HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: T600044-MCP200v1 6/2/356    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kinoshita-Kikuta, E. Articles by Koike, T. Search for Related Content PubMed PubMed Citation Articles by Kinoshita-Kikuta, E. Articles by Koike, T. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 6:356-366, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Technology

Label-free Kinase Profiling Using Phosphate Affinity Polyacrylamide Gel Electrophoresis^*,S

Emiko Kinoshita-Kikuta, Yuri Aoki, Eiji Kinoshita and Tohru Koike

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

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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-3ß, 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.

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-OPO₃^2–) as a bridging ligand. A Mn(II) homologue (Mn²⁺-Phos-tag) can capture R-OPO₃^2– anions, such as phosphoserine and phosphotyrosine, at alkaline pH (9) (see the structure of R-OPO₃^2–-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-3ß (GSK-3ß), 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

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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-3ß, 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, [-³²P]ATP, Hyperfilm ß-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 x 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-3ß, cdk5/p35, PKA, MAPK, and CKII, a reaction buffer (20 µl) containing 25 mM Tris-HCl (pH 7.5), 5.0 mM ß -glycerol phosphate, 12 mM MgCl₂, 2.0 mM dithiothreitol, 0.10 mM sodium orthovanadate, 50 µ M ATP, and 37 kBq [-³²P]ATP was used. The amount of each kinase in the buffer was 2.0 µg of GSK-3ß, 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 ß -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 [-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 [ -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-3ß, 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 ß -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

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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-3ß, 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 R_f value of 0.6 (Fig. 1a). The R_f 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-3ß. 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-3ß (R_f = 0.32 in lane 2), PKA (R_f = 0.15 and 0.28 in lane 4), and MAPK (R_f < 0.1 in lane 5), were observed. The migration of the nonphosphorylated Tau protein and GSK-3ß (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; K_i = 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.

View larger version (52K): [in this window] [in a new window]   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-3ß, 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.

View larger version (18K): [in this window] [in a new window]   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.

View larger version (42K): [in this window] [in a new window]   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-3ß. 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.

View larger version (25K): [in this window] [in a new window]   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-3ß. 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.

View larger version (72K): [in this window] [in a new window]   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.

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 R_f 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 k_obs (min^–1) for the Abl kinase reaction, i.e. a pseudo-first-order reaction, were calculated using a kinetic equation of k_obs x 60 = ln[ C_o] – ln[ C_t] where C_o is the initial concentration of Abltide-GST and C_t is the concentration of the remaining nonphosphorylated Abltide-GST after 60-min incubation. The relationship between the concentrations of Glivec and the k_obs 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.

View larger version (19K): [in this window] [in a new window]   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

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-3ß, 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

 
Received, August 1, 2006, and in revised form, October 30, 2006.

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-3ß, glycogen synthase kinase-3ß; 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.

To whom correspondence may be addressed. Tel.: 81-82-257-5281; Fax: 81-82-257-5336; E-mail: kinoeiji{at}hiroshima-u.ac.jp

To whom correspondence may be addressed. Tel.: 81-82-257-5281; Fax: 81-82-257-5336; E-mail: tkoike{at}hiroshima-u.ac.jp

	REFERENCES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Hunter, T. (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225 –236[CrossRef][Medline]

2. Hunter, T. (2000) Signaling—2000 and beyond. Cell 100, 113 –127[CrossRef][Medline]

3. Mann, M., Ong, S. E., Grønborg, M., Steen, H., Jensen, O. N., and Pandey, A. (2002) Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 20, 261 –268[CrossRef][Medline]

4. Hunter, T. (2002) Tyrosine phosphorylation in cell signaling and disease. Keio J. Med. 51, 61 –71[Medline]

5. Cohen, P. (2002) Protein kinases—the major drug targets of the twenty-first century? Nat. Rev. Drug Discov. 1, 309 –315[CrossRef][Medline]

6. Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Yoshida, H., Watanabe, A., Titani, K., and Ihara, Y. (1995) Hyperphosphorylation of Tau in PHF. Neurobiol. Aging 16, 365 –380[CrossRef][Medline]

7. Lee, V. M.-Y., Goedert, M., and Trojanowski, J. Q. (2001) Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24, 1121 –1159[CrossRef][Medline]

8. Min, D.-H., Su, J., and Mrksich, M. (2004) Profiling kinase activities by using a peptide chip and mass spectrometry. Angew. Chem. Int. Ed. Engl. 43, 5973 –5977

