PP2A Regulatory Subunit Research Articles

Corrigendum: Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

Corrigendum: Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

β-Adrenergic regulation of cardiac type 2A protein phosphatase through phosphorylation of regulatory subunit B56δ at S573

BackgroundType 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ. Methods and resultsPhosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the β1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. ConclusionsIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.

PP2A Regulatory Subunit B55γ is a Gatekeeper of Osteoblast Maturation and Lineage Maintenance.

Bone marrow mesenchymal stem cells (BM-MSCs) mediate skeletal remodeling by differentiating into osteoblasts. However, this remodeling is impaired with aging as well as following long-term glucocorticoid treatment, resulting in osteoporosis. In this study, we report a novel factor of osteoblast differentiation-PP2A regulatory subunit B55γ. We show that B55γ is induced by glucocorticoid receptor (GR) in human primary BM-MSCs during differentiation to osteoblast, but not to adipocytes, and is required for osteoblast morphogenesis and mineralization. Moreover, B55γ knockdown under osteogenic conditions leads to the accumulation of lipid droplets in a subset of BM-MSCs. Treatment of BM-MSCs with only GR ligand dexamethasone induces B55γ transcript, but not protein, and causes widespread generation of lipid droplets. These data indicate that B55γ is an essential factor of osteoblast lineage, acting as a gatekeeper downstream of master regulators. This opens a new direction of research for understanding mechanisms of glucocorticoid-induced osteoporosis and bone marrow adiposity.

Abstract 5762: PR55-alpha associated PP2A promotes pancreatic tumorigenesis

Abstract PP2A holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Loss of function analysis using PP2A catalytic inhibitors or inhibition via tumor viral proteins suggest PP2A as a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α, a PP2A regulatory subunit, in pancreatic cancer cells compared to normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared to adjacent normal pancreatic tissues (P<0.0001) and correlated with poor survival of pancreatic cancer patients (P<0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cells resulted in diminished phosphorylation of both AKT and ERK1/2 and decreased protein levels of β-catenin. Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice showed a marked reduction in tumorigenicity (P<0.001) and distant metastases. These results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK and Wnt. Citation Format: Ying Yan, Ashley L. Hein, Parthasarathy Seshacharyulu, Satyanarayana Rachagani, Yuri M. Sheinin, Michel M. Ouellette, Moorthy P. Ponnusamy, Surinder K. Batra. PR55-alpha associated PP2A promotes pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5762. doi:10.1158/1538-7445.AM2017-5762

Insufficient activation of Akt upon reperfusion because of its novel modification by reduced PP2A-B55α contributes to enlargement of infarct size by chronic kidney disease.

Chronic kidney disease (CKD) increases myocardial infarct size by an unknown mechanism. Here we examined the hypothesis that impairment of protective PI3K-PDK1-Akt and/or mTORC-Akt signaling upon reperfusion contributes to CKD-induced enlargement of infarct size. CKD was induced in rats by 5/6 nephrectomy (SNx group) 4weeks before myocardial infarction experiments, and sham-operated rats served as controls (Sham group). Infarct size as a percentage of area at risk after ischemia/reperfusion was significantly larger in the SNx group than in the Sham group (56.3±4.6 vs. 41.4±2.0%). In SNx group, myocardial p-Akt-Thr308 level at baseline was elevated, and reperfusion-induced phosphorylation of p-Akt-Ser473, p-p70s6K and p-GSK-3β was significantly suppressed. Inhibition of Akt-Ser473 phosphorylation upon reperfusion by Ku-0063794 significantly increased infarct size in the Sham group but not in the SNx group. There was no difference between the two groups in activities of mTORC2 and PDK1 and protein level of PTEN. However, the PP2A regulatory subunit B55α, which specifically targets Akt-Thr308, was reduced by 24% in the SNx group. Knockdown of B55α by siRNA increased baseline p-Akt-Thr308 and blunted Akt-Ser473 phosphorylation in response to insulin-like growth factor-1 (IGF-1) in H9c2 cells. A blunted response of Akt-Ser473 to IGF-1 was also observed in HEK293 cells transfected with a p-Thr308-mimetic Akt mutant (T308D). These results indicate that increased Akt-Thr308 phosphorylation by down-regulation of B55α inhibits Akt-Ser473 phosphorylation upon reperfusion in CKD and that the impaired Akt activation by insufficient Ser473 phosphorylation upon reperfusion contributes to infarct size enlargement by CKD.

