Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B'' (delta) regulatory subunit in vitro and identification of the phosphorylation sites.
Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.
Usui
H
H
Department of Biochemistry, Hiroshima University School of Medicine, Japan.
Inoue
R
R
Tanabe
O
O
Nishito
Y
Y
Shimizu
M
M
Hayashi
H
H
Kagamiyama
H
H
Takeda
M
M
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D017421:Sequence Analysis; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D019319:Okadaic Acid; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2