Eukaryotic-like Phosphoprotein Phosphatase (PPP) enzyme evolution: interactions with environmental toxins and regulatory proteins.

Phosphoprotein phosphatases (PPPs) are a ubiquitous class of enzymes which dephosphorylate serine and threonine residues on substrate proteins involved in a wide variety of cellular processes. The active site of PPP enzymes are highly conserved with key residues coordinating the substrate phosphoryl group (the two R-clamp) and two metal ions necessary for catalysis. Because of the diverse number of roles that these enzymes play it is no surprise that they are highly regulated in the cell, often accomplished by binding regulatory subunits. These regulatory subunits are able to dictate substrate specificity, localization, and activity of the bound catalytic subunit. Eukaryotic PPP subtypes have been previously shown to manifest varying degrees of sensitivity to environmental toxins. We present here an evolutionary model which now rationalizes this data. Our re-examination of published structural evidence reveals that Eukaryotic PPP toxin-binding residues also interact with substrate binding residues (the two R-clamp) and ancient regulatory proteins. Such functional interactions could have stabilized PPP sequence early in Eukaryotic evolution, providing a stable target which was co-opted by toxins and their producer organisms.

The origin and radiation of the phosphoprotein phosphatase (PPP) enzymes of Eukaryotes.

Phosphoprotein phosphatase (PPP) enzymes are ubiquitous proteins involved in cellular signaling pathways and other functions. Here we have traced the origin of the PPP sequences of Eukaryotes and their radiation. Using a bacterial PPP Hidden Markov Model (HMM) we uncovered "BacterialPPP-Like" sequences in Archaea. A HMM derived from eukaryotic PPP enzymes revealed additional, unique sequences in Archaea and Bacteria that were more like the eukaryotic PPP enzymes then the bacterial PPPs. These sequences formed the basis of phylogenetic tree inference and sequence structural analysis allowing the history of these sequence types to be elucidated. Our phylogenetic tree data strongly suggest that eukaryotic PPPs ultimately arose from ancestors in the Asgard archaea. We have clarified the radiation of PPPs within Eukaryotes, substantially expanding the range of known organisms with PPP subtypes (Bsu1, PP7, PPEF/RdgC) previously thought to have a more restricted distribution. Surprisingly, sequences from the Methanosarcinaceae (Euryarchaeota) form a strongly supported sister group to eukaryotic PPPs in our phylogenetic analysis. This strongly suggests an intimate association between an Asgard ancestor and that of the Methanosarcinaceae. This is highly reminiscent of the syntrophic association recently demonstrated between the cultured Lokiarchaeal species Prometheoarchaeum and a methanogenic bacterial species.

Origin of the Phosphoprotein Phosphatase (PPP) sequence family in Bacteria: Critical ancestral sequence changes, radiation patterns and substrate binding features.

Background: Phosphoprotein phosphatases (PPP) belong to the PPP Sequence family, which in turn belongs to the broader metallophosphoesterase (MPE) superfamily. The relationship between the PPP Sequence family and other members of the MPE superfamily remains unresolved, in particular what transitions took place in an ancestral MPE to ultimately produce the phosphoprotein specific phosphatases (PPPs). Methods: We use structural and sequence alignment data, phylogenetic tree analysis, sequence signature (Weblogo) analysis, in silico protein-peptide modeling data, and in silico mutagenesis to trace a likely route of evolution from MPEs to the PPP Sequence family. Hidden Markov Model (HMM) based iterative database search strategies were utilized to identify PPP Sequence Family members from numerous bacterial groups. Results: Using Mre11 as proxy for an ancestral nuclease-like MPE we trace a possible evolutionary route that alters a single active site substrate binding His-residue to yield a new substrate binding accessory, the "2-Arg-Clamp". The 2-Arg-Clamp is not found in MPEs, but is present in all PPP Sequence family members, where the phosphomonesterase reaction predominates. Variation in position of the clamp arginines and a supplemental sequence loop likely provide substrate specificity for each PPP Sequence family group. Conclusions: Loss of a key substrate binding His-in MPEs opened the path to bind novel substrates and evolution of the 2-Arg-Clamp, a sequence change seen in both bacterial and eukaryotic phosphoprotein phosphatases.General significance: We establish a likely evolutionary route from nuclease-like MPE to PPP Sequence family enzymes, that includes the phosphoprotein phosphatases.

