Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin.

BACKGROUND: Nitrate reductase (NR) in leaves is rapidly inactivated in the dark by a two-step mechanism in which phosphorylation of NR on the serine at position 543 (Ser543) promotes binding to nitrate reductase inhibitor protein (NIP). The eukaryotic 14-3-3 proteins bind to many mammalian signalling components (Raf-1, Bcr, phosphoinositide 3-kinase, protein kinase C, polyomavirus middle-T antigen and Cdc25), and are implicated in the timing of mitosis, DNA-damage checkpoint control, exocytosis, and activation of the plant plasma-membrane H+-ATPase by fusicoccin. Their dimeric, saddle-shaped structures support the proposal that 14-3-3 proteins are 'adaptors' linking different signalling proteins, but their precise functions are still a mystery. RESULTS: We purified NIP to homogeneity and established by means of amino-acid sequencing that it is a mixture of several 14-3-3 isoforms. Mammalian and yeast 14-3-3 proteins were just as effective as NIP at inhibiting phosphorylated NR. The sequence around Ser543, the phosphorylation site in NR, is strikingly similar to the sequences around the phosphoserine residues (Ser259 and Ser621) of mammalian Raf-1 that interact with 14-3-3 proteins. We found that NIP activity was blocked by a synthetic phosphopeptide corresponding to residues 251-266 of Raf. Fusicoccin also blocked NIP activity, and plant plasma-membrane H+-ATPases were activated by either fusicoccin, the phosphoserine259-Raf-1 peptide, or protein phosphatase 2A. CONCLUSIONS: Our findings establish that the mechanism of inactivation of NR involves the phosphorylation of Ser 543 followed by interaction with one or more plant 14-3-3 proteins. These results support the idea of a common mechanism for binding of 14-3-3 to its targets in all eukaryotes, and suggest that the phosphoserine259-Raf-1 peptide and fusicoccin may be of general use for disrupting the interaction of 14-3-3 with its target proteins. We propose that the plant plasma-membrane H+-ATPase is regulated in an analogous manner to NR-NIP, and speculate that 14-3-3 proteins provide a link between 'sensing' the activity state of NR and signalling to other cellular processes in plants.

Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines.

BACKGROUND: Mitogen-activated protein (MAP) kinase is central to a signal transduction pathway that triggers cell proliferation or differentiation. Activation of the p42mapk isoform requires its phosphorylation at two residues, Thr 183 and Tyr 185, and this phosphorylation is catalysed by MAP kinase kinase (MAPKK). Relatively little is known, however, about the enzymes that dephosphorylate these residues, thereby inactivating the pathway. Recently, the CL100 phosphatase has been shown to inactivate p42mapk in vitro by dephosphorylating Thr 183 and Tyr 185 at similar rates. CL100, the product of an immediate early gene, is synthesized within one hour of stimulating cells with growth factors or exposure to oxidative stress or heat shock. Incubation of NIH 3T3 fibroblasts with cycloheximide prevents both synthesis of CL100 and inactivation of p42mapk after stimulation with serum. RESULTS: Depleting cells of CL100 and preventing its induction using cycloheximide stopped the inactivation of p42mapk in Swiss 3T3 fibroblasts following stimulation with epidermal growth factor (EGF), but had no effect on the rapid inactivation of p42mapk in response to EGF in adipose (3T3-L1) or chromaffin (PC12) cells or in response to platelet-derived growth factor (PDGF) in endothelial (PAE) cells. Moreover, maximal induction of CL100 mRNA and a CL100-like activity did not trigger inactivation of p42mapk, which was sustained at a high level after stimulation of PC12 cells with nerve growth factor, PAE cells with serum, or Swiss 3T3 cells with PDGF. Dephosphorylation of Tyr 185 but not Thr 183 of p42mapk was suppressed by vanadate in EGF-stimulated PC12 cells; dephosphorylation of Thr 183, by contrast, was elicited by a vanadate-insensitive activity. Protein phosphatase-2A was the only vanadate-insensitive phosphatase acting on Thr 183 of p42mapk or on MAPKK to be detected in PC12 cell extracts. Phosphorylation of Thr 183 also inhibited the dephosphorylation of Tyr 185 in vitro by the major vanadate-sensitive Tyr 185-specific phosphatase, explaining why dephosphorylation of Thr 183 is rate-limiting for p42mapk inactivation in PC12 cells after stimulation with EGF. CONCLUSIONS: The rapid inactivation of p42mapk initiated five minutes after stimulation of endothelial, adipose and chromaffin cells with growth factor is not catalysed by CL100, but rather by protein phosphatase 2A and by a protein tyrosine phosphatase distinct from CL100. Induction of CL100 is not accompanied by the inactivation of p42mapk in a number of situations.

