Oxidative Inhibition of Protein Phosphatase 2A Activity: Role of Catalytic Subunit Disulfides

A molecular basis for the inhibition of brain protein phosphatase 2A (PP2A) activity by oxidative stress was examined in a high-speed supernatant (HSS) fraction from rat cerebral cortex. PP2A activity was subject to substantial disulfide reducing agent-reversible inhibition in the HSS fraction. Results of gel electrophoresis support the conclusions that inhibition of PP2A activity was associated with the both the disulfide cross-linking of the catalytic subunit (PP2A_C) of the enzyme to other brain proteins and with the formation of an apparent novel intramolecular disulfide bond in PP2A_C. Additional findings that the vicinal dithiol cross-linking reagent phenylarsine oxide (PAO) produced a potent dithiothreitol-reversible inhibition of PP2A activity suggest that the cross-linking of PP2A_C vicinal thiols to form an intramolecular disulfide bond may be sufficient to inhibit PP2A activity under oxidative stress. We propose that the dithiol–disulfide equilibrium of a vicinal thiol pair of PP2A_C may confer redox sensitivity on cellular PP2A.     

Binding Specificity of Protein Phosphatase 2A Core Enzyme for Regulatory B Subunits and T Antigens

The core enzyme of protein phosphatase 2A is composed of a regulatory subunit A and a catalytic subunit C. It is controlled by three types of regulatory B subunits (B, B′, and B") and by tumor (T) antigens, which are unrelated by sequence but bind to overlapping regions on the A subunit. To find out whether the different B subunits and T antigens bind to identical or distinct amino acids of the A subunit, mutants were generated and their abilities to bind B subunits and T antigens were tested. We found that some amino acids are involved in the binding of all types of B subunits, whereas others are specifically involved in the binding of one or two types of B subunits. T-antigen-binding specificity does not correlate with that of a particular type of B subunit.     

Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation

The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.     

A Protein Phosphatase 2A Catalytic Subunit Modulates Blue Light-Induced Chloroplast Avoidance Movements through Regulating Actin Cytoskeleton in Arabidopsis

Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.     

A High-Affinity Binding Protein for the Regulatory Subunit of cAMP-Dependent Protein Kinase II in the Centrosome of Human Cells

In the human lymphoblastic cell line KE 37, Northern blot analysis with cDNA probes for human regulatory subunits RIIα and RIIβ of the cAMP-dependent protein kinase (A-kinase) type II and immunoblotting or immunoprecipitation studies with several antibodies directed against RIIα and RIIβ show that these two isoforms are expressed. The major isoform α is mostly cytosolic, whereas the β isoform appears concentrated in the Golgi-centrosomal area, as judged by immunofluorescence and cell fractionation. Using a ³²P-labeled RII overlay on Western blots, a 350-kDa RII-binding protein (AKAP 350) was specifically identified in centrosomes isolated from this cell line, whereas a Golgi fraction has previously been demonstrated to contain an 85-kDa RII-binding protein (AKAP 85). AKAP 350 is highly insoluble and can partially be extracted from centrosomes as a complex of AKAP 350 and RII subunit. AKAP 350 was identified as a specific centrosomal protein previously demonstrated in the pericentriolar material. The potential significance of a specific subcellular distribution for different RII-binding proteins in nonneuronal cells is discussed.     

The Ser/Thr Phosphatase PP2A Regulatory Subunit Widerborst Inhibits Notch Signaling

Drosophila Enhancer of split M8, an effector of Notch signaling, is regulated by protein kinase CK2. The phosphatase PP2A is thought to play an opposing (inhibitory) role, but the identity of the regulatory subunit was unknown. The studies described here reveal a role for the PP2A regulatory subunit widerborst (wdb) in three developmental contexts; the bristle, wing and the R8 photoreceptors of the eye. wdb overexpression elicits bristle and wing defects akin to reduced Notch signaling, whereas hypomorphic mutations in this PP2A subunit elicit opposite effects. We have also evaluated wdb functions using mutations in Notch and E(spl) that affect the eye. We find that the eye and R8 defects of the well-known N^spl mutation are enhanced by a hypomorphic allele of wdb, whereas they are strongly rescued by wdb overexpression. Similarly, ectopic wdb rescues the eye and R8 defects of the E(spl)D mutation, which affects the m8 gene. In addition, wdb overexpression also rescues the bristle defects of ectopically expressed M8, or the eye and R8 defects of its CK2 phosphomimetic variant M8-S159D. The latter finding suggests that PP2A may target M8 at highly conserved residues in the vicinity of the CK2 site, whose phosphorylation controls repression of Atonal and the R8 fate. Together, the studies identify PP2A-Wdb as a participant in Notch signaling, and suggest that M8 activity is controlled by phosphorylation and dephosphorylation. The conservation of the phosphorylation sites between Drosophila E(spl) and the HES/HER proteins from mammals, reptiles, amphibians, birds and fish raises the prospect that this mode of regulation is widespread.     

