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.

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.

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 β-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.

Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.

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.

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.

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.

Conformation and protein interactions of intramolecular DNA and phosphorothioate four-way junctions.

The objectives of this study are to evaluate the structure and protein recognition features of branched DNA four-way junctions in an effort to explore the therapeutic potential of these molecules. The classic immobile DNA 4WJ, J1, is used as a matrix to design novel intramolecular junctions including natural and phosphorothioate bonds. Here we have inserted H2-type mini-hairpins into the helical termini of the arms of J1 to generate four novel intramolecular four-way junctions.

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.

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

In Arabidopsis () 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.

A Framework to Investigate Peroxisomal Protein Phosphorylation in Arabidopsis.

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.

Protein Kinases and Phosphatases of the Plastid and Their Potential Role in Starch Metabolism.

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.

Aurora B opposes PP1 function in mitosis by phosphorylating the conserved PP1-binding RVxF motif in PP1 regulatory proteins.

Protein phosphatase 1 (PP1) is a highly conserved protein phosphatase that performs most of the serine- and threonine-dephosphorylation reactions in eukaryotes and opposes the actions of a diverse set of serine and threonine (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.

Fast XANES fluorescence imaging using a Maia detector.

A new fast X-ray absorption spectroscopy scanning method was recently implemented at the Hard X-ray Microprobe endstation P06, PETRA III, DESY, utilizing a Maia detector. Spectromicroscopy maps were acquired with spectra for X-ray absorption near-edge structure (XANES) acquisition in the sub-second regime. The method combines XANES measurements with raster-scanning of the sample through the focused beam.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in .

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 Relative quantification of the changes in the lysine acetylation levels was determined on a proteome-wide scale after treatment of leaves with deacetylase inhibitors apicidin and trichostatin A.

AtSLP2 is an intronless protein phosphatase that co-expresses with intronless mitochondrial pentatricopeptide repeat (PPR) and tetratricopeptide (TPR) protein encoding genes.

Shewanella-like PPP family phosphatases (SLPs) are a unique lineage of eukaryote PPP-family phosphatases of bacterial origin which are not found in metazoans. Their absence in metazoans is marked by their ancient bacterial origins and presence in plants. 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.

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.

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.

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.

"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.

Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.

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.

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.

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.