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1).

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1-6) indicating that at least 4-6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS-PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC(50)=0.65 nM), tautomycin (IC(50)=0.06 nM), microcystin-LR (IC(50)=0.01 nM), nodularin (IC(50)=0.035 nM), calyculin A (IC(50)=0.09 nM), okadaic acid (IC(50)=20 nM) and cantharidin (IC(50)=60 nM). The enzyme was also inhibited by fostriecin (IC(50)=22 microM), NaF (IC(50)=2.1 mM), Pi (IC(50)=9.5 mM), and PPi (IC(50)=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.

Evidence for an interaction between cytosolic aldolase and the ATP-and pyrophosphate-dependent phosphofructokinases in carrot storage roots.

Immunoaffinity chromatography was employed to identify potential plant cytosolic aldolase (ALDc) binding proteins. A clarified homogenate of carrot storage root was chromatographed on a column of protein-A-Sepharose that had been covalently coupled to anti-(carrot root ALDc) immunoglobulin G. The column was washed with phosphate-buffered saline (PBS), followed by step-wise elution with increasing concentrations of NaCl in PBS. Several proteins were eluted following application of the salt gradient. Western blotting identified the major eluting proteins to be the PPi-dependent phosphofructokinase (PFP) and the cytosolic form of the ATP-dependent phosphofructokinase (PFKc), enzymes that are metabolically sequential to ALDc. The results suggest that ALDc may specifically interact with PFP and PFKc in carrots.

Purification of the hepatic glycogen-associated form of protein phosphatase-1 by microcystin-Sepharose affinity chromatography.

The form of protein phosphatase-1 associated with hepatic glycogen (PP1G) was purified to near homogeneity from rat liver by affinity chromatography on microcystin-Sepharose and gel-filtration. The enzyme is a heterodimer consisting of the catalytic subunit of PP1 (the alpha and beta isoforms) complexed to a 33 kDa glycogen-binding (GL) subunit. The GL subunit binds phosphorylase a with high affinity, and is responsible for the enhanced dephosphorylation of glycogen synthase by PP1G and its allosteric inhibition by phosphorylase a.

The major myosin phosphatase in skeletal muscle is a complex between the beta-isoform of protein phosphatase 1 and the MYPT2 gene product.

Myosin is dephosphorylated by distinct forms of protein phosphatase 1 (PP1) in smooth muscle and skeletal muscle that are composed of PP1 complexed to different regulatory subunits. The smooth muscle myosin phosphatase (smPP1M) has been characterised previously and is composed of PP1beta complexed to M110 and M21 subunits that enhance the dephosphorylation of smooth muscle myosin, but not skeletal muscle myosin. In contrast, the regulatory subunit(s) of skeletal muscle myosin phosphatase (skPP1M) greatly enhance(s) the dephosphorylation of skeletal muscle myosin. Here we identify a regulatory subunit of skPP1M as the product of the MYPT2 gene, a protein whose sequence is 61% identical to the M110 subunit of smPP1M. Surprisingly, the M21 subunit of smPP1M appears to be produced from the same gene that encodes MYPT2.

