Technology

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis

Emiko Kinoshita-Kikuta, Yuri Aoki, Eiji Kinoshita and Tohru Koike
Molecular & Cellular Proteomics February 1, 2007, First published on November 5, 2006, 6 (2) 356-366; https://doi.org/10.1074/mcp.T600044-MCP200

Abstract

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.

Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells (1, 2). It occurs on several amino acid residues, including histidine, aspartic acid, glutamic acid, lysine, arginine, and cysteine on which it is very labile and difficult to detect, whereas more stable and well studied phosphorylation takes place on the three specific residues serine, threonine, and tyrosine (3). The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Many of the genetic changes that play a causal role in the cancer phenotype involve mutations of protein kinases and phosphatases (4). There has been considerable progress in the development of selective inhibitors for the protein kinase and phosphatase involved in disease (5). Some of these inhibitors have been recently approved for use in humans for the treatment of cancer. Furthermore the activities of several protein kinases are dysregulated, leading to a hyperphosphorylation state of the microtubule-associated protein Tau, which is a classical hallmark of Alzheimer disease, a neurodegenerative disorder (6, 7). The phosphorylation site and stoichiometry of the Tau protein are correlated with the pathological characteristics of the disease.

Methods for the determination of the phosphorylation status of a protein are thus very important with respect to the evaluation of the basis for understanding the molecular origins of diseases and for drug design. A conventionally used method for defining a particular phosphorylation event is the incorporation of a radioactive label, i.e. a 32P or 33P isotope, in a phosphorylated protein, which is followed by PAGE and autoradiography. The phosphorylation state of the target protein is detected and quantified as radioactivity. A newer, non-radioactive method using poly- and monoclonal antibodies has been well established for the detection of site-specific phosphorylation. The anti-phosphoprotein antibody can be used in many analytical procedures such as the enzyme-linked immunosorbent assay, Western blotting, immunocytochemistry, and immunoprecipitation. Recently a few high throughput methods for defining a number of phosphorylation events were developed using a peptide chip followed by MS (8) and surface plasmon resonance imaging (9). Chemical labeling of the phosphate group has also been used for phosphospecific site mapping in peptide mass fingerprinting and subsequent MS analysis (10–13).

Recently we reported that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olate acts as a phosphate-binding tag (Phos-tag)1 in an aqueous solution (14–18). The Phos-tag molecule has a vacancy on two metal ions that is suitable for accessing a phosphomonoester dianion (R-OPO32−) as a bridging ligand. A Mn(II) homologue (Mn2+-Phos-tag) can capture R-OPO32− anions, such as phosphoserine and phosphotyrosine, at alkaline pH (∼9) (see the structure of R-OPO32−-bound Mn2+-Phos-tag in Supplemental Fig. S1). This finding has contributed to the development of phosphate affinity electrophoresis for the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts (17). We utilized an acrylamide-pendant Mn2+-Phos-tag as a novel additive, i.e. a copolymer of the separating gel in SDS-PAGE. The Mn2+-Phos-tag SDS-PAGE offers the following significant advantages. (i) Radioactive and chemical labels are avoided. (ii) The time course quantitative ratio of the phosphorylated and nonphosphorylated proteins can be determined. (iii) The phosphate binding specificity is independent of the amino acid sequence context. (iv) A downstream procedure, such as Western blotting analysis, is applicable. (v) The procedure is almost identical to that of the general SDS-PAGE system.

Herein we describe three novel applications of Mn2+-Phos-tag SDS-PAGE. The first is in vitro kinase activity profiling for the analysis of the phosphoprotein isotypes derived from various kinase reactions. The activity profiles of six kinds of kinases, glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (cdk5)/p35, protein kinase A (PKA), mitogen-activated protein kinase (MAPK), casein kinase II (CKII), and calmodulin-dependent protein kinase II (CaMKII), were determined using the substrate Tau protein. The second application is in vivo kinase activity profiling for the analysis of extracellular signal-dependent protein phosphorylation. The time-dependent alterations of epidermal growth factor (EGF)-induced phosphorylation levels of Shc and MAPK1/2 were demonstrated using the lysate of A431 human epidermoid carcinoma cells. The third application is in vitro kinase inhibition profiling for the quantitative analysis of a kinase-specific inhibitor. The inhibitory profile of a tyrosine kinase, Abl (a histidine-tagged recombinant mouse Abl kinase), was demonstrated 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 (a 2-phenylaminopyrimidine derivative, STI-571) used for the treatment of chronic myeloid leukemia (19–22).