"PP2C7s", Genes Most Highly Elaborated in Photosynthetic Organisms, Reveal the Bacterial Origin and Stepwise Evolution of PPM/PP2C Protein Phosphatases.

Mg+2/Mn+2-dependent type 2C protein phosphatases (PP2Cs) are ubiquitous in eukaryotes, mediating diverse cellular signaling processes through metal ion catalyzed dephosphorylation of target proteins. We have identified a distinct PP2C sequence class ("PP2C7s") which is nearly universally distributed in Eukaryotes, and therefore apparently ancient. PP2C7s are by far most prominent and diverse in plants and green algae. Combining phylogenetic analysis, subcellular localization predictions, and a distillation of publically available gene expression data, we have traced the evolutionary trajectory of this gene family in photosynthetic eukaryotes, demonstrating two major sequence assemblages featuring a succession of increasingly derived sub-clades. These display predominant expression moving from an ancestral pattern in photosynthetic tissues toward non-photosynthetic, specialized and reproductive structures. Gene co-expression network composition strongly suggests a shifting pattern of PP2C7 gene functions, including possible regulation of starch metabolism for one homologue set in Arabidopsis and rice. Distinct plant PP2C7 sub-clades demonstrate novel amino terminal protein sequences upon motif analysis, consistent with a shifting pattern of regulation of protein function. More broadly, neither the major events in PP2C sequence evolution, nor the origin of the diversity of metal binding characteristics currently observed in different PP2C lineages, are clearly understood. Identification of the PP2C7 sequence clade has allowed us to provide a better understanding of both of these issues. Phylogenetic analysis and sequence comparisons using Hidden Markov Models strongly suggest that PP2Cs originated in Bacteria (Group II PP2C sequences), entered Eukaryotes through the ancestral mitochondrial endosymbiosis, elaborated in Eukaryotes, then re-entered Bacteria through an inter-domain gene transfer, ultimately producing bacterial Group I PP2C sequences. A key evolutionary event, occurring first in ancient Eukaryotes, was the acquisition of a conserved aspartate in classic Motif 5. This has been inherited subsequently by PP2C7s, eukaryotic PP2Cs and bacterial Group I PP2Cs, where it is crucial to the formation of a third metal binding pocket, and catalysis.

The mitotic PP2A regulator ENSA/ARPP-19 is remarkably conserved across plants and most eukaryotes.

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase of eukaryotes. PP2A containing the B55 subunit is a key regulator of mitosis and must be inhibited by phosphorylated α-endosulfine (ENSA) or cyclic AMP-regulated 19 kDa phosphoprotein (ARPP-19) to allow passage through mitosis. Exit from mitosis then requires dephosphorylation of ENSA/ARPP-19 to relieve inhibition of PP2A/B55. ENSA/ARPP-19 has been characterized in several vertebrates and budding yeast, but little is known about its presence in plants and the majority of other eukaryotes. Here we show that three isoforms of ENSA/ARPP-19 are present in the Arabidopsis thaliana genome with distinct expression profiles across various plant tissues. The ENSA/ARPP-19 proteins, and in particular their key inhibitory sequence FDSGDY (FDSADW in plants), is remarkably conserved across plants and most eukaryotes suggesting an ancient origin and conserved function to control PP2A activity.

Evolution of bacterial-like phosphoprotein phosphatases in photosynthetic eukaryotes features ancestral mitochondrial or archaeal origin and possible lateral gene transfer.

