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The presence of Ser/Thr protein phosphatase activities in higher plants was recognized some years ago (1-3). The molecular cloning of the cDNAs encoding the catalytic subunits of several of these phosphatases made clear two points: First, that plant Ser/Thr phosphatases were very similar in sequence to those found in other organisms. Second, that they constitute an extremely complex gene family. For instance, at least six genes encoding proteins highly related to the catalytic subunit of PP1 have been identified in Arabidopsis thaliana (4,5), whereas only four appear to exist in mammals and only one in other organisms, such as budding yeast. The author's laboratory has proven the existence in Arabidopsis thaliana of at least five isoforms of the catalytic subunit of PP2A (6,7), whereas two forms have been identified in mammals (8,9) and only one in other organisms (10-12). On the basis of their amino acid sequence, Arabidopsis thaliana PP2A isoforms can be classified into two subfamilies. The first subfamily is composed of PP2A-1, PP2A-2, and PP2A-5 and the second is composed of PP2A-3 and PP2A-4 (4,5,13). The sequence identity among the members of each family is very high (97-98%), whereas members of different subfamilies are about 80% identical. Plant PP2A isoforms are strongly related to the PP2A proteins from other organisms (about 80% identical their mammalian counterparts). A protein phosphatase related to type 2A phosphatases, protein phosphatase X (PPX), was initially identified in rabbit (14), although it has also been found in insects. [ABSTRACT FROM AUTHOR]

Ten years ago, the purification of mammalian protein phosphatase 1 (PP1) was done routinely in only a handful of laboratories around the world. It typically involved the sacrifice of five rabbits and a cumbersome procedure that lasted 5 or 6 d, not to mention the requirement for heavy equipment, such as large-capacity centrifuges and tissue blenders. If all went well, you were rewarded with 1 or 2 mg of a close to homogeneously pure PP1 preparation. As in so many other instances, the classical purification of PP1 from its natural source has been supplanted by the recombinant DNA approach. This usually involves several steps, i.e., cloning of the gene or cDNA, subcloning the cDNA into a suitable vector, expressing the cDNA in a suitable host, and finally, purifying the recombinant protein. [ABSTRACT FROM AUTHOR]

Expression of recombinant proteins using plasmid vectors in various kinds of cells has been widely used for the following three purposes: 1.To obtain a large amount of a purified specific protein in a relatively short time,2.To investigate the phenotypes of the cells caused by a high expression of a protein whose physiological functions are not fully known, and3.To determine whether posttranslational modification of proteins demonstrated in vitro is also observed in vivo. [ABSTRACT FROM AUTHOR]

Various isoforms of the catalytic subunit of Protein Phosphatase-1 (Ser/Thr phosphatase of type-1, PP1) were discovered by cDNA cloning (1). PP1α, PP1γ1 and PP1δ (also called β) are found in virtually all mammalian cells, whereas PP1γ2 is testis-specific (2,3). Although PP1α, PP1γ1, and PP1δ are products of different genes, they are about 90% identical (2) and migrate as a single protein band on SDS-electrophoresis. For instance, the 37 kDa PP1 purified from rabbit muscle is a mixture of the three isoforms (4,5). The only sequence differences among the isoforms are in the C-termini (2). Consequently, only antibodies to peptides derived from these regions are isoform-specific (5,6). Based on the involvement of PP1 in many different regulatory pathways, differential subcellular localizations for the isoforms may be envisaged, These studies require isoform-specific antibodies, such as those produced and purified using the procedures described herein. The authors also show how the antibodies can be used to distinguish the PP1 isoforms in immunoblot, immunoprecipitate, two-dimensional electrophoresis, and immunofluorescence. [ABSTRACT FROM AUTHOR]

