Ca2+/calmodulin-activated protein phosphatase (PP2B) of Saccharomyces cerevisiae. PP2B activity is not essential for growth.

Protein phosphatase (PP2B) whose activity is stimulated 12-20-fold by Ca2+/calmodulin (CaM) was partially purified by CaM-Sepharose and heparin-agarose chromatographies from cell extract of the yeast Saccharomyces cerevisiae. PP2B activity was not detectable in a mutant in which two genes (CMP1 and CMP2) encoding homologs of mammalian PP2B catalytic subunit were disrupted. We have previously shown that the double gene disruption has no significant effect on the growth of yeast [1991, Mol. Gen. Genet. 227, 52-59]. The results indicated that CMP1 and CMP2 are the only genes that encode the PP2B catalytic polypeptide in S. cerevisiae, and PP2B activity is not essential for the growth of the yeast under normal conditions.     

A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity

Using a combination of tandem affinity purification tagging and mass spectrometry, we characterized a novel, evolutionarily conserved protein phosphatase 4 (PP4)-containing complex (PP4cs, protein phosphatase 4, cisplatin-sensitive complex) that plays a critical role in the eukaryotic DNA damage response. PP4cs is comprised of the catalytic subunit PP4C; a known regulatory subunit, PP4R2; and a novel protein that we termed PP4R3. The Saccharomyces cerevisiae PP4R3 ortholog Psy2 was identified previously in a screen for sensitivity to the DNA-damaging agent and anticancer drug cisplatin. We demonstrated that deletion of any of the PP4cs complex orthologs in S. cerevisiae elicited cisplatin hypersensitivity. Furthermore human PP4R3 complemented the yeast psy2 deletion, and Drosophila melanogaster lacking functional PP4R3 (flfl) exhibited cisplatin hypersensitivity, suggesting a highly conserved role for PP4cs in DNA damage repair. Finally we found that PP4R3 may target PP4cs to the DNA damage repair machinery at least in part via an interaction with Rad53 (CHK2).     

Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo

The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase. We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase. Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity. Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit. Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit. These phenotypes can be reversed by overexpression of the B regulatory subunit. These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.     

The scaffolding A/Tpd3 subunit and high phosphatase activity are dispensable for Cdc55 function in the Saccharomyces cerevisiae spindle checkpoint and in cytokinesis

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional enzyme whose trimeric form consists of a scaffolding A subunit, a catalytic C subunit, and one of several regulatory B subunits (B, B', and B'). The adenovirus E4orf4 protein associates with PP2A by directly binding the B or B' subunits. An interaction with an active PP2A containing the B subunit, or its homologue in yeast, Cdc55, is required for E4orf4-induced apoptosis in mammalian cells and for induction of growth arrest in Saccharomyces cerevisiae. In this work, Cdc55 was randomly mutagenized by low-fidelity PCR amplification, and Cdc55 mutants that lost the ability to transduce the E4orf4 toxic signal in yeast were selected. The mutations obtained by this protocol inhibited the association of Cdc55 with E4orf4, or with the PP2A-AC subunits, or both. Functional analysis revealed that a mutant that does not bind Tpd3, the yeast A subunit, as well as wild type Cdc55 in a tpd3Delta background, can form a heterodimer with the catalytic subunit. This association requires C subunit carboxyl methylation. The residual phosphatase activity associated with Cdc55 in the absence of Tpd3 is sufficient to maintain a partially active spindle checkpoint and to prevent cytokinesis defects.     

The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.

In higher eukaryotes, the activity and specificity of the type 1 protein serine-threonine phosphatase (PP1) catalytic subunit is thought to be controlled by its association with a number of regulatory or targeting subunits. Here we describe the characterization of a gene encoding one such potential polypeptide in the yeast Saccharomyces cerevisiae. The gene which we have isolated (termed SDS22) encodes a product with a high degree of sequence identity to the fission yeast sds22 protein, a known regulator of the mitotic function of PP1 in Schizosaccharomyces pombe. Using two different criteria, we have demonstrated that Sds22p and the catalytic subunit of PP1 (Glc7p) interact in yeast cells. We have also generated a temperature-sensitive allele of GLC7 (glc7-12) which causes a block to the completion of mitosis at the restrictive temperature. Additional copies of SDS22 lead to allele-specific suppression of the glc7-12 mutant, strongly suggesting that the interaction between the two proteins is of functional significance. Sds22p is therefore likely to be the second example of a PP1 regulatory subunit identified in S. cerevisiae.     

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.     

Expression of human protein phosphatase-1 in Saccharomyces cerevisiae highlights the role of phosphatase isoforms in regulating eukaryotic functions

Human (PP1) isoforms, PP1alpha, PP1beta, PP1gamma1, and PP1gamma2, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1alpha-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1alpha-expressing yeast accumulated less glycogen than PP1beta-or PP1gamma1-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1alpha/beta subunits highlighted a critical role for their unique N termini in defining PP1alpha and PP1beta functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.     

Cdc55p-mediated E4orf4 growth inhibition in Saccharomyces cerevisiae is mediated only in part via the catalytic subunit of protein phosphatase 2A

The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2A(BAC)), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2A(BAC) holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2A(BAC) heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2A(BAC) heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.     

Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p

Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine phosphatase known to play important roles in cell cycle regulation. Association of different B-type targeting subunits with the heterodimeric core (A/C) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. We have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pph21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approaches were taken. First, S. cerevisiae cells were generated whose survival depended on the expression of different carboxyl-terminal Pph21p mutants. Second, the major S. cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PP2A C subunit carboxyl-terminal leucine was identified, and cells deleted for this methyltransferase were utilized for our studies. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-terminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p. Loss of Cdc55p association was accompanied in each case by a large reduction in binding of the yeast A subunit, Tpd3p, to Pph21p. Moreover, decreased Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylation also greatly reduced the association of another yeast B-type subunit, Rts1p. Thus, methylation of Pph21p is important for formation of PP2A trimeric and dimeric complexes, and consequently, for PP2A function. Taken together, our results indicate that methylation and phosphorylation may be mechanisms by which the cell dynamically regulates PP2A complex formation and function.     

Protein phosphatase methyltransferase 1 (Ppm1p) is the sole activity responsible for modification of the major forms of protein phosphatase 2A in yeast.

Protein phosphatase 2A (PP2A) is a major threonine/serine phosphatase that is involved in regulating a variety of cellular processes. It has been shown in both yeast and mammals that the PP2A catalytic subunit (PP2Ac) is methyl-esterified at the conserved C-terminal Leu residue. The recent characterization of a mammalian PP2A carboxyl methyltransferase has led to the identification of two ORFs in Saccharomyces cerevisiae as potential orthologues of the mammalian PP2A methyltransferase: protein phosphatase methyltransferase 1 (PPM1) and protein phosphatase methyltransferase 2 (PPM2). To experimentally identify the PP2A methyltransferase in yeast, we obtained deletion mutants of PPM1 and PPM2 and then constructed double mutants. Using in vivo-labeling techniques, we demonstrate that only the PPM1 gene is required for PP2Ac methylation at the C-terminus. Because yeast has at least three homologues of PP2Ac (PPH21, PPH22, and PPH3), we then asked whether all of these catalytic subunits are methylated by the PPM1 and/or PPM2 putative methyltransferases. We modified the segment corresponding to the N-terminal coding region of all three PP2Ac genomic genes with a hemagglutinin (HA) tag in the parent, ppm1, ppm2, and ppm1ppm2 mutant genetic backgrounds. Using immuoprecipitation with anti-HA antibodies followed by methyl ester analysis, we showed that only in the ppm1 mutant were both Pph21p and Pph22p not methylated. We did not detect any methylesterification of Pph3p under our conditions. Our results indicate that PPM1 is the sole methyltransferase responsible for methylating the two major homologues of PP2Ac in yeast. The function of the PPM2 gene product remains unclear.     

Mutation of the C-terminal leucine residue of PP2Ac inhibits PR55/B subunit binding and confers supersensitivity to microtubule destabilization in Saccharomyces cerevisiae

Protein phosphatase 2A is ubiquitous among eukaryotes and exists as a family of holoenzymes in which the catalytic subunit. PP2Ac, binds a variety of regulatory subunits. Using the yeast Saccharomyces cerevisia, we have investigated the role of the phylogenetically invariant C-terminal leucine residue of PP2Ac, which, in mammalian cells, undergoes reversible methylation and modulates binding of the PR55/B subunit. In S. cerevisiae, the C-terminal Leu-377 residue of Pph22p (equivalent to human PP2Ac Leu-309) was dispensable for cell growth under optimum conditions and its removal, or substitution by alanine, did not inhibit PP2A activity in vitro. However, Leu-377 is required for binding of the yeast PR55/B subunit, Cdc55p, by Pph22p, though apparently not for the binding of Rts1p, the yeast PR61/B' subunit. Furthermore, mutation of this leucine enhanced the sensitivity of cells to microtubule destabilization, a defect characteristic of cdc55delta mutant cells, which are impaired for spindle checkpoint function. These results demonstrate that the regulation of PP2A, mediated by PR55/B binding to the highly conserved PP2Ac C-terminus, is critical for cell viability under conditions of microtubule damage and support a role for PP2A in exit from mitosis.     

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.     

A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.

Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.     

Assembly and structure of protein phosphatase 2A

Protein phosphatase 2A (PP2A) represents a conserved family of important protein serine/threonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, medi-ated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interac-tion with a large n...     

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa

We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A B subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.     

Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.

The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits. The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells. Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C. Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer. Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits. Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues. The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis. Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP). Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.     

Generation of active protein phosphatase 2A is coupled to holoenzyme assembly

Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.