Identification of manganese-toxicity-responsive genes in roots of two citrus species differing in manganese tolerance using cDNA-AFLP

We identified more Mn-toxicity-responsive genes from Mn-intolerant Citrus grandis than from Mn-tolerant Citrus sinensis roots. These findings increased our understanding of the molecular mechanisms on plant Mn toxicity and Mn tolerance. Manganese (Mn) toxicity is the most important factor limiting crop production after aluminum toxicity in acidic soils. However, little is known about Mn-toxicity-induced alterations of gene expression profiles in woody plants. Using cDNA-AFLP, we identified 87 and 63 Mn-toxicity-responsive genes from Mn-intolerant ‘Sour pummelo’ (Citrus grandis) and Mn-tolerant ‘Xuegan’ (Citrus sinensis) roots. Among these genes, only 22 genes with the same accession number were shared by both. Protein phosphorylation/dephosphorylation-related genes were upregulated in C. sinensis roots, and downregulated in C. grandis roots except for one differentially expressed gene. Sulfur metabolism-related genes were repressed only in Mn-toxic C. grandis roots. Obviously, great differences existed in Mn-toxicity-induced alterations of gene expression profiles between C. sinensis and C. grandis roots. Genes related to protein phosphorylation/dephosphorylation (i.e., cyclin-dependent kinase-activating kinase assembly factor-related protein and PP2A regulatory subunit TAP46), cellular transport (i.e., Ca-transporting ATPase 1), and nucleic acid (i.e., ethylene-responsive transcription factor ERF109-like, structural maintenance of chromosomes protein 4-like, RNA-binding protein and DEAD-box ATP-dependent RNA helicase 21), cell wall (i.e., pectin methylesterase 1 and invertase/pectin methylesterase inhibitor family protein) and fatty acid (i.e., carboxylesterase 20) metabolisms might play a role in C. sinensis Mn tolerance. In addition, cell wall materials were increased in Mn-toxic C. sinensis and C. grandis roots, especially in the former. Interestingly, lignin content was increased in Mn-toxic C. sinensis roots, while a reverse trend was displayed in Mn-toxic C. grandis roots. In conclusion, our results provided novel clues to the molecular mechanisms on Mn toxicity and Mn tolerance in higher plants.

Protein phosphatase 2A regulatory subunit B56β modulates erythroid differentiation

Anemia due to attenuated erythroid terminal differentiation is one of the most common hematological disorders occurring at all stages of life. We previously demonstrated that catalytic subunit α of protein phosphatase 2A (PP2Acα) modulates fetal liver erythropoiesis. However the corresponding PP2A regulatory subunit in this process remains unknown. In this study, we report that chemical inhibition of PP2A activity with okadaic acid impairs hemin-induced erythroid differentiation. Interestingly, B56 family member B56β is the only regulatory subunit whose expression is induced by both erythropoietin in fetal liver cells and hemin in erythroleukemia K562 cells. Finally, knockdown of B56β attenuates hemin-induced K562 erythroid differentiation. Collectively, our data identify B56β as the potential functional regulatory subunit of PP2A in erythroid differentiation, shedding light on new target for precise modulation of PP2A activity for treatment of anemia and related diseases.

Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.

Septins are a conserved family of GTPases that form hetero–oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspBmt-T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A. fumigatus hyphal septation.

PR55α Subunit of Protein Phosphatase 2A Supports the Tumorigenic and Metastatic Potential of Pancreatic Cancer Cells by Sustaining Hyperactive Oncogenic Signaling.

The protein phosphatase 2 (PP2A) holoenzyme consists of a catalytic subunit, a scaffold subunit, and a regulatory subunit. Based on loss-of-function analysis using PP2A catalytic inhibitors or inhibition via tumor viral antigens, limited studies suggest that PP2A is a putative tumor suppressor. However, PP2A has also been shown to facilitate the activation of oncogenic signaling pathways when associated with specific regulatory subunits. In this study, we investigated the possible oncogenic role of PP2A in pancreatic cancer. We found a striking increase in the expression of PR55α (PPP2R2A), a PP2A regulatory subunit, in pancreatic cancer cells compared with normal pancreatic epithelial cells. Consistently, PR55α expression was markedly elevated in pancreatic ductal adenocarcinoma tissues compared with adjacent normal pancreatic tissues (P < 0.0001) and correlated with poor survival of pancreatic cancer patients (P < 0.0003). RNAi-mediated depletion of PR55α in pancreatic cancer cell lines resulted in diminished phosphorylation of both AKT and ERK1/2 (MAPK3/1) and decreased protein levels of β-catenin (CTNNB1). Accordingly, pancreatic cancer cells with reduced PR55α expression exhibited significantly impaired properties of transformation, including attenuated cell growth, clonogenicity, mobility, and anchorage-independent growth. Moreover, orthotopic implantation of PR55α-depleted pancreatic cancer cells into nude mice resulted in markedly reduced tumorigenicity (P < 0.001) and distant metastases. Together, these results suggest that PR55α promotes pancreatic cancer development by sustaining hyperactivity of multiple oncogenic signaling pathways, including AKT, ERK, and Wnt. These studies also provide a basis for exploring PR55α as a diagnostic or therapeutic target in pancreatic cancer. Cancer Res; 76(8); 2243-53. ©2016 AACR.