Purification of type 1 protein (serine/threonine) phosphatases by microcystin-Sepharose affinity chromatography.

A microcystin (MC)-Sepharose column was prepared by addition of 2-aminoethanethiol to the alpha, beta-unsaturated carbonyl of the N-methyldehydroalanine residue of MC-LR, followed by reaction of the introduced amino group with N-hydroxysuccinimide-activated CH-Sepharose. The MC-Sepharose bound protein phosphatase-1 (PP1) with high capacity and purified human PP1 gamma in one step from E. coli extracts. It was also used to purify forms of PP1 bound to myofibrils from skeletal muscle. Two of these comprised PP1 complexed to N-terminal fragments of the M-subunit which enhance its myosin phosphatase activity, while the third comprised PP1 and an N-terminal fragment of the glycogen-binding (G)-subunit.

Amino acid sequence and expression of the hepatic glycogen-binding (GL)-subunit of protein phosphatase-1.

A full-length cDNA encoding the putative hepatic glycogen-binding (GL) subunit of protein phosphatase-1 (PP1) was isolated from a rat liver library. The deduced amino acid sequence (284 residues, 32.6 kDa) was 23% identical (39% similar) to the N-terminal region of the glycogen-binding (GM) subunit of PP1 from striated muscle. The similarities between GM and GL were most striking between residues 63-86, 144-166 and 186-227 of human GM (approximately 40% identity), nearly all the identities with the putative yeast homologue GAC1 being located between 144-166 and 186-227. The cDNA was expressed in E. coli, and the expressed protein transformed the properties of PP1 to those characteristic of the hepatic glycogen-associated enzyme. These experiments establish that the cloned protein is GL.

Updating the evidence relating to physical activity intervention studies in older people.

For older adults, physical activity (PA) plays a central role in the prevention and management of chronic disease, and has the potential to reduce physical decline, maintain functional ability and prevent injuries. This review provides an update of the evidence relating to the effectiveness of PA intervention studies (both general PA and trials specific to progressive resistance training (PRT)) for older adults. The following electronic databases were searched for articles published since 1999: Medline, PubMed, EMBASE, CINAHL and Sport Discus. For the PRT section, a 2002 Cochrane review was also used. Eight general PA intervention studies were included in this review, ranging from one-on-one counselling in general practice to the community-wide promotion of walking. The aim of most of the trials was to increase moderate and/or vigorous activity levels. Most of the studies reviewed had some degree of success in getting older people to be more active. However, a major limitation was the use of self-report measures of PA. The review of PRT interventions included 21 trials. Participants in half the studies had either functional limitations or a chronic condition. Most trials were conducted in a supervised setting using specialised equipment. Increased strength and improvement in basic functional tasks were generally reported, but there was a paucity of strong evidence linking PRT with reduced physical disability and improved health-related quality of life. While considerable progress is being made in this area, further population-based studies that include home and whole-community interventions are required.

RRP1B is a metastasis modifier that regulates the expression of alternative mRNA isoforms through interactions with SRSF1.

RRP1B (ribosomal RNA processing 1 homolog B) was first identified as a metastasis susceptibility gene in breast cancer through its ability to modulate gene expression in a manner that can be used to accurately predict prognosis in breast cancer. However, the mechanism(s) by which RRP1B modulates gene expression is currently unclear. Many RRP1B binding candidates are involved in alternative splicing, a mechanism of gene expression regulation that is increasingly recognized to be involved in cancer progression and metastasis. One such target is SRSF1 (serine/arginine-rich splicing factor 1) (SF2/ASF, splicing factor 2/alternative splicing factor), an essential splicing regulator that also functions as an oncoprotein. Earlier studies demonstrated that splicing and transcription occur concurrently and are coupled processes. Given that RRP1B regulates transcriptional activity, we hypothesized that RRP1B also regulates the expression of alternative mRNA isoforms through its interaction with SRSF1. Interaction between RRP1B and SRSF1 was verified by coimmunoprecipitation and coimmunofluorescence. Treatment of cells with transcriptional inhibitors significantly increased this interaction, demonstrating that the association of these two proteins is transcriptionally regulated. To assess the role of RRP1B in the regulation of alternative isoform expression, RNA-sequencing data were generated from control and Rrp1b-knockdown cells. Knockdown of Rrp1b induced a significant change in isoform expression in over 600 genes compared with control cell lines. This was verified by quantitative reverse-transcription PCR using isoform-specific primers. Pathway enrichment analyses identified cell cycle and checkpoint regulation to be those most affected by Rrp1b knockdown. These data suggest that RRP1B suppresses metastatic progression by altering the transcriptome through its interaction with splicing regulators such as SRSF1.