9. Inamori, K., Kyo, M., Nishiya, Y., Inoue, Y., Sonoda, T., Kinoshita, E., Koike, T., and Katayama, Y. (2005) Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule. Anal. Chem. 77, 3979 –3985[Medline]

10. Ahn, N. G., and Resing, K. A. (2001) Toward the phosphoproteome. Nat. Biotechnol. 19, 317 –318[CrossRef][Medline]

11. Zhou, H., Watts, J. D., and Aebersold, R. (2001) A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375 –378[CrossRef][Medline]

12. Oda, Y., Nagasu, T., and Chait, B. T. (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379 –382[CrossRef][Medline]

13. Knight, Z. A., Schilling, B., Row, R. H., Kenski, D. M., Gibson, B. W., and Shokat, K. M. (2003) Phosphospecific proteolysis for mapping sites of protein phosphorylation. Nat. Biotechnol. 21, 1047 –1054[CrossRef][Medline]

14. Takeda, H., Kawasaki, A., Takahashi, M., Yamada, A., and Koike, T. (2003) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule. Rapid Commun. Mass Spectrom. 17, 2075 –2081[CrossRef][Medline]

15. Kinoshita, E., Takahashi, M., Takeda, H., Shiro, M., and Koike, T. (2004) Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex. Dalton Trans. 1189 –1193

16. Kinoshita, E., Yamada, A., Takeda, H., Kinoshita-Kikuta, E., and Koike, T. (2005) Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins. J. Sep. Sci. 28, 155 –162[CrossRef][Medline]

17. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., and Koike, T. (2006) Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol. Cell. Proteomics 5, 749 –757[Abstract/Free Full Text]

18. Kinoshita-Kikuta, E., Kinoshita, E., Yamada, A., Endo, M., and Koike, T. (2006) Enrichment of phosphorylated proteins from cell lysate using a novel phosphate-affinity chromatography at physiological pH. Proteomics 6, 5088 –5095[CrossRef][Medline]

19. Buchdunger, E., Zimmermann, J., Mett, H., Meyer, T., Müller, M., Druker, B. J., and Lydon, N. B. (1996) Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res. 56, 100 –104[Abstract/Free Full Text]

20. Carroll, M., Ohno-Jones, S., Tamura, S., Buchdunger, E., Zimmermann, J., Lydon, N. B., Gilliland, D. G., and Druker, B. J. (1997) CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood 90, 4947 –4952[Abstract/Free Full Text]

21. Schindler, T., Bornmann, W., Pellicena, P., Miller, W. T., Clarkson, B., and Kuriyan, J. (2000) Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289, 1938 –1942[Abstract/Free Full Text]

22. Brasher, B. B., and Van Etten, R. A. (2000) c-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines. J. Biol. Chem. 275, 35631 –35637[Abstract/Free Full Text]

23. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254[CrossRef][Medline]

24. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 –685[CrossRef][Medline]

25. Berggren, K., Chernokalskaya, E., Steinberg, T. H., Kemper, C., Lopez, M. F., Diwu, Z., Haugland, R. P., and Patton, W. F. (2000) Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex. Electrophoresis 21, 2509 –2521[CrossRef][Medline]

26. Baudier, J., and Cole, R. D. (1987) Phosphorylation of tau proteins to a state like that in Alzheimer’s brain is catalyzed by a calcium/calmodulin-dependent kinase and modulated by phospholipids. J. Biol. Chem. 262, 17577 –17583[Abstract/Free Full Text]

27. Steiner, B., Mandelkow, E.-M., Biernat, J., Gustke, N., Meyer, H. E., Schmidt, B., Mieskes, G., Söling, H. D., Drechsel, D., Kirschner, M. W., Goedert, M., and Mandelkow, E. (1990) Phosphorylation of microtubule-associated protein tau: identification of the site for Ca²⁺-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles. EMBO J. 9, 3539 –3544[Medline]

28. Litersky, J. M., and Johnson, G. V. (1992) Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain. J. Biol. Chem. 267, 1563 –1568[Abstract/Free Full Text]

29. Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T., and Imahori, K. (1992) Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments. J. Biol. Chem. 267, 10897 –10901[Abstract/Free Full Text]

30. Drewes, G., Lichtenberg-Kraag, B., Döring, F., Mandelkow, E.-M., Biernat, J., Goris, J., Dorée, M., and Mandelkow, E. (1992) Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state. EMBO J. 11, 2131 –2138[Medline]

