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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.

Phosphoprotein phosphatases (PPPs) execute >90% of serine/threonine dephosphorylation in cells and tissues. While the role of PPPs in cell biology and diseases such as cancer, cardiac hypertrophy and Alzheimer’s disease is well established, the molecular mechanisms governing and governed by PPPs still await discovery. Here we describe a chemical proteomic strategy, phosphatase inhibitor beads and mass spectrometry (PIB-MS), that enables the identification and quantification of PPPs and their posttranslational modifications in as little as 12 h. Using a specific but nonselective PPP inhibitor immobilized on beads, PIB-MS enables the efficient affinity-capture, identification and quantification of endogenous PPPs and associated proteins (‘PPPome’) from cells and tissues. PIB-MS captures functional, endogenous PPP subunit interactions and enables discovery of new binding partners. It performs PPP enrichment without exogenous expression of tagged proteins or specific antibodies. Because PPPs are among the most conserved proteins across evolution, PIB-MS can be employed in any cell line, tissue or organism. This protocol describes a proteomic approach for efficient affinity capture, identification and quantification of endogenous phosphoprotein phosphatases and associated proteins from cells and tissues.

Reversible protein phosphorylation regulates the transitions between different phases of the cell cycle ensuring proper segregation of the duplicated genome into two daughter cells. Protein kinases and protein phosphatases establish the appropriate phosphorylation stoichiometries in diverse substrates maintaining genomic stability as a cell undergoes this complex process. Along with regulating common substrates, these opposing enzymes regulate one another by fine-tuning each other's activity both spatially and temporally throughout mitosis. Protein phosphatase catalytic subunits work together with regulatory proteins, which control their localization, activity, and specificity. Protein phosphatase 1 (PP1) recognizes its regulatory proteins via a short linear interaction motif (SLIM) called the "RVxF" motif. A subset of proteins with these "RVxF" motifs contain a phosphorylatable amino acid (S/T) at the 'x' position.Here, we describe methods to generate, affinity purify and utilize phospho-specific antibodies to monitor phosphorylation sites during the cell cycle and the appropriate use of mitotic kinase inhibitors. More specifically, we employ phospho-specific antibodies, which recognize phosphorylated RVp[S/T]F motif-containing proteins, to monitor the phosphorylation status of these motifs throughout the cell cycle. Furthermore, we use mitotic kinase inhibitors to examine the effect of kinase inhibition on the phosphorylation status of multiple RV[S/T]F motifs using these phospho-specific antibodies.

Protein phosphatase one (PP1) is a major eukaryotic serine/threonine protein phosphatase whose activity is controlled by targeting or regulatory subunits. Currently, very few plant protein phosphatase one regulatory subunits are known. Here, Arabidopsis GL2 EXPRESSION MODULATOR (GEM) was identified and confirmed as a protein phosphatase one binding partner. GEM is a phosphoprotein, contains a highly conserved phosphoinositide binding GRAM domain and a classic protein phosphatase one binding RVXF motif. Lipid overlays show GEM has the ability to interact with phosphoinositides through its GRAM domain. GEM is the first plant specific protein phosphatase one interactor to be discovered.

The plant Shewanella-like PPP protein phosphatases, SLP1 and SLP2, are localized to the chloroplast and mitochondria, respectively. Originally uncovered through bioinformatics, these enzymes have been proven to be bona fide protein serine/threonine phosphatases that originated in bacteria but display limited distribution across eukaryotes. They are remarkably conserved in plants, suggesting they play fundamental roles in plant chloroplast and mitochondrial biology. SLP1 appears to reside in the stromal or soluble fraction of chloroplasts and is not expressed in non-photosynthetic plastids. SLP2 is found in peroxisomes but is predominately localized to mitochondria and specifically in the mitochondrial intermembrane space. Like many proteins destined for the mitochondrial intermembrane space, SLP2 is retained here by the formation of disulfide bonds generated by association with oxidoreductase Mia40. We review the unique aspects of these plant enzymes and discuss the use of quantitative mass spectrometry (MS) to discover protein phosphatase substrates.

