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

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

Protein phosphatases in MAPK signalling: we keep learning from yeast

Because of their key role in cell signalling, a rigorous regulation of mitogen-activated protein kinases (MAPKs) is essential in eukaryotic physiology. Whereas the use of binding motifs and scaffold proteins guarantees the selective activation of a specific MAPK pathway, activating kinases and downregulating phosphatases control the appropriate intensity and timing of MAPK activation. Tyrosine, serine/threonine and dual-specificity phosphatases co-ordinately dephosphorylate and thereby inactivate MAPKs. In budding yeast, enzymes that belong to these three types of phosphatases have been shown to counteract the MAPKs that govern the cellular response to varied extracellular stimuli. Studies carried out with these yeast phosphatases have expanded our knowledge of essential key aspects of the biology of these negative regulators, such as their function, the mechanisms that operate in their modulation by MAPK pathways and their binding to MAPK substrates. Furthermore, yeast MAPK phosphatases have been shown to play additional and essential roles in MAPK-mediated signalling, controlling MAPK localization or cross-talk among pathways. This review stresses the importance of these negative regulators in eukaryotic signalling by discussing the recent developments and perspectives in the study of yeast MAPK phosphatases.     

Regulation of the osmoregulatory HOG MAPK cascade in yeast

The budding yeast Saccharomyces cerevisiae has at least five signal pathways containing a MAP kinase (MAPK) cascade. The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity environment. This mini-review surveys recent developments in regulation of the HOG pathway with specific emphasis on the roles of protein phosphatases and protein subcellular localization. The Hog1 MAPK in the HOG pathway is negatively regulated jointly by the protein tyrosine phosphatases Ptp2/Ptp3 and the type 2 protein phosphatases Ptc1/Ptc2/Ptc3. Specificities of these phosphatases are determined by docking interactions as well as their cellular localizations. The subcellular localizations of the osmosensors (Sln1 and Sho1), kinases (Pbs2, Hog1), and phosphatases in the HOG pathway are intricately regulated to achieve their specific functions.     

Genetic analyses of yeast protein serine/threonine phosphatases

Abstract Protein phosphorylation is an important regulatory phenomenon in yeasts just as in other eukaryotic cells and controls a wide variety of cellular processes. The importance of protein phosphatases as well as protein kinases as key elements in such control is becoming increasingly clear. Over the past four years since the first yeast protein phosphatase gene was isolated, many more such genes have been described and the number of genes encoding protein phosphatase catalytic subunits in Saccharomyces cerevisiae has comfortably entered double figures. Given the genetic approaches available, yeasts offer powerful systems for addressing the cellular roles of these enzymes. This review summarises the results of genetic studies aimed at determining the functions of protein serine/threoninc phosphatases in yeast.     

Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome.

Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.     

Identification of PTM5 protein interaction partners, a MADS-box gene involved in aspen tree vegetative development

In a past article, our lab described the identification and characterization of a novel vegetative MADS-box gene from quaking aspen trees, Populus tremuloides MADS-box 5 (PTM5). PTM5 was shown to be a member of the SOC1/TM3 class of MADS-box genes with a seasonal expression pattern specific to developing vascular tissues including the vascular cambium, the precursor to all woody branches, stems, and roots. Since the proper function of MADS-box proteins is dependent on specific interactions with other regulatory proteins, we further examined PTM5 protein-protein interactions as a means to better understand its function. Through yeast two-hybrid analyses, it was demonstrated that, like other SOC1/TM3 class proteins, PTM5 is capable of interacting with itself as well as other MADS-box proteins from aspen. In addition, yeast two-hybrid library screening revealed that PTM5 interacts with two non-MADS proteins, an actin depolymerizing factor (PtADF) and a novel leucine-rich repeat protein (PtLRR). In situ RNA localization was used to verify the overlapping expression patterns of these genes, and transgenic studies showed that over-expression of PTM5 in aspen causes alterations in root vasculature and root biomass development consistent with the cell growth and expansion functions of related ADF and LRR genes. These results suggest that the interaction of vegetative MADS-box genes with specific protein cofactors is a key step in the mechanisms that control woody tissue development in trees.     