EXPERIMENTAL PROCEDURES

Materials—

The acrylamide-pendant Phos-tag ligand was obtained from the Phos-tag consortium (Japan). The histidine-tagged recombinant human Tau isoform consisting of 441 amino acid residues, histidine-tagged recombinant human GSK-3β, recombinant mouse PKA catalytic subunit, PKA-specific competitive peptide inhibitor (PKI-(14–22)-amide), recombinant human histone H1.2, phosphorylated site-specific Ser(P)199 and Ser(P)214 Tau antibodies, sodium deoxycholate, and Na3VO4 were purchased from Calbiochem. The recombinant human cdk5/p35, recombinant mouse MAPK2, recombinant human CKII, rat forebrain CaMKII, recombinant bovine calmodulin, histidine-tagged recombinant mouse Abl, recombinant Abltide-GST, anti-Shc antibody, and anti-MAPK1/2 antibody were purchased from Upstate Biotechnology (Lake Placid, NY). The phosphorylated site-specific Thr(P)212, Thr(P)231, Ser(P)396, and Ser(P)404 Tau antibodies were purchased from BioSource (Camarillo, CA). The phosphorylated site-specific Tyr(P)239/240 and Tyr(P)317 Shc antibodies and the Thr(P)202/Tyr(P)204 MAPK1/2 antibody were purchased from Cell Signaling Technology (Danvers, MA). Bovine intestinal mucosa alkaline phosphatase, NaCl, and EGF were purchased from Sigma. The ECL Advance Western blotting detection kit, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody, [γ-32P]ATP, Hyperfilm β-max, and a liquid scintillator (ACSII) were purchased from GE Healthcare. The developing fluid (RENDOL) and fixing fluid (RENFIX) were purchased from Fujifilm (Tokyo, Japan). A PVDF membrane (Fluorotrans W) was purchased from Nihon Pall (Tokyo, Japan). The 3MM paper was purchased from Whatman Japan (Tokyo, Japan). The SYPRO Ruby protein gel stain, RPMI 1640 cell culture medium, fetal bovine serum, penicillin, and streptomycin were purchased from Invitrogen. The Sharpline low range markers for protein molecular weight and Can Get Signal Immunoreaction Enhancer Solution were purchased from Toyobo (Osaka, Japan). Silver gel stain (Sil-Best stain for protein/PAGE), leupeptin, aprotinin, pepstatin, NaF, Nonidet P-40, and PMSF were purchased from Nacalai Tesque (Kyoto, Japan). A protein assay kit was purchased from Bio-Rad. Glivec was supplied by Novartis (Basel, Switzerland). All reagents and solvents were of the highest commercial quality and were used without further purification.

Cell Culture, EGF Stimulation, and Preparation of the Cell Lysate—

The A431 human epidermoid carcinoma cell line was supplied by the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer at Tohoku University (Japan). The cells were grown in an RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin under a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The cells (106) were placed into the same medium in a 30-mm culture plate. After the cells were allowed to adhere to the plate (9 h), the medium was removed, and a serum-free medium was added. After incubating for 16 h, the cells were stimulated with 250 ng/ml EGF for 0 (no treatment with EGF), 2, 5, 10, 30, 60, 120, and 240 min. To terminate the stimulation, the medium was removed, and the remaining cells were rinsed with Tris-buffered saline (20 mm Tris-HCl (pH 7.6) and 138 mm NaCl) at room temperature. After the saline was removed, the culture plate was placed on ice. The cells were exposed to 50 μl of cold RIPA buffer consisting of 50 mm Tris-HCl (pH 7.4), 0.15 m NaCl, 0.25% (w/v) sodium deoxycholate, 1.0% (v/v) Nonidet P-40, 1.0 mm EDTA, 1.0 mm PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1.0 mm Na3VO4, and 1.0 mm NaF. The plate was gently rocked for 15 min on ice, and the adherent cells were then removed from the plate with a cell scraper. The resultant suspension was transferred to a microcentrifuge tube. The plate was washed with 50 μl of RIPA buffer, and the washing solution was combined with the first suspension in a microcentrifuge tube. The mixed sample was incubated for 60 min on ice and centrifuged at 13,000 × g for 10 min at 4 °C. The supernatant fluid was used as the cell lysate. The concentration of the solubilized cellular proteins was adjusted to 2.0 mg/ml with an appropriate amount of RIPA buffer. The quantification of protein was performed according to Bradford’s method (23) with a Bio-Rad protein assay kit. Each sample was mixed with a half-volume of SDS-PAGE loading buffer (195 mm Tris-HCl (pH 6.8), 3.0% (w/v) SDS, 15% (v/v) 2-mercaptoethanol, 30% (v/v) glycerol, and 0.10% (w/v) bromphenol blue) and was heated at 95 °C for 5 min before SDS-PAGE analysis.

SDS-PAGE—

Polyacrylamide gel electrophoresis was conducted according to Laemmli’s method (24) and was usually performed at 30 mA/gel and room temperature in a 1-mm-thick, 9-cm-wide, 9-cm-long gel on a PAGE apparatus (Model AE6500; Atto, Tokyo, Japan). The gel consisted of 1.8 ml of a stacking gel (4.0% (w/v) polyacrylamide, 125 mm Tris-HCl (pH 6.8), and 0.10% (w/v) SDS) and 6.3 ml of a separating gel (7.5–12.5% (w/v) polyacrylamide, 375 mm Tris-HCl (pH 8.8), and 0.10% (w/v) SDS). For Mn2+-Phos-tag SDS-PAGE, an acrylamide-pendant Phos-tag ligand (25–100 μm) and 2 eq of MnCl2 were added to the separating gel before polymerization. An acrylamide stock solution was prepared as a mixture of acrylamide to N,N′-methylenebisacrylamide at a 29:1 ratio. The electrophoresis running buffer (pH 8.4) was 25 mm Tris and 192 mm glycine containing 0.10% (w/v) SDS.