Centrosomal protein of 192 kDa (CEP192) is a scaffolding protein that recruits the mitotic protein kinases Aurora A and PLK1 to the centrosome. Here we demonstrate that CEP192 also recruits the type one protein phosphatase (PP1) via a highly conserved KHVTF docking motif. The threonine of the KHVTF motif is phosphorylated during mitosis and protein kinase inhibition studies suggest this to be a PLK1-dependent process.

In Arabidopsis (Arabidopsis thaliana) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by β-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1. Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the β-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1, and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3. We propose that LSF1 binds β-amylases at the starch granule surface, thereby promoting starch degradation.

Reversible protein phosphorylation catalyzed by protein kinases and phosphatases represents the most prolific and well-characterized posttranslational modification known. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) Shewanella-like protein phosphatase 2 (AtSLP2) is a bona fide Ser/Thr protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (AtMIA40), forming a protein complex. Interaction with AtMIA40 is necessary for the phosphatase activity of AtSLP2 and is dependent on the formation of disulfide bridges on AtSLP2. Furthermore, by utilizing atslp2 null mutant, AtSLP2 complemented and AtSLP2 overexpressing plants, we identify a function for the AtSLP2-AtMIA40 complex in negatively regulating gibberellic acid-related processes during seed germination. Results presented here characterize a mitochondrial IMS-localized protein phosphatase identified in photosynthetic eukaryotes as well as a protein phosphatase target of the highly conserved eukaryotic MIA40 IMS oxidoreductase.

Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants.

Peroxisomes perform essential roles in a range of cellular processes, highlighted by lipid metabolism, reactive species detoxification, and response to a variety of stimuli. The ability of peroxisomes to grow, divide, respond to changing cellular needs, interact with other organelles, and adjust their proteome as required, suggest that, like other organelles, their specialized roles are highly regulated. Similar to most other cellular processes, there is an emerging role for protein phosphorylation to regulate these events. In this review, we establish a knowledge framework of key players that control protein phosphorylation events in the plant peroxisome (i.e., the protein kinases and phosphatases), and highlight a vastly expanded set of (phospho)substrates.

A “tug-of-war” between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases. We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the “PPPome”). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.

Phospho-proteomic studies have confirmed that phosphorylation is a common mechanism to regulate protein function in the chloroplast, including the enzymes of starch metabolism. In addition to the photosynthetic machinery protein kinases (STN7 and STN8) and their cognate protein phosphatases PPH1 (TAP38) and PBCP, multiple other protein kinases and phosphatases have now been localized to the chloroplast. Here, we build a framework for understanding protein kinases and phosphatases, their regulation, and potential roles in starch metabolism. We also catalog mapped phosphorylation sites on proteins of chloroplast starch metabolism to illustrate the potential and mostly unknown roles of protein phosphorylation in the regulation of starch biology.

Protein phosphatase 1 (PP1) is a highly conserved protein phosphatase that performs the majority of serine and threonine (Ser-Thr) dephosphorylation reactions in eukaryotes and opposes the actions of a diverse set of Ser-Thr protein kinases. PP1 gains substrate specificity through binding to a large number (> 200) of regulatory proteins that control PP1 localization, activity, and interactions with substrates. PP1 recognizes the well-characterized RVxF binding motif that is present in many of these regulatory proteins, thus generating a multitude of distinct PP1 holoenzymes. Here we show that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), were phosphorylated specifically during mitosis and that this phosphorylation event abrogated the interaction of PP1 with the regulatory protein. We determined that this phosphorylation was primarily governed by the mitotic protein kinase Aurora B and that high phosphorylation site stoichiometry of these sites maintained phosphorylation of PP1 substrates during mitosis by disrupting the assembly of PP1 holoenzymes. We generated an antibody that recognizes the phosphorylated form of the RV[S/T]F motif (RVp[S/T]F) and used it to identify known PP1 regulatory proteins (KNL1, CDCA2, and RIF1) as well as multiple proteins that could potentially act as PP1 binding partners (UBR5, ASPM, SEH1, and ELYS) governed by this mechanism. Taken together, the work presented here suggests a general regulatory mechanism by which the coordinated activities of Aurora B and PP1 control mitotic progression.

