dithiothreitol, 0.01% Tween 20, and 25 µM MUP as a substrate, as described previously [16]. The reaction was started by the addition of phosphatases and terminated by the addition of 100 µl of the stop solution containing 50 mM Tris-HCl (pH 8.0), 4% (w/v) SDS and 20 mM EDTA. The 4-methylumbelliferone released from MUP was measured with a CytoFluor 4000TC

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fluorescence microplate reader (Applied Biosystems) using an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

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Results

Efficient phosphorylation of TandeMBP by the protein kinase mixture

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We previously developed TandeMBP as a useful substrate for a protein kinase assay [9]. Because TandeMBP was efficiently phosphorylated by various Ser/Thr protein kinases [9], we

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hypothesized that P-TandeMBP would be an efficient protein substrate for a variety of protein phosphatases without 32P radiolabeling. Initially, we searched for potential phosphorylation sites of TandeMBP by multifunctional protein kinases (CaMKI, CK1, and ERK2) on the basis of phosphorylation consensus sequences (CaMKI: R-X-X-S/T; CK1: E/D-X-X-S/T and

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phosphoS/T-X-X-S/T; ERK2: S/T-P) [11, 21, 22]. The potential phosphorylation sites of TandeMBP were seven for CaMKI, five for CK1 (with the exception of sites phosphoS/T-X-X-S/T), and six for ERK2, as shown in Fig. 1. To investigate whether

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TandeMBP was effectively phosphorylated by the protein kinase mixture, we compared phosphorylation levels of TandeMBP by CaMKIδ, CK1δ, ERK2 and their protein kinase

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mixture. As shown in Fig. 2, TandeMBP served as an efficient substrate for the protein kinase mixture. When TandeMBP was incubated with the protein kinase mixture, the phosphorylation levels of TandeMBP were maximal at 10 h (Fig. 2A). After 10 h incubation, the phosphate incorporation into one mol of TandeMBP was 2.4 (CaMKI), 7.1 (CK1), 3.3 (ERK2) and 9.3 (protein kinase mixture) mol of phosphate (Fig. 2B). These results indicated that TandeMBP was phosphorylated efficiently by the protein kinase mixture.

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Preparation of P-TandeMBP as a substrate for protein phosphatases Next, we developed a preparation method of P-TandeMBP for the protein phosphatase assay. The in vitro phosphatase assay using P-TandeMBP combined with the malachite green

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assay, which monitors inorganic free phosphate, was expected to be non-radioactive method for detecting protein phosphatase activity. When TandeMBP was phosphorylated by the protein kinase mixture, P-TandeMBP, protein kinases, ATP, Pi, and ADP were included in the

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reaction mixture. For quantitation of Pi by the malachite green assay, elimination of protein kinases, ATP, Pi, and ADP was essential to suppress the background level. Therefore, we

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performed the precipitation of P-TandeMBP by TCA and its re-solubilization with HCl, as shown in Fig. 3A. The following four steps describe the preparation of P-TandeMBP. TandeMBP was incubated with the protein kinase mixture (CaMKIδ, CK1δ and ERK2) and ATP at 30 °C for 10 h to phosphorylate TandeMBP (STEP 1). After incubation, P-TandeMBP

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and protein kinases were precipitated by TCA, and the supernatant including ATP, Pi, and ADP was removed (STEP 2). P-TandeMBP was dissolved in HCl, and protein kinases were precipitated by centrifugation (STEP 3). The supernatant was harvested and neutralized with

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Tris (STEP 4). When each fraction was analyzed by Western blotting using an anti-His6 antibody, P-TandeMBP was confirmed to be efficiently purified by the above four steps (Fig.

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3B, left panel). In addition, when P-TandeMBP was dephosphorylated by λPPase, a down-shifted band as a non-phosphorylated form of TandeMBP was detected (Fig. 3B, right panel). When the Pi concentration in the final preparation of P-TandeMBP solution was measured using the malachite green assay, Pi was hardly detected (Fig. 3C).

Comparison of P-TandeMBP and other substrates on the phosphatase activity of various protein phosphatases

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Because various substrates have been used for the protein phosphatase assay, we compared the dephosphorylation activities of protein phosphatases. As representative protein phosphatases, we used eight different PPM family phosphatases [PPM1A (PP2Cα), PPM1B

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(PP2Cβ), PPM1D (PP2Cδ), PPM1F (CaMKP), PPM1G (PP2Cγ), PPM1H (NERPP-2C), PPM1K (PP2Cκ), PPM1M (PP2Cη)] and one PPP family phosphatase (PP5). When

P-TandeMBP and pp10 were used for the protein phosphatase assay, the dephosphorylation

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activity was determined by monitoring the absorbance at 620 nm. In contrast, using MUP, the phosphatase activity was measured as fluorescence units. For comparison, the activity of

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PPM1A was calculated to be 100% in two different assays. As shown in Fig. 4, P-TandeMBP was a better substrate than pp10 and MUP for all protein phosphatases examined. In particular, using P-TandeMBP, the relative phosphatase activity of PPM1G was approximately five times higher than that of PPM1A. Next, we examined kinetic parameters of P-TandeMBP, pp10 and

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Erk2(179–189) peptide for PPM1A and PPM1G. The values of Vmax of the three substrates for PPM1A were almost similar level, while Vmax of P-TandeMBP for PPM1G was higher than those of pp10 and Erk2(179–189) peptide (Table 3). Moreover, the values of Km of

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P-TaneMBP for PPM1A and PPM1G were lower than those of pp10 and Erk2(179–189) peptide. These results suggested that P-TandeMBP was good substrate for PPM1A and

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PPM1G than pp10 and Erk2(179–189) peptide.

Comparison of P-TandeMBP and other substrates on the dephosphorylation activity of a gel filtration fraction from mouse brain Metal ions play a crucial role for the activation of PPM and PPP family phosphatases [23]. The activities of several phosphatases, such as PPM1F are strictly dependent on Mn2+ or Mg2+ [24]. Therefore, in the presence of Mn2+ or Mg2+, we measured protein phosphatase activities

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using P-TandeMBP, pp10 and MUP. A mouse brain extract was fractionated by gel filtration, and the phosphatase activities in the gel filtration fractions were analyzed. As shown in Fig. 5A, two peaks of phosphatase activities were mainly detected using P-TandeMBP in the

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presence of Mn2+. A weak activity was observed at more than 200 kDa (fractions 6−8), while marked activity was found at approx. 160 kDa (fractions 17−31). In the case of MUP, mainly three peaks were detected, comprising a weak activity at approximately 130 kDa (fractions

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22−30) and significant activities at ~40 and ~30 kDa (fractions 33−45). Using pp10, marked activity was not detected. In the presence of Mg2+, a peak of strong phosphatase activity was

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detected at 160 kDa (fractions 17−30) using P-TandeMBP, whereas a marked peak was not detected using pp10 and MUP (Fig. 5B). These results suggest that P-TandeMBP is a useful substrate to detect protein phosphatase activities from tissue extracts.

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Discussion

In the present study, we developed a preparation method of P-TandeMBP and confirmed

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the following advantages of P-TandeMBP as a substrate for protein phosphatases. First, in combination with the malachite green assay, P-TandeMBP could be used to detect phosphatase

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activities without 32P radioactive materials. Although 32P-labeled proteins have often been used as substrates for protein phosphatases, the preparation of 32P-labeled proteins is usually complicated owing to the phosphorylation of proteins with [γ-32P]ATP. Moreover, 32P radioactive materials have a short half-life and specific facilities or areas for their use are required. Second, P-TandeMBP served as an efficient substrate for various protein phosphatases, such as PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, PPM1M and PP5.

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Most of the phosphopeptides and phosphoproteins that have been used as specific substrates for protein phosphatases possess a few phosphorylated residues, whereas P-TandeMBP has at least nine phosphorylated residues with different surrounding sequences. Therefore,

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P-TandeMBP should be a useful tool to detect a wide variety of protein phosphatase activities in cells and tissue extracts.

Third, P-TandeMBP should be a useful tool for detecting various phosphatase activities

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when combined with other known substrates. The metal coordinating structures of PPM family members have been elucidated. Among them, the crystal structure of PPM1A with

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Mn2+ was first reported in 1996 [25]. In addition, we reported previously that the activities of phosphatases, such as PPM1A and PPM1F, were strictly dependent on metal ions, Mn2+ and Mg2+ [8]. Since the structure of protein phosphatases may alter in the presence of metal ions, their activities and specificities are influenced by these ions. As shown in Fig. 5, the

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phosphatase activities determined by MUP were strongly affected by the coordinating metal ion Mg2+ when compared with those determined by P-TandeMBP with Mn2+ or Mg2+. One possibility is that P-TandeMBP possesses at least nine phosphorylated residues instead of one

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phosphate group in MUP. Therefore, P-TandeMBP can be considered a good substrate for protein phosphatases coordinating Mn2+ or Mg2+ ions.