Abstract 17404: Intramolecular Inhibition of Ser473 Phosphorylation by Up-regulated Thr308 Phosphorylation in Akt Contributes to Enlargement of Infarct Size by Chronic Renal Failure

Objective: We previously showed that chronic renal failure (CRF) enlarges infarct size after ischemia/reperfusion (I/R) through impairment of reperfusion-induced Akt activation. Here we examined molecular mechanisms by which CRF impairs Akt activation, focusing on interplay between two phosphorylation sites in Akt, Thr308 and Ser473. Methods and results: Rats received 5/6 nephrectomy (SNx) or a sham operation (Sham). Development of CRF in SNx was confirmed by a 2.2-fold increase in serum creatinine level at 4 weeks after the operation. Infarct size after 20-min coronary occlusion/2-hr reperfusion was larger in SNx than in Sham (53.2±5.8 vs. 41.1±2.4% of risk area, p&lt;0.05), indicating impaired tolerance against I/R injury in CRF. Immunoblot analyses revealed that the level of phospho-Akt-Thr308 was higher by 375% in SNx than in Sham under baseline conditions, while the levels of phospho-Akt-Ser473 were similar in the two groups. Upon reperfusion, phosphorylation of Ser473 and that of Thr308 were similarly enhanced in Sham. However, reperfusion-induced phosphorylation of Ser473, but not that of Thr308, was blunted by 32.8% in SNx compared with that in Sham. Reduction in Akt activity after reperfusion in SNx was indicated by lower levels of phospho-p70S6K and phospho-GSK3β compared with those in Sham. The levels of PTEN, a negative regulator of Akt, and PDK1, a promoter of Thr308 phosphorylation, were similar in SNx and Sham. However, the level of PP2A regulatory subunit B55α, which specifically dephosphorylates Thr308, was lower by 23.6% in SNx than in Sham. To determine whether the constitutive phosphorylation of Thr308 mitigates reperfusion-induced phosphorylation of Ser473, HA-tagged wild-type Akt (HA-WT) and phospho-Thr308-mimetic mutant Akt (HA-T308D) were transiently transfected into HEK293 cells. IGF-1 (10 nmol/L)-induced phosphorylation of Ser473 in HA-T308D was lower by 30.5% than that in HA-WT, whereas that in endogenous Akt at Ser473 was comparable between cells overexpressing HA-T308D and HA-WT. Conclusion: Insufficient activation of Akt by its intramolecular interplay between Thr308 and Ser473 phosphorylation due to down-regulated B55α contributes to increased myocardial susceptibility to I/R injury in CRF.

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 Nspl 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.

The Protein Phosphatase 2A Regulatory Subunit B56γ Mediates Suppression of T Cell Receptor (TCR)-induced Nuclear Factor-κB (NF-κB) Activity

NF-κB is an important transcription factor in the immune system, and aberrant NF-κB activity contributes to malignant diseases and autoimmunity. In T cells, NF-κB is activated upon TCR stimulation, and signal transduction to NF-κB activation is triggered by a cascade of phosphorylation events. However, fine-tuning and termination of TCR signaling are only partially understood. Phosphatases oppose the role of kinases by removing phosphate moieties. The catalytic activity of the protein phosphatase PP2A has been implicated in the regulation of NF-κB. PP2A acts in trimeric complexes in which the catalytic subunit is promiscuous and the regulatory subunit confers substrate specificity. To understand and eventually target NF-κB-specific PP2A functions it is essential to define the regulatory PP2A subunit involved. So far, the regulatory PP2A subunit that mediates NF-κB suppression in T cells remained undefined. By performing a siRNA screen in Jurkat T cells harboring a NF-κB-responsive luciferase reporter, we identified the PP2A regulatory subunit B56γ as negative regulator of NF-κB in TCR signaling. B56γ was strongly up-regulated upon primary human T cell activation, and B56γ silencing induced increased IκB kinase (IKK) and IκBα phosphorylation upon TCR stimulation. B56γ silencing enhanced NF-κB activity, resulting in increased NF-κB target gene expression including the T cell cytokine IL-2. In addition, T cell proliferation was increased upon B56γ silencing. These data help to understand the physiology of PP2A function in T cells and the pathophysiology of diseases involving PP2A and NF-κB.

An In Vivo Screen Implicates PPP2R2D as an Inhibitor of T-Cell Function

The PP2A regulatory subunit PPP2R2D inhibits T-cell proliferation and survival within tumors.