Quantification of Proteins in a Polyacrylamide Gel—

Silver staining was conducted using Sil-Best stain for protein/PAGE according to the manufacturer’s instructions. For autoradiography, the gel was dried in vacuum and exposed to an x-ray film at −80 °C for 48 h. For SYPRO Ruby staining (25), the gel was fixed in an aqueous solution containing 10% (v/v) MeOH and 7.0% (v/v) acetic acid for 30 min. The fixed gel was stained in a solution of SYPRO Ruby protein gel stain for 2 h and then washed in 10% (v/v) MeOH and 7.0% (v/v) acetic acid for 2 h. SYPRO Ruby dye-bound proteins were detected as 575-nm emission signals on 473-nm excitation using an FLA 5000 laser scanner (Fujifilm). The gel images obtained by silver staining, autoradiography, and SYPRO Ruby staining were analyzed using Multi Gauge software (Fujifilm).

Western Blotting Analysis—

After Mn2+-Phos-tag SDS-PAGE, the gel was soaked in a solution containing 25 mm Tris, 192 mm glycine, 10% (v/v) MeOH, and 1.0 mm EDTA for 10 min and then soaked in a solution containing 25 mm Tris, 192 mm glycine, and 10% (v/v) MeOH for 30 min. The gel was electroblotted to a PVDF membrane for 16 h using a blotting system (Nippon Eido Model NA-1511C, Tokyo, Japan) at 4.0 V/cm. The blotting membrane was soaked in an aqueous solution containing 10 mm Tris-HCl (pH 7.5), 0.10 m NaCl, and 0.10% (v/v) Tween 20 (TBS-T solution) for 1 h and then blocked by 1.0% (w/v) bovine serum albumin in TBS-T solution for 1 h. For immunoblotting detection of each protein substrate, the membrane was probed with a solution (1 ml/30 cm2) containing each antibody in a plastic bag for 1 h. The antibody solutions were prepared by dilution of the commercially available products with TBS-T solution at 1:1000 for anti-phosphorylated MAPK1/2 antibody against Thr(P)202/Tyr(P)204; 1:2000 for anti-Shc antibody and anti-phosphorylated Tau antibody against Ser(P)199; 1:5000 for anti-MAPK1/2 antibody and anti-phosphorylated Tau antibodies against Thr(P)212, Ser(P)214, and Ser(P)396; and 1:10,000 for anti-phosphorylated Tau antibodies against Thr(P)231 and Ser(P)404. The membrane was washed twice with a TBS-T solution (2 ml/cm2) for 10 min in each case, probed with HRP-conjugated anti-rabbit IgG antibody (at 1:10,000 dilution in TBS-T solution, 1 ml/30 cm2) in a plastic bag for 1 h, and washed twice with TBS-T solution (2 ml/cm2) for 10 min in each case. To reduce the nonspecific binding of anti-phosphorylated Shc antibodies against Tyr(P)239/240 and Tyr(P)317, Can Get Signal Solution 1 was used at 1:2000 dilution, and HRP-conjugated anti-rabbit IgG antibody was diluted at 1:10,000 with Can Get Signal Solution 2. The ECL signal was then observed using a LAS 3000 image analyzer (Fujifilm). For reprobing of the blotting membrane, the membrane was incubated with a stripping buffer (5 ml/cm2) consisting of 62.5 mm Tris-HCl (pH 6.8), 2.0% (w/v) SDS, and 0.10 m 2-mercaptoethanol for 20 min at 50 °C and washed three times with TBS-T solution (5 ml/cm2) for 1 h at room temperature in each case. The remaining proteins on the membrane were reprobed with the other antibody by the procedure described above.

Kinase Activity Profiling Using Tau Protein—

The in vitro phosphorylation assay was carried out using the recombinant Tau protein (4.1 μg) at 30 °C. For phosphorylation by GSK-3β, cdk5/p35, PKA, MAPK, and CKII, a reaction buffer (20 μl) containing 25 mm Tris-HCl (pH 7.5), 5.0 mm β -glycerol phosphate, 12 mm MgCl2, 2.0 mm dithiothreitol, 0.10 mm sodium orthovanadate, 50 μ m ATP, and 37 kBq [γ-32P]ATP was used. The amount of each kinase in the buffer was 2.0 μg of GSK-3β, 0.10 μg of cdk5/p35, 2500 units of PKA, 0.20 μg of MAPK, and 0.25 μg of CKII. For phosphorylation by CaMKII (50 ng), a reaction buffer (20 μl) containing 20 mm MOPS (pH 7.2), 25 mm β -glycerol phosphate, 15 mm MgCl2, 1.0 mm dithiothreitol, 1.0 mm sodium orthovanadate, 1.0 mm CaCl2, 20 μg/ml calmodulin, 50 μm ATP, and 37 kBq [γ-32P]ATP was used. After incubation for various reaction times (0–300 min), 3.0 μl of the reaction mixture was taken out and added to an SDS-PAGE loading buffer (1.5 μl) to stop the kinase reaction. An aliquot (1.2 μl) of the resulting solution was subjected to Mn2+-Phos-tag SDS-PAGE followed by silver gel staining and autoradiography. For Western blotting analysis, the phosphorylation assay was conducted in the absence of [γ -32P]ATP using a similar reaction buffer (55 μl) containing 6.9 μg of Tau. The amount of each kinase was 6.0 μg of GSK-3β, 0.37 μg of cdk5/p35, and 5000 units of PKA. After incubation, 8.0 μl of the reaction mixture was taken out and added to the SDS-PAGE loading buffer (4.0 μl). The aliquot (3.0 μl) of the resulting solution was subjected to Mn2+-Phos-tag PAGE analysis followed by Western blotting.