Despite belonging to the phosphoserine- and phosphothreonine-specific phosphoprotein phosphatase (PPP) family, Arabidopsis thaliana Rhizobiales-like phosphatase 2 (RLPH2) strongly prefers substrates bearing phosphorylated tyrosine residues. We solved the structures of RLPH2 crystallized in the presence or absence of sodium tungstate. These structures revealed the presence of a central domain that forms a binding site for two divalent metal ions that closely resembles that of other PPP-family enzymes. Unique structural elements from two flanking domains suggest a mechanism for the selective dephosphorylation of phosphotyrosine residues. Cocrystallization with the phosphate mimetic tungstate also suggests how positively charged residues that are highly conserved in the RLPH2 class form an additional pocket that is specific for a phosphothreonine residue located near the phosphotyrosine residue that is bound to the active site. Site-directed mutagenesis confirmed that this auxiliary recognition element facilitates the recruitment of dual-phosphorylated substrates containing a pTxpY motif.

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.

Shewanella-like PPP family phosphatases (SLPs) are a unique lineage of eukaryote PPP-family phosphatases of bacterial origin which are not found in metazoans.1,2 Their absence in metazoans is marked by their ancient bacterial origins and presence in plants.1 Recently, we found that the SLP2 phosphatase ortholog of Arabidopsis thaliana localized to the mitochondrial intermembrane space (IMS) where it was determined to be activated by mitochondrial intermembrane space protein 40 (MIA40) to regulate seed germination.3 Through examination of atslp2 knockout (accelerated germination) and 35S::AtSLP2 over-expressing (delayed germination) plants it was found that AtSLP2 influences Arabidopsis thaliana germination rates via gibberellic acid (GA) biosynthesis.3 However, the exact mechanism by which this occurs remains unresolved. To identify potential partners of AtSLP2 in regulating germination through GA, we undertook a gene co-expression network analysis using RNA-sequencing data available through Genevestigator (https://genevestigator.com/gv/).

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions., Molecular Systems Biology, 13 (10), ISSN:1744-4292

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.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.

Starch is the major carbohydrate reserve in plants, and is degraded for growth at night. Starch breakdown requires reversible glucan phosphorylation at the granule surface by novel dikinases and phosphatases. The dual-specificity phosphatase starch excess 4 (SEX4) is required for glucan desphosphorylation; however, regulation of the enzymatic activity of SEX4 is not well understood. We show that SEX4 switches between reduced (active) and oxidized (inactive) states, suggesting that SEX4 is redox-regulated. Although only partial reactivation of SEX4 was achieved using artificial reductants (e.g. dithiothreitol), use of numerous chloroplastic thioredoxins recovered activity completely, suggesting that thioredoxins could reduce SEX4 in vivo. Analysis of peptides from oxidized and reduced SEX4 identified a disulfide linkage between the catalytic cysteine at position 198 (Cys198) and the cysteine at position 130 (Cys130) within the phosphatase domain. The position of these cysteines was structurally analogous to that for known redox-regulated dual-specificity phosphatases, suggesting a common mechanism of reversible oxidation amongst these phosphatases. Mutation of Cys130 renders SEX4 more sensitive to oxidative inactivation and less responsive to reductive reactivation. Together, these results provide the first biochemical evidence for a redox-dependent structural switch that regulates SEX4 activity, which represents the first plant phosphatase known to undergo reversible oxidation via disulfide bond formation like its mammalian counterparts.

