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.

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.

"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.

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.

Phosphoglucan phosphatase function sheds light on starch degradation.

Phosphoglucan phosphatases are novel enzymes that remove phosphates from complex carbohydrates. In plants, these proteins are vital components in the remobilization of leaf starch at night. Breakdown of starch is initiated through reversible glucan phosphorylation to disrupt the semi-crystalline starch structure at the granule surface.

Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein.

The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic 'RVxF' motif and suppresses the general phosphatase activity of the enzyme.

Phosphonate analogs of 2-oxoglutarate perturb metabolism and gene expression in illuminated Arabidopsis leaves.

Although the role of the 2-oxoglutarate dehydrogenase complex (2-OGDHC) has previously been demonstrated in plant heterotrophic tissues its role in photosynthetically active tissues remains poorly understood. By using a combination of metabolite and transcript profiles we here investigated the function of 2-OGDHC in leaves of Arabidopsis thaliana via use of specific phosphonate inhibitors of the enzyme. Incubation of leaf disks with the inhibitors revealed that they produced the anticipated effects on the in situ enzyme activity.

Isolation of human mitotic protein phosphatase complexes: identification of a complex between protein phosphatase 1 and the RNA helicase Ddx21.

Metazoan mitosis requires remodelling of sub-cellular structures to ensure proper division of cellular and genetic material. Faults often lead to genomic instability, cell cycle arrests and disease onset. These key structural changes are under tight spatial-temporal and post-translational control, with crucial roles for reversible protein phosphorylation.

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.

Insight into the redox regulation of the phosphoglucan phosphatase SEX4 involved in starch degradation.

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.

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).

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

Background: Phosphorylated phosphatidylinositol (PtdIns) lipids, produced and modified by PtdIns kinases and phosphatases, are critical to the regulation of diverse cellular functions. The myotubularin PtdIns-phosphate phosphatases have been well characterized in yeast and especially animals, where multiple isoforms, both catalytically active and inactive, occur. Myotubularin mutations bring about disruption of cellular membrane trafficking, and in humans, disease.

Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates gamma-H2AX.

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.

PII in higher plants: a modern role for an ancient protein.

PII in prokaryotic organisms is a crucial integrator of cellular carbon, nitrogen and energy levels. In higher plants, however, its role remains significantly less clear. Previous findings suggest that PII-N-acetylglutamate kinase (NAGK) complex formation controls l-arginine biosynthesis, whereas other work implicates PII in regulating chloroplastic NO2(-) uptake.

Evolution of protein phosphatases in plants and animals.

Protein phosphorylation appears to be a universal mechanism of protein regulation. Genomics has provided the means to compile inventories of protein phosphatases across a wide selection of organisms and this has supplied insights into the evolution of this group of enzymes. Protein phosphatases evolved independently several times yielding the groups we observe today.

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

Background: Starch accumulation and degradation in chloroplasts is accomplished by a suite of over 30 enzymes. Recent work has emphasized the importance of multi-protein complexes amongst the metabolic enzymes, and the action of associated non-enzymatic regulatory proteins. Arabidopsis At5g39790 encodes a protein of unknown function whose sequence was previously demonstrated to contain a putative carbohydrate-binding domain.

Displacement affinity chromatography of protein phosphatase one (PP1) complexes.

Background: Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif.

Results: We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose.

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

In addition to the major serine/threonine-specific phosphoprotein phosphatase, Mg(2+)-dependent phosphoprotein phosphatase, and protein tyrosine phosphatase families, there are novel protein phosphatases, including enzymes with aspartic acid-based catalysis and subfamilies of protein tyrosine phosphatases, whose evolutionary history and representation in plants is poorly characterized. We have searched the protein data sets encoded by the well-finished nuclear genomes of the higher plants Arabidopsis (Arabidopsis thaliana) and Oryza sativa, and the latest draft data sets from the tree Populus trichocarpa and the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, for homologs to several classes of novel protein phosphatases. The Arabidopsis proteins, in combination with previously published data, provide a complete inventory of known types of protein phosphatases in this organism.

Structural basis for the regulation of N-acetylglutamate kinase by PII in Arabidopsis thaliana.

PII is a highly conserved regulatory protein found in organisms across the three domains of life. In cyanobacteria and plants, PII relieves the feedback inhibition of the rate-limiting step in arginine biosynthesis catalyzed by N-acetylglutamate kinase (NAGK). To understand the molecular structural basis of enzyme regulation by PII, we have determined a 2.

The nuclear PP1 interacting protein ZAP3 (ZAP) is a putative nucleoside kinase that complexes with SAM68, CIA, NF110/45, and HNRNP-G.

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.

Emerging roles of nuclear protein phosphatases.

The phosphorylation state of any protein represents a balance of the actions of specific protein kinases and protein phosphatases. Many protein phosphatases are highly enriched in, or exclusive to, the nuclear compartment, where they dephosphorylate key substrates to regulate various nuclear processes. In this review we will discuss recent findings that define the role of nuclear protein phosphatases in controlling transforming growth factor-beta (TGFbeta) and bone-morphogenetic protein (BMP) signalling, the DNA-damage response, RNA processing, cell-cycle progression and gene transcription.

Crystal structure of Arabidopsis PII reveals novel structural elements unique to 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.

Purification of PP2Ac from bovine heart.

The catalytic subunit of PP2A (PP2Ac) can be purified in milligram quantities from bovine heart using ethanol precipitation, ammonium sulfate precipitation, ion exchange and size exclusion chromatography. The detailed procedure is described to purify PP2Ac over 4 d.

Utilizing protein phosphatase inhibitors to define PP2A as a regulator of ataxia-telangiectasia mutated.

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to DNA double-strand breaks caused by ionizing radiation. Ionizing radiation induces the autophosphorylation of ATM on serine 1981; however, the precise mechanisms that regulate ATM autophosphorylation are not fully understood. By treating cells with okadaic acid, a cell-permeable protein phosphatase inhibitor, together with assays to quantify the activity of particular protein phosphatases, we have demonstrated that the autophosphorylation of ATM on serine 1981 is regulated by a protein phosphatase 2A-like activity.