Protein phosphorylation is a reversible regulatory process catalyzed by the opposing reactions of protein kinases and phosphatases, which are central to the proper functioning of the cell. Dysfunction of members in either the protein kinase or phosphatase family can have wide-ranging deleterious effects in both metazoans and plants alike. Previously, three bacterial-like phosphoprotein phosphatase classes were uncovered in eukaryotes and named according to the bacterial sequences with which they have the greatest similarity: Shewanella-like (SLP), Rhizobiales-like (RLPH), and ApaH-like (ALPH) phosphatases. Utilizing the wealth of data resulting from recently sequenced complete eukaryotic genomes, we conducted database searching by hidden Markov models, multiple sequence alignment, and phylogenetic tree inference with Bayesian and maximum likelihood methods to elucidate the pattern of evolution of eukaryotic bacterial-like phosphoprotein phosphatase sequences, which are predominantly distributed in photosynthetic eukaryotes. We uncovered a pattern of ancestral mitochondrial (SLP and RLPH) or archaeal (ALPH) gene entry into eukaryotes, supplemented by possible instances of lateral gene transfer between bacteria and eukaryotes. In addition to the previously known green algal and plant SLP1 and SLP2 protein forms, a more ancestral third form (SLP3) was found in green algae. Data from in silico subcellular localization predictions revealed class-specific differences in plants likely to result in distinct functions, and for SLP sequences, distinctive and possibly functionally significant differences between plants and nonphotosynthetic eukaryotes. Conserved carboxyl-terminal sequence motifs with class-specific patterns of residue substitutions, most prominent in photosynthetic organisms, raise the possibility of complex interactions with regulatory proteins.

Bacterial-like PPP protein phosphatases: novel sequence alterations in pathogenic eukaryotes and peculiar features of bacterial sequence similarity.

Reversible phosphorylation is a widespread modification affecting the great majority of eukaryotic cellular proteins, and whose effects influence nearly every cellular function. Protein phosphatases are increasingly recognized as exquisitely regulated contributors to these changes. The PPP (phosphoprotein phosphatase) family comprises enzymes, which catalyze dephosphorylation at serine and threonine residues. Nearly a decade ago, "bacterial-like" enzymes were recognized with similarity to proteins from various bacterial sources: SLPs (Shewanella-like phosphatases), RLPHs (Rhizobiales-like phosphatases), and ALPHs (ApaH-like phosphatases). A recent article from our laboratory appearing in Plant Physiology characterizes their extensive organismal distribution, abundance in plant species, predicted subcellular localization, motif organization, and sequence evolution. One salient observation is the distinct evolutionary trajectory followed by SLP genes and proteins in photosynthetic eukaryotes vs. animal and plant pathogens derived from photosynthetic ancestors. We present here a closer look at sequence data that emphasizes the distinctiveness of pathogen SLP proteins and that suggests that they might represent novel drug targets. A second observation in our original report was the high degree of similarity between the bacterial-like PPPs of eukaryotes and closely related proteins of the "eukaryotic-like" phyla Myxococcales and Planctomycetes. We here reflect on the possible implications of these observations and their importance for future research.

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile.

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

A phylogenetic survey of myotubularin genes of eukaryotes: distribution, protein structure, evolution, and gene expression.

BACKGROUND: Phosphorylated phosphatidylinositol (PtdIns) lipids, produced and modified by PtdIns kinases and phosphatases, are critical to the regulation of diverse cellular functions. The myotubularin PtdIns-phosphate phosphatases have been well characterized in yeast and especially animals, where multiple isoforms, both catalytically active and inactive, occur. Myotubularin mutations bring about disruption of cellular membrane trafficking, and in humans, disease. Previous studies have suggested that myotubularins are widely distributed amongst eukaryotes, but key evolutionary questions concerning the origin of different myotubularin isoforms remain unanswered, and little is known about the function of these proteins in most organisms. RESULTS: We have identified 80 myotubularin homologues amidst the completely sequenced genomes of 30 organisms spanning four eukaryotic supergroups. We have mapped domain architecture, and inferred evolutionary histories. We have documented an expansion in the Amoebozoa of a family of inactive myotubularins with a novel domain architecture, which we dub "IMLRK" (inactive myotubularin/LRR/ROCO/kinase). There is an especially large myotubularin gene family in the pathogen Entamoeba histolytica, the majority of them IMLRK proteins. We have analyzed published patterns of gene expression in this organism which indicate that myotubularins may be important to critical life cycle stage transitions and host infection. CONCLUSIONS: This study presents an overall framework of eukaryotic myotubularin gene evolution. Inactive myotubularin homologues with distinct domain architectures appear to have arisen on three separate occasions in different eukaryotic lineages. The large and distinctive set of myotubularin genes found in an important pathogen species suggest that in this organism myotubularins might present important new targets for basic research and perhaps novel therapeutic strategies.

