Immunological characterization of phosphoprotein phosphatases

Phosphoprotein phosphatases regulate the biological activities of proteins through their involvement in cyclic phosphorylation/dephosphorylation cascades. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 and types 2A, 2B, and 2C. To elucidate the relationship between the different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes were tested for immunological antigenic relatedness. Two heterologous antibody preparations were employed for this purpose. One was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (calcineurin, holoenzyme). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. The antibody to the type 2A phosphatase reacted with the catalytic subunits of every type 2 enzyme tested, including both the catalytic and Ca2+-binding subunits of the Ca2+/calmodulin-dependent type 2B phosphatase (calcineurin), bovine cardiac type 2A phosphatase, and turkey gizzard smooth muscle phosphatase-1 (type 2A1). It did not react with any type 1 phosphatase (catalytic subunit or ATP-Mg-dependent). The antigenic relatedness of calcineurin and the bovine cardiac type 2A phosphatase (Mr 38,000) was demonstrated further by protein blot analysis showing that the anti-calcineurin antibody cross-reacted with both enzymes. The mutual cross-reactivity poses an intriguing problem because these enzymes are so different in their molecular structures and modes of regulation. The degree of evolutionary conservation exhibited by the antigenic cross-reactivity of the type 2 enzymes from a broad range of species and tissues suggests a strong selective pressure on maintaining one or more features of these important regulatory enzymes.     

蛋白磷酸酶PP2A的结构及其肿瘤抑制因子功能

蛋白磷酸酶在细胞的生命活动中起着十分重要的作用,蛋白磷酸酶2A(protein phosphatase 2A, PP2A)作为蛋白磷酸酶家族中十分重要的一员,它几乎与所有真核细胞的生命活动都有密不可分的关系．2006年,PP2A核心酶和全酶晶体结构的陆续破解对于深入了解PP2A自身的结构和亚基之间的相互作用,以及其与结合蛋白作用的机制都有重大的影响．随着PP2A与肿瘤相关性的一系列新研究成果的不断涌现,PP2A在肿瘤发生和细胞迁移中也彰显出十分关键的作用．重点介绍PP2A的组成与结构、催化亚基的特殊修饰、亚基之间的相互作用关系以及PP2A作为一种新的肿瘤抑制因子的生物学功能．     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.     

The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...     

The tetratricopeptide repeat domain and a C-terminal region control the activity of Ser/Thr protein phosphatase 5.

Protein Ser/Thr phosphatase 5 is a 58-kDa protein containing a catalytic domain structurally related to the catalytic subunits of protein phosphatases 1, 2A, and 2B and an extended N-terminal domain with three tetratricopeptide repeats. The activity of this enzyme is stimulated 4-14-fold in vitro by polyunsaturated fatty acids and anionic phospholipids. The structural basis for lipid activation of protein phosphatase 5 was examined by limited proteolysis and site-directed mutagenesis. Trypsinolysis removed the tetratricopeptide repeat domain and increased activity to approximately half that of lipid-stimulated, full-length enzyme. Subtilisin removed the tetratricopeptide repeat domain and 10 residues from the C terminus, creating a catalytic fragment with activity that was equal to or greater than that of lipid-stimulated, full-length enzyme. Catalytic fragments generated by proteolysis were no longer stimulated by lipid, and degradation of the tetratricopeptide repeat domain was decreased by association with lipid. A truncated mutant missing 13 C-terminal residues was also insensitive to lipid and was as active as full-length, lipid-stimulated enzyme. These results suggest that the C-terminal and N-terminal domain act in a coordinated manner to suppress the activity of protein phosphatase 5 and mediate its activation by lipid. These regions may be targets for the regulation of protein phosphatase 5 activity in vivo.     

Multiple pathways regulated by the tumor suppressor PP2A in transformation

Reversible protein phosphorylation plays a central role in regulating intracellular signaling. Dysregulation of the mechanisms that regulate phosphorylation plays a direct role in cancer initiation and maintenance. Although abundant evidence supports the role of kinase oncogenes in cancer development, recent work has illuminated the role of specific protein phosphatases in malignant transformation. Protein phosphatase 2A (PP2A) is the major serine-threonine phosphatase in mammalian cells. Inactivation of PP2A by viral oncoproteins, mutation of specific subunits or overexpression of endogenous inhibitors contributes to cell transformation by regulating specific phosphorylation events. Here, we review recent progress in our understanding of how PP2A regulates mitogenic signaling pathways in cancer pathogenesis and how PP2A activity is modulated in human cancers.     

