Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B" (δ) 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 mu M okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B ", respectively. The K-m value of A-kinase for CAB " was 0.17 +/- 0.01 mu M 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. (C) 1998 Federation of European Biochemical Societies.