Kinase Inhibition Profiling of Abl—

The in vitro inhibition assay for tyrosine kinase Abl was carried out at 30 °C for 1.0 h. The reaction mixture (6.0 μl) consisted of 18 mm MOPS (pH 7.2), 23 mm β -glycerol phosphate, 4.5 mm EGTA, 0.90 mm sodium orthovanadate, 0.90 mm dithiothreitol, 15 mm MgCl2, 0.10 mm ATP, 0.10 μg of Abltide-GST, 20 ng of recombinant mouse Abl, and various concentrations of Glivec (0–100 μm). Each reaction was stopped by the addition of the SDS-PAGE loading buffer (3.0 μl), and the resulting solution was subjected to Mn2+-Phos-tag SDS-PAGE followed by SYPRO Ruby staining.

RESULTS

Determination of in Vitro Kinase Activities toward Tau Protein—

In the first kinase activity profiling using Mn2+-Phos-tag SDS-PAGE, we characterized six kinds of Ser/Thr kinases in the phosphorylation of a recombinant human Tau protein. For normal SDS-PAGE (Fig. 1, a and b) and Mn2+-Phos-tag SDS-PAGE (Fig. 1, c and d) followed by silver gel staining and autoradiography, each kinase reaction product using GSK-3β, cdk5/p35, PKA, MAPK, CKII, and CaMKII was sequentially applied to lanes 2–7. Nonphosphorylated Tau was applied to lane 1 as a control. In the normal SDS-PAGE, nonphosphorylated and phosphorylated Tau were observed as the migration bands at an Rf value of ∼0.6 (Fig. 1a). The Rf value was estimated as the relative ratio against bromphenol blue dye. The electrophoresis migration of phosphorylated Tau has been reported to be a little slower than that of nonphosphorylated Tau in a normal SDS-PAGE gel (26–35). The slightly up-shifted bands by phosphorylation with those kinases (Fig. 1a) are consistent with previous results. The faster migration band shown in lane 2 (Fig. 1a, arrow) was assigned to GSK-3β. The corresponding autoradiogram image (Fig. 1b) shows that all kinase reactions progressed successfully. Although no up-shifted band of Tau in the CKII reaction was observed on the normal SDS-PAGE gel, the phosphorylation was confirmed by autoradiography (Fig. 1b, lane 6). In contrast to the normal SDS-PAGE, a number of characteristic slower migration bands were observed on the Mn2+-Phos-tag SDS-PAGE gel (Fig. 1c). Some faint bands (indicated by arrows) assigned to the commercially available kinases, GSK-3β (Rf = 0.32 in lane 2), PKA (Rf = 0.15 and 0.28 in lane 4), and MAPK (Rf < 0.1 in lane 5), were observed. The migration of the nonphosphorylated Tau protein and GSK-3β (Fig. 1c, lanes 1 and 2) became slower than that in normal SDS-PAGE (Fig. 1a, lanes 1 and 2) possibly due to an electrostatic interaction between cationic Mn2+-Phos-tag and anionic SDS-bound proteins as described previously (17). The corresponding autoradiogram image (Fig. 1d) demonstrated that the radioactive 32P isotope was incorporated in the up-shifted proteins. The 32P signal intensities were different from those for the silver-stained image (Fig. 1c). The treatment of the multiphosphorylated proteins with alkaline phosphatase gave a single migration band of nonphosphorylated Tau. When a 0.10 μm concentration of a PKA-specific competitive peptide inhibitor (PKI-(14–22)-amide; Ki = 1.7 nm) was added to each kinase reaction mixture, the up-shifted Tau bands disappeared only in the PKA reaction under the same experimental conditions (data not shown). These facts show that the multiple bands obtained by each kinase reaction should correspond to kinase-specific phosphorylated Tau proteins.

Fig. 1.
Fig. 1.

Normal SDS-PAGE and Mn2+-Phos-tag SDS-PAGE of kinase products of the Tau protein.a, a silver-stained image of normal 7.5% (w/v) polyacrylamide SDS-PAGE. b, an autoradiogram image of the same gel as that used in a. c, a silver-stained image of 80 μm polyacrylamide-bound Mn2+-Phos-tag 7.5% (w/v) polyacrylamide SDS-PAGE. d, an autoradiogram image of the same gel as that used in c. Lane 1 contains the nonphosphorylated Tau protein (0.17 μg). Each lane (lanes 2–7) contains the kinase reaction product of the Tau protein (0.17 μg) using GSK-3β, cdk5/p35, PKA, MAPK, CKII, and CaMKII, respectively. The incubation time for each kinase reaction was 300, 60, 60, 300, 300, and 300 min, respectively. Arrows indicate faint bands assigned to the commercially available kinase.

To determine the relationship between the stoichiometry of phosphate incorporation and the degree of mobility shift (Rf values), the ratios of the 32P signal intensities to the density of silver staining (32P-SI/DSS values) of each electrophoresis band shown in Fig. 1, c and d, were evaluated by densitographic analysis. The 32P-SI/DSS value is an index of the number of phosphate groups in one molecule of Tau. The plots of the 32P-SI/DSS values against the Rf values are shown in Fig. 2. Although there was an increase in the 32P-SI/DSS values, the Rf values decreased in each kinase reaction except for the GSK-3β reaction. The reverse relationships between the 32P-SI/DSS values and the Rf values were considerably different among those kinase reactions. These results show that the degree of mobility shift of a phosphoprotein is possibly due to not only the stoichiometry of phosphate incorporation but also other factors such as the kinase-specific phosphorylation sites.