Overexpression of the protein phosphatase 2A regulatory subunit Bgamma promotes neuronal differentiation by activating the MAP kinase (MAPK) cascade

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional regulator of cellular signaling. Variable regulatory subunits associate with a core dimer of scaffolding and catalytic subunits and are postulated to dictate substrate specificity and subcellular location of the heterotrimeric PP2A holoenzyme. The role of brain-specific regulatory subunits in neuronal differentiation and signaling was investigated in the PC6-3 subline of PC12 cells. Endogenous Bbeta, Bgamma, and B'beta protein expression was induced during nerve growth factor (NGF)-mediated neuronal differentiation. Transient expression of Bgamma, but not other PP2A regulatory subunits, facilitated neurite outgrowth in the absence and presence of NGF. Tetracycline-inducible expression of Bgamma caused growth arrest and neurofilament expression, further evidence that PP2A/Bgamma can promote differentiation. In PC6-3 cells, but not non-neuronal cell lines, Bgamma specifically promoted long lasting activation of the mitogen-activated protein (MAP) kinase cascade, a key mediator of neuronal differentiation. Pharmacological and dominant-negative inhibition and kinase assays indicate that Bgamma promotes neuritogenesis by stimulating the MAP kinase cascade downstream of the TrkA NGF receptor but upstream or at the level of the B-Raf kinase. Mutational analyses demonstrate that the divergent N terminus is critical for Bgamma activity. These studies implicate PP2A/Bgamma as a positive regulator of MAP kinase signaling in neurons.     

Critical role for protein phosphatase 2A heterotrimers in mammalian cell survival

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (alpha/beta), whereas three gene families (B, B', and B') with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding Aalpha subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. Aalpha RNAi decreased total PP2A activity as well as protein levels of C, B, and B' but not B' subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of Aalpha triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant Aalpha cDNAs rescued RNAi-induced loss of the C subunit, and Aalpha point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type Abeta and Aalpha mutants capable of binding to at least one family of regulatory subunits were able to delay Aalpha RNAi-induced death of PC12 cells. However, only the expression of wild-type Aalpha restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the Aalpha subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.     

Protein phosphatase 2A: a panoply of enzymes

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.     

The protein phosphatases involved in cellular regulation. Glycolysis, gluconeogenesis and aromatic amino acid breakdown in rat liver

The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-bisphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phosphofructo-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation.     

Interaction between protein phosphatase 5 and the A subunit of protein phosphatase 2A: evidence for a heterotrimeric form of protein phosphatase 5

Members of the phosphoprotein phosphatase family of serine/threonine phosphatases are thought to exist in different native oligomeric complexes. Protein phosphatase 2A (PP2A) is composed of a catalytic subunit (PP2Ac) that complexes with an A subunit, which in turn also interacts with one of many B subunits that regulate substrate specificity and/or (sub)cellular localization of the enzyme. Another family member, protein phosphatase 5 (PP5), contains a tetratricopeptide repeat domain at its N terminus, which has been suggested to mediate interactions with other proteins. PP5 was not thought to interact with partners homologous to the A or B subunits that exist within PP2A. However, our results indicate that this may not be the case. A yeast two-hybrid screen revealed an interaction between PP5 and the A subunit of PP2A. This interaction was confirmed for endogenous proteins in vivo using immunoprecipitation analysis and for recombinant proteins by in vitro binding experiments. Our results also indicate that the tetratricopeptide repeat domain of PP5 is required and sufficient for this interaction. In addition, immunoprecipitated PP5 contains associated B subunits. Thus, our results suggest that PP5 can exist in a PP2A-like heterotrimeric form containing both A and B subunits.     

Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing.

We have used a combination of highly specific protein phosphatase inhibitors and purified mammalian protein phosphatases to show that at least two separate Ser/Thr protein phosphatase activities are required for pre-mRNA splicing, but not for spliceosome assembly. Okadaic acid, tautomycin, and microcystin-LR, which are potent and specific inhibitors of PP1 and PP2A, two of the four major types of Ser/Thr-specific phosphatase catalytic subunits, block both catalytic steps of the pre-mRNA splicing mechanism in HeLa nuclear extracts. Inhibition of PP2A inhibits the second step of splicing predominantly while inhibition of both PP1 and PP2A blocks both steps, indicating a differential contribution of PP1 and PP2A activities to the two separate catalytic steps of splicing. Splicing activity is restored to toxin-inhibited extracts by the addition of highly purified mammalian PP1 or PP2A. Protein phosphatase activity was not required for efficient assembly of splicing complexes containing each of the U1, U2, U4/U6 and U5 snRNPs. The data indicate that reversible protein phosphorylation may play an important role in regulating the pre-mRNA splicing mechanism.     

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.     