Evolution of protein phosphatases in plants and animals.

Protein phosphorylation appears to be a universal mechanism of protein regulation. Genomics has provided the means to compile inventories of protein phosphatases across a wide selection of organisms and this has supplied insights into the evolution of this group of enzymes. Protein phosphatases evolved independently several times yielding the groups we observe today. Starting from a core catalytic domain, phosphatases evolved by a series of gene duplication events and by adopting the use of regulatory subunits and/or fusion with novel functional modules or domains. Recent analyses also suggest that the serine/threonine specific enzymes are more ancient than the PTPs (protein tyrosine phosphatases). It is likely that the latter played a key role at the onset of metazoan evolution in conjunction with the tremendous expansion of tyrosine kinases and PTPs at this point. In the present review, we discuss the evolution of the PTPs, the serine/threonine specific PPP (phosphoprotein phosphatase) and PPM (metallo-dependent protein phosphatase) families and the more recently discovered phosphatases that utilize an aspartate-based catalytic mechanism. We will also highlight examples of convergent evolution and several phosphatases which are unique to plants.

Arabidopsis At5g39790 encodes a chloroplast-localized, carbohydrate-binding, coiled-coil domain-containing putative scaffold protein.

BACKGROUND: Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain. RESULTS: We here show that At5g39790 is chloroplast-localized, and binds starch, with a preference for amylose. The protein persists in starch binding under conditions of pH, redox and Mg(+2) concentrations characteristic of both the day and night chloroplast cycles. Bioinformatic analysis demonstrates a diurnal pattern of gene expression, with an accumulation of transcript during the light cycle and decline during the dark cycle. A corresponding diurnal pattern of change in protein levels in leaves is also observed. Sequence analysis shows that At5g39790 has a strongly-predicted coiled-coil domain. Similar analysis of the set of starch metabolic enzymes shows that several have strong to moderate coiled-coil potential. Gene expression analysis shows strongly correlated patterns of co-expression between At5g39790 and several starch metabolic enzymes. CONCLUSION: We propose that At5g39790 is a regulatory scaffold protein, persistently binding the starch granule, where it is positioned to interact by its coiled-coil domain with several potential starch metabolic enzyme binding-partners.

Evolutionary radiation pattern of novel protein phosphatases revealed by analysis of protein data from the completely sequenced genomes of humans, green algae, and higher plants.

In addition to the major serine/threonine-specific phosphoprotein phosphatase, Mg(2+)-dependent phosphoprotein phosphatase, and protein tyrosine phosphatase families, there are novel protein phosphatases, including enzymes with aspartic acid-based catalysis and subfamilies of protein tyrosine phosphatases, whose evolutionary history and representation in plants is poorly characterized. We have searched the protein data sets encoded by the well-finished nuclear genomes of the higher plants Arabidopsis (Arabidopsis thaliana) and Oryza sativa, and the latest draft data sets from the tree Populus trichocarpa and the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, for homologs to several classes of novel protein phosphatases. The Arabidopsis proteins, in combination with previously published data, provide a complete inventory of known types of protein phosphatases in this organism. Phylogenetic analysis of these proteins reveals a pattern of evolution where a diverse set of protein phosphatases was present early in the history of eukaryotes, and the division of plant and animal evolution resulted in two distinct sets of protein phosphatases. The green algae occupy an intermediate position, and show similarity to both plants and animals, depending on the protein. Of specific interest are the lack of cell division cycle (CDC) phosphatases CDC25 and CDC14, and the seeming adaptation of CDC14 as a protein interaction domain in higher plants. In addition, there is a dramatic increase in proteins containing RNA polymerase C-terminal domain phosphatase-like catalytic domains in the higher plants. Expression analysis of Arabidopsis phosphatase genes differentially amplified in plants (specifically the C-terminal domain phosphatase-like phosphatases) shows patterns of tissue-specific expression with a statistically significant number of correlated genes encoding putative signal transduction proteins.