Centromere Binding and a Conserved Role in Chromosome Stability for SUMO-Dependent Ubiquitin Ligases

The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.

Immunoproteomics of HER2-Positive and HER2-Negative Breast Cancer Patients with Positive Lymph Nodes

Identification of more and more novel tumor antigens and autoantibodies will lead to the earlier diagnosis, better prognosis prediction, and more efficient therapy of cancer in the future. Immunoproteomics techniques have successfully been used for finding novel cancer biomarkers in different subgroups of cancer patients. HER2 is a marker for an aggressive breast cancer, particularly in node-positive (NP) cases. The aim of our study was to identify antigens eliciting a humoral immune response in HER2+ and HER2- NP breast cancers by two-dimensional electrophoresis (2D), Western blotting, and mass spectrometry. Sera from 18 women with newly diagnosed NP breast cancer (9 HER2+ and 9 HER2-) and 9 healthy volunteers were individually investigated for the presence of antibodies to MCF7 breast cancer cell line proteome. Reactive spots in 2D blots were matched to stained 2D gels. Twenty-eight of matched spots were identified by mass spectrometry. Among them were LDH-A, glyceraldehydes-3-phosphate dehydrogenase, enolase-α, phosphoglycerate dehydrogenase, proteasome 26S non-ATPase subunit 13, triosephosphate isomerase, hnRNP K, hsp27, hsp90, prohibitin, nucleophosmin, 14-3-3ɛ, PP2A regulatory subunit, and ribonuclease inhibitor-angiogenin. The five former antigens were more commonly reacted with sera from HER2+ cases, and the three latter antigens were more commonly reacted with sera from HER2- cases. Noteworthy, the antigenicity of the 28 spots showed a few differences when SBR3 cell line was used as the source of antigens. Although some of the identified antigens were previously defined as tumor antigens, others were novel. Further investigations for their utilizations as markers for breast cancer diagnosis, progression, and therapy are warranted.

Clk2 and B56β Mediate Insulin-Regulated Assembly of the PP2A Phosphatase Holoenzyme Complex on Akt

Akt mediates important cellular decisions involved in growth, survival, and metabolism. The mechanisms by which Akt is phosphorylated and activated in response to growth factors or insulin have been extensively studied, but the molecular regulatory components and dynamics of Akt attenuation are poorly understood. Here we show that a downstream target of insulin-induced Akt activation, Clk2, triggers Akt dephosphorylation through the PP2A phosphatase complex. Clk2 phosphorylates the PP2A regulatory subunit B56β (PPP2R5B, B'β), which is a critical regulatory step in the assembly of the PP2A holoenzyme complex on Akt leading to dephosphorylation of both S473 and T308 Akt sites. Since Akt plays a pivotal role in cellular signaling, these results have important implications for our understanding of Akt regulation in many biological processes.

The PP2A Regulatory Subunit Tap46, a Component of the TOR Signaling Pathway, Modulates Growth and Metabolism in Plants

Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway.

Dephosphorylation of Carma1 by PP2A negatively regulates T-cell activation

The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane. We have identified the serine-threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.

Phosphorylation/dephosphorylation of human SULT4A1: Role of Erk1 and PP2A

SULT4A1 is a cytosolic sulfotransferase that shares little homology with other human sulfotransferases but is highly conserved between species. It is found in neurons located in several regions of the brain. Recently, the stability of SULT4A1 was shown to be regulated by Pin1, a peptidyl-prolyl cis-trans isomerase implicated in several neurodegenerative diseases. Since Pin1 binds preferentially to phosphoproteins, these findings suggested that SULT4A1 is post-translationally modified. In this study, we show that the Thr 11 residue of SULT4A1, which is involved in Pin1 binding is phosphorylated. MEK inhibition was shown to abolish Pin1 mediated degradation of SULT4A1 while in vitro phosphorylation assays using alanine substitution mutants of SULT4A1 demonstrated phosphorylation of Thr 11 by ERK1. We also show that dephosphorylation was catalyzed by the protein phosphatase 2A. The PP2A regulatory subunit, Bβ was identified from a yeast-2-hybrid screen of human brain cDNA as a SULT4A1 interacting protein. This was further confirmed by GST pull-downs and immunoprecipitation. Other members of the B subunit (αδγ) did not interact with SULT4A1. Taken together, these studies indicate that SULT4A1 stability is regulated by post-translational modification that involves the ERK pathway and PP2A. The phosphorylation of SULT4A1 allows interaction with Pin1, which then promotes degradation of the sulfotransferase.

Does Protein Kinase A–Mediated Phosphorylation of the Cardiac Ryanodine Receptor Play Any Role in Adrenergic Regulation of Calcium Handling in Health and Disease?

See related articles, pages 1743–1752 Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative …