The structure of the PP2A regulatory subunit B56 gamma: the remaining piece of the PP2A jigsaw puzzle

The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56 gamma. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56 gamma and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56 gamma first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly.     

含B56调节亚基的PP2A全酶在肿瘤信号通路中的调控作用

蛋白磷酸酶2A（protein phosphatase 2A,PP2A）是细胞中广泛表达的异三聚体全酶,调节许多重要的信号通路,它的表达异常所致的信号通路紊乱会引发肿瘤和促进肿瘤的发展.PP2A在特定的状态下能够发挥抑癌因子的作用,这种抑癌特性由B调节亚基与底物的相互作用来决定,因此B调节亚基在PP2A的抑癌功能中起关键作用.     

PP2A:B56epsilon is required for eye induction and eye field separation

Eye induction and eye field separation are the earliest events during vertebrate eye development. Both of these processes occur much earlier than the formation of optic vesicles. The insulin-like growth factor (IGF) pathway appears to be essential for eye induction, yet it remains unclear how IGF downstream pathways are involved in eye induction. As a consequence of eye induction, a single eye anlage is specified in the anterior neural plate. Subsequently, this single eye anlage is divided into two symmetric eye fields in response to Sonic Hedgehog (Shh) secreted from the prechordal mesoderm. Here, we report that B56epsilon regulatory subunit of protein phosphatase 2A (PP2A) is involved in Xenopus eye induction and subsequent eye field separation. We provide evidence that B56epsilon is required for the IGF/PI3K/Akt pathway and that interfering with the PI3K/Akt pathway inhibits eye induction. In addition, we show that B56epsilon regulates the Hedgehog (Hh) pathway during eye field separation. Thus, B56epsilon is involved in multiple signaling pathways and plays critical roles during early development.     

Silencing of the PP2A catalytic subunit causes HER-2/neu positive breast cancer cells to undergo apoptosis

Protein phosphatase 2A (PP2A), in its activated form as a phosphatase, is a tumour suppressor. However, when PP2A is phosphorylated at the tyrosine residue (pY307), it loses its phosphatase activity and becomes inactivated. In our previous study, we found a higher expression of pY307-PP2A in HER-2/neu positive breast tumour samples and significantly correlated to tumour progression, and in this context, it could function as a proto-oncogene. The above and subsequent findings led us to postulate that the critical role of PP2A in maintaining the balance between cell survival and cell death may be linked to its phosphorylation status at its Y307 residue. Hence, we further investigated the effects of knocking down the PP2A catalytic subunit which contains the Y307 amino acid residue in two HER-2/neu positive breast cancer cell lines, BT474 and SKBR3. We showed that this causes the silenced HER-2/neu breast cancer cells to undergo apoptosis and furthermore, that such apoptosis is mediated by p38 MAPK-caspase 3/PARP activation. Understanding the role of PP2A in HER2/neu positive cells might thus provide insight into new targets for breast cancer therapy.     

A highly conserved pocket on PP2A‐B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.     

Determination of serine/threonine protein phosphatase type 2B PP2B in lymphocytes by HPLC

Current cyclosporin (CsA) and tacrolimus (FK506) monitoring methods are based on blood concentrations determination, but the optimal sampling times are not clearly defined. An alternative pharmacodynamic approach has recently been introduced, based on assaying lymphocytes activity of calcineurin (PP2B), a phosphatase that is partially inhibited by CsA and FK506. However, the princeps method uses large amounts of radioactive [32P]substrate, raising a number of safety issues. Here we describe an alternative method for PP2B activity determination, based on HPLC coupled with spectrophotometric detection. A 19-amino-acid peptide is phosphorylated by a protein kinase, and further dephosphorylated by lymphocyte PP2B in the presence of okadaic acid. The two peptides are separated by using reverse-phase chromatography with a detection wavelength of 205 nm. After lymphocyte isolation by density-gradient centrifugation, PP2B activity is derived from the dephosphorylated peptide concentration measured during the first 6 min of the enzymatic reaction. This technique gives reproducible results and good analytical sensitivity with 10(6) lymphocytes. With an average isolation rate of 59.6%, only 7 ml of blood is required, making the method suitable for lymphopenic patients. Moreover, PP2B activity is stable in blood samples kept for 24h at room temperature and in isolated lymphocytes stored for 48 h at -20 degrees C. We validated the method by comparing median PP2B activity in 10 healthy volunteers (285.0+/-46.5 pmol/min/10(6)lymphocytes) and in 10 liver transplant patients (147.8+/-71.0 pmol/min/10(6)lymphocytes) (p<0.001). The relation between calcineurin activity and tacrolimus blood concentrations was also studied.     

Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.     

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30

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Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α: ROLE OF B56α IN NUCLEAR EXPORT OF THE CATALYTIC SUBUNIT*

Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.     

Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down‐regulation of BmPP2A expression. Furthermore, up‐regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down‐regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti‐BmNPV mechanism, and a possible target for the breeding of silkworm‐resistant strains.     

PP2A function toward mitotic kinases and substrates during the cell cycle

To maintain cellular homeostasis against the demands of the extracellular environment, a precise regulation of kinases and phosphatases is essential. In cell cycle regulation mechanisms, activation of the cyclin-dependent kinase (CDK1) and cyclin B complex (CDK1:cyclin B) causes a remarkable change in protein phosphorylation. Activation of CDK1:cyclin B is regulated by two auto-amplification loops-CDK1:cyclin B activates Cdc25, its own activating phosphatase, and inhibits Wee1, its own inhibiting kinase. Recent biological evidence has revealed that the inhibition of its counteracting phosphatase activity also occurs, and it is parallel to CDK1:cyclin B activation during mitosis. Phosphatase regulation of mitotic kinases and their substrates is essential to ensure that the progression of the cell cycle is ordered. Outlining how the mutual control of kinases and phosphatases governs the localization and timing of cell division will give us a new understanding about cell cycle regulation. [BMB Reports 2013; 46(6): 289-294]     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

Oncoprotein CIP2A is stabilized via interaction with tumor suppressor PP2A/B56

Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two‐hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N‐terminal CIP2A fragment (amino acids 1–560) at 3.0 Å resolution, and by subsequent structure‐based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N‐terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure–function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy.     

Increased activity of phosphatase PP2A in the presence of the PlA2 polymorphism of alphaIIbbeta3

Polymorphisms in αIIbβ3 are important genetic factors that alter platelet biology and have been associated with susceptibility to thromboembolic disorders. To define the molecular mechanisms that lead to variance in thrombotic diathesis dictated by the β3 polymorphism, we examined regulation of intracellular signaling by αIIbβ3, and studied the effects of a common β subunit PlA2 polymorphism. We found that PP2A regulates αIIbβ3 control of the ERK signaling in a polymorphism specific fashion. In CHO cells, exogenous expression of αIIbβ3 reduced ATP-stimulated ERK phosphorylation and more so for PlA2 than PlA1. Interestingly, reduced level of ERK phosphorylation correlated with an increase in PP2A activity, with higher activity associated with PlA2 than PlA1. We tested the effect of PP2A on αIIbβ3-dependent adhesion, and found that PP2A overexpression increased cell adhesion, while phosphatase inhibitors decreased cell adhesion. We propose that PlA2 alters cell signaling at least in part by increasing β3-associated PP2A activity.     

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.