Fig. 2.
Fig. 2.

Relationship between the phosphate incorporation ratio and the mobility shift degree in Mn2+-Phos-tag SDS-PAGE. Plots of the phosphate incorporation ratios (32P signal intensity to the density of silver staining of each electrophoresis band in Fig. 1, c and d; 32P-SI/DSS values) against the Rf values are shown.

Next we determined the time course of Tau phosphorylation by the kinases for 0–300 min using Mn2+-Phos-tag SDS-PAGE. The diverse isotypes of phosphorylated Tau in the kinase reactions are shown in Fig. 3, a–f. The silver staining density, i.e. the amount of protein, demonstrates that the up-shifted bands increased time-dependently in all kinase reactions. The corresponding autoradiogram intensity, i.e. the number of phosphate groups, shows that the up-shifted proteins were radioactive 32P derivatives. The time course band patterns of the phosphorylated Tau proteins are characteristic of the kinase reactions. Furthermore the total radioactivity of each lane was measured by using a scintillation counter, and the counting values (cpm) per lane were then plotted against the kinase reaction times (Fig. 3g). The cpm values for the kinase reactions increased rapidly and leveled off at 300 min under the experimental conditions. Thus, the time course experiments showed the final phosphorylation status for each kinase reaction as the characteristic migration bands at 300 min (Fig. 3, a–f).

Fig. 3.
Fig. 3.

Kinase assays of the Tau protein using six kinds of kinases by Mn2+-Phos-tag SDS-PAGE followed by silver staining, autoradiography, and scintillation counting.a, incubation with GSK-3β. b, incubation with cdk5/p35. c, incubation with PKA. d, incubation with MAPK. e, incubation with CKII. f, incubation with CaMKII. The incubation times for each kinase reaction were 0 (no treatment with kinases), 10, 30, 60, 120, 180, and 300 min. Each lane contains the kinase reaction product of the Tau protein (0.17 μg). The Mn2+-Phos-tag SDS-PAGE gels (80 μm polyacrylamide-bound Mn2+-Phos-tag and 7.5% (w/v) polyacrylamide) were subjected to silver gel staining and subsequent autoradiography. g, plots of the liquid scintillation counting (cpm) of each lane against the incubation time. Arrows indicate faint bands assigned to the commercially available kinase.

Although the Tau isoform used has 79 phosphate acceptors, i.e. serine and threonine residues, ∼30 residues have been reported as phosphorylation sites of the native Tau protein under biological conditions (7). To assign each electrophoresis migration band to the phosphorylation site of Tau, we performed Western blotting analysis using site-specific anti-phosphorylated Tau antibodies after Mn2+-Phos-tag SDS-PAGE. Fig. 4 shows three typical results using six kinds of anti-phosphorylated Tau antibodies for Ser(P)199, Thr(P)212, Ser(P)214, Thr(P)231, Ser(P)396, and Ser(P)404 residues. Mn2+-Phos-tag SDS-PAGE, Western transfer, probing with an antibody, and ECL detection were performed with the same experimental procedures. The nonphosphorylated Tau gave no ECL signal derived from those antibodies (Fig. 4, leftmost lane of each panel). As for the GSK-3β reaction (Fig. 4a), up-shifted bands responding to antibodies for Ser(P)199, Thr(P)231, Ser(P)396, and Ser(P)404 were observed. The ECL signals for Ser(P)199, Ser(P)396, and Ser(P)404 increased analogously at Rf values of ∼0.4. The resulting phosphoserine isoforms gave a small change in the migration rate. In contrast, the anti-Thr(P)231 antibody responded to a much slower migration band (Rf value = 0.02) at the final stage. The highly up-shifted band showed no cross-activity with the anti-Ser(P)199, -Ser(P)396, and -Ser(P)404 antibodies. These facts show that the phosphorylation of the Thr231 residue by GSK-3β should require prior phosphorylation except at the Ser199, Ser396, and Ser404 residues. Actually it has been reported that GSK-3β typically requires priming phosphorylation of the Ser235 to phosphorylate the Thr231 in in vivo assay using human embryonic kidney cells cotransfected with Tau and GSK-3β (36). As for the cdk5/p35 reaction (Fig. 4b), up-shifted bands responding to antibodies for Ser(P)199, Thr(P)212, Thr(P)231, and Ser(P)404 were observed. The ECL signals varied more widely and were more up-shifted (Rf values of 0.25–0.40) than those for the reaction with GSK-3β. The differences indicate that cdk5/p35 may be a less site-specific kinase promoting the multiphosphorylation of the Tau protein at Ser199, Thr212, Thr231, and Ser404. In this case, the anti-Thr(P)231 antibody responding to a much slower migration showed cross-activity with the anti-Thr(P)212 antibody. As for the PKA reaction (Fig. 4c), the responses to the antibodies were in contrast to those with GSK-3β (Fig. 4a). The phosphorylated Tau bands by PKA were observed in higher positions than those by GSK-3β. The lower bands (Fig. 4c, open triangles, for Ser(P)214) were assigned to phosphorylated proteins derived from a low molecular weight contaminant in the Tau used. Furthermore the up-shifted bands showed cross-activity with the anti-Thr(P)212 and -Ser(P)214 antibodies but not with the anti-Ser(P)199, -Thr(P)231, -Ser(P)396, and -Ser(P)404 antibodies. These contrastive results demonstrated that kinase-specific phosphorylation sites have a strong influence on the Rf of the phosphorylated Tau isoforms in Mn2+-Phos-tag SDS-PAGE. The same Western blotting analyses of phosphorylated Tau by the other kinases (MAPK, CKII, and CaMKII) showed similar kinase-specific responses to those antibodies (Supplemental Fig. S2).

