Structure of the protein phosphatase 2A holoenzyme

Protein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B'/B56/PR61. Surprisingly, the B'/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B'/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B'/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A.     

Structural basis of PP2A inhibition by small t antigen

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST.     

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.     

Altered phosphorylation of cytoskeletal proteins in mutant protein phosphatase 2A transgenic mice

Protein phosphatase 2A (PP2A) is a family of heterotrimeric enzymes with diverse functions under physiologic and pathologic conditions such as Alzheimer's disease. All PP2A holoenzymes have in common a catalytic subunit C and a structural scaffolding subunit A. These core subunits assemble with various regulatory B subunits to form heterotrimers with distinct functions in the cell. Substrate specificity of PP2A in vitro is determined by regulatory subunits with leucine 309 of the catalytic subunit C playing a crucial role in the recruitment of regulatory subunits into the complex. Here we expressed a mutant form of Calpha, L309A, in brain and Harderian (lacrimal) gland of transgenic mice. We found an altered recruitment of regulatory subunits into the complex, demonstrating a role for the carboxyterminal leucine of Calpha in regulating holoenzyme assembly in vivo. This was associated with an increased phosphorylation of tau in brain and an impaired dephosphorylation of vimentin demonstrating that both cytoskeletal proteins are in vivo substrates of distinct PP2A holoenzyme complexes.     

A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.     

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.     

Targeting of PP2A enzymes by viral proteins and cancer signalling

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. PP2A proteins are made of a core dimer, composed of a catalytic (C) subunit and a structural (A) subunit, in association with a third variable -regulatory (B) subunit. Although initially considered as a constitutive housekeeping enzyme, PP2A is indeed highly regulated by post-translational modifications of its catalytic subunit or by the identity of a regulatory type B subunit, which determines substrate specificity, subcellular localization and enzymatic activity of a defined holoenzyme. During the two last decades, multiple studies of structural and functional regulation of PP2A holoenzymes by viral proteins led to the identification of critical pathways for both viral biology and tumorigenesis. To date a dozen of different viruses (ADN/ARN or retrovirus) have been identified that encode viral proteins associated to PP2A. In this review, we analyze a biological strategy, used by various viruses based on the targeting of PP2A enzymes by viral proteins, in order to specifically deregulate cellular pathways of their hosts. The impact of such PP2A targeting for biomedical search, and in further therapeutic developments against cancer, will also be discussed.     

Structure of protein phosphatase 2A core enzyme bound to tumor-inducing toxins

The serine/threonine phosphatase protein phosphatase 2A (PP2A) plays an essential role in many aspects of cellular functions and has been shown to be an important tumor suppressor. The core enzyme of PP2A comprises a 65 kDa scaffolding subunit and a 36 kDa catalytic subunit. Here we report the crystal structures of the PP2A core enzyme bound to two of its inhibitors, the tumor-inducing agents okadaic acid and microcystin-LR, at 2.6 and 2.8 A resolution, respectively. The catalytic subunit recognizes one end of the elongated scaffolding subunit by interacting with the conserved ridges of HEAT repeats 11-15. Formation of the core enzyme forces the scaffolding subunit to undergo pronounced structural rearrangement. The scaffolding subunit exhibits considerable conformational flexibility, which is proposed to play an essential role in PP2A function. These structures, together with biochemical analyses, reveal significant insights into PP2A function and serve as a framework for deciphering the diverse roles of PP2A in cellular physiology.     

Structural and biochemical insights into the regulation of protein phosphatase 2A by small t antigen of SV40

The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.     

A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. Retrotransposon insertion was found to produce an N-terminally truncated form (Deltagamma1) of the B56gamma1 regulatory subunit isoform of protein phosphatase (PP) 2A in BL6 cells, but not in F10 cells. We found an interaction of paxillin with PP2A C and B56gamma subunits by co-immunoprecipitation. B56gamma1 co-localized with paxillin at focal adhesions, suggesting a role for this isoform in targeting PP2A to paxillin. In this regard, Deltagamma1 behaved similarly to B56gamma1. However, the Deltagamma1-containing PP2A heterotrimer was insufficient for the dephosphorylation of paxillin. Transfection with Deltagamma1 enhanced paxillin phosphorylation on serine residues and recruitment into focal adhesions, and cell spreading with an actin network. In addition, Deltagamma1 rendered F10 cells as highly metastatic as BL6 cells. These results suggest that mutations in PP2A regulatory subunits may cause malignant progression.     

Phosphoregulation of MgcRacGAP in mitosis involves Aurora B and Cdk1 protein kinases and the PP2A phosphatase

MgcRacGAP, a Rho GAP essential to cytokinesis, works both as a Rho GTPase regulator and as a scaffolding protein. MgcRacGAP interacts with MKLP1 to form the centralspindlin complex and associates with the RhoGEF Ect2. The GAP activity of MgcRacGAP is regulated by Aurora B phosphorylation. We have isolated B56epsilon, a PP2A regulatory subunit, as a new MgcRacGAP partner. We report here that (i) MgcRacGAP is phosphorylated by Aurora B and Cdk1, (ii) PP2A dephosphorylates Aurora B and Cdk1 phosphorylated sites and (iii) inhibition of PP2A abrogates MgcRacGAP/Ect2 interaction. Therefore, PP2A may regulate cytokinesis by dephosphorylating MgcRacGAP and its interacting partners.     

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)

The prototypical form of the Ser/Thr phosphatase PP2A is a heterotrimeric complex consisting of catalytic subunit (C), and A and B regulatory subunits. C-terminal methylation of PP2A-C influences holoenzyme assembly. Using late gestation development in the rat as an in vivo model of liver growth, we found that PP2A-C protein and activity levels were higher in fetal compared to adult liver extracts. However, unmethylated PP2A-C was much higher in the adult extracts. In MonoQ fractionation, unmethylated C eluted separately from methylated C, which was present predominantly in ABC heterotrimers. Gel filtration chromatography revealed that some unmethylated C was present as free catalytic subunit in adult liver. In addition, a significant proportion of PP2A was in inactive forms that may involve novel regulatory subunits. Our results indicate that methylation of PP2A-C appears to be a primary determinant for the biogenesis of PP2A heterotrimers.     

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.     

Light-driven translocation of the protein phosphatase 2A complex regulates light/dark dephosphorylation of phosducin and rhodopsin

In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B' subunit, B56 epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B' subunit (sequenced to be B56 alpha) or with the B alpha regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56 epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (approximately 20 ng/mouse retina) and 9 times more of the C subunit (approximately 4 ng/mouse retina) than of the B56 epsilon subunit (approximately 0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56 epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.     

B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK

The protein phosphatase 2A (PP2A) acts on several kinases in the extracellular signal-regulated kinase (ERK) signaling pathway but whether a specific holoenzyme dephosphorylates ERK and whether this activity is controlled during mitogenic stimulation is unknown. By using both RNA interference and overexpression of PP2A B regulatory subunits, we show that B56, but not B, family members of PP2A increase ERK dephosphorylation, without affecting its activation by MEK. Induction of the early gene product and ERK substrate IEX-1 (ier3) by growth factors leads to opposite effects and reverses B56-PP2A-mediated ERK dephosphorylation. IEX-1 binds to B56 subunits and pERK independently, enhances B56 phosphorylation by ERK at a conserved Ser/Pro site in this complex and triggers dissociation from the catalytic subunit. This is the first demonstration of the involvement of B56-containing PP2A in ERK dephosphorylation and of a B56-specific cellular protein inhibitor regulating its activity in an ERK-dependent fashion. In addition, our results raise a new paradigm in ERK signaling in which ERK associated to a substrate can transphosphorylate nearby proteins.     

The function of PP2A/B56 in non-metazoan multicellular development

DB56, the Dictyostelium B56 homolog, displayed high sequence homology to other eukaryotic B56 subunits of the PP2A and a specific association with the PP2A catalytic subunit. Cells lacking DB56, psrA(-), displayed higher PP2A phosphatase activity compared with the wild type, approximately 10 hr of delayed expression of ecmA and ecmB prestalk markers, and inefficient culmination. The prespore marker cotB declined as wild-type cells culminate, but no such decline was observed from psrA(-) cells. Interestingly, psrA(-) cells exhibited higher GSK3 kinase activity. Furthermore, the expression of the dominant negative GSK3 mutant (K84/85M) in psrA(-) cells improved both prestalk and prespore expression patterns similarly to wild-type cells. However, culmination was not restored in psrA(-) cells expressing dominant negative GSK3, which suggests that PP2A/DB56 has an extra target during terminal differentiation. This report shows that PP2A/DB56 controls not only metazoan development, but also non-metazoan cell fate decision processes.     

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.     

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.