Protein phosphorylation, catalyzed by the opposing actions of protein kinases and phosphatases, is a cornerstone of cellular signaling and regulation. Since their discovery, protein phosphatases have emerged as highly regulated enzymes with specificity that rivals their counteracting kinase partners. However, despite years of focused characterization in mammalian and yeast systems, many protein phosphatases in plants remain poorly or incompletely characterized. Here, we describe a bioinformatic, biochemical, and cellular examination of an ancient, Bacterial-like subclass of the phosphoprotein phosphatase (PPP) family designated the Shewanella-like protein phosphatases (SLP phosphatases). The SLP phosphatase subcluster is highly conserved in all plants, mosses, and green algae, with members also found in select fungi, protists, and bacteria. As in other plant species, the nucleus-encoded Arabidopsis (Arabidopsis thaliana) SLP phosphatases (AtSLP1 and AtSLP2) lack genetic redundancy and phylogenetically cluster into two distinct groups that maintain different subcellular localizations, with SLP1 being chloroplastic and SLP2 being cytosolic. Using heterologously expressed and purified protein, the enzymatic properties of both AtSLP1 and AtSLP2 were examined, revealing unique metal cation preferences in addition to a complete insensitivity to the classic serine/threonine PPP protein phosphatase inhibitors okadaic acid and microcystin. The unique properties and high conservation of the plant SLP phosphatases, coupled to their exclusion from animals, red algae, cyanobacteria, archaea, and most bacteria, render understanding the function(s) of this new subclass of PPP family protein phosphatases of particular interest.

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.

Protein phosphatase 1 (PP1) accumulates within the nucleolus, which plays a central role in regulation of cell growth and proliferation. Using a powerful combination of imaging and quantitative proteomics, we identify RRP1B as a nucleolar PP1 targeting subunit that sits at the hub of the pre-60S ribosomal subunit processing complex., A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions.

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G(2)/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (gamma-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of gamma-H2AX, the dissolution of IR-induced foci, and release from the G(2)/M checkpoint in vivo.

The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.

The PII carbon/nitrogen sensing protein was discovered in Escherichia coli (Migula 1895) Castellani and Chalmers 1919, over 40 years ago. Orthologues have been discovered in three kingdoms of life making it one of the most ancient and conserved signaling proteins known. Recent advances in the field have established its primary binding partner in plants as N-acetyl glutamate kinase and the crystal structure has revealed features unique to plants that likely contribute to its function in vivo. Here, we review the properties, function, and novel structural features of this chloroplast-localized metabolic sensor of higher plants.

The 1.9 A resolution crystal structure of PII from Arabidopsis thaliana reveals for the first time the molecular structure of a widely conserved regulator of carbon and nitrogen metabolism from a eukaryote. The structure provides a framework for understanding the arrangement of highly conserved residues shared with PII proteins from bacteria, archaea, and red algae as well as residues conserved only in plant PII. Most strikingly, a highly conserved segment at the N-terminus that is found only in plant PII forms numerous interactions with the alpha2 helix and projects from the surface of the homotrimer opposite to that occupied by the T-loop. In addition, solvent-exposed residues near the T-loop are highly conserved in plants but differ in prokaryotes. Several residues at the C-terminus that are also highly conserved only in plants contribute part of the ATP-binding site and likely participate in an ATP-induced conformational change. Structures of PII also reveal how citrate and malonate bind near the triphosphate binding site occupied by ATP in bacterial and archaeal PII proteins.