Fig. 4.
Fig. 4.

Kinase assays of the Tau protein using three kinds of kinases by Mn2+-Phos-tag SDS-PAGE followed by Western blotting.a, incubation with GSK-3β. b, incubation with cdk5/p35. c, incubation with PKA. The incubation times for each kinase reaction were 0 (no treatment with kinases), 10, 30, 60, 120, 180, and 300 min. Each lane contains the kinase reaction product of the Tau protein (0.25 μg). The Mn2+-Phos-tag SDS-PAGE gels (80 μm polyacrylamide-bound Mn2+-Phos-tag and 7.5% (w/v) polyacrylamide) were subjected to Western blotting analysis using the site-specific Ser(P)199, Thr(P)212, Ser(P)214, Thr(P)231, Ser(P)396, and Ser(P)404 Tau antibodies. Open triangles indicate low molecular weight contaminants derived from the Tau used.

Determination of in Vivo Signal-dependent Kinase Activities toward Cellular Proteins—

We extended the utility of Mn2+-Phos-tag SDS-PAGE to the mobility shift analysis of cellular proteins phosphorylated by a specific manner of EGF stimulation in A431 human epidermoid carcinoma cells. The EGF-dependent protein phosphorylation in the A431 cell has been well established (17, 18); therefore, we selected the cell and analyzed the motility of phosphorylated Shc and MAPK, which are typical cellular protein substrates in EGF signaling. The A431 cells were treated with 250 ng/ml EGF for 0 (without treatment), 2, 5, 10, 30, 60, 120, and 240 min and then lysed with RIPA buffer. These lysate samples were individually handled and sequentially applied to the lanes of the gel for SDS-PAGE. From the results of the normal SDS-PAGE followed by Western blotting analysis using anti-Shc and anti-MAPK1/2 antibodies (Fig. 5a, left panels), it was confirmed that the amount of each isoform of Shc (66, 52, and 46 kDa) and MAPK1/2 (44 and 42 kDa) was almost constant during the incubation. As for Shc, slightly up-shifted bands were observed in the EGF-treated samples. To determine the time-dependent changes of phosphorylation levels, the same samples were analyzed with anti-phosphorylated Shc (Tyr(P)239/Tyr(P)240 and Tyr(P)317) and anti-phosphorylated MAPK1/2 (Thr(P)202/Tyr(P)204) antibodies (Fig. 5a, center and right panels). The phosphorylation levels of both proteins increased rapidly for 10 min, whereas the phosphorylation level of Shc was maintained for 240 min and that of MAPK decreased gradually for 30–240 min.

Fig. 5.
Fig. 5.

Analyses of phosphorylation of Shc and MAPK in A431 cells stimulated with EGF using normal SDS-PAGE and Mn2+-Phos-tag SDS-PAGE followed by Western blotting.a, normal SDS-PAGE (7.5% (w/v) polyacrylamide) followed by Western blotting using the anti-Shc antibody, anti-phosphorylated Shc for Tyr(P)239/Tyr(P)240, and anti-phosphorylated Shc for Tyr(P)317 antibodies (upper panels) as well as the anti-MAPK1/2 antibody and anti-phosphorylated MAPK for the Thr(P)202/Tyr(P)204 antibody (lower panels). b, Mn2+-Phos-tag SDS-PAGE (25 μm polyacrylamide-bound Mn2+-Phos-tag and 7.5% (w/v) polyacrylamide) followed by Western blotting using the anti-Shc antibody, anti-phosphorylated Shc for Tyr(P)239/Tyr(P)240, and anti-phosphorylated Shc for Tyr(P)317 antibodies (upper panels) as well as the anti-MAPK1/2 antibody and anti-phosphorylated MAPK for the Thr(P)202/Tyr(P)204 antibody (lower panels). The incubation times with EGF (250 ng/ml) were 0 (without EGF), 2, 5, 10, 30, 60, 120, and 240 min. Each lane contains 15 μg of cellular proteins.

Next we subjected the same lysate samples to the analysis of Mn2+-Phos-tag SDS-PAGE. We first used 80 μm Mn2+-Phos-tag SDS-PAGE (7.5% (w/v) polyacrylamide), the same condition as that used for in vitro kinase profiling for the Tau protein. However, the cellular proteins (>10 kDa) showed much slower migration at Rf <0.5 (data not shown). Thus, we adopted a lower concentration of 25 μm Mn2+-Phos-tag for the Shc and MAPK analyses. The obtained gel staining image of the cell lysate proteins in 25 μm Mn2+-Phos-tag SDS-PAGE (7.5% (w/v) polyacrylamide) showed an appropriate migration without disordering (waving or tailing protein bands) (Supplemental Fig. S3). From the results of Mn2+-Phos-tag SDS-PAGE followed by Western blotting analysis using the anti-Shc and anti-MAPK1/2 antibodies, multiple characteristic, slower migration bands were observed in the EGF-treated samples (Fig. 5b, left panels). The time-dependent appearance of the highly up-shifted bands was consistent with the phosphorylation status shown in the normal SDS-PAGE (Fig. 5a, center and right panels). Analyses of the same samples with the anti-phosphoprotein antibodies used for the normal SDS-PAGE disclosed that the up-shifted bands were various phosphoprotein isotypes (Fig. 5b, center and right panels). Thus, the time course changes of the signal intensities of the up-shifted bands would give detailed information on the separation of the phosphoprotein isotypes in a phosphorylation state and the order of a stepwise phosphorylation event in vivo.

Determination of in Vitro Abl Kinase Inhibition—

Recently we reported a visualization method for protein monophosphorylation using Mn2+-Phos-tag SDS-PAGE (17). The method has enabled the simultaneous and quantitative determination of phosphorylated and corresponding nonphosphorylated proteins in a polyacrylamide gel. Here we apply Mn2+-Phos-tag SDS-PAGE to kinase inhibition profiling using the tyrosine kinase Abl, the substrate Abltide-GST (a recombinant fusion protein containing the peptide EAIYAAPFAKKK with an N-terminal GST tag), and the specific inhibitor Glivec, which acts as an ATP competitor at the catalytic domain of Abl (21). The residue for the phosphorylation is the Tyr in the Abltide sequence. The inhibition analysis was conducted with 0.10 μg of Abltide-GST, 20 ng of Abl, and 0.10 mm ATP by using Mn2+-Phos-tag SDS-PAGE followed by SYPRO Ruby gel staining. In the absence of the inhibitor, the phosphorylated Abltide was produced by the kinase reaction at 30 °C for 60 min in ∼50% yield. After separation of the reaction mixture by Mn2+-Phos-tag SDS-PAGE, monophosphorylated and nonphosphorylated Abltide-GST appeared as two migration bands at Rf values of 0.25 and 0.38, respectively, with almost the same fluorescence intensity. The gel image is shown in the leftmost lane of Fig. 6a. In the presence of an increasing concentration of Glivec (0.10–100 μm), dose-dependent inhibition was observed as shown in Fig. 6a. The fluorescence intensity of phosphorylated Abltide-GST (the higher band) decreased with an increase in that of nonphosphorylated Abltide-GST (the lower band). The total intensity of both bands was a constant value, indicating no side reaction such as degradation of the protein. In addition, no inhibition activity of Glivec (1.0 mm) was observed in the phosphorylation of human histone H1.2 by PKA (Supplemental Fig. S4). The observed first-order rate constants kobs (min−1) for the Abl kinase reaction, i.e. a pseudo-first-order reaction, were calculated using a kinetic equation of kobs × 60 = ln[ Co] − ln[ Ct] where Co is the initial concentration of Abltide-GST and Ct is the concentration of the remaining nonphosphorylated Abltide-GST after 60-min incubation. The relationship between the concentrations of Glivec and the kobs values showed a sigmoidal curve with an inflection point from which an IC50 value of 1.6 μm was estimated (Fig. 6b). Thus, Mn2+-Phos-tag SDS-PAGE enabled a quantitative inhibition analysis for a kinase reaction using a kinase-specific substrate such as a fusion protein.

Fig. 6.
Fig. 6.

Kinase inhibition assay of a tyrosine kinase, Abl, using the substrate Abltide-GST and the specific inhibitor Glivec.a, Mn2+-Phos-tag SDS-PAGE (100 μm polyacrylamide-bound Mn2+-Phos-tag and 12.5% (w/v) polyacrylamide) of reaction mixtures of phosphorylated (higher band) and nonphosphorylated Abltide-GST (lower band) by Abl in the absence and presence of Glivec (0.10, 0.20, 0.40, 0.80, 1.6, 3.2, 6.4, 13, 25, 50, and 100 μm). Each lane contains the kinase reaction product of Abltide-GST (0.10 μg). Nonphosphorylated Abltide-GST (0.10 μg) was applied as a control in the rightmost lane. b, inhibition curve of the Abl kinase reaction in the presence of Glivec. The observed rate constants kobs (min−1) were plotted against the concentrations of Glivec (μm) where a logarithmic scale was used for the x axis.

DISCUSSION

In this report, we have described three kinds of protein profiling using Mn2+-Phos-tag SDS-PAGE without any special apparatuses, radioisotopes, or chemical labels. The method is based on the mobility shift of phosphorylated proteins from the nonphosphorylated counterpart, a kinase substrate; thus, the amounts of phosphorylated and nonphosphorylated proteins can be simultaneously determined using general colorimetric staining or immunoblotting. If protein phosphorylation occurs at one residue of a target protein, the monophosphorylated and nonphosphorylated proteins are separated as two migration bands on Mn2+-Phos-tag SDS-PAGE. In the case of multiphosphorylation, the phosphorylated products appear as multiple bands, depending on the phosphorylation status, such as the number and positions of the phosphate groups.

The first application is in vitro kinase activity profiling for the analysis of varied phosphoprotein isotypes in a phosphorylation status. We determined the activity profiles of six kinds of Alzheimer disease-related kinases, GSK-3β, cdk5/p35, PKA, MAPK, CKII, and CaMKII, using a substrate protein, Tau, which has a number of phosphorylation sites. Each kinase induced the kinase-specific gel shifting pattern from nonphosphorylated Tau due to differences in the phosphorylation sites and stoichiometry. To investigate the relationship between the phosphorylation status and the biological function, antibodies or radioisotopes have been most often used; however, these approaches are limited for the separation analysis of varied phosphoprotein isotypes in a phosphorylation status. Our established method enabled the detection of the isotypes generated by various kinase activities in a polyacrylamide gel. The following immunoblotting using the site-specific anti-phosphorylated Tau antibodies disclosed the order of stepwise phosphorylation of Tau (Fig. 4 and Supplemental Fig. S2). We believe that great progress in phosphoproteomics would be attained by combining this application and existing methods, such as advanced mass spectrometry. A typical MS-MS peptide fragment analysis of the phosphorylated Tau separated by Mn2+-Phos-tag SDS-PAGE is shown in Supplemental Fig. S5. Similar kinase activity profiling on the other kinase system would give novel information on the relationship between protein phosphorylation and the various biological responses.