31. Mandelkow, E.-M., Drewes, G., Biernat, J., Gustke, N., Van Lint, J., Vandenheede, J. R., and Mandelkow, E. (1992) Glycogen synthase kinase-3 and the Alzheimer-like state of microtubule-associated protein tau. FEBS Lett. 314, 315 –321[CrossRef][Medline]

32. Scott, C. W., Spreen, R. C., Herman, J. L., Chow, F. P., Davison, M. D., Young, J., and Caputo, C. B. (1993) Phosphorylation of recombinant tau by cAMP-dependent protein kinase. J. Biol. Chem. 268, 1166 –1173[Abstract/Free Full Text]

33. Baumann, K., Mandelkow, E.-M., Biernat, J., Piwnica-Worms, H., and Mandelkow, E. (1993) Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5. FEBS Lett. 336, 417 –424[CrossRef][Medline]

34. Litersky, J. M., Johnson, G. V. W., Jakes, R., Goedert, M., Lee, M., and Seubert, P. (1996) Tau protein is phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II within its microtubule-binding domains at Ser-262 and Ser-356. Biochem. J. 316, 655 –660

35. Liu, F., Iqbal, K., Grundke-Iqbal, I., and Gong, C. X. (2002) Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3ß. FEBS Lett. 530, 209 –214[CrossRef][Medline]

36. Li, T., and Paudel, H. K. (2006) Glycogen synthase kinase 3ß phosphorylates Alzheimer’s disease-specific Ser³⁹⁶ of microtubule-associated protein tau by a sequential mechanism. Biochemistry 45, 3125 –3133[CrossRef][Medline]

37. Watanabe, A., Hasegawa, M., Suzuki, M., Takio, K., Moroshima-Kawashima, M., Titani, K., Arai, T., Kosik, K. S., and Ihara, Y. (1993) In vivo phosphorylation sites in fetal and adult rat tau. J. Biol. Chem. 268, 25712 –25717[Abstract/Free Full Text]

38. Greenwood, J. A., Scott, C. W., Spreen, R. C., Caputo, C. B., and Johnson, G. V. W. (1994) Casein kinase II preferentially phosphorylates human tau isoforms containing an amino-terminal insert. Identification of threonine 39 as the primary phosphate acceptor. J. Biol. Chem. 269, 4373 –4380[Abstract/Free Full Text]

39. Pullen, N., Dennis, P. B., Andjelkovic, M., Dufner, A., Kozma, S. C., Hemmings, B. A., and Thomas, G. (1998) Phosphorylation and activation of p70^s6k by PDK1. Science 279, 707 –710[Abstract/Free Full Text]

40. Liu, F., Zaidi, T., Iqbal, K., Grundke-Iqbal, I., and Gong, C.-X. (2002) Aberrant glycosylation modulates phosphorylation of tau by protein kinase A and dephosphorylation of tau by protein phosphatase 2A and 5. Neuroscience 115, 829 –837[CrossRef][Medline]

41. Sakchaisri, K., Asano, S., Yu, L.-R., Shulewitz, M. J., Park, C. J., Park, J.-E., Cho, Y.-W., Veenstra, T. D., Thorner, J., and Lee, K. S. (2004) Coupling morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. U. S. A. 101, 4124 –4129[Abstract/Free Full Text]

42. Misonou, H., Mohapatra, D. P., Park, E. W., Leung, V., Zhen, D., Misonou, K., Anderson, A. E., and Trimmer, J. S. (2004) Regulation of ion channel localization and phosphorylation by neuronal activity. Nat. Neurosci. 7, 711 –718[CrossRef][Medline]

43. Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyama, T., and Kondo, T. (2005) Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science 308, 414 –415[Abstract/Free Full Text]

44. Tak, Y.-S., Tanaka, Y., Endo, S., Kamimura, Y., and Araki, H. (2006) A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11. EMBO J. 25, 1987 –199[CrossRef][Medline]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S.-F. Tseng, Z.-J. Shen, H.-J. Tsai, Y.-H. Lin, and S.-C. Teng Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13 Nucleic Acids Res., June 1, 2009; 37(11): 3602 - 3611. [Abstract] [Full Text] [PDF]

				C. A. Ydenberg and M. D. Rose Antagonistic regulation of Fus2p nuclear localization by pheromone signaling and the cell cycle J. Cell Biol., February 9, 2009; 184(3): 409 - 422. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: T600044-MCP200v1 6/2/356    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kinoshita-Kikuta, E. Articles by Koike, T. Search for Related Content PubMed PubMed Citation Articles by Kinoshita-Kikuta, E. Articles by Koike, T. Social Bookmarking                      What's this?