Four stress-sensing kinases phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to activate the integrated stress response (ISR). In animals, the ISR is antagonised by selective eIF2α phosphatases comprising a catalytic protein phosphatase 1 (PP1) subunit in complex with a PPP1R15-type regulatory subunit. An unbiased search for additional conserved components of the PPP1R15-PP1 phosphatase identified monomeric G-actin. Like PP1, G-actin associated with the functional core of PPP1R15 family members and G-actin depletion, by the marine toxin jasplakinolide, destabilised the endogenous PPP1R15A-PP1 complex. The abundance of the ternary PPP1R15-PP1-G-actin complex was responsive to global changes in the polymeric status of actin, as was its eIF2α-directed phosphatase activity, while localised G-actin depletion at sites enriched for PPP1R15 enhanced eIF2α phosphorylation and the downstream ISR. G-actin's role as a stabilizer of the PPP1R15-containing holophosphatase provides a mechanism for integrating signals regulating actin dynamics with stresses that trigger the ISR. DOI: http://dx.doi.org/10.7554/eLife.04872.001, eLife digest For a cell to build a protein, it must first copy the instructions contained within a gene. A complex molecular machine called a ribosome then reads these instructions and translates them into a protein. This translation process involves a number of steps. Proteins called eukaryotic translation initiation factors (or eIFs for short) coordinate the first step in the process, which is known as ‘initiation’. The eIFs also provide the cell with ways to control how quickly it makes proteins. For example, when a cell is stressed, either by starvation or toxins, it adds a phosphate group onto part of an eIF protein, called eIF2α. This modification makes this eIF protein less able to initiate translation, and so the cell builds fewer proteins and conserves more of its resources during times of stress. Once the stressful conditions are over, the phosphate group is removed from eIF2α by an enzyme called a phosphatase. This phosphatase contains two subunits: one that recognizes eIF2α and another that removes the phosphate group. However, experiments that attempted to recreate this phosphatase activity using just these two subunits in a test tube failed to generate a working enzyme that specifically targeted the phosphate group of eIF2α. This suggests that in cells this enzyme contains an additional unknown subunit. Now, Chambers, Dalton et al. (and Chen et al.) report the identity of a ‘missing’ third subunit as a protein known as globular-actin or G-actin. Chambers, Dalton et al. engineered human and fruit fly cells to add ‘molecular handles’ on the two known subunits of the phosphatase enzyme. These handles could then be used to essentially pull these proteins out of the mixture of molecules within a cell and see what other proteins came along too. Both of the known subunits ‘pulled’ G-actin along with them; this suggested that it could be the missing part of the phosphatase enzyme. Further experiments confirmed that G-actin works together with the other two subunits to specifically remove the phosphate group from eIF2α in mouse cells that had been stressed using a harmful chemical. Individual G-actin proteins can bind together to form long filaments, and signals that encourage a cell to divide or move also trigger the formation of actin filaments. This reduces the activity of the phosphatase enzyme by depriving it of a crucial component, i.e., free G-actin proteins. As such, the new mechanism described by Chambers, Dalton et al. suggests how growth and movement signals might also change a cell's sensitivity to stress. These findings may hopefully enable stressed cells to be targeted by drugs to treat disease; but future work is needed to clarify under what circumstances the integration of such signals into the stress response is beneficial to the cell. DOI: http://dx.doi.org/10.7554/eLife.04872.002

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.

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.

The PII proteins are key mediators of the cellular response to carbon and nitrogen status and are found in all domains of life. In eukaryotes, PII has only been identified in red algae and plants, and in these organisms, PII localizes to the plastid. PII proteins perform their role by assessing cellular carbon, nitrogen, and energy status and conferring this information to other proteins through protein-protein interaction. We have used affinity chromatography and mass spectrometry to identify the PII-binding proteins of Arabidopsis thaliana. The major PII-interacting protein is the chloroplast-localized enzyme N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. The interaction of PII with N-acetyl glutamate kinase was confirmed through pull-down, gel filtration, and isothermal titration calorimetry experiments, and binding was shown to be enhanced in the presence of the downstream product, arginine. Enzyme kinetic analysis showed that PII increases N-acetyl glutamate kinase activity slightly, but the primary function of binding is to relieve inhibition of enzyme activity by the pathway product, arginine. Knowing the identity of PII-binding proteins across a spectrum of photosynthetic and non-photosynthetic organisms provides a framework for a more complete understanding of the function of this highly conserved signaling protein.

The phosphoinositide-3-OH-kinase-related kinases (PIKKs) are atypical protein kinases exclusive to eukaryotes. They mediate the cellular response to a range of stresses, including genome and RNA surveillance and availability of nutrients for growth. Orthologues of five out of the six PIKK family members are present in plant genomes. Recent studies in plant PIKKs have revealed features unique to, and in common with, other PIKKs. This review summarizes the basic knowledge of these proteins in mammals and yeast in comparison with what is known for Arabidopsis and other plants.

As proteins represent the functional center piece of all biology, proteomics represents a field of research central to the greater plant research community. From discussions with those attending the European Proteomics Association (EuPA) 2012 meeting in Glasgow, Scotland, plant proteomics can be seen as expanding and evolving to investigate novel aspects of diverse organisms and to apply the latest technologies to plant research. Moving forward, targeted plant proteomics holds much promise in unraveling many of the complexities of plants. Highlighted are discussions on quantitative and non-quantitative plant proteomics, covalent modifications and the unique challenges of proteomics in plants.

