Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits.

The alpha form of the A subunit of human protein phosphatase 2A was expressed in insect cells following infection with a recombinant baculovirus. A alpha was expressed as a soluble protein that comprised approximately 10% of total cellular protein. The expressed A alpha subunit was purified by chromatography on amino-hexyl-Sepharose and Mono Q with a yield of 2 mg/500-ml culture. The recombinant protein had the same apparent molecular mass as the bovine cardiac protein and was devoid of myosin light chain phosphatase activity. Biological activity of expressed A was assessed by assays of complex formation with the catalytic (C) and B subunits, purified from bovine cardiac tissue, and by inhibition of phosphatase activity. Purified A alpha had a high apparent affinity for C (IC50 = 0.10 nM) and bound with a stoichiometry of 1 mol of A/mol of C. Interaction of A alpha with the catalytic subunit caused a maximal inhibition of myosin light chain and phosphorylase phosphatase activities of 50 and 79%, respectively. The AC complex prepared by reconstitution of recombinant A alpha with C had the same electrophoretic mobility in nondenaturing polyacrylamide gels and the same elution volume when chromatographed on a size exclusion column as the native AC complex purified from cardiac muscle. Similar chromatographic profiles were also observed for the heterotrimer reconstituted from recombinant A alpha, purified B and C, and the native bovine cardiac heterotrimeric holoenzyme. Cross-linking of the native enzyme and the reconstituted heterotrimer generated the same pattern of high molecular weight species. Immunological analyses of these complexes demonstrated that distinct cross-linked forms composed of ABC, AC, AB, and BC were obtained. These results suggest that each of the three subunits of protein phosphatase 2A forms direct contacts with both of the others.     

Effects of regulatory subunits on the kinetics of protein phosphatase 2A

Both the scaffold (A) and the regulatory (R) subunits of protein phosphatase 2A regulate enzyme activity and specificity. Heterotrimeric enzymes containing different R-subunits differ in their specific activities for substrates. Kinetic parameters for the dephosphorylation of a phosphopeptide by different oligomeric forms of PP2A were determined to begin to elucidate the molecular basis of regulatory subunit effects on phosphatase activity. Using steady state kinetics and the pH dependence of kinetic parameters, we have explored the effect of the A- and R-subunits on the kinetic and chemical mechanism of PP2A. The regulatory subunits affected a broad range of kinetic parameters. The C-subunit and AC dimer were qualitatively similar with respect to the product inhibition patterns and the pH dependence of kinetic parameters. However, a 22-fold decrease in rate and a 4.7-fold decrease in K(m) can be attributed to the presence of the A-subunit. The presence of the R2alpha (Balpha or PR55alpha) subunit caused an additional decrease in K(m) and changed the kinetic mechanism of peptide dephosphorylation. The R2alpha-subunit also caused significant changes in the pH dependence of kinetic parameters as compared to the free C subunit or AC heterodimer. The data support an important role for the regulatory subunits in determining both the affinity of PP2A heterotrimers for peptide substrates and the mechanism by which they are dephosphorylated.     

Cloning and characterization of B delta, a novel regulatory subunit of protein phosphatase 2A.

Variable regulatory subunits of protein phosphatase 2A (PP2A) modulate activity, substrate selectivity and subcellular targeting of the enzyme. We have cloned a novel member of the B type regulatory subunit family, B delta, which is most highly related to B alpha. B delta shares with B alpha epitopes previously used to generate subunit-specific antibodies. Like B alpha, but unlike B beta and B gamma which are highly brain-enriched, B delta mRNA and protein expression in tissues is widespread. B delta is a cytosolic subunit of PP2A with a subcellular localization different from B alpha and may therefore target a pool of PP2A holoenzymes to specific substrates.     

Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1.

The diverse forms of protein phosphatase 1 in vivo result from the association of its catalytic subunit (PP1c) with different regulatory subunits, one of which is the G-subunit (G(M)) that targets PP1c to glycogen particles in muscle. Here we report the structure, at 3.0 A resolution, of PP1c in complex with a 13 residue peptide (G(M[63-75])) of G(M). The residues in G(M[63-75]) that interact with PP1c are those in the Arg/Lys-Val/Ile-Xaa-Phe motif that is present in almost every other identified mammalian PP1-binding subunit. Disrupting this motif in the G(M[63-75]) peptide and the M(110[1-38]) peptide (which mimics the myofibrillar targeting M110 subunit in stimulating the dephosphorylation of myosin) prevents these peptides from interacting with PP1. A short peptide from the PP1-binding protein p53BP2 that contains the RVXF motif also interacts with PP1c. These findings identify a recognition site on PP1c, invariant from yeast to humans, for a critical structural motif on regulatory subunits. This explains why the binding of PP1 to its regulatory subunits is mutually exclusive, and suggests a novel approach for identifying the functions of PP1-binding proteins whose roles are unknown.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.     

Genomic structure and localization of the human protein phosphatase 2A BRgamma regulatory subunit.

In the course of our analysis of genomic sequence from the human chromosome 4p16.1 region harboring both the Wolfram and Ellis van Creveld syndrome genes we have identified a sequence with high homology (98% at the amino acid level) to the rat cDNA coding for the protein phosphatase 2A BRgamma (PP2ABRgamma) regulatory subunit. Although the human cDNAs for both the BRalpha and BRbeta isoforms have been described previously, the BRgamma subunit has not yet been identified in humans. Here we describe the precise genomic organization and genetic localization of the human PP2ABRgamma gene.     

Transgenic expression of protein phosphatase 2A regulatory subunit B56gamma disrupts distal lung differentiation

The distal epithelium of the developing lung exhibits high-level expression of protein phosphatase 2A (PP2A), a vital signaling enzyme. Here we report the discovery that in the lung, the PP2A regulatory subunit B56gamma is expressed in a discrete developmental period, with the highest protein levels at embryonic day (e) 17, but no detectable protein in the newborn or adult. By in situ hybridization, B56gamma was highly expressed in the distal epithelium of newly forming airways and in mesenchymal cells. In contrast, expression of B56gamma was quite low in the bronchial epithelium and vascular smooth muscle. Transgenic expression of B56gamma using the lung-specific promoter for surfactant protein C (SP-C) resulted in neonatal death. Examination of lungs from SP-C-B56gamma transgenic e18 fetuses revealed proximal airways and normal blood vessels, but the tissue was densely populated with epithelial-type cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway, beta-catenin, was developmentally regulated in the normal lung and was absent in lung tissue from B-56gamma transgenic fetuses. We propose that B56gamma is expressed at a particular stage of lung development to modulate PP2A action on the Wnt/beta-catenin signaling pathway during lung airway morphogenesis.     

Protein phosphatase 2A regulatory subunits and cancer

The serine/threonine protein phosphatase (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of a number of major signaling pathways whose deregulation can contribute to cancer. The specificity and activity of PP2A are highly regulated through the interaction of a family of regulatory B subunits with the substrates. Accumulating evidence indicates that PP2A acts as a tumor suppressor. In this review we summarize the known effects of specific PP2A holoenzymes and their roles in cancer relevant pathways. In particular we highlight PP2A function in the regulation of MAPK and Wnt signaling.     

Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains

Protein phosphatase 6 (PP6) is an essential Ser/Thr phosphatase conserved among eukaryotes. The Saccharomyces cerevisiae homologue of PP6 called Sit4 depends on association with SAPS domain subunits. This study used a human SAPS domain subunit FLAG-PP6R1 to identify endogenous interacting proteins. Mass spectrometry identified coprecipitating proteins as PP6 catalytic subunit and three ankyrin repeat proteins (Ankrd28, Ankrd44, and Ankrd52). These proteins have extensive sequence identity to one another but segregate into separate branches on a phylogenetic tree for vertebrate species, suggesting individual biological functions. Tagged Ankrd28 coprecipitated with PP6, not with PP2A or PP4, and with SAPS domain subunits PP6R1 and PP6R3. Tagged PP6 coprecipitated endogenous SAPS domain subunits and Ankrd28. The C-terminal region of PP6R1 was sufficient to coprecipitate Ankrd28, but not PP6, demonstrating that PP6R1 acts as a scaffold with separate regions for binding to PP6 and to Ankrd28. Endogenous PP6 holoenzymes with PP6R1 and PP6R3 subunits were resolved by DEAE chromatography and eluted together with Ankrd28 at Mr > 440 kDa from Superose 12. Knockdown of PP6R1 or Ankrd28, but not PP6R3, produced equivalent enhancement of IkappaBepsilon degradation in response to TNFalpha. The results suggest that PP6 functions as a heterotrimer, composed of the PP6 catalytic subunit bound to a SAPS domain scaffold subunit that associates with Ankrd28. We propose that the SAPS and ankyrin repeat regulatory subunits determine the function and specificity of PP6.     

Role of B regulatory subunits of protein phosphatase type 2A in myosin II assembly control in Dictyostelium discoideum

In Dictyostelium discoideum, myosin II resides predominantly in a soluble pool as the result of phosphorylation of the myosin heavy chain (MHC), and dephosphorylation of the MHC is required for myosin II filament assembly, recruitment to the cytoskeleton, and force production. Protein phosphatase type 2A (PP2A) was identified in earlier studies in Dictyostelium as a key biochemical activity that can drive MHC dephosphorylation. We report here gene targeting and cell biological studies addressing the roles of candidate PP2A B regulatory subunits (phr2aBα and phr2aBβ) in myosin II assembly control in vivo. Dictyostelium phr2aBα- and phr2aBβ-null cells show delayed development, reduction in the assembly of myosin II in cytoskeletal ghost assays, and defects in cytokinesis when grown in suspension compared to parental cell lines. These results demonstrate that the PP2A B subunits phr2aBα and phr2aBβ contribute to myosin II assembly control in vivo, with phr2aBα having the predominant role facilitating MHC dephosphorylation to facilitate filament assembly.     

Molecular characterization of the B' regulatory subunit gene family of Arabidopsis protein phosphatase 2A.

Type 2A serine/threonine protein phosphatases (PP2A) have been implicated as important mediators of a diverse array of reversible protein phosphorylation events in plants. We have identified a novel Arabidopsis gene (AtB' delta) which encodes a 55-kDa B' type regulatory subunit of PP2A. The protein encoded by this gene is 57-63% identical and 69-74% similar to the previously identified AtB' genes. The AtB' delta gene appears to be expressed in all Arabidopsis organs indicating its protein product has a basic housekeeping function in plant cells. Unlike certain mRNAs derived from the AtB' gamma gene, AtB' delta mRNAs do not fluctuate significantly in response to heat stress. Further analysis of cDNA sequences derived from the AtB' genes identified an alternatively spliced cDNA derived from AtB' gamma. This cDNA differs from the previously identified AtB' gamma cDNA by the absence of a 133-bp region in its 5' untranslated region. The missing 133-bp region appears to constitute an unspliced intron and its presence in the AtB' gamma gene was confirmed by PCR using Arabidopsis genomic DNA as a template. AtB' gamma mRNA containing the 133-bp intron accumulate in all Arabidopsis organs and their levels fluctuate differentially in response to heat stress. The 133-bp insert contains two short open reading frames and hence might serve as a translational control mechanism affecting AtB' gamma protein synthesis. Finally we show, using both the yeast two hybrid system and in vitro binding assays, that the B' subunit of Arabidopsis PP2A is able to associate with other PP2A subunits, supporting the notion that the B' protein serves as a regulator of PP2A activity in plants.     

蛋白磷酸酶2A的亚基功能

蛋白磷酸酶2A(protein phosphatase 2A,PP2A)是一种由催化亚基C、结构亚基A和多种功能特异的调节亚基B组成的全酶复合物,其在基因表达、细胞增殖分化和信号转导等方面有重要调控作用.各种不同亚基组成功能各异的PP2A全酶,调控不同的细胞功能.各亚基在PP2A功能调控中均起关键作用.本文重点介绍PP2A各个亚基在PP2A生物学功能实现中的作用.     

Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits

Phosphoprotein phosphatase 2A (PP2A) is a major phosphoserine/threonine protein phosphatase in all eukaryotes. It has been isolated as a heterotrimeric holoenzyme composed of a 65 kDa A subunit, which serves as a scaffold for the association of the 36 kDa catalytic C subunit, and a variety of B subunits that control phosphatase specificity. The C subunit is reversibly methyl esterified by specific methyltransferase and methylesterase enzymes at a completely conserved C-terminal leucine residue. Here we show that methylation plays an essential role in promoting PP2A holoenzyme assembly and that demethylation has an opposing effect. Changes in methylation indirectly regulate PP2A phosphatase activity by controlling the binding of regulatory B subunits to AC dimers.     

Overlapping binding sites in protein phosphatase 2A for association with regulatory A and alpha-4 (mTap42) subunits

Diverse functions of protein Ser/Thr phosphatases depend on the distribution of the catalytic subunits among multiple regulatory subunits. In cells protein phosphatase 2A catalytic subunit (PP2Ac) mostly binds to a scaffold subunit (A subunit or PR65); however, PP2Ac alternatively binds to alpha-4, a subunit related to yeast Tap42 protein, which also associates with phosphatases PP4 or PP6. We mapped alpha-4 binding to PP2Ac to the helical domain, residues 19-165. We mutated selected residues and transiently expressed epitope-tagged PP2Ac to assay for association with A and alpha-4 subunits by co-precipitation. The disabling H118N mutation at the active site or the presence of the active site inhibitor microcystin-LR did not interfere with binding of PP2Ac to either the A subunit or alpha-4, showing that these are allosteric regulators. Positively charged side chains Lys(41), Arg(49), and Lys(74) on the back surface of PP2Ac are unique to PP2Ac, compared with phosphatases PP4, PP6, and PP1. Substitution of one, two, or three of these residues with Ala produced a progressive loss of binding to the A subunit, with a corresponding increase in binding to alpha-4. Conversely, mutation of Glu(42) in PP2Ac essentially eliminated PP2Ac binding to alpha-4, with an increase in binding to the A subunit. Reciprocal changes in binding because of mutations indicate competitive distribution of PP2Ac between these regulatory subunits and demonstrate that the mutated catalytic subunits retained a native conformation. Furthermore, neither the Lys(41)-Arg(49)-Lys(74) nor Glu(42) mutations affected the phosphatase-specific activity or binding to microcystin-agarose. Binding of PP2Ac to microcystin and to alpha-4 increased with temperature, consistent with an activation energy barrier for these interactions. Our results reveal that the A subunit and alpha-4 (mTap42) require charged residues in separate but overlapping surface regions to associate with the back side of PP2Ac and modulate phosphatase activity.     

Protein phosphatase 2A carboxymethylation and regulatory B subunits differentially regulate mast cell degranulation

Asthma is characterised by antigen-mediated mast cell degranulation resulting in secretion of inflammatory mediators. Protein phosphatase 2A (PP2A) is a serine/threonine protein phosphatase composed of a catalytic (PP2A-C) subunit together with a core scaffold (PP2A-A) subunit and a variable, regulatory (PP2A-B) subunit. Previous studies utilising pharmacological inhibition of protein phosphatases have suggested a positive regulatory role for PP2A in mast cell degranulation. In support of this we find that a high okadaic acid concentration (1 μM) inhibits mast cell degranulation. Strikingly, we now show that a low concentration of okadaic acid (0.1 μM) has the opposite effect, resulting in enhanced degranulation. Selective downregulation of the PP2A-Cα subunit by short hairpin RNA also enhanced degranulation of RBL-2H3 mast cells, suggesting that the primary role of PP2A is to negatively regulate degranulation. PP2A-B subunits are responsible for substrate specificity, and carboxymethylation of the PP2A-C subunit alters B subunit binding. We show here that carboxymethylation of PP2A-C is dynamically altered during degranulation and inhibition of methylation decreases degranulation. Moreover downregulation of the PP2A-Bα subunit resulted in decreased MK2 phosphorylation and degranulation, whilst downregulation of the PP2A-B′δ subunit enhanced p38 MAPK phosphorylation and degranulation. Taken together these data show that PP2A is both a positive and negative regulator of mast cell degranulation, and this differential role is regulated by carboxymethylation and specific PP2A-B subunit binding.