Masao Takeda

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″ (δ) 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 μM 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 μM 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.

Molecular cloning of a 74-kDa regulatory subunit (B″ or δ) of human protein phosphatase 2A

Based on amino acid sequence data of a 74‐kDa regulatory subunit (B″ or δ) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M r 66 138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74‐kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, an SH3 accessible proline‐rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Fission yeast homologues of the B' subunit of protein phosphatase 2A: multiple roles in mitotic cell division and functional interaction with calcineurin.

Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B′, and PR72. Specific functions for each variable subunit are not well understood.

Direct metal analyses of Mn2+-dependent and -independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+-independent one

A Mn2+‐dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C′ subunit and a 63 kDa regulatory A′ subunit, was purified from human erythrocyte cytosol. C′ and A′ produced V8‐ and papain‐peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+‐independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C′A and CA′ revealed that the metal dependency resided in C′ and not in A′. In CA, 0.87±0.12 mol zinc and 0.35±0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C′A′. Pre‐incubation of C′ with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+‐independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+‐ and Fe2+‐metalloenzyme and that C′ is the apoenzyme.

Molecular heterogeneity of the cDNA encoding a 74-kDa regulatory subunit (B″ or δ) of human protein phosphatase 2A1

Two cDNAs for possible splicing variants of a 74‐kDa regulatory subunit (B″ or δ) of human protein phosphatase 2A, were isolated. These variants were identified from human cerebral cortex by library screening and PCR, and designated δ1 and δ3 isoforms, while the previously reported isoform [Tanabe et al. (1996) FEBS Lett. 379, 107–111] was designated δ2. Compared with the δ2 isoform, the δ1 isoform contained a 32‐residue insertion beginning at residue 84, and consisted of 602 amino acids in all. The δ3 isoform lacked a 74‐residue sequence corresponding to residues 1083 of the δ2 isoform, and consisted of 496 amino acids. Using isoform‐specific antipeptide antisera, the 74‐kDa subunit (B″ or δ) originally purified from human erythrocytes was identified as the δ1 isoform.

Comparison of tyrosine phosphorylation of proteins by membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma

When membrane fractions from mouse liver. Ehrlich ascites tumor and MH 134 hepatoma were incubated with [γ‐32]ATP at O°C in the presence of MnCly ZnCl; and NaV03, proteins were phosphorylated on tyrosines to a larger extent in liver membranes than in tumor membranes. Separation of labelled proteins by SDS‐gel electrophoresis showed phosphorylated alkali‐resistant bands of 170, 140, 130, 80, 56, 53 and 46 kDa proteins in Ehrlich ascites tumor membranes; liver membranes exhibited more strongly phosphorylated bands of 170, 56, 53 and 46 kDa proteins. Epidermal growth factor stimulated the tyrosine phosphorylation of only a 170 kDa protein, which was more significant in liver membranes. Liver membranes exhibited slightly higher levels of tyrosine protein kinase activity compared to tumor membranes.

Dissociation and reassociation of a pig heart phosphoprotein phosphatase

Abstract A pig heart phosphoprotein phosphatase with a molecular weight of 224,000 was dissociated in the presence of 40 % ethanol into an active component (C) of molecular weight 31,000 and components (R) of higher molecular weight. After removal of the ethanol, C and R reassociated and formed an enzyme of molecular weight 188,000. C alone could not form the enzyme. The newly formed enzyme had substrate specificity and response to Mg acetate similar to those of the original large form of the enzyme and was clearly distinguishable from C. The ability of R to associate with C was supressed by treatment of R with trypsin or heat (60°C, 2 min), but not with RNase or DNase.

Isolation of an inactive component from pig heart phosphoprotein phosphatase and its reassociation with an active component.

Treatment of a pig heart phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) of Mr 224 000 with 40% ethanol followed by gel-filtration on Sephadex G-150, dissociated the enzyme into an active component of Mr 31 000 and an inactive component of Mr 80 000. The inactive component reassociated with the active component, resulting in the formation of an enzyme form of Mr 123 000. A large excess of either component in the reassociation produced only this enzyme form. The ability of the inactive component to associate with the active component was lost by treatment of the inactive component with trypsin and heat (60 degrees C, 2 min) but not with DNAase and RNAase. Effects of the inactive component on the activities of the active component by the association were as followings. The inactive component: (1) stimulated slightly the 32P-H2B histone phosphatase activity in the presence of either NaCl or Mg(CH3COO)2 but inhibited strongly in the absence of the salts; (2) stimulated the 32P-H1 histone phosphatase activity in the presence of the salts; (3) inhibited the phosphorylase a phosphatase activity in the presence and absence of the salts; (4) enhanced the response to the stimulatory effects of the salts on the dephosphorylation of 32P-histone; and (5) protected the phosphorylase a phosphatase activity from inhibition by the salts.

Effects of polyamine hydrochlorides and salts on phosphoprotein phosphatase

Polyamine hydrochlorides, NaCl and magnesium acetate stimulated the enzymatic dephosphorylation of phosphorylated H2B histone by two forms (large form, mol. wt. 250 000; small form, mol. wt. 30 000) of a pig heart phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16). These ionic compounds stimulated the large form of the enzyme 5--9-fold but stimulated the small form of theenzyme only 2-fold. With phosphorylated H2B histone as substrate, these effectors caused an increase in both Km and V values of the two forms of the enzyme. On the other hand, when a tryptic phosphodecapeptide derived from phosphorylated H2B histone was used as substrate, these effectors were always inhibitory apparently non-competitively with respect to the substrate. Using phosphorylated H1 histone as substrate, these effectors stimulated the large form of the enzyme 2-fold but inhibited the small form. With phosphorylase a as substrate, the reactions were also inhibited by these effectors irrespective of the enzyme employed. With respect to phosphorylase a, this inhibition was apparently of a competitive type for the large form and a non-competitive type for the small form of the enzyme.