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Fourth, P-TandeMBP can be conveniently prepared in four steps (phosphorylation of

TandeMBP, TCA precipitation, HCl treatment and neutralization) and retains high stability. Protein kinases (CaMKIδ, CK1δ, and ERK2) used to phosphorylate TandeMBP can be prepared easily by E. coli protein expression systems [10-12]. TandeMBP can be easily purified by extraction with HCl, as well as MBP [9]. We confirmed the phosphate groups of P-TandeMBP were highly stable after at least 1 year in the −80 °C freezer because we checked the background level of Pi in the P-TandeMBP solution (data not shown).

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In conclusion, we have reported the preparation method of P-TandeMBP and how P-TandeMBP can be used in an in vitro phosphatase assay without radioactive materials to

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analyze various protein phosphatase activities.

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research [Grant No.

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from the Japan Society for the Promotion of Science.

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26750372 (to Y. Sugiyama), 25350978 (to I. Kameshita) and 15K07842 (to N. Sueyoshi)]

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Enzymol. 38 (1974) 287−290.

[14] N. Goshima, Y. Kawamura, A. Fukumoto, A. Miura, R. Honma, R. Satoh, A Wakamatsu, J. Yamamoto, K. Kimura, T. Nishikawa, T. Andoh, Y. Iida, K. Ishikawa, E. Ito, N. Kagawa, C.

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Kaminaga, K. Kanehori, B. Kawakami, K. Kenmochi, R. Kimura, M. Kobayashi, T. Kuroita, H. Kuwayama, Y. Maruyama, K. Matsuo, K. Minami, M. Mitsubori, M. Mori, R. Morishita, A.

AC C

Murase, A. Nishikawa, S. Nishikawa, T. Okamoto, N. Sakagami, Y. Sakamoto, Y. Sasaki, T. Seki, S. Sono, A. Sugiyama, T. Sumiya, T. Takayama, Y. Takayama, H. Takeda, T. Togashi, K. Yahata, H. Yamada, Y. Yanagisawa, Y. Endo, F. Imamoto, Y. Kisu, S. Tanaka, T. Isogai, J. Imai, S. Watanabe, N. Nomura, Human protein factory for converting the transcriptome into an in vitro–expressed proteome, Nat. Methods 5 (2008) 1011−1017. [15] I. Kameshita, H. Baba, Y. Umeda, N. Sueyoshi, In-gel protein phosphatase assay using fluorogenic substrates, Anal. Biochem. 400 (2010) 118−122.

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[16] H. Baba, N. Sueyoshi, Y. Shigeri, A. Ishida, I. Kameshita, Regulation of Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) by oxidation/reduction at Cys-359, Arch. Biochem. Biophys. 526 (2012) 9−15.

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[17] M. Sekiguchi, S. Katayama, N. Hatano, Y. Shigeri, N. Sueyoshi, I. Kameshita,

Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase

257−267.

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associated with X-linked neurodevelopmental disorder, Arch. Biochem. Biophys. 535 (2013)

[18] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of

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bacteriophage T4, Nature 227 (1970) 680−685.

[19] Y. Sugiyama, S. Katayama, I. Kameshita, K. Morisawa, T. Higuchi, H. Todaka, E. Kinoshita, E. Kinoshita-Kikuta, T. Koike, T. Taniguchi, S. Sakamoto, Expression and phosphorylation state analysis of intracellular protein kinases using Multi-PK antibody and

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Phos-tag SDS-PAGE, MethodsX 2 (2015) 469−474.

[20] A.A. Baykov, O.A. Evtushenko, S.M. Avaeva, A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay,

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Anal. Biochem. 171 (1988) 266−270.

[21] R.R. White, K. Young-Guen, M. Taing, D.S. Lawrence, A.M. Edelman, Definition of

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optimal substrate recognition motifs of Ca2+-calmodulin-dependent protein kinase IV and II reveals shared and distinctive features, J. Biol. Chem. 273 (1998) 3166−3172. [22] R.J. Davis, The mitogen-activated protein kinase signal transduction pathway, J. Biol. Chem. 268 (1993) 14553−14556. [23] Y. Shi, Serine/threonine phosphatases: mechanism through structure, Cell 139 (2009) 468−484. [24] A. Ishida, I. Kameshita, H. Fujisawa, A novel protein phosphatase that dephosphorylates

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and regulates Ca2+/calmodulin-dependent protein kinase II, J. Biol. Chem. 273 (1998) 1904−1910. [25] A.K. Das, N.R. Helps, P.T.W. Cohen, D. Barford, Crystal structure of the.protein

AC C

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serine/threonine phosphatase 2C at 2.0 Å resolution, EMBO J. 15 (1996) 6798−6809.