Insulin signaling is mediated by a cascade of phosphorylation/dephosphory-lation reactions via the stimulation of specific serine/threonine (ser/thr) kinases and phosphatases (1-7). Dephosphorylations catalyzed by ser/thr phosphatases result in the activation or inhibition of several intracellular enzymes and proteins that govern the final steps of insulin action (2). The best examples are glycogen synthase (GS), pyruvate dehydrogenase (PDH), acetyl-CoA carboxylase, hormone-sensitive lipoprotein lipase, and Glut-4 (the insulin regulatable muscle/fat glucose transporter) (1-10). Dephosphorylation increases the activity of GS, PDH, and Glut-4, and inhibits phosphorylase kinase (2,8-10). Protein phosphatase 1 (PP1) appears to be the key enzyme responsible for connecting the insulin-initiated phosphorylation cascade with the dephosphorylation of insulin-sensitive substrates (2). The exact in vivo molecular mechanism by which insulin regulates the activity of this enzyme remains unclear. A large proportion of the spontaneously active PP1 is found associated with the particulate fraction of the cell, notably with glycogen, membranes, and myofibrils (11). Therefore, it appears that the catalytic activity of PP1 is determined by its interaction with regulatory subunits that target the enzyme to particular locations in the cell and alter its substrate specificity (11). [ABSTRACT FROM AUTHOR]

The genome of the yeast S. cerevisiae contains a fairly large number of genes encoding Ser/Thr protein phosphatases. Among these genes can be found the homologs of the classical phosphatases described in mammalian tissues in the early 1980s. For instance, the catalytic subunit of PP1 is encoded by the gene GLC7, whereas two forms of PP2A are encoded by genes PPH21 and PPH22. As it happens in other organisms, the yeast catalytic subunits of PP1, PP2A, and PP2B (calcineurin) are quite related in sequence. In addition to these phosphatases, yeast cells contain other phosphatase genes that, while related to PP1 or PP2A in their primary structure, are functionally different. The main features of these genes (1-13) are described in Table 1. [ABSTRACT FROM AUTHOR]

Synthetic lethal screening is a useful genetic strategy that has a variety of applications in identifying new genes in yeast or other organisms. The method starts with a gene, YFG (your favorite gene), and isolates yeast mutants that need that gene for viability. These mutants are subsequently used to identify the mutant yeast gene or isolate genes from other species that complement the yeast mutation. This method has proven successful in identifying yeast homologs based on the function of foreign genes (1). In synthetic lethal screening, only functional homology is required, rather than DNA or amino acid homology. Therefore, this approach is also called "Cloning by Function." Additionally, synthetic lethal screening has also been productive in identifying genes, which function within the same or parallel pathways as YFG in complicated regulatory systems (2-4). [ABSTRACT FROM AUTHOR]

The protein serine/threonine phosphatase 2A (PP2A) family of holoenzymes has been implicated in the regulation of many cellular signaling molecules including metabolic enzymes, cell-surface receptors, cytosohc protein kinases, and transcription factors (reviewed in refs. 1-5). PP2A is a multimeric enzyme, composed of a catalytic subunit associated with two regulatory subunits (reviewed in refs. 1-5). The core structure of PP2A is a heterodimer consisting of the 36 kDa catalytic subunit (C) tightly complexed with the 65 kDa A regulatory subunit (A). This heterodimer complexes with one of multiple B sub-units (ranging from 54-130 kDa). Currently, three B subunit families have been identified (B or B55, B′ or B56, and B″ or B72). Whereas no amino acid sequence homology exists between the different families, isoforms within the same family are highly homologous. The physiological significance of this heterogeneity is not known; however, recent data suggest that the B regulatory subunit has a role in determining the substrate selectivity of the catalytic sub-unit. It also is postulated that the B regulatory subunit participates in localization of the catalytic subunit to distinct cellular microenvironments (reviewed in refs. 1-5). [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A (PP2A) is one of the major intracellular serine/threonine protein phosphatases. PP2A in vivo is an ABC heterotrimer whose substrate specificity is regulated in large part by the variable B subunit. Several additional polypeptides have been identified that modulate the activity of the heterotrimer. It seems likely that more regulatory subunits and interacting polypeptides exist that may be of low abundance or expressed in a tissue-specific manner. One attractive approach to finding these novel interacting polypeptides is to utilize the two-hybrid interaction method. [ABSTRACT FROM AUTHOR]

A large variety of methods exist to identify protein-protein interactions. The two hybrid method is unique in that interactions are assayed in vivo. The method can be used "analytically" or "preparatively." In the analytical application, one surveys interactions between two proteins known to interact by comparing interaction of mutant proteins. The preparative two hybrid method allows one to identify putative proteins that interact with a given protein (called the "bait") by screening libraries. This chapter will focus only on the analytical application of the two hybrid assay to analyze interactions of mutant protein phosphatase 1 (PP1) molecules with some PP1 noncatalytic subunits. For examples of the latter screening method, readers are referred to original papers that employed the method to isolate PP1 interacting proteins (1-3). [ABSTRACT FROM AUTHOR]