Ionizing radiation induces autophosphorylation of the ataxia-telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, we show that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.

Microtubules play an essential role in the growth and development of plants and are known to be involved in regulating many cellular processes ranging from translation to signaling. In this article, we describe the proteomic characterization of Arabidopsis tubulin-binding proteins that were purified using tubulin affinity chromatography. Microtubule co-sedimentation assays indicated that most, if not all, of the proteins in the tubulin-binding protein fraction possessed microtubule-binding activity. Two-dimensional gel electrophoresis of the tubulin-binding protein fraction was performed, and 86 protein spots were excised and analyzed for protein identification. A total of 122 proteins were identified with high confidence using LC-MS/MS. These proteins were grouped into six categories based on their predicted functions: microtubule-associated proteins, translation factors, RNA-binding proteins, signaling proteins, metabolic enzymes, and proteins with other functions. Almost one-half of the proteins identified in this fraction were related to proteins that have previously been reported to interact with microtubules. This study represents the first large-scale proteomic identification of eukaryotic cytoskeleton-binding proteins, and provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.

Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms alpha1, alpha2, beta, and gamma1, plus the Aalpha and several of the B and B' subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M110. The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing.

Although the signal sensing protein PII is well known to play a central role in bacterial nitrogen metabolism, the structure and function of PII in plants remains only partially understood. Comparative modeling was undertaken based on the high degree of amino acid identity between Escherichia coli and Arabidopsis PII. The mature Arabidopsis PII predicted structure superimposes very well onto the E. coli PII structure (Calpha root mean square deviation < 0.4 A). The model of the highly conserved T-loop suggests a molecular mechanism by which the plant PII may regulate putative post-translational modification in response to metabolite binding. Consistent with the presence of key conserved residues necessary for trimer formation, gel filtration showed the oligomeric structure of Arabidopsis thaliana PII to be a homotrimer. We have demonstrated that Arabidopsis PII binds to the small molecules, ATP, ADP, 2KG, and with lesser affinity to OAA, using isothermal titration calorimetry. We have determined the metabolite dissociation constants and compared these with known physiological concentrations of these metabolites in the plant to identify the Arabidopsis PII effector molecules and their possible roles. We predict that the plant PII is likely continually bound by ATP, and its ligand-bound state only varying with respect to the degree of 2KG binding. Based on our in vitro binding studies, the function of plant PII as a 2KG sensor is suggested.

A simple and fast procedure that allows the separation of small (1–3 kDa) peptides on glycine-SDS gels is described. Peptides were separated by glycine-SDS/PAGE as a result ofin situ complexation of peptide/SDS during electrophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamide mini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.

Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature.

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.

Reversible protein phosphorylation is of central importance to the proper cellular functioning of all living organisms. Catalyzed by the opposing reactions of protein kinases and phosphatases, dysfunction in reversible protein phosphorylation can result in a wide variety of cellular aberrations. In eukaryotic organisms there exists four classes of protein phosphatases, of which the PPP-family protein phosphatases have documented susceptibility to a range of protein and small molecule inhibitors. These inhibitors have been of great importance to the biochemical characterization of PPP-family protein phosphatases since their discovery, but also maintain in natura biological significance with their endogenous regulatory properties (protein inhibitors) and toxicity (small molecule inhibitors). Recently, two unique PPP-family protein phosphatases, named the Shewanella-like protein phosphatases (SLP phosphatases), from Arabidopsis thaliana were characterized and found to be phylogenetically similar to the PPP-family protein phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), while completely lacking sensitivity to the classic PPP-family phosphatase small molecule inhibitors okadaic acid and microcystin-LR. SLP phosphatases were also found to be absent in metazoans, but present in a wide range of bacteria, fungi and protozoa responsible for human disease. The unique biochemical properties and evolutionary heritage of SLP phosphatases suggests they could not only be potential biotechnology targets for agriculture, but may also prove to be of interest for future therapeutic drug development.