Visualization of Subunit Interactions and Ternary Complexes of Protein Phosphatase 2A in Mammalian Cells

Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.     

Identification and Functional Characterization of Inhibitor-3, a Regulatory Subunit of Protein Phosphatase 1 in Plants

Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine protein phosphatase, and mediates diverse cellular processes in animal systems via the association of a catalytic subunit (PP1c) with multiple regulatory subunits that determine the catalytic activity, the subcellular localization, and the substrate specificity. However, no regulatory subunit of PP1 has been identified in plants so far. In this study, we identified inhibitor-3 (Inh3) as a regulatory subunit of PP1 and characterized a functional role of Inh3 in Vicia faba and Arabidopsis (Arabidopsis thaliana). We found Inh3 as one of the proteins interacting with PP1c using a yeast two-hybrid system. Biochemical analyses demonstrated that Arabidopsis Inh3 (AtInh3) bound to PP1c via the RVxF motif of AtInh3, a consensus PP1c-binding sequence both in vitro and in vivo. AtInh3 inhibited the PP1c phosphatase activity in the nanomolar range in vitro. AtInh3 was localized in both the nucleus and cytoplasm, and it colocalized with Arabidopsis PP1c in these compartments. Disruption mutants of AtINH3 delayed the progression of early embryogenesis, arrested embryo development at the globular stage, and eventually caused embryo lethality. Furthermore, reduction of AtINH3 expression by RNA interference led to a decrease in fertility. Transformation of the lethal mutant of inh3 with wild-type AtINH3 restored the phenotype, whereas that with the AtINH3 gene having a mutation in the RVxF motif did not. These results define Inh3 as a regulatory subunit of PP1 in plants and suggest that Inh3 plays a crucial role in early embryogenesis in Arabidopsis.     

Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.     

Proteomics Analysis of the Cardiac Myofilament Subproteome Reveals Dynamic Alterations in Phosphatase Subunit Distribution

Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were “skinned,” and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56α. Immunoblot analysis of myocyte fractions confirmed that β-adrenergic stimulation by isoproterenol decreased the B56α content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56α from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following β-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.Myofilament proteins comprise the fundamental contractile apparatus of the heart, the cardiac sarcomere. They are subdivided into thin filament proteins, including actin, tropomyosin, the troponin complex (troponin C, troponin I, and troponin T), and thick filament proteins, including myosin heavy chains, myosin light chains, and myosin-binding protein C. Although calcium is the principal regulator of cardiac contraction through the excitation-contraction coupling process that culminates in calcium binding to troponin C, myofilament function is also significantly modulated by phosphorylation of constituent proteins, such as cardiac troponin I (cTnI),1 cardiac myosin-binding protein C (cMyBP-C), and myosin regulatory light chain (MLC-2). “Skinned” myocyte preparations from rodent hearts, in which the sarcolemmal envelope is disrupted through the use of detergents, have been invaluable in providing mechanistic information on the functional consequences of myofilament protein phosphorylation following exposure to neurohormonal stimuli that activate pertinent kinases prior to skinning or direct exposure to such kinases in active form after skinning (for recent examples, see studies on the phosphorylation of cTnI (1–3), cMyBP-C (4–6), and MLC-2 (7–9)). Nevertheless, to date, only a few myofilament proteins have been studied using proteomics (10–19), and a detailed proteomic characterization of the myofilament subproteome and its associated proteins from skinned myocytes has not been performed. In the present analysis, we used an LTQ Orbitrap XL equipped with ETD (20) to analyze the subproteome of skinned cardiomyocytes with or without prior stimulation. Endothelin-1 and isoproterenol were used to activate the endothelin receptor/protein kinase C and β-adrenoreceptor/protein kinase A pathway, respectively (21, 22). Importantly, the mass accuracy of the Orbitrap mass analyzer helped to distinguish true phosphorylation sites from false assignments, and the sensitivity of the ion trap provided novel insights into the translocation of phosphatase regulatory and targeting subunits following β-adrenergic stimulation.     

