Peroxisomal protein phosphatase PP2A-B' theta interacts with and piggybacks SINA-like 10 E3 ligase into peroxisomes.

Protein phosphatase 2A (PP2A) is targeted to the plant peroxisome via a C-terminal SSL sequence on its regulatory B' theta (θ) subunit. To date the substrates of peroxisomal PP2A are unknown but are thought to be recruited by the regulatory B'θ subunit. Employing yeast two hybrid screening, we have identified Arabidopsis E3 ligase SINA-like 10 as a B'θ binding partner. The E3 ligase SINA-like 10 was found to harbor the PP2A B'-binding Short Linear interaction Motif or SLiM, LxxIxE. This interaction was further verified both in vitro and in vivo using direct pulldown assays and bimolecular fluorescence complementation. Utilizing peroxisomal targeted and a cytosolic version of B'θ (lacking its C-terminal peroxisomal targeting sequence SSL>) bimolecular fluorescence complementation suggests an interaction to occur in the cytosol followed by piggybacking E3 ligase SINA-like 10 into peroxisomes. These results identify a first peroxisomal PP2A interactor, which also obtains a PP2A B'-binding SLiM.

LIKE SEX4 1 Acts as a ß-Amylase-Binding Scaffold on Starch Granules during Starch Degradation.

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 ß-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 ß-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 ß-amylases at the starch granule surface, thereby promoting starch degradation.

GL2 EXPRESSION MODULATOR, a plant specific protein phosphatase one interactor that binds phosphoinositides.

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.

A Quantitative Chemical Proteomic Strategy for Profiling Phosphoprotein Phosphatases from Yeast to Humans.

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.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis.

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.

Activation of Mitochondrial Protein Phosphatase SLP2 by MIA40 Regulates Seed Germination.

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.

Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

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.

Recruitment of PP1 to the centrosomal scaffold protein CEP192.

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.

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.

Quantitative fragmentome mapping reveals novel, domain-specific partners for the modular protein RepoMan (recruits PP1 onto mitotic chromatin at anaphase).

RepoMan is a protein phosphatase 1 (PP1) regulatory subunit that targets the phosphatase to key substrates throughout the cell cycle. Most work to date has focused on the mitotic roles of RepoMan/PP1, although equally important interphase role(s) have been demonstrated. Initial mapping of the interactome of nuclear RepoMan, both endogenous and tagged, was complicated by various factors, including antibody cross-reactivity and low sensitivity of the detection of chromatin-associated partners above the high background of proteins that bind nonspecifically to affinity matrices. We therefore adapted the powerful combination of fluorescence imaging with labeling-based quantitative proteomics to map the "fragmentomes" of specific regions of RepoMan. These regions demonstrate distinct localization patterns and turnover dynamics that reflect underlying binding events. The increased sensitivity and signal-to-noise ratio provided by this unique approach facilitated identification of a large number of novel RepoMan interactors, several of which were rigorously validated in follow-up experiments, including the association of RepoMan/PP1 with a specific PP2A-B56γ complex, interaction with ribosomal proteins and import factors involved in their nucleocytoplasmic transport and interaction with proteins involved in the response to DNA damage. This same strategy can be used to investigate the cellular roles of other modular proteins.

The human mitotic kinesin KIF18A binds protein phosphatase 1 (PP1) through a highly conserved docking motif.

Protein phosphatase 1 (PP1), a serine/threonine protein phosphatase, controls diverse key cellular events. PP1 catalytic subunits form complexes with a variety of interacting proteins that control its ability to dephosphorylate substrates. Here we show that the human mitotic kinesin-8, KIF18A, directly interacts with PP1γ through a conserved RVxF motif. Our phylogenetic analyses of the kinesins further uncovered the broad conservation of this interaction potential within the otherwise highly diverse motor-protein superfamily. This suggests an ancestral origin of PP1 recruitment to KIF18A and a strategic role in human mitotic cells.

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.

Molecular mechanisms underlying the interaction of protein phosphatase-1c with ASPP proteins.

