scholarly journals Phosphoproteins Involved in the Signal Transduction of Cryptogein, an Elicitor of Defense Reactions in Tobacco

2000 ◽  
Vol 13 (8) ◽  
pp. 821-829 ◽  
Author(s):  
Fatma Lecourieux-Ouaked ◽  
Alain Pugin ◽  
Angela Lebrun-Garcia
Keyword(s):  
Signal Transduction ◽  
Calcium Influx ◽  
Active Oxygen Species ◽  
Defense Responses ◽  
Specific Protein ◽  
Calyculin A ◽  
Phosphorylated Proteins ◽  
Defense Reactions

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaïc acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involved early in the elicitor signaling pathway, we analyzed by 2-D electrophoresis the in vivo phosphorylation status of proteins after cryptogein, staurosporine, and calyculin A treatments of tobacco cells (5 min). Of about 100 phospholabeled polypeptides, 19 showed increased 32P incorporation after 5 min of cryptogein treatment. Phosphorylation of 12 of the 19 polypeptides depended upon calcium influx. Staurosporine inhibited the phosphorylations induced by cryptogein whereas calyculin A activated the phosphorylation of 18 of these polypeptides. This study highlighted the role of PKs and/or constitutive active PPs whose activation and inhibition, respectively, resulted in an increased phosphorylation of proteins that may be involved in cryptogein signal transduction. Identification of the phosphoproteins is in progress and will increase our knowledge of signal transduction pathways implicated in plant defense responses.

scholarly journals The Endopolygalacturonase 1 from Botrytis cinerea Activates Grapevine Defense Reactions Unrelated to Its Enzymatic Activity

2003 ◽  
Vol 16 (6) ◽  
pp. 553-564 ◽  
Author(s):  
Benoît Poinssot ◽  
Elodie Vandelle ◽  
Marc Bentéjac ◽  
Marielle Adrian ◽  
Caroline Levis ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Calcium Influx ◽  
Active Oxygen Species ◽  
Degree Of Polymerization ◽  
Mass Spectrometry Analysis ◽  
Gene Transcript ◽  
Transcript Accumulation ◽  
Spectrometry Analysis ◽  
Defense Reactions

A purified glycoprotein from Botrytis cinerea(strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

scholarly journals cROStalk for Life: Uncovering ROS Signaling in Plants and Animal Systems, from Gametogenesis to Early Embryonic Development

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 525
Author(s):  
Valentina Lodde ◽  
Piero Morandini ◽  
Alex Costa ◽  
Irene Murgia ◽  
Ignacio Ezquer
Keyword(s):  
Signal Transduction ◽  
Signaling Pathways ◽  
Analytical Techniques ◽  
Antioxidant Systems ◽  
Developmental Studies ◽  
Science Field ◽  
Ros Signaling ◽  
Reproductive Processes

This review explores the role of reactive oxygen species (ROS)/Ca2+ in communication within reproductive structures in plants and animals. Many concepts have been described during the last years regarding how biosynthesis, generation products, antioxidant systems, and signal transduction involve ROS signaling, as well as its possible link with developmental processes and response to biotic and abiotic stresses. In this review, we first addressed classic key concepts in ROS and Ca2+ signaling in plants, both at the subcellular, cellular, and organ level. In the plant science field, during the last decades, new techniques have facilitated the in vivo monitoring of ROS signaling cascades. We will describe these powerful techniques in plants and compare them to those existing in animals. Development of new analytical techniques will facilitate the understanding of ROS signaling and their signal transduction pathways in plants and mammals. Many among those signaling pathways already have been studied in animals; therefore, a specific effort should be made to integrate this knowledge into plant biology. We here discuss examples of how changes in the ROS and Ca2+ signaling pathways can affect differentiation processes in plants, focusing specifically on reproductive processes where the ROS and Ca2+ signaling pathways influence the gametophyte functioning, sexual reproduction, and embryo formation in plants and animals. The study field regarding the role of ROS and Ca2+ in signal transduction is evolving continuously, which is why we reviewed the recent literature and propose here the potential targets affecting ROS in reproductive processes. We discuss the opportunities to integrate comparative developmental studies and experimental approaches into studies on the role of ROS/ Ca2+ in both plant and animal developmental biology studies, to further elucidate these crucial signaling pathways.

CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases

1996 ◽  
Vol 271 (2) ◽  
pp. C650-C657 ◽  
Author(s):  
W. W. Reenstra ◽  
K. Yurko-Mauro ◽  
A. Dam ◽  
S. Raman ◽  
S. Shorten
Keyword(s):  
Chloride Channel ◽  
Kinase Inhibitor ◽  
Maximal Activity ◽  
Calyculin A ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Nih 3T3 ◽  
Cystic Fibrosis Transmembrane

We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995] that genistein, a tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in NIH/3T3 cells that have been stably transfected with an expression vector for the CFTR (NIH-CFTR cells). In this study, we present evidence suggesting that both genistein and the serine/threonine protein phosphatase (PPase) inhibitor calyculin A activate the CFTR by inhibiting PPase activity. As measured by 125I efflux, genistein and calyculin A stimulate the CFTR to approximately 50% of the maximal activity with forskolin. Neither agonist increases CFTR activity at saturating forskolin concentrations, but genistein and calyculin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A(PKA) activity. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phosphorylation by all three agonists. Proteolytic digestion of in vivo phosphorylated CFTR suggests that the CFTR is phosphorylated on the same sites during stimulation with genistein and forskolin but on different sites stimulation with calyculin A. The data suggest that genistein and calyculin A inhibit different PPase activities, allowing CFTR phosphorylation and partial stimulation, by a basal PKA activity.

Overexpression of a directed mutant of 14-3-3ω in Arabidopsis leaves affects phosphorylation and protein content of nitrate reductaseThis paper is one of a selection published in a Special Issue comprising papers presented at the 50th Annual Meeting of the Canadian Society of Plant Physiologists (CSPP) held at the University of Ottawa, Ontario, in June 2008.

Botany ◽  
2009 ◽  
Vol 87 (7) ◽  
pp. 691-701 ◽  
Author(s):  
Man-Ho Oh ◽  
Joan L. Huber ◽  
Wei Shen ◽  
Gurdeep S. Athwal ◽  
Xia Wu ◽  
...  
Keyword(s):  
Defense Responses ◽  
Cellular Proteins ◽  
Signaling Proteins ◽  
Glutathione S Transferase ◽  
Independent Manner ◽  
Client Proteins ◽  
The University

The 14-3-3 family of proteins are highly conserved signaling proteins in eukaryotes that bind to their client proteins, usually through specific phosphorylated target sequences. While the 14-3-3 proteins are thought to interact with a wide array of cellular proteins, there have been few studies addressing the in-vivo role of 14-3-3. As one approach to study this in-vivo role, we generated transgenic Arabidopsis plants constitutively overexpressing a directed mutant of 14-3-3 isoform ω that inhibits phosphorylated nitrate reductase (pNR) in a largely divalent-cation-independent manner in vitro. The transgenic plants had increased relative phosphorylation of NR at the regulatory Ser-534 site and decreased NR activity measured in the presence of 5 mmol·L–1 MgCl2 relative to nontransgenic plants. In addition, total NR protein was increased and the protein half-life was increased about two-fold. Two-dimensional difference gel electrophoresis analysis of proteins extracted from leaves of plants expressing the mutant 14-3-3 identified numerous cellular proteins that were altered in abundance. In particular, several β-glucosidase and glutathione S-transferase isoforms were decreased in abundance relative to wild type plants suggesting a possible alteration in stress or defense responses.

scholarly journals Role of Escherichia coli Nitrogen Regulatory Genes in the Nitrogen Response of the Azotobacter vinelandiiNifL-NifA Complex

2001 ◽  
Vol 183 (10) ◽  
pp. 3076-3082 ◽  
Author(s):  
Francisca Reyes-Ramirez ◽  
Richard Little ◽  
Ray Dixon
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Nitrogen Control ◽  
Fixed Nitrogen ◽  
Nitrogen Response ◽  
E Coli ◽  
Nitrogen Excess

ABSTRACT The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (nif)-specific transcriptional activator NifA in Azotobacter vinelandii in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the A. vinelandii NifL-NifA system responds to fixed nitrogen in vivo is unknown, the glnK gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of A. vinelandii glnK in this response is difficult to establish because of the essential nature of this gene. We have shown previously that A. vinelandii NifL is able to respond to fixed nitrogen to control NifA activity when expressed inEscherichia coli. In this study, we investigated the role of the E. coli PII-like signal transduction proteins in nitrogen control of the A. vinelandii NifL-NifA regulatory system in vivo. In contrast to recent findings with Klebsiella pneumoniae NifL, our results indicate that neither the E. coli PII nor GlnK protein is required to relieve inhibition byA. vinelandii NifL under nitrogen-limiting conditions. Moreover, disruption of both the E. coli glnB andntrC genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that glnB ntrC and glnB glnK ntrC mutant strains accumulate high levels of intracellular 2-oxoglutarate under conditions of nitrogen excess. These findings are in accord with our recent in vitro observations (R. Little, F. Reyes-Ramirez, Y. Zhang, W. Van Heeswijk, and R. Dixon, EMBO J. 19:6041–6050, 2000) and suggest a model in which nitrogen control of the A. vinelandii NifL-NifA system is achieved through the response to the level of 2-oxoglutarate and an interaction with PII-like proteins under conditions of nitrogen excess.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Proteomic identification of the differentially expressed and phosphorylated proteins in 20-hydroxyecdysone (20E) signal transduction pathway in salivary gland of Drosophila melanogaster