Purification and characterization of cytosolic aldolase from carrot storage root.

A single fructose-1,6-bisphosphate (FBP) aldolase has been detected in extracts from carrot storage roots (Daucus carota L.). The enzyme was purified 850-fold to electrophoretic homogeneity and a final specific activity of 26.3 mumols of FBP utilized/min per mg of protein. SDS/PAGE of the final preparation revealed a single protein-staining band of 40 kDa. The native molecular mass was determined by analytical gel filtration to be 159 kDa, indicating that the enzyme is a homotetramer. Denaturing isoelectric focusing revealed two predominant protein-staining bands, with pI values of 5.6 and 5.7. The enzyme is a class I aldolase, since EDTA or metal ions had no effect on its activity. The enzyme was relatively heat-stable, had an activation energy (Ea) of 68.3 kJ.mol-1, and had an absorption coefficient of 8.08 x 10(4) M-1.cm-1 at 280 nm. Km values for FBP and sedoheptulose 1,7-bisphosphate (SBP) were both determined to be 6 microM (pH optima 7.4). The specificity constant with FBP was 2.6 times that obtained with SBP. Ribose 5-phosphate, 6-phosphogluconate, MgAMP, glucose 1-phosphate and phosphoenolpyruvate (PEP) were inhibitors. PEP was a mixed-type inhibitor with respect to FBP (Ki = 3.2 mM, K'i = 5.1 mM). No activators were found. Rabbit anti-(carrot aldolase) polyclonal antibodies immunoprecipitated the activity of both carrot root aldolase and spinach leaf cytosolic aldolase, but not that of spinach leaf plastid aldolase. Western-blot analysis also revealed cross-reactivity with cytosolic, but not plastid, spinach leaf aldolase, indicating that the single carrot root aldolase is cytosolic.

Binding of glycolytic enzymes to a particulate fraction in carrot and sugar beet storage roots : dependence on metabolic state.

Numerous studies have demonstrated a rapid increase in the respiration rate during aging of slices of tuber and storage roots. To determine the molecular mechanisms of this phenomenon, the role of enzyme binding to the subcellular particulate fraction has been assessed in carrot (Daucus carota L.) and sugar beet (Beta vulgaris L.). Soluble versus particulate fractions were separated by centrifugation at 16,000g and both fractions assayed for the activities of six glycolytic enzymes. Preparations from sliced and aged tissues showed elevated percentages of five enzymes associated with the particulate fraction as compared with controls. The stimulation of respiration which occurs during aging of underground storage organ slices may result, in part, from an association of enzymes with the particulate fraction of the cell promoting an elevated glycolytic rate.

Peptide mapping by CNBr fragmentation using a sodium dodecyl sulfate-polyacrylamide minigel system.

The method of peptide mapping by sodium dodecyl sulfate-gel electrophoresis following partial protein fragmentation with cyanogen bromide was adapted for a polyacrylamide minigel system. The combined use of the discontinuous gel electrophoresis system of J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271) and a vertical polyacrylamide minigel system produced the following advantages over other procedures: (a) the ability to resolve cyanogen bromide cleavage fragments over a broad molecular mass range while yielding very sharp protein staining bands; (b) well-defined peptide maps are produced with as little as 2 micrograms of protein; (c) less time is required to perform fragmentation with cyanogen bromide, to equilibrate the gel slices in sodium dodecyl sulfate buffer, as well as to perform the electrophoresis; and (d) the cyanogen bromide fragmentation patterns are highly reproducible.