The serine/threonine PP-1c (protein phosphatase-1 catalytic subunit) is regulated by association with multiple regulatory subunits. Human ASPPs (apoptosis-stimulating proteins of p53) comprise three family members: ASPP1, ASPP2 and iASPP (inhibitory ASPP), which is uniquely overexpressed in many cancers. While ASPP2 and iASPP are known to bind PP-1c, we now identify novel and distinct molecular interactions that allow all three ASPPs to bind differentially to PP-1c isoforms and p53. iASPP lacks a PP-1c-binding RVXF motif; however, we show it interacts with PP-1c via a RARL sequence with a Kd value of 26 nM. Molecular modelling and mutagenesis of PP-1c-ASPP protein complexes identified two additional modes of interaction. First, two positively charged residues, Lys260 and Arg261 on PP-1c, interact with all ASPP family members. Secondly, the C-terminus of the PP-1c α, ß and γ isoforms contain a type-2 SH3 (Src homology 3) poly-proline motif (PxxPxR), which binds directly to the SH3 domains of ASPP1, ASPP2 and iASPP. In PP-1cγ this comprises residues 309-314 (PVTPPR). When the Px(T)PxR motif is deleted or mutated via insertion of a phosphorylation site mimic (T311D), PP-1c fails to bind to all three ASPP proteins. Overall, we provide the first direct evidence for PP-1c binding via its C-terminus to an SH3 protein domain.

Arabidopsis thaliana histone deacetylase 14 (HDA14) is an α-tubulin deacetylase that associates with PP2A and enriches in the microtubule fraction with the putative histone acetyltransferase ELP3.

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/ß-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.

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.

Two ancient bacterial-like PPP family phosphatases from Arabidopsis are highly conserved plant proteins that possess unique properties.

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.

Identification and characterization of AtI-2, an Arabidopsis homologue of an ancient protein phosphatase 1 (PP1) regulatory subunit.

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.

Identification of Arabidopsis Protein Kinases That Harbor Functional Type 1 Peroxisomal Targeting Signals.

Peroxisomes are eukaryotic specific organelles that perform diverse metabolic functions including fatty acid ß-oxidation, reactive species metabolism, photorespiration, and responses to stress. However, the potential regulation of these functions by post-translational modifications, including protein phosphorylation, has had limited study. Recently, we identified and catalogued a large number of peroxisomal phosphorylated proteins, implicating the presence of protein kinases in this organelle. Here, we employed available prediction models coupled with sequence conservation analysis to identify 31 protein kinases from the Arabidopsis kinome (all protein kinases) that contain a putative, non-canonical peroxisomal targeting signal type 1 (PTS1). From this, twelve C-terminal domain-PTS1s were demonstrated to be functional in vivo, targeting enhanced yellow fluorescent protein to peroxisomes, increasing the list of presumptive peroxisomal protein kinases to nineteen. Of the twelve protein kinases with functional PTS1s, we obtained full length clones for eight and demonstrated that seven target to peroxisomes in vivo. Screening homozygous mutants of the presumptive nineteen protein kinases revealed one candidate (GPK1) that harbors a sugar-dependence phenotype, suggesting it is involved in regulating peroxisomal fatty acid ß-oxidation. These results present new opportunities for investigating the regulation of peroxisome functions.

Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability.

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase regulating many cellular processes. PP1alpha and -gamma are closely related isoforms with distinct localization patterns, shown here by time-lapse microscopy of stably expressed fluorescent protein fusions. A pool of PP1gamma is selectively loaded onto chromatin at anaphase. Using stable isotope labeling and proteomics, we identified a novel PP1 binding protein, Repo-Man, which selectively recruits PP1gamma onto mitotic chromatin at anaphase and into the following interphase. This approach revealed both novel and known PP1 binding proteins, quantitating their relative distribution between PP1alpha and -gamma in vivo. When overexpressed, Repo-Man can also recruit PP1alpha to chromatin. Mutating Repo-Man's PP1 binding domain does not disrupt chromatin binding but abolishes recruitment of PP1 onto chromatin. RNA interference-induced knockdown of Repo-Man caused large-scale cell death by apoptosis, as did overexpression of this dominant-negative mutant. The data indicate that Repo-Man forms an essential complex with PP1gamma and is required for the recruitment of PP1 to chromatin.

Use of Mitotic Protein Kinase Inhibitors and Phospho-Specific Antibodies to Monitor Protein Phosphorylation During the Cell Cycle.

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.