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Volumn 276, Issue 5314, 1997, Pages 952-955

Regulation of protein phosphatase 2A by direct interaction with casein kinase 2α

Author keywords

[No Author keywords available]

Indexed keywords

CASEIN KINASE; PHOSPHOPROTEIN PHOSPHATASE 2A;

EID: 0030979386     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5314.952     Document Type: Article
Times cited : (252)

References (37)
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    • 6 ) were transferred with Lipofectamine (Gibco) with 18 μg of the relevant CK2α-HA expression vector (6), serum-deprived for 20 hours, then lysed in 1 ml of buffer B [20 mM tris-HCl (pH 7.4), 50 mM NaCl, 50 mM NaF, 1% Triton X-100, 14 mM β-mercaptoethanol, aprotinin (25 μg/ml), leupeptin (25 μg/ml), 100 nM okadaic acid, and 0.1 mM activated vanadate]. Clarified lysates were immunoprecipitated with 1 μl of monoclonal antibody (12CA5) to HA and 25 μl of protein A-Sepharose. Immunoprecipitates were washed three times in buffer B and once in tris-buffered saline [20 mM tris-HCl (pH 7.4), 150 mM NaCl] containing 100 nM okadaic acid and subjected to chemilumi nescent immunoblotting.
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    • 2. Reactions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography.
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    • Wild-type and mutant chicken CK2α cDNAs were subcloned (as HA-tagged derivatives) in frame with GST and expressed in Escherichia coli BL21 (DE3). GST fusions were purified by glutathione-Sepharose chromatography as recommended by the manufacturer (Pharmacia). The purified GST fusions (200 ng) were incubated with purified PP2A dimer (200 ng, UBI) in 100 μl of kinase buffer for 20 min at 25°C. The mixture was adsorbed to glutathione-Sepharose in buffer A [20 mM tris-HCl (pH 7.4), 50 mM NaCl, 0.1% Triton X-100, 14 mM β-mercaptoethanol] for 30 min at 4°C. The beads were washed three times in buffer A, eluted into Laemmli buffer, and analyzed by chemiluminescent immunoblotting.
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    • All probabilities were computed by Student's t test with Scheffe's correction for multiple comparisons.
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    • note
    • 6) were transfected by calcium phosphate precipitation with a mixture of 2 μg of CK2α, 8 μg of His-MEK1, and 12 μg of carrier plasmid. Cells were lysed in buffer B containing 100 mM NaCl (14), and His-MEK1 was adsorbed onto 20 μl of nickeltrinitriloacetate resin (Invitrogen) for 1 hour at 4°C, washed three times in lysis buffer, and eluted into 50 μl of lysis buffer containing BSA (1 mg/ml) and 0.25 M imidazole (pH 7.4).
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    • note
    • We thank S. A. Courtneidge for anti-PP2A and peptide; A. Brunet and J. Pouysségur for the HA-MAPK and MEKD constructs; U. R. Rapp for anti-Raf; J. Ghysdael for the Raf 1 baculovirus; T. Sturgill for the SrcF527 and v-Ha-Ras baculoviruses; A. Berns and C. Ovitt for the pGKHygro plasmid; and J. Ghysdael, D. Job, and our laboratory colleagues for critically reviewing the manuscript. J.-K.H. was supported by the Ministère de l'Enseignement Supérieur et de la Recherche and by the Ligue Nationale contre le Cancer. Supported by Commissariat àl'Energie Atomique, INSERM, and CNRS.


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