High-yield purification of potato tuber pyrophosphate: fructose-6-phosphate 1-phosphotransferase.

The procedure of Yuan et al. (1988, Biochem. Biophys. Res. Commun. 154, 111-117) for the isolation of potato pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) has been modified so that a high yield of homogeneous enzyme could be obtained. Modifications included a lower temperature heat step, a lower percentage initial polyethylene glycol fractionation step (0 to 4%, w/v), stepwise elution following an increase from 30 to 50 mM pyrophosphate during affinity chromatography on Whatman P11 phosphocellulose, anion-exchange chromatography using Q-Sepharose "Fast Flow," and gel filtration chromatography with Superose 6 "Prep grade." Our procedure resulted in an overall 42% yield and a final specific activity of 87 mumol fructose 1,6-bisphosphate produced per minute per milligram protein. Rabbit anti-(potato PFP) polyclonal antibodies effectively immunoprecipitated the activity of both the pure enzyme and the enzyme from a crude extract. Western blot analysis demonstrated that the antibodies were monospecific for PFP. A survey of various potato cultivars demonstrated significant differences in PFP activity with respect to fresh weight. This observation should be taken into consideration before any purification of potato PFP is undertaken.

Copurification of cytosolic fructose-1,6-bisphosphatase and cytosolic aldolase from endosperm of germinating castor oil seeds.

The cytosolic isozymes of fructose-1,6-bisphosphatase (FBPasec) and aldolase (ALDc) from germinating castor oil seed endosperm (COS) (Ricinus communis L.; cv Hale) were purified to homogeneity and final specific activities 49 and 2.8 (mumol product produced/min)/mg protein, respectively. Nondenaturing polyacrylamide gel electrophoresis of the final FBPasec preparation resolved a single protein-staining band which comigrated with FBPase activity. Two protein-staining bands of 41 and 39 kDa that occurred in an approximate 1:1 ratio were observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final FBPasec preparation. Rabbit anti-(FBPasec) immune serum immunoprecipitated the activities of FBPasec, but not that of the plastidic isozyme of FBPase from germinated COS. Immunoblot analysis utilizing affinity purified anti-(COS FBPasec) immunoglobulin G established that the 39-kDa subunit of FBP-asec did not arise via proteolytic cleavage of the 41-kDa subunit during tissue extraction and enzyme purification. However, FBPasec was susceptible to degradation by endogenous protease(s) during incubation of an acidic (pH 5.9) clarified COS extract at 25 degrees C. This proteolysis caused the production of a 32-kDa antigenic polypeptide and resulted in FBPase inactivation. Gel filtration indicated that purified FBPasec exists in at least 8 different oligomeric forms ranging in size from > 2 million to < 34 kDa. The majority of FBPasec, however, eluted as a 143-kDa heterotetramer. Sodium dodecyl sulfate gel electrophoresis of the final ALDc preparation yielded a single 40-kDa protein-staining polypeptide that cross-reacted with anti-(carrot ALDc) IgG. FBPasec copurified with ALDc through polyethylene glycol fractionation, Q-Sepharose, and phosphocellulose chromatographies, and the intensity of the fluorescence emission spectrum of ALDc was greatly reduced in the presence of COS FBPasec, but not rabbit muscle FBPase. These findings suggest that these two metabolically sequential enzymes might specifically interact in the cytosol of the highly gluconeogenic germinating COS. Our results also demonstrate that endogenous nonspecific acid phosphatase activity can interfere with the spectrophotometric assay for FBPase and can thus result in overestimations of FBPase activity in impure plant extracts.

Potato tuber pyrophosphate-dependent phosphofructokinase: effect of thiols and polyalcohols on its intrinsic fluorescence, oligomeric structure, and activity in dilute solutions.