Modular evolution of phosphorylation-based signalling systems

Phosphorylation sites are formed by protein kinases ('writers'), frequently exert their effects following recognition by phospho-binding proteins ('readers') and are removed by protein phosphatases ('erasers'). This writer-reader-eraser toolkit allows phosphorylation events to control a broad range of regulatory processes, and has been pivotal in the evolution of new functions required for the development of multi-cellular animals. The proteins that comprise this system of protein kinases, phospho-binding targets and phosphatases are typically modular in organization, in the sense that they are composed of multiple globular domains and smaller peptide motifs with binding or catalytic properties. The linkage of these binding and catalytic modules in new ways through genetic recombination, and the selection of particular domain combinations, has promoted the evolution of novel, biologically useful processes. Conversely, the joining of domains in aberrant combinations can subvert cell signalling and be causative in diseases such as cancer. Major inventions such as phosphotyrosine (pTyr)-mediated signalling that flourished in the first multi-cellular animals and their immediate predecessors resulted from stepwise evolutionary progression. This involved changes in the binding properties of interaction domains such as SH2 and their linkage to new domain types, and alterations in the catalytic specificities of kinases and phosphatases. This review will focus on the modular aspects of signalling networks and the mechanism by which they may have evolved.     

Protein phosphatases PP2A,PP4 and PP6: mediators and regulators in development and responses to environmental cues

The three closely related groups of serine/threonine protein phosphatases PP2A, PP4 and PP6 are conserved throughout eukaryotes. The catalytic subunits are present in trimeric and dimeric complexes with scaffolding and regulatory subunits that control activity and confer substrate specificity to the protein phosphatases. In Arabidopsis, three scaffolding (A subunits) and 17 regulatory (B subunits) proteins form complexes with five PP2A catalytic subunits giving up to 255 possible combinations. Three SAP‐domain proteins act as regulatory subunits of PP6. Based on sequence similarities with proteins in yeast and mammals, two putative PP4 regulatory subunits are recognized in Arabidopsis. Recent breakthroughs have been made concerning the functions of some of the PP2A and PP6 regulatory subunits, for example the FASS/TON2 in regulation of the cellular skeleton, B′ subunits in brassinosteroid signalling and SAL proteins in regulation of auxin transport. Reverse genetics is starting to reveal also many more physiological functions of other subunits. A system with key regulatory proteins (TAP46, TIP41, PTPA, LCMT1, PME‐1) is present in all eukaryotes to stabilize, activate and inactivate the catalytic subunits. In this review, we present the status of knowledge concerning physiological functions of PP2A, PP4 and PP6 in Arabidopsis, and relate these to yeast and mammals.     

Identification of Yeast Factors Involved in the Replication of Mungbean Yellow Mosaic India Virus Using Yeast Temperature-Sensitive Mutants

正Dear Editor,The geminiviruses are small single-stranded plant DNA viruses belonging to the family Geminiviridae, which cause serious diseases in many economically important     

Analysis of the small GTPase gene superfamily of Arabidopsis

Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. These proteins function as molecular switches that cycle between "active" and "inactive" states, and this cycle is linked to the binding and hydrolysis of GTP. The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs. Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases. We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis. We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems.     

Type 2C protein phosphatases in fungi

Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.     

Brain protein serine/threonine phosphatases.

All of the known protein serine/threonine phosphatases are expressed in the brain. These enzymes participate in a variety of signaling pathways that modulate neuronal activity. The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins. Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling. The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

Protein feature based identification of cell cycle regulated proteins in yeast

DNA microarrays have been used extensively to identify cell cycle regulated genes in yeast; however, the overlap in the genes identified is surprisingly small. We show that certain protein features can be used to distinguish cell cycle regulated genes from other genes with high confidence (features include protein phosphorylation, glycosylation, subcellular location and instability/degradation). We demonstrate that co-expressed, periodic genes encode proteins which share combinations of features, and provide an overview of the proteome dynamics during the cycle. A large set of novel putative cell cycle regulated proteins were identified, many of which have no known function.