The second application is in vivo kinase activity profiling for the analysis of protein phosphorylation involved in intracellular signal transduction. The time course changes of EGF-induced phosphorylation levels of Shc and MAPK1/2 in A431 cells were visualized as highly up-shifted migration bands by subsequent immunoblotting with anti-Shc and anti-MAPK1/2 antibodies. We demonstrated the utility of this Mn2+-Phos-tag SDS-PAGE to separate phosphoproteins, phosphorylated in vivo in a stimulus-specific manner, from a nonphosphorylated counterpart in the presence of other cellular proteins. This method might offer an advantage in profiling the phosphorylation state of low abundance substrates in a complex biological sample when it is not feasible to analyze the phosphorylation events by MS. Gel shifting in SDS-PAGE has traditionally been utilized to determine whether a protein is phosphorylated; however, many phosphoproteins do not shift reliably, and many cellular proteins that are known to be phosphorylated do not exhibit gel shifting in general SDS-PAGE gels. In our normal SDS-PAGE condition, no up-shifted band of phosphorylated MAPK was observed (Fig. 5a, left panel of MAPK1/2). In contrast, Mn2+-Phos-tag SDS-PAGE was able to induce dramatic gel shifting of varied phosphoprotein isotypes. Use of the method is worthy of consideration for hypothesis-free inquisition of the phosphorylation state of cellular proteins.

The third application is in vitro kinase inhibition profiling for the quantitative screening of kinase-specific inhibitors. We demonstrated the inhibition profile of the tyrosine kinase Abl using the substrate Abltide-GST and the specific inhibitor Glivec. The dose-dependent inhibition of Glivec was determined by an alteration in the ratio of the monophosphorylated substrate (the slower migration band) and the nonphosphorylated counterpart (the faster one) in an SDS-PAGE gel. The obtained inhibition curve showed an IC50 value of 1.6 μ m in the presence of 0.10 mm ATP. These data indicate that this application enables quantitative analysis of kinase activities to evaluate the inhibition kinetics. Typical IC50 values of Glivec with Abl in vitro have been reported to be 0.038 μm (19), 0.13 μm (8), and 0.44 μm (22). Differences in the reported values may reflect differences in Abl concentrations, ATP concentrations, or substrates. This kinase inhibition profiling might help in developing tools for therapeutic intervention. A similar procedure would be applicable to phosphatase inhibition profiling using an appropriate phosphorylated protein.

Protein phosphorylation, which is one of the most important post-translational modifications, dramatically enhances the diversity of genetically encoded proteins. Many different isotypes by phosphorylation site and stoichiometry appear during a number of biological processes (37–44). Hyperphosphorylation of a certain protein sometimes gives cells or tissues abnormal functions and often introduces pathogenic processes. It has been extremely difficult to pursue the role of variable isotypes during such processes because current methods treat only crude samples containing the complex isotypes. Therefore, the techniques for the separation of the different isotypes of phosphoproteins are very important in phosphoproteome studies in biological and medical fields. Efficient separation by using phosphate affinity electrophoresis, i.e. Mn2+-Phos-tag SDS-PAGE, should increase the sensitivity of the detection of hierarchical protein phosphorylation and dephosphorylation; thus, the method could assist in mapping low abundance phosphorylation events and would be a useful tool in the study of the complicated kinase-phosphatase network.

Footnotes

  • Published, MCP Papers in Press, November 5, 2006, DOI 10.1074/mcp.T600044-MCP200

  • ↵ 1 The abbreviations used are: Phos-tag, phosphate-binding tag; GSK-3β, glycogen synthase kinase-3β; cdk5, cyclin-dependent kinase 5; PKA, protein kinase A; MAPK, mitogen-activated protein kinase; CKII, casein kinase II; CaMKII, calmodulin-dependent protein kinase II; EGF, epidermal growth factor; HRP, horseradish peroxidase; 32P-SI/DSS value, ratio of the 32P signal intensities to the density of silver staining.

  • ↵* This work was supported by Grant-in-aid for Scientific Research (B) 15390013 from Japan Society for the Promotion of Science, Grant-in-aid for Exploratory Research 18659030 from Ministry of Education, Culture, Sports, Science and Technology (MEXT), Grant-in-aid for Young Scientists (B) 17790034 from MEXT, Grant-in-aid for Young Scientists (B) 18790120 from MEXT, a research grant from the Fujii Foundation, and a research grant for Feasibility Study from Japan Science and Technology Innovation Plaza Hiroshima. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.

    • Received August 1, 2006.
    • Revision received October 30, 2006.

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Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis
Emiko Kinoshita-Kikuta, Yuri Aoki, Eiji Kinoshita, Tohru Koike
Molecular & Cellular Proteomics February 1, 2007, First published on November 5, 2006, 6 (2) 356-366; DOI: 10.1074/mcp.T600044-MCP200
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