The effect of dilution of homogeneous potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90; PFP) on the enzyme's intrinsic fluorescence, activity, and oligomeric structure has been examined. A rapid decrease in PFP's intrinsic fluorescence occurred in response to dilution. The decay follows double-exponential kinetics and was accompanied by a reduction in catalytic activity (measured in the glycolytic direction). Gel filtration-HPLC indicated a concomitant deaggregation of the native alpha 4 beta 4 heterooctamer into the inactive free alpha- and beta-subunits, followed by random aggregation of the subunits into an inactive, high M(r) conglomerate. The addition of 2 mM dithiothreitol, 2 mM 2-mercaptoethanol, or 5% (w/v) polyethylene glycol, but not any of the substrates, Mg2+, or fructose 2,6-bisphosphate, prevented this process. When purified PFP was stored for 1 week at -20 degrees C in the presence of 50% (v/v) glycerol partial degradation of its alpha-subunit occurred. This resulted in a labile enzyme that was more susceptible to subunit dissociation. The intrinsic fluorescence of the degraded PFP could be stabilized by 5% (w/v) polyethylene glycol, but not by 2 mM dithiothreitol or 2-mercaptoethanol. It is proposed that the current assay procedures for PFP, which normally involve considerable dilution in the absence of added sulfhydryl reducing agents or polyhydroxy compounds, may underestimate the actual activity of the enzyme. This has important implications for the assessment of the functions and regulation of PFP in vivo.

The Tendency toward Defective Decision Making within Self-Managing Teams: The Relevance of Groupthink for the 21st Century.

Groupthink theory has continued relevance to organizations because of the organizational trend toward self-managing work teams. A typology is developed linking the key differentiating characteristics of self-managing teams to groupthink antecedents of group cohesion, structural faults of the organization, and provocative situational context. Building upon this framework, we more specifically examine variables that will impact the occurrence of groupthink within self-managing teams. Implications for the prevention of groupthink in self-managing teams are discussed. Copyright 1998 Academic Press.

Categorization of hospital emergency services--developing valid criteria.

In a pilot project sponsored by the California State Emergency Medical Services Authority, validated, verifiable criteria were developed for the vertical categorization of hospital emergency services in 11 different groupings of medical and surgical emergencies. We describe the development of an assessment process and categorization criteria to identify the most appropriate receiving facility for interfacility transfer and, in selected instances, field triage of patients with different levels of severity of illness or injury. We propose that this facility assessment project be used in the critical care planning process for the eventual vertical categorization of hospital emergency services in California and as a template for similar projects in other states.

Phosphate Starvation Inducible ;Bypasses' of Adenylate and Phosphate Dependent Glycolytic Enzymes in Brassica nigra Suspension Cells.

When Brassica nigra leaf petiole suspension cells were subjected to 7 days of inorganic phosphate (Pi) starvation the extractable activity of: (a) pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, phosphoenolpyruvate phosphatase, and phosphoenolpyruvate carboxylase increased at least fivefold, (b) phosphorylating NAD-glyceraldehyde 3-phosphate dehydrogenase decreased about sixfold, and (c) ATP:fructose 6-phosphate 1-phosphotransferase, 3-phosphoglycerate kinase, pyruvate kinase, or NAD malic enzyme was not altered. Pi deprivation also resulted in significant reductions in extractable levels of Pi, ATP, ADP, fructose 2,6-bisphosphate, and soluble protein, but caused a sixfold elevation in free amino acid concentrations. No change in inorganic pyrophosphate concentration was observed following Pi starvation. It is hypothesized that pyrophosphate:fructose 6-phosphate 1-phosphotransferase, nonphosphorylating NADP-glyceraldehyde 3-phosphate dehydrogenase, and phosphoenolpyruvate phosphatase bypass nucleotide phosphate or Pi-dependent glycolytic reactions during sustained periods of Pi depletion.