Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms.

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.     

The structure of Tap42/alpha4 reveals a tetratricopeptide repeat-like fold and provides insights into PP2A regulation

Physiological functions of protein phosphatase 2A (PP2A) are determined via the association of its catalytic subunit (PP2Ac) with diverse regulatory subunits. The predominant form of PP2Ac assembles into a heterotrimer comprising the scaffolding PR65/A subunit together with a variable regulatory B subunit. A distinct population of PP2Ac associates with the Tap42/alpha4 subunit, an interaction mutually exclusive with that of PR65/A. Tap42/alpha4 is also an interacting subunit of the PP2Ac-related phosphatases, PP4 and PP6. Tap42/alpha4, an essential protein in yeast and suppressor of apoptosis in mammals, contributes to critical cellular functions including the Tor signaling pathway. Here, we describe the crystal structure of the PP2Ac-interaction domain of Saccharomyces cerevisiae Tap42. The structure reveals an all alpha-helical protein with striking similarity to 14-3-3 and tetratricopeptide repeat (TPR) proteins. Mutational analyses of structurally conserved regions of Tap42/alpha4 identified a positively charged region critical for its interactions with PP2Ac. We propose a scaffolding function for Tap42/alpha4 whereby the interaction of PP2Ac at its N-terminus promotes the dephosphorylation of substrates recruited to the C-terminal region of the molecule.     

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.     

Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta

Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Acalpha and cbeta in High Five insect cells. The recombinant PP2Acalpha and cbeta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acalpha or cbeta was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aalpha/calpha and Aalpha/cbeta) with PR65alpha/Aalpha. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acalpha and cbeta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.     

Eukaryotic translation termination factor 1 associates with protein phosphatase 2A and targets it to ribosomes

Purification of type 2A protein phosphatase (PP2A) from rabbit skeletal muscle resulted in the isolation of a trimeric phosphatase which is composed of a catalytic (PP2Ac), a structural (PR65alpha/Aalpha), and a regulatory (PR55alpha/Balpha) subunit, together with translation termination factor 1 (eRF1) and another protein of 55 kD (EMBO J., 15, 101-112). Yeast two-hybrid system analysis demonstrated that the eRF1 interacted with PP2Acalpha but not with PR65alpha/Aalpha or PR55alpha/Balpha. The N-terminal region of PP2Acalpha, comprising 50 amino acid residues, and the C-terminal part of eRF1, corresponding to an internal region between amino acids 338-381, were found to be necessary for eRF1--PP2Acalpha interaction in yeast. Immunoprecipitations using 12CA5 antibodies and extracts from COS1 cells transiently transfected with eRF1 tagged with 9-amino acid epitope from influenza hemagglutinin (HA) demonstrated the presence of eRF1--PP2Acalpha--PR65alpha/Aalpha complex in these cells. In addition, polysomes obtained from COS1 cells overexpressing HA--eRF1 displayed several-fold higher PP2A activity than control polysomes. No effect of either PP2Ac or dimeric and trimeric PP2A holoenzymes on the rate of translation termination was detected using an in vitro reconstituted translation termination assay. In summary, eRF1 appears to represent a novel PP2A-targeting subunit that brings this phosphatase in contact with putative ribosomal substrate(s). It remains to be established whether termination of translation requires dephosphorylation of participating protein factor(s).     

Endotoxin Conditioning Induces VCP/p97-mediated and Inducible Nitric-oxide Synthase-dependent Tyr284 Nitration in Protein Phosphatase 2A

Endotoxins activate Toll-like receptors and reprogram cells to be refractory to secondary exposure. Here we found that activation of different Toll-like receptors elicited a time- and dose-dependent increase in the levels of the protein phosphatase 2A catalytic subunit (PP2Ac) but not its partner A subunit. We purified the lipopolysaccharide-induced form of PP2A by chromatography plus immunoprecipitation and used mass spectrometry to identify VCP/p97 as a novel partner for PP2Ac. Endogenous VCP/p97 and PP2Ac were co-immunoprecipitated from primary murine macrophages and human lymphocytes. GST-VCP/p97 bound purified PP2A in pulldown assays, showing direct protein-protein interaction. Endotoxin conditioning of macrophages induced formation of 3-nitrotyrosine in the PP2Ac associated with VCP/p97, a response severely reduced in macrophages from iNOS knock-out mice. The reaction of purified PP2A with peroxynitrite dissociated the A subunit, and 3-nitro-Tyr²⁸⁴ was identified in PP2Ac by mass spectrometry. Myc-PP2Ac (Y284F) expressed in cells was resistant to peroxynitrite-induced nitration and reduction of A subunit binding. Transient expression of either VCP/p97 or PP2Ac was sufficient to elevate levels of the dual specificity phosphatase DUSP1, reduce p38 MAPK activation, and suppress tumor necrosis factor-α release. We propose that VCP/p97-mediated Tyr nitration of PP2A increases the levels of phosphatases PP2A and DUSP1 to contribute to the refractory response of conditioned cells.     

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.     

Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression

Protein phosphatase 2A (PP2A) appears to be involved in the regulation of many cellular processes. Control mechanisms that lead to the activation (and deactivation) of the various holoenzymes to initiate appropriate dephosphorylation events remain obscure. The core components of all PP2A holoenzymes are the catalytic (PP2Ac) and 63-65- kD regulatory (PR65) subunits. Monospecific and affinity-purified antibodies against both PP2Ac and PR65 show that these proteins are ubiquitously localized in the cytoplasm and the nucleus in nontransformed fibroblasts. As determined by quantitative immunofluorescence the core subunits of PP2A are twofold more concentrated in the nucleus than in the cytoplasm. Detailed analysis of synchronized cells reveals striking changes in the nuclear to cytoplasmic ratio of PP2Ac-specific immunoreactivity albeit the total amounts of neither PP2Ac nor PR65 in each compartment alters significantly during the cell cycle. Our results imply that differential methylation of PP2Ac occurs at the G0/G1 and G1/S boundaries. Specifically a demethylated form of PP2Ac is found in the cytoplasm of G1 cells, and in the nucleus of S and G2 cells. In addition nuclear PP2A holoenzymes appear to undergo conformational changes at the G0/G1 and G1/S boundaries. During mitosis PP2A is lost from the nuclear compartment, and unlike protein phosphatase 1 shows no specific association with the condensed chromatin.     

The catalytic subunit of protein phosphatase 2A associates with the translation termination factor eRF1.

By a number of criteria, we have demonstrated that the translation termination factor eRF1 (eukaryotic release factor 1) associates with protein phosphatase 2A (PP2A). Trimeric PP2A1 was purified from rabbit skeletal muscle using an affinity purification step. In addition to the 36 kDa catalytic subunit (PP2Ac) and established regulatory subunits of 65 kDa (PR65) and 55 kDa (PR55), purified preparations contained two proteins with apparent Mrs of 54 and 55 kDa. Protein microsequencing revealed that the 55 kDa component is a novel protein, whereas the 54 kDa protein was identified as eRF1, a protein that functions in translational termination as a polypeptide chain release factor. Using the yeast two-hybrid system, human eRF1 was shown to interact specifically with PP2Ac, but not with the PR65 or PR55 subunits. By deletion analysis, the binding domains were found to be located within the 50 N-terminal amino acids of PP2Ac, and between amino acid residues 338 and 381 in the C-terminal part of human eRF1. This association also occurs in vivo, since PP2A can be co-immunoprecipitated with eRF1 from mammalian cells. We observed a significant increase in the amount of PP2A associated with the polysomes when eRF1 was transiently expressed in COS1 cells, and eRF1 immunoprecipitated from those fractions contained associated PP2A. Since we did not observe any dramatic effects of PP2A on the polypeptide chain release activity of eRF1 (or vice versa), we postulate that eRF1 also functions to recruit PP2A into polysomes, thus bringing the phosphatase into contact with putative targets among the components of the translational apparatus.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

PP4R4/KIAA1622 forms a novel stable cytosolic complex with phosphoprotein phosphatase 4

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor alpha signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c.PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c.PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and gammaH2AX, but this activity is lower than that associated with the PP4c.PP4R2.PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2.PP4R3, and alpha4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.     

Identification of proteins interacting with the catalytic subunit of PP2A by proteomics

The protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in the regulation of multiple signaling pathways including the Wnt/beta-catenin and the ERK pathways. To understand the complex signaling networking associated with PP2A, we searched proteins interacting with the catalytic subunit of protein phosphatase 2A (PP2Ac) by a pull-down analysis followed by 2-D gel electrophoresis and proteomic analyses. The probability of identification of the proteins interacting with PP2Ac was increased by searching proteins differently interacting with PP2Ac according to stimulation of Wnt3a, which regulates both the Wnt/beta-catenin and the ERK pathways. Around 100 proteins, pulled-down by His-tagged PP2Ac, were identified in 2-D gels stained with CBB. By MALDI-TOF-MS analyses of 45 protein spots, we identified several proteins that were previously known to interact with PP2A, such as Axin and CaMK IV. In addition, we also identified many proteins that potentially interact with PP2Ac. The interactions of several candidate proteins, such as tuberous sclerosis complex 2, RhoB, R-Ras, and Nm23H2, with PP2Ac, were confirmed by in vitro binding analyses and/or coimmunoprecipitation experiments.     

Identification and characterization of an alternatively spliced isoform of the human protein phosphatase 2Aα catalytic subunit

PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.     

Phenylarsine Oxide Binding Reveals Redox-Active and Potential Regulatory Vicinal Thiols on the Catalytic Subunit of Protein Phosphatase 2A

Our earlier finding that the activity of protein phosphatase 2A from rat brain is inhibited by micromolar concentrations of the dithiol cross-linking reagent phenylarsine oxide (PAO) has encouraged the hypothesis that the catalytic subunit (PP2Ac) of PP2A contains one or more pairs of closely-spaced (vicinal) thiol pairs that may contribute to regulation of the enzyme. The results of the present study demonstrate using immobilized PAO-affinity chromatography that PP2Ac from rat brain formed stable DTT-sensitive adducts with PAO with or without associated regulatory subunits. In addition, a subset of the PAO-binding vicinal thiols of PP2Ac was readily oxidized to disulfide bonds in vitro. Importantly, a small fraction of PP2Ac was still found to contain disulfide bonds after applying stringent conditions designed to prevent protein disulfide bond formation during homogenization and fractionation of the brains. These findings establish the presence of potentially regulatory and redox-active PAO-binding vicinal thiols on the catalytic subunit of PP2A and suggest that a population of PP2Ac may contain disulfide bonds in vivo.     

Interaction of nucleoredoxin with protein phosphatase 2A

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.     

Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris.

Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N-termini, were introduced into the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriacetate/agarose. In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 microg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a [1.7 micromol.min-1.(mg protein)-1] and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state-specific antibodies, recombinant PP2Ac was carboxymethylated at the last C-terminal leucine residue. Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine-agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.