Protein phosphatases are important for the maintenance of functional activity of many proteins. Phosphorylation of proteins by protein kinases is a common posttranslational modification that modulates the signaling capability of the target polypeptides. Neurotransmitter receptors, which belong to the super-family of GTP (G) protein-coupled receptors, are uncoupled from their signaling pathway(s) after phosphorylation by receptor-specific protein kinases. [ABSTRACT FROM AUTHOR]

One species of protein phosphatase 2C (PP2C) in the yeast S. cerevisiae is encoded by the gene PTC1/TPD1 (1,2). This gene encodes a protein that is highly conserved in all eukaryotes. It is 38% identical to the rat protein over the entire sequence, with identity reaching up to 80% in distinct regions. TPD1 was shown to encode protein phosphatase 2C activity based on two lines of evidence: first, Ptc1/Tpd1 protein expressed in E. coli exhibits readily detectable Mg2+ or Mn2+ dependent protein phosphatase activity with 32P-labeled casein as a substrate. Second, this activity does not require Ca2+ and is resistant to okadaic acid at concentrations capable of inhibiting all the other mam families of protein phosphatases in eukaryotic organisms. These are the primary distinguishing enzymatic characteristics of mammalian PP2C (3). Yeast has at least two other PP2C species since extracts made from cells deleted for PTC1/TPD1 exhibit substantial PP2C activity (1,2). Putative genes for this activity have been cloned (1). [ABSTRACT FROM AUTHOR]

A major component of the biochemical mechanisms involved in the photoresponse in both invertebrate and vertebrate organisms is the phosphorylation and dephosphorylation of specific protein intermediates whose phosphorylation state is transitory in response to light, darkness, and other stimuli (seeref. 1 for review), and results from changes in the relative activities of specific protein kinases and phosphatases. Of the known protein serinethreonine protein phosphatases, type 2A (PP2A) dephosphorylates many of the identified phosphoproteins involved in the light-dependent processes in photoreceptors from both vertebrates and invertebrate organisms For example, in vertebrates rod photoreceptors, dephosphorylation of rhodopsin by PP2A (2-5) is necessary for the restoration of increased light sensitivity (dark adaptation) [ABSTRACT FROM AUTHOR]

Unlike the other serine/threonine protein phosphatases (for reviews seerefs. 1-3), type 2C (PPM) is a monomeric enzyme and its activity depends on the presence of Mg2+ (4). Two major isoforms of protein phosphatase type 2C (PP2C) were isolated from various vertebrate organisms and tissues: PP2Cα and PP2Cβ (formerly called PP2C1 and PP2C2) with a molecular mass of 43-48 kDa (5,6). The isoforms cannot be distinguished by substrate specificity. In addition, neither a specific activator nor an inhibitor is known. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A (PP2A) is a protein serine/threonine phosphatase that regulates the activities of key rate-limiting enzymes and proteins involved in diverse cellular processes (1-4). However, although much is known about the subunit composition and sequence of different forms of the enzyme (1-7), little information is available on its regulation. Nonetheless, evidence has emerged that the catalytic subunit of PP2A is subject to posttranslational modifications including phosphorylation on threonines (8-10) and tyrosines (11), and methylation at Leu309 (12-14). Phosphorylation at either threonine or tyrosine residues inactivates PP2A (8-10). By contrast, methylation appears to activate the phosphatase (14). [ABSTRACT FROM AUTHOR]

This chapter deals with a family of Ser/Thr protein phosphatases that are known as type-1 protein phosphatases or PP1 (1,2). They all share one of five known isoforms of the same catalytic subunit (PP1C) of 36-38 kDa, designated with the subscripts α1, α2, δ, λ1, and λ2. In addition, each PP1 holoenzyme contains one or two regulatory subunits. The latter are multifunctional and not only determine the substrate specificity and the activity of the holoenzyme, but also anchor the phosphatase to the cellular compartment that harbors its physiological substrate(s). The targeting role of the noncatalytic subunits has formed the basis for the nomenclature of the various PP1 holoenzymes (Table 1). [ABSTRACT FROM AUTHOR]