2010 ◽  
Author(s):  
◽  
Yaning Sun
Keyword(s):  
Signal Transduction ◽  
Drosophila Melanogaster ◽  
Salivary Gland ◽  
Intracellular Localization ◽  
Signal Transduction Pathway ◽  
Phosphorylated Proteins ◽  
Rnai Technology ◽  
Pkc Isoforms ◽  
First Time

Protein kinase C (PKC) plays important role in 20-hydroxyecdysone (20E) signal transduction, however, little is known about the exact role of PKC in this process. In my research, PKC-regulated phosphorylation in 20E signal transduction is investigated in the salivary gland of Drosophila melanogaster. Our experiments demonstrate that PKCregulated phosphorylation is responsible for the intracellular localization of ecdysone receptor (EcR) and its heterodimeric partner, ultraspiracle protein (USP), which is possibly due to the forming of receptor complex with chaperons. We also confirmed PKC-regulated phosphorylation is required in 20E induced protein expression and identified 14 proteins induced by 20E but inhibited by PKC inhibitor. Using 2D Western blot and phospho-(Ser) PKC substrate, we were able to identify four phosphorylated PKC substrates in 20E signal transduction process, which may function in 20E-induced gene transcription/translation process or in ecdysteroid transporting. In addition, PKC isoforms in the salivary gland were also investigated by RNA interference (RNAi). For the first time, we showed the successful application of RNAi technology by soaking the salivary glands of D. melanogaster with dsRNAs.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Development ◽  
2021 ◽  
Vol 148 (6) ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela M. M. Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Specific Protein ◽  
Cellular Processes ◽  
Cell Compartments ◽  
Peptide Tags ◽  
Genetic Level ◽  
Protein Binders ◽  
And Function

ABSTRACT Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research, and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies have significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analysing the role of POIs in dynamic cellular processes, such as cell migration or cell division, would benefit from more-direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we have selected a number of short-tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POI binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be used for POI manipulation in vivo.

scholarly journals Piezo1 links mechanical forces to red blood cell volume

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Stuart M Cahalan ◽  
Viktor Lukacs ◽  
Sanjeev S Ranade ◽  
Shu Chien ◽  
Michael Bandell ◽  
...  
Keyword(s):  
Blood Cell ◽  
Calcium Influx ◽  
Mechanical Stretch ◽  
Conditional Knockout ◽  
Volume Control ◽  
Mechanical Forces ◽  
Gardos Channel

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. In this study, we show that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, we show that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis.

scholarly journals Role of type 1 and type 2A phosphatases in signal transduction of platelet-activating-factor-stimulated rabbit platelets

1994 ◽  
Vol 301 (2) ◽  
pp. 531-537 ◽  
Author(s):  
C T Murphy ◽  
J Westwick
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Platelet Activating Factor ◽  
Signal Transduction Pathway ◽  
Calyculin A ◽  
Signal Molecule ◽  
Kinase C

Calyculin A, the potent inhibitor of type 1 (PP1) and type 2A (PP2A) phosphatases, has been employed in order to investigate the role of endogenously activated PP1/PP2A in the signal-transduction pathway of platelet-activating-factor (PAF)-stimulated platelets. Calyculin A alone caused an increase in protein phosphorylation in unstimulated platelets, with the detection of a number of newly phosphorylated proteins, whereas in PAF-stimulated platelets phosphorylation of the major substrates of protein kinase C and myosin light-chain kinase were no longer transient, but phosphorylation was sustained. PP1/PP2A appear to play a role in Ca2+ homoeostasis, as inhibition of PP1/PP2A caused an inhibition of Ca2+ mobilization and Ca2+ influx through the plasma membrane in PAF-stimulated platelets. The effect of calyculin A on Ca2+ mobilization correlated with the observed inhibition of the production of the signal molecule Ins(1,4,5)P3. The release reaction (which is a Ca(2+)-dependent event) was also inhibited by calyculin A. The results are discussed in relation to the possible role of protein kinase C in mediating the events leading to the effects observed with calyculin A.

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