The protein phosphatases involved in cellular regulation. Antibody to protein phosphatase-2A as a probe of phosphatase structure and function

Antibody prepared against the catalytic subunit of protein phosphatase-2A from rabbit skeletal muscle, could completely inhibit this enzyme, but did not significantly affect the activities of protein phosphatases-1, 2B and 2C. The antibody was used to establish the following points. The three forms of protein phosphatase-2A that can be resolved by ion-exchange chromatography, termed 2A0, 2A1, and 2A2, share the same catalytic subunit. The antigenic sites on the catalytic subunit of protein phosphatase-2A remain accessible to the antibody, when the catalytic subunit is complexed with the other subunits of protein phosphatases-2A0, 2A1 and 2A2. The catalytic subunits of protein phosphatase-2A from rabbit skeletal muscle and rabbit liver are very similar, as judged by immunotitration experiments. Protein phosphatase-1 and protein phosphatase-2A account for virtually all the phosphorylase phosphatase activity in dilute tissue extracts prepared from skeletal muscle, liver, heart, brain and kidney, and for essentially all the glycogen synthase phosphatase activity in dilute skeletal muscle and liver extracts. Protein phosphatase-2A is almost absent from the protein-glycogen complex prepared from skeletal muscle or liver extracts. Protein phosphatase-2A accounts for a major proportion of the phosphatase activity in dilute liver extracts towards 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, 6-phosphofructo-1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase and phenylalanine hydroxylase, the major phosphorylated enzymes involved in the hormonal control of hepatic glycolysis and gluconeogenesis.     

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.     

Inactivation and Reactivation of the Multifunctional Calmodulin-Dependent Protein Kinase from Brain by Autophosphorylation and Dephosphorylation: Involvement of Protein Phosphatases from Brain

The multifunctional calmodulin-dependent protein kinase (calmodulin-kinase) from rat brain was autophosphorylated in a Ca2+- and calmodulin-dependent manner. The activity of the autophosphorylated enzyme was independent of Ca2+ and calmodulin. Calmodulin-kinase was dephosphorylated by protein phosphatase C from bovine brain, which is the catalytic subunits of protein phosphatases 1 and 2A. The holoenzyme of protein phosphatase 2A was also involved in the dephosphorylation of the enzyme. The autophosphorylated sites of calmodulin-kinase were universally dephosphorylated by protein phosphatase C. Calmodulin-kinase was inactivated and reactivated by autophosphorylation and dephosphorylation, respectively. Furthermore, the regulation of calmodulin-kinase by autophosphorylation and dephosphorylation was observed using calmodulin-kinase from canine heart. These results suggest that the activity of calmodulin-kinase is regulated by autophosphorylation and dephosphorylation, and that the regulation is the universal phenomenon for many other calmodulin-kinases in various tissues.     

Protein serine/threonine phosphatases: life, death, and sleeping

Protein serine/threonine phosphatases control key biological pathways including early embryonic development, cell proliferation, cell death, circadian rhythm and cancer. Recent studies have provided important insights into how several of the many phosphatase regulators, through their interaction with a conserved phosphatase catalytic subunit, control the activity of critical substrates in these diverse pathways. Recent co-crystal structures provided a major insight into how the diverse protein serine/threonine regulators rein in the otherwise promiscuous catalytic subunits.     

Identification of the phosphatase deinhibitor protein phosphatases in rabbit skeletal muscle.

In rabbit skeletal muscle the polycation-stimulated (PCS) protein phosphatases [Merlevede (1985) Adv. Protein Phosphatases 1, 1-18] are the only phosphatases displaying significant activity toward the deinhibitor protein. Among them, the PCSH protein phosphatase represents more than 80% of the measurable deinhibitor phosphatase activity associated with the PCS phosphatases. The deinhibitor phosphatase activity co-purifies with the PCSH phosphatase to apparent homogeneity. In the last purification step two forms of PCSH phosphatase were separated (PCSH1, containing 62, 55 and 34 kDa subunits, and PCSH2, containing 62 and 35 kDa subunits), both showing the same deinhibitor/phosphorylase phosphatase activity ratio. The activity of the PCSH phosphatase toward the deinhibitor is not stimulated by polycations such as protamine, histone H1 or polylysine, unlike the stimulation observed with phosphorylase as the substrate. The phosphorylase phosphatase activity of PCSH phosphatase is inhibited by ATP, PPi and Pi, whereas the deinhibitor phosphatase activity of the enzyme is much less sensitive to these agents.     

Characterization of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5

Background  

Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.     

Bioinformatic identification of novel protein phosphatases in the dog genome

Protein kinases and protein phosphatases constitute about 2-4% of the genes in a typical eukaryotic genome. Protein phosphatases are important players in many cellular processes such as proliferation, differentiation, cell adhesion, and motility. In this study, we identified, classified, and analyzed protein phosphatase complement of the dog genome. In this article, we report the identification of at least 178 putative protein phosphatases in dog which include 51 PSTPs, 112 PTPs, and 15 Asp-based protein phosphatases. Interestingly, we found at least five novel protein phosphatases in dog, namely DUSP5L, DUSP18L, MTMR9L, MTMR12L, and PPP6CL which are not present in human, mouse, rat, and cow. In addition, we found PTP4A1-rt, a retro-transposed copy of the PTP4A1 gene, in chromosome 27. Furthermore, we modeled three-dimensional structures of the catalytic domains of these putative protein phosphatases and aligned them to see the structural similarities between them. We docked PPP2CA with okadaic acid and calculated the value of affinity energy as -8.8?kcal/mol. Our nucleotide substitution rate study revealed that apparently none of the phosphatase family is under significantly higher evolutionary pressure.