Numerous studies have been reported that demonstrate differences in protein function dependent upon the phosphorylation state of the protein. For many of these studies, a logical next step involves studying the enzyme(s) responsible for placing the phosphates on, as well as taking them off. When performing enzyme studies, it is advantageous to have the substrate protein in as pure a state as possible, and in a form that readily lends itself to manipulation and subsequent analysis. In situations where eukaryotic cell lysate is both the source of the enzyme and the protein substrate, multiple steps may be required to isolate the substrate to facilitate biochemical analysis of the reaction. [ABSTRACT FROM AUTHOR]

Type 1 and 2A protein phosphatases (PPases) account for the majority of enzyme activity dephosphorylating phospho-serine and -threonines in living cells. Their action is opposed to that of a large number of serine/threonine protein kinases. In recent years, the enigma of how the action of many protein kinases is reversed by a limited number of protein phosphatases in a regulated manner has been solved by a combination of biochemical and molecular cloning procedures: Both PP1 and PP2A were found to exist as multimeric holoenzymes in vivo, where a limited number of catalytic subunits are complexed to a vast number of regulatory subunits. The latter are now considered to be the determinants of substrate specificity and intracellular localization of the catalytic subunits (1,2). [ABSTRACT FROM AUTHOR]

There are multiple regulatory devices of protein phosphatase type 2A (PP2A), known on the biochemical level: association of regulatory subunits, interaction with other proteins, covalent modification by phosphorylation on tyrosyl and threonine residues, methylation of the carboxy terminus (seeref. 1 for a recent review). In this chapter still another device will be described and discussed. Although PP2A is generally known as a phosphatase that specifically dephosphorylates seryl and threonyl residues, it can also operate as a phosphotyrosyl phosphatase, and this activity can be regulated independently. Historically, PP2A was the first phosphatase described that could remove phosphate from phosphotyrosyl residues (2,3) and is, therefore, a "dual specificity" phosphatase "avant la lettre." The in vitro characterization of the dual specificity of PP2A indicated that the phosphoseryl and phosphotyrosyl phosphatase activities exhibited distinct catalytic properties and thermostability (2,4); they were either conversely affected by free ATP or pyrophosphate (5), or concurrently stimulated by tubulin (6). Further observations led to the isolation of a protein that specifically stimulates the phosphotyrosyl phosphatase activity of PP2A without affecting its Ser-P/Thr-P phosphatase activity (3,7-9). This phosphotyrosyl phosphatase activator or PTPA was shown to be highly specific for the dimeric form of PP2A (PP2AD) (7). PTPA stimulates the PTPase activity of PP2AD in a time-dependent enzyme-like reaction requiring ATP, Mg2+ as essential cofactor that cannot ble ATP analogs (7).be replaced by nonhydrolysa [ABSTRACT FROM AUTHOR]

A variety of studies in recent years have indicated that modulation of protein phosphatases perturbs brain function. For example, a mutation in PP1 is associated with defective habituation and associative learning in drosophila (1), and the activity of PPl and/or PP2A is essential for induction and maintenance of long term depression (LTD) in hippocampal neurons (2). Recent studies link reduced PP2A activity with hyperphosphorylation of Tau in Alzheimer's disease (3). A number of studies using inhibitors of PP1 and PP2A have also implicated a role for PP1 and PP2A in regulating neurotransmitter release. Studies from this laboratory suggest a role for these enzymes in priming or modulation rather than triggering of release (4. At the molecular level, regulation of PP1 and PP2A has been implicated in modulating the activity of several receptors and ion channels. For example, inhibition with okadaic acid leads to stimulation of non-NMDA-type glutamate receptors (5) and activation of NMDA-type receptors induces specific dephosphorylation and inactivation of DARPP-32, a neuronal homolog of PPl-inhibitor 1 (6). Given the ubiquitous distribution and substrate overlap of the catalytic subunits of PP1 and PP2A, both between cell types and within cells, the current challenge is to delineate and localize the individual mechanisms regulating PP1 and PP2A in the nervous system. [ABSTRACT FROM AUTHOR]