Genome-scale discovery and characterization of class-specific protein sequences: an example using the protein phosphatases of Arabidopsis thaliana.

The increasing pace of acquisition of fully sequenced genomes makes desirable a program of discovery and characterization of protein sequences of biologically significant structural classes. An example is protein phosphatases, involved in modulating reversible protein phosphorylation events underlying the whole gamut of cellular biology. The ready availability of software that can be downloaded to run on a personal computer, or accessed on a server via the Web, allows appropriate sequences to be collected and analyzed. A process is outlined here that has been successfully employed in the description of the genomic complement of protein phosphatase catalytic subunits from the model plant Arabidopsis thaliana. However, the methods are general and readily adapted to deal with any desired class of protein, from any organism of interest.

A chloroplast-localized dual-specificity protein phosphatase in Arabidopsis contains a phylogenetically dispersed and ancient carbohydrate-binding domain, which binds the polysaccharide starch.

Dual-specificity protein phosphatases (DSPs) are important regulators of a wide variety of protein kinase signaling cascades in animals, fungi and plants. We previously identified a cluster of putative DSPs in Arabidopsis (including At3g52180 and At3g01510) in which the phosphatase domain is related to that of laforin, the human protein mutated in Lafora epilepsy. In animal and fungal systems, the laforin DSP and the beta-regulatory subunits of AMP-regulated protein kinase (AMPK) and Snf-1 have all been demonstrated to bind to glycogen by a glycogen-binding domain (GBD). We present a bioinformatic analysis which shows that these DSPs from Arabidopsis, together with other related plant DSPs, share with the above animal and fungal proteins a widespread and ancient carbohydrate-binding domain. We demonstrate that DSP At3g52180 binds to purified starch through its predicted carbohydrate-binding region, and that mutation of key conserved residues reduces this binding. Consistent with its ability to bind exogenous starch, DSP At3g52180 was found associated with starch purified from Arabidopsis plants and suspension cells. Immunolocalization experiments revealed a co-localization with chlorophyll, placing DSP At3g52180 in the chloroplast. Gene-expression data from different stages of the light-dark cycle and across a wide variety of tissues show a strong correlation between the pattern displayed by transcripts of the At3g52180 locus and that of genes encoding key starch degradative enzymes. Taken together, these data suggest the hypothesis that plant DSPs could be part of a protein assemblage at the starch granule, where they would be ideally situated to regulate starch metabolism through reversible phosphorylation events.

Arabidopsis proteins containing similarity to the universal stress protein domain of bacteria.

We have collected a set of 44 Arabidopsis proteins with similarity to the USPA (universal stress protein A of Escherichia coli) domain of bacteria. The USPA domain is found either in small proteins, or it makes up the N-terminal portion of a larger protein, usually a protein kinase. Phylogenetic tree analysis based upon a multiple sequence alignment of the USPA domains shows that these domains of protein kinases 1.3.1 and 1.3.2 form distinct groups, as do the protein kinases 1.4.1. This indicates that their USPA domain structures have diverged appreciably and suggests that they may subserve distinct cellular functions. Two USPA fold classes have been proposed: one based on Methanococcus jannaschii MJ0577 (1MJH) that binds ATP, and the other based on the Haemophilus influenzae universal stress protein (1JMV), highly similar to E. coli UspA, which does not bind ATP. A set of common residues involved in ATP binding in 1MJH and conserved in similar bacterial sequences is also found in a distinct cluster of Arabidopsis sequences. Threading analysis, which examines aspects of secondary and tertiary structure, confirms this Arabidopsis sequence cluster as highly similar to 1MJH. This structural approach can distinguish between the characteristic fold differences of 1MJH-like and 1JMV-like bacterial proteins and was used to assign the complete set of candidate Arabidopsis proteins to one of these fold classes. It is clear that all the plant sequences have arisen from a 1MJH-like ancestor.

The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis.

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-M(r) protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.