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

Abstract

Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory BQ (N N) subunit, was phosphorylated at serine residues of BQ in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 W WM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of BQ, respectively. The K m value of A-kinase for CABQ was 0.17 þ 0.01 W WM in the presence of OA. The major in vitro phosphorylation sites of BQ 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 BQ did not dissociate BQ from CA, and stimulated the molecular activity of CABQ toward phosphorylated H1 and H2B histones, 3.8-and 1.4-fold, respectively, but not toward phosphorylase a.z 1998 Federation of European Biochemical Societies.

A phosphatase cascade by which rewarding stimuli control nucleosomal response

“…Fig.13) and can phosphorylate nuclear DARPP-32 and promote its export. Stimulation of D1R triggers rapid phosphorylation of Thr-34 responsible for cytoplasmic effects of DARPP-32, and slower dephosphorylation of Ser-97 by activating PP2A through cAMP/PKA-mediated phosphorylation of its B56δ subunit 26,27 , thus decreasing the nuclear export of DARPP-32. Although phosphorylation has been reported to regulate cyto-nuclear shuttling of a number of proteins 35 , this is a rare example of facilitation of CRM1-mediated nuclear export by phosphorylation 36,37 .…”

“…Fig.9a), and is activated by cAMP-dependent phosphorylation 26,27 . When B56δ was co-expressed with DARPP-32 in HEK293 cells, it promoted dephosphorylation of Ser-97 in response to forskolin (Fig.3e, Supp.…”

“…Student t-test: cocaine: **p<0.01 (n=16); morphine: *p<0.01 (n=8). (c) Conditioned place preference to cocaine was prevented in S97A mice: Scores are differences between times spent in the cocaine-paired compartment after and before conditioning (n=7-8): genotype-treatment interaction, F (1,26) =3.16, NS; genotype effect, F (1,26) =3.76, NS; treatment effect, F (1,26) =14.21, p<0.01; **p<0.01 vs. saline. (d) Mice trained to nose poke for food reward were subjected to progressive ratio (PR) schedule and the breaking point determined as the number of pokes at which mice stopped working: Student t-test :…”

Amplification of dopaminergic signaling by a positive feedback loop

“…Regulatory subunits are subdivided into PR55 B, PR56 BЈ, PR72 BЉ, and PR61 families (20,21). Usui et al reported that the regulatory BЈ␦ subunit (or B56␦), originally purified from erythrocytes and named the BЉ (␦) subunit, is phosphorylated by PKA and that phosphorylated forms of BЈ␦ stimulate the catalytic activity of PP-2A in a substrate-specific manner (22,23). Immunoblot analysis using BЈ␦ antibody revealed that the level of expression of the protein was slightly higher in striatum than in neocortex (A.N., unpublished observations), the region in the central nervous system in which the level of expression of BЈ␦ was reported to be most abundant (24).…”

The Paired-like Homeodomain Protein, Arix, Mediates Protein Kinase A-stimulated Dopamine β-Hydroxylase Gene Transcription through Its Phosphorylation Status

“…The reaction products were analyzed by SDS-PAGE, followed by autoradiography. For the in vitro kinase assay using [␥- 32 P]ATP, bacterially produced and purified His-tagged Arix proteins (1 g) (14) were incubated at 30°C for 5 min with 10 ng of rPKA in the kinase reaction buffer containing 12.5 mM Tris-Cl (pH 8), 0.1 mM ATP, 10 mM MgCl 2 , 0.25 mg/ml bovine serum albumin, and 0.5 Ci [␥- 32 P]ATP. The reaction was stopped by adding EDTA at the final concentration of 80 mM on ice.…”