If it was not for the protein kinases, protein phosphatases would probably not have a right to exist. Naturally, protein kinases most often respond to an extracellular signal and catalyze a reaction that bears a positive signal—the incorporation of phosphate into a protein—a process that can be studied in a straightforward manner. It is only after the protein kinase has completed its task, that a protein phosphatase might or might not be called to action. In fact, protein phosphatases were considered to be housekeeping, if not boring, enzymes. Thus, protein dephosphorylation was—for decades—treated as a mere afterthought. This view had to be revised during the last few years, since the use of cell-permeable protein phosphatase inhibitors in many biological systems has suggested that phosphatases play an active role in most cellular processes. [ABSTRACT FROM AUTHOR]

With the discovery of numerous eukaryotic protein serine/threonine phosphatases, a classification scheme was developed that exploited some of the biochemical properties of these enzymes to expedite their identification (1). These properties included the preferential dephosphorylation of either α or β-subunits of phosphorylase kinase, the metal ion dependency displayed by some phosphatases for enzyme activity and their inhibition by two mammalian thermostable proteins, inhibitor-1 (I-1) and inhibitor-2 (I-2). [ABSTRACT FROM AUTHOR]

This chapter describes the use of phosphatase-inhibitory compounds to study gene regulation. Serine/threonine protein phosphatase inhibitors can be divided into two groups: The first group (see Table 1) comprises environmental toxins and other natural products that are mostly produced by micro-organisms, such as blue-green algae or soil bacteria. They are structurally very diverse molecules that share the ability to inhibit phosphatase activity (1-3). The best known and most widely used compound of this group is okadaic acid, a polyether fatty acid derivative that is produced by marine dinoflagellates (4). Its use in research became quite widespread during the last few years, and the number of publications describing its effects have increased enormously. In addition, a number of natural and synthetic compounds have been discovered recently, which are useful as inhibitors of protein phosphatase type 2B (also called calcineurin) (5,6) see Table 1). [ABSTRACT FROM AUTHOR]

Protein phosphatases type 1 (PP1) and type 2A (PP2A) are the only activities known in mammalian tissues to dephosphorylate glycogen phosphorylase a. Phosphorylase was the first enzyme demonstrated to undergo regulation of catalytic activity via reversible covalent modification involving phosphorylation. It exists in a dephosphorylated, b form, which is catalytically active only in the presence of its allosteric activator, AMP, and a phosphorylated, a form, which is catalytically active in the absence of AMP. The conversion of phosphorylase b to a is catalyzed by phosphorylase kinase, a process that results in the phosphorylation of a single serine residue (Ser-14) in each identical subunit of the native dimer. This is one of the advantages of using phosphorylase a as a protein phosphatase substrate to study PP1 and PP2A activities in mammalian and nonmammalian eucaryotic cells. Other protein substrates, such as glycogen synthase and phosphorylase kinase, contain multiple phosphorylation sites, the reversible covalent modification of which are not always correlated with changes in enzymic activity. Other advantages include the commercial availability of both phosphorylase and phosphorylase kinase Phosphorylase b can also be conveniently isolated in gram quantities from 1 kg of either fresh or frozen rabbit skeletal muscle by a procedure that can be completed within a week and does not involve any chromatography steps (1,2). Phosphorylase kinase can be isolated within a 24 h period from fresh rabbit skeletal muscle (3). [ABSTRACT FROM AUTHOR]

As this chapter deals with the protein-serine/threonine phosphatases of prokaryotic organisms, some comments on the role of prokaryotes in the study of these important enzymes would appear to be in order. Prokaryotic organisms dominate the living world. They represent by the largest source of biomass on the planet, forming the indispensable foundation of the food chain upon which all other living organisms depend. They are the exclusive agents for carrying out biological nitrogen fixation, and are responsible for the majority of the photosynthetic activity that generates the oxygen we breath. In absolute numbers, in number of species, in range of habitat, and in the spectrum of their metabolic activities, the prokaryotes far outpace their eukaryotic brethren. More immediately, in humans prokaryotes perform essential functions in the digestion and assimilation of nutrients, whereas infection by bacterial pathogens can lead to illness or death. [ABSTRACT FROM AUTHOR]