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Figure legends

Fig. 1. Amino acid sequence of TandeMBP. The potential phosphorylation sites for CaMKI

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(white), CK1 (black), and ERK2 (gray) are indicated by arrowheads.

Fig. 2. Phosphorylation of TandeMBP by protein kinases. (A) TandeMBP (200 ng) was

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incubated with 20 ng of CaMKIδ (filled circle), CK1δ (square), ERK2 (triangle), or the protein kinase mixture (open circle) in the presence of 100 µM [γ-32P]ATP. After incubation at

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30 °C for 2, 4, 6, 8, 10, and 12 h, the 32P-phosphate incorporation into TandeMBP was measured by a liquid scintillation counter. (B, C) TandeMBP was phosphorylated by protein kinases for 10 h. The incorporation of 32P-phosphate into TandeMBP was measured by a liquid scintillation counter. Three independent experiments were carried out and the data

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represent means ± SD (B). The phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography (C).

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Fig. 3. Preparation of P-TandeMBP. (A) Outline of the preparation of P-TandeMBP. The sequential procedure consists of phosphorylation of TandeMBP by the protein kinase mixture

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(CaMKIδ, CK1δ, and ERK2: PK mixture), removal of ATP, Pi, and ADP, extraction of P-TandeMBP, and neutralization with Tris. (B) The preparation of P-TandeMBP was carried out as described in Materials and methods. Approximately 100 ng of TandeMBP, TandeMBP incubated with the protein kinase mixture (Input), supernatant (sup), and pellet (ppt) after 0.2 N HCl extraction were analyzed by SDS-PAGE followed by Western blotting using an anti-His6 antibody (left panel). P-TandeMBP (100 ng) was incubated in the presence (+) or absence (–) of λPPase (100 ng) and analyzed by Western blotting with an anti-His6 antibody

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(right panel). (C) The concentration of Pi in the solutions was determined using the malachite green assay.

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Fig. 4. Comparison of P-TandeMBP and other substrates on various protein phosphatase activities. Dephosphorylation activities of various phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, PPM1M, and PP5) were determined by the phosphatase

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assay using P-TandeMBP, pp10, and MUP as described in Materials and methods.

Phosphatase activities were expressed as relative values by setting the activity of PPM1A as

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100%. Three independent experiments were carried out and the data represent means ± SD.

Fig. 5. Phosphatase activities of gel filtration fractions from mouse brain using several phosphatase substrates. The mouse brain extract was applied to a Superdex column and

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1.25-ml fractions (53 fractions in total) were collected over a 40-min run. Each fraction was incubated with P-TandeMBP, pp10 and MUP in a reaction mixture including 2 mM MnCl2 (A)

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methods.

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or 5 mM MgCl2 (B), and phosphatase activities were determined as described in Materials and

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Table 1. List of the primers used for construction of plasmids Name

Sequence

Restriction enzyme

5'-TTTTCTAGAATGGGAGCATTTTTAGACAAGCCA-3'

XbaI

PPM1A -as

5'-AAACTCGAGCCACATATCATCTGTTGATGTAGAGTCAG-3'

XhoI

PPM1B-s

5'-TTTTCTAGAATGGGTGCATTTTTGGATAAACCC-3'

XbaI

PPM1B -as

5'-AAACTCGAGTATTTTTTCACCACTCATCTTTGTCCC-3'

XhoI

PPM1D-s

5'-TTTGCTAGCATGGCGGGGCTGTACTCGC-3'

NheI

PPM1D-as

5'-AAAGTCGACATAGCAAACACAAACAGTTTTCCTGTG-3'

SalI

PPM1G-s

5'-TTTGCTAGCATGGGTGCCTACCTCTCCCAG-3'

NheI

PPM1G-as

5'-AAAGTCGACATAGTCTCGCTTGGCCTTCTTCTT-3'

SalI

PPM1H-s

5'-TTTGCTAGCCATGCTCACTCGAGTGAAATCTGCC-3'

NheI

PPM1H -as

5'-AAAGTCGACTGACAGCTTGTTTCCATGTATTAAAGG-3'

SalI

PPM1K -s

5'-TTTGCTAGCATGTCAACAGCTGCCTTAATTACTTTG-3'

NheI

PPM1K-as

5'-AAACTCGAGGGCCCATCGTCCACTGGAG-3'

XhoI

PPM1M-s

5'-TTTGCTAGCATGTCCGCCGGCTGGTTC-3'

NheI

PPM1M-as

5'-AAACTCGAGGTGGTCACTCTCTTGGCCC-3'

XhoI

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PPM1A-s

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Under lines indicate the sites of restriction enzymes.