Mutations in the Saccharomyces Cerevisiae Type 2a Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22(ts) alleles for PP2A function displayed a rapid arrest of proliferation. Ts(-) pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts(-) pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts(-) pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts(-) pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts(-) pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts(-) pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.     

Genetic Interactions between Reg1/Hex2 and Glc7, the Gene Encoding the Protein Phosphatase Type 1 Catalytic Subunit in Saccharomyces Cerevisiae

Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes. Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogen-deficient phenotype of the glc7-1 allele. Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size. However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7(Y-170) allele, lack of growth at 14°. The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SNF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation. We propose that REG1 has a role in controlling glycogen accumulation.     

Classification and Phylogenetic Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit Family

The members of the PKA regulatory subunit family (PKA-R family) were analyzed by multiple sequence alignment and clustering based on phylogenetic tree construction. According to the phylogenetic trees generated from multiple sequence alignment of the complete sequences, the PKA-R family was divided into four subfamilies (types I to IV). Members of each subfamily were exclusively from animals (types I and II), fungi (type III), and alveolates (type IV). Application of the same methodology to the cAMP-binding domains, and subsequently to the region delimited by β-strands 6 and 7 of the crystal structures of bovine RIα and rat RIIβ (the phosphate-binding cassette; PBC), proved that this highly conserved region was enough to classify unequivocally the members of the PKA-R family. A single signature sequence, F–G–E–[LIV]–A–L–[LIMV]–x(3)–[PV]–R–[ANQV]–A, corresponding to the PBC was identified which is characteristic of the PKA-R family and is sufficient to distinguish it from other members of the cyclic nucleotide-binding protein superfamily. Specific determinants for the A and B domains of each R-subunit type were also identified. Conserved residues defining the signature motif are important for interaction with cAMP or for positioning the residues that directly interact with cAMP. Conversely, residues that define subfamilies or domain types are not conserved and are mostly located on the loop that connects α-helix B′ and β strand 7. Received: 2 November 2000/Accepted: 14 June 2001     

Solution Structure of the Oncogenic MIEN1 Protein Reveals a Thioredoxin-Like Fold with a Redox-Active Motif

The novel tumor biomarker MIEN1, identified by representational difference analysis, is overexpressed in breast cancer and prostate cancer. MIEN1 is considered an oncogenic protein, because MIEN1 overexpression functionally enhances migration and invasion of tumor cells via modulating the activity of AKT. However, the structure and molecular function of MIEN1 is little understood. Here, we report the solution structure of MIEN1, which adopts a thioredoxin-like fold with a redox-active motif. Comparison of backbone chemical shifts showed that most of the residues for both oxidized and reduced MIEN1 possessed the same backbone conformation, with differences limited to the active motif and regions in proximity. The redox potential of this disulfide bond was measured as −225 mV, which compares well with that of disulfides for other thioredoxin-like proteins. Overall, our results suggest that MIEN1 may have an important regulatory role in phosphorylation of AKT with its redox potential.     

微囊藻毒素的毒性效应与蛋白磷酸酶2A

蛋白磷酸酶2A(protein phosphatase 2A,PP2A)是蛋白磷酸酶家族的主要成员,在蛋白质可逆磷酸化过程中与蛋白激酶一样起着举足轻重的作用。自然界存在很多天然毒素可特异性地作用于PP2A从而影响体内蛋白质的可逆磷酸化,其中微囊藻毒素由于急性肝毒性和强促癌活性日益引起关注。尽管确切的机制仍未探明,但从目前的研究来看,微囊藻毒素产生毒性的机制可能与其引起细胞氧化应激、DNA损伤、细胞骨架的破坏以及诱导细胞凋亡相关。而PP2A在氧化应激、DNA损伤修复及维持细胞骨架稳态中起着重要作用,并能调控凋亡相关激酶CaMKII和Bcl-2家族蛋白,这对更好地理解微囊藻毒素LR如何通过影响PP2A而产生毒作用提供了新思路。     

豌豆核酮糖1,5-二磷酸羧化酶/加氧酶亚基结合蛋白的纯化及特性

用~(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD光谱具有典型的α-螺旋结构的光谱特性,α-螺旋度为39％。此外,以纯化的豌豆结合蛋白制备了多克隆抗体。琼脂糖双扩散实验显示,结合蛋白的抗体与结合蛋白产生一条沉淀线,而与豌豆的RubisCO无沉淀反应,这表明所得到的抗体是高度专一的。