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Table 2. Culture conditions for expression of PPM family phosphatases IPTG (mM)

Temperature (°C)

PPM1A

None

25

24

PPM1B

0.1

23

24

PPM1D

0.1

18

6

PPM1G

0.1

37

PPM1H

None

25

PPM1K

0.1

18

PPM1M

None

18

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Incubation time (h)

4

12 12 24

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Phosphatase

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Table 3. Kinetic parameters of P-TandeMBP, pp10 and Erk2(179–189) peptide for PPM1A and PPM1G PPM1A Vmax

Km

Vmax

(µM)

(µmol/min/mg)

(µM)

(µmol/min/mg)

P-TandeMBP

4.16±0.34

1.42±0.07

2.85±0.75

pp10

10.15±1.50

1.22±0.05

72.28±19.35

Erk2(179–189)

11.38±0.39

1.29±0.04

12.18±2.90

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Km

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2.91±0.50

0.97±0.17

0.18±0.02

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Substrate

PPM1G

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MASQKRPSQRSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFSGDRGAPKRGSGKDSHTRTTHYGSLP

:70

71:

QKSQHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQKPGFGYGGRASDYKSAHKGFK

:140

141:

GAYDAQGTLSKIFKLGGRDSRSGSPMARRGSMASQKRPSQRSKYLATASTMDHARHGFLPRHRDTGILDS

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1:

MBP (iso 5)

:210

MBP (iso 6)

IGRFFSGDRGAPKRGSGKVPWLKQSRSPLPSHARSRPGLCHMYKDSHTRTTHYGSLPQKSQHGRTQDENP

281:

VVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGGRDSRSGSPMARR

:325

CaMKI

CK1

:280

ERK2

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211:

Fig. 1 Sugiyama et al.

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10 9 8 7 6 5 4 3 2 1 0

PK mixture

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CK1δ

ERK2

CaMKIδ

2

4

6 8 10 Time (h)

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0

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(mol phosphate / mol TandeMBP)

32P-incorporated

A

14

CaMKIδ CK1δ ERK2 PK mix

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(kDa)

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10 9 8 7 6 5 4 3 2 1 0

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(mol phosphate / mol TandeMBP)

C

32P-incorporated

B

12

CaMKIδ

CK1δ

ERK2

45 29

PK mix

Autoradiography

Fig. 2 Sugiyama et al.

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A

STEP 1

STEP 3

STEP 2

STEP 4

Input +TCA remove supernatant

30℃ 10 h

HCl treatment

harvest supernatant

neutralization with Tris

46

P-TandeMBP

PKs P-TandeMBP TandeMBP

31

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λPPase

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73

P-TandeMBP (sup)

C

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sup ppt

PK mixture (ppt)

60 50 Pi (nmol) / ml

HCl treatment

P-TandeMBP + PK mixture

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TandeMBP

Input

(kDa)

TandeMBP

B

P-TandeMBP + PK mixture + ATP, Pi, ADP

removal of PK mixture

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TandeMBP + PK mixture + ATP

solubilization of P-TandeMBP

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PPase assay removal of ATP, Pi, ADP

phosphorylation of TandeMBP

40 30 20 10

λPPase

0 Anti-His6

Input

sup

Fig. 3 Sugiyama et al.

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600 500

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400

■ pp10 MUP

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300

□ P-TandeMBP

200

0 PPM1B

PPM1D

PPM1F

PPM1G

PPM1H

PPM1K

PPM1M

PP5

EP

PPM1A

TE D

100

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Relative phosphatase activity (%)

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Fig. 4 Sugiyama et al.

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81

SC Fraction No.

81

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43

43

(kDa)

RFU (Relative fluorescence units)

(kDa)

Fig. 5 Sugiyama et al.

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Fraction No.

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200

200

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Mn2+

P-TandeMBP pp10 MUP

Mg2+

P-TandeMBP pp10 MUP

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Phosphatase activity (nmol/min/ml)

A

B

Phosphatase activity (nmol/min/